dexniguldipine and niguldipine

The biological behavior of osteosarcoma in dogs is similar to that in humans and the dog has been suggested as a model for the disease in humans. Because occult metastatic disease is common at presentation, systemic therapy is necessary. The dihydropyridine, dexniguldipine hydrochloride (B859-35), is a potent inhibitor of protein-kinase-C(PKC)-stimulated cell proliferation and has shown therapeutic activity in experimentally induced neuroendocrine hamster lung tumors and in a mammary cancer cell line. In human osteosarcoma cell lines, PKC activity can be down-regulated, resulting in increased sensitivity to cisplatin. Since these results supported the involvement of PKC inhibitors in the therapeutic management of osteosarcoma, we performed a prospective, randomized clinical trial using dogs with naturally occurring appendicular osteosarcoma to determine the therapeutic potential of dexniguldipine. Dogs received either no drug treatment (control group, n = 8), standard treatment (e.g., cisplatin, n = 14), or dexniguldipine treatment (n = 14) following amputation. Dexniguldipine- and cisplatin-treated dogs had a longer median remission duration and survival time than untreated dogs (P < 0.05); however, dexniguldipine-treated dogs had a shorter survival time than cisplatin-treated dogs (P < 0.05). The results of this study demonstrate that dexniguldipine has significant activity in the inhibition of canine osteosarcoma micrometastases. The identification of a tumor model that may be responsive to this class of antiproliferative agents warrants further clinical investigation to determine the optimum dosage of dexniguldipine and the role it may have in the therapeutic management of canine osteosarcoma.

Topics: Amputation, Surgical; Animals; Antineoplastic Agents; Bone Neoplasms; Cisplatin; Combined Modality Therapy; Dihydropyridines; Dogs; Female; Male; Osteosarcoma; Prospective Studies

Dexniguldipine (DNIG) is the R-enantiomer of the dihydropyridine derivate niguldipine. DNIG showed a binding affinity to the P-glycoprotein (P-gp) and therefore it is to be assumed to block the P-gp pumping mechanism. This open phase I study was conducted to determine the maximal tolerated dose (MTD) and safety of intravenously administered DNIG alone and in combination with vinblastine in patients with a metastatic or locally advanced cancer. Additionally, serum levels of DNIG were assessed and compared between dosage groups to investigate the intravenous dose linearity. The study was divided into two parts concerning DNIG administration. In part I the patients received DNIG for four hours daily over four consecutive days and additionally 0.15 mg/kg vinblastine at day 3. Treatment was started with 1 mg/kg/4h, and whenever the drug was well tolerated the dosage was increased. In part II the patients received up to three courses of a four-hour infusion (5 and 7 mg/kg/4h) of DNIG followed by a continuous infusion for 48 hours (5 and 7 mg/kg/24h). Twenty-six patients entered this trial and were given at least one infusion of DNIG; vinblastine was given immediately after the 4-hour infusion. One to seven courses and dosages from 1-11 mg/kg were administered. In five patients the dose limiting toxicity was seen in cardiovascular adverse events such as a drop in blood pressure, decreased heart rate and in one patient an AV block III. Most frequent adverse events were nausea, dizziness, vomiting, peripheral paresthesia, atactic gait, mild constipation, polyuria, hypocalcemia; all disappeared within 24 hours after discontinuation of infusion. A linear increase in DNIG serum concentration with increasing doses was found following intravenous infusion of DNIG over a four-hour period. Long-term infusion regimes over a period of two or five days resulted in reasonably constant DNIG serum levels. MTD was determined at 5 mg/kg/4h. It is to be assumed that the MTD for continuous infusion of DNIG is higher than 5 mg/kg/24h, but this was not followed up in the study and must be the aim of a later trial.

Topics: Adult; Aged; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; Dihydropyridines; Drug Therapy, Combination; Female; Humans; Infusions, Intravenous; Male; Middle Aged; Neoplasm Metastasis; Neoplasms; Vinblastine

Dexniguldipine-HCl is a new dihydropyridine compound that exerts selective antiproliferative activity in a variety of tumor models and, in addition, has a high potency in overcoming multidrug resistance. The purpose of this trial was to determine the toxicity and pharmacokinetics of dexniguldipine and to establish a recommended dose for phase II trials. A total of 37 patients with cancer were treated with oral dexniguldipine in increasing doses for up to 7 days. The main parameters evaluated were subjective tolerance and laboratory and cardiovascular parameters (blood pressure and ECG). Blood samples were drawn for analysis of the drug's pharmacokinetics. Dizziness and nausea were the major adverse events observed in seven patients, but episodes were generally mild and not clearly dose-related. Vomiting occurred in one patient. Hypotensive effects and orthostatic dysregulation were observed in some patients but were not considered to be dose-limiting. Therefore, no dose-limiting toxicity was found and the maximally tolerable dose could not be determined. Pharmacokinetic data showed wide interindividual variation and a dose-dependent increase in steady-state serum concentrations at doses of up to 1,000 mg daily, with no clear further increase being observed at higher doses. Consistently high concentrations were achieved with the 2,500-mg dose. Despite the lack of dose-limiting toxicity, higher doses of dexniguldipine do not appear to be useful for clinical evaluation because of the pharmacokinetic properties of the compound: therefore, 2,500 mg/day is recommended as the daily dose for phase II trials.

Topics: Administration, Oral; Antineoplastic Agents; Clinical Trials, Phase II as Topic; Dihydropyridines; Dose-Response Relationship, Drug; Headache; Humans; Metabolic Clearance Rate; Nausea; Neoplasms; Vertigo

Dexniguldipine-HCl is a new dihydropyridine derivative with antineoplastic activity and potency for overcoming multidrug resistance. In this pharmacokinetic study the bioavailability of 3 doses of an oral formulation of dexniguldipine was to be determined. Fourteen patients with malignant disease not eligible for higher priority treatment and sufficient general condition were included. In 12 patients all pharmacokinetic investigations were available for evaluation. A single 4-h infusion of 2 mg per kg body weight of dexniguldipine was given as reference. Thereafter 3 increasing oral dosages (750, 1,500, 2,250 mg/d) were given on a 3-time daily basis for 3 consecutive weeks. On day 7 (under steady state conditions) of each period, a pharmacokinetic profile was done. Absolute bioavailability at the 3-dose levels was 3, 4, and 5%, respectively, thus slightly increasing with dose, but generally low. After intravenous administration terminal half life was 22.4 h, clearance 36.9 l/h and volume of distribution 1,193 1. Toxicity was tolerable with main adverse events being loss of appetite, nausea, and vomiting. Cardiovascular effects and a decrease in serum calcium were reported in several patients. Patients were allowed to continue treatment if a benefit was expected, and 2 patients showed tumor regression during treatment. One patient with renal cell carcinoma achieved a partial remission. Bioavailability of this oral formulation seems too low for routine clinical use, despite the fact that clinical effects have been observed.

Topics: Administration, Oral; Adolescent; Adult; Aged; Antineoplastic Agents; Biological Availability; Dihydropyridines; Dose-Response Relationship, Drug; Drug Evaluation; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Female; Humans; Infusions, Intravenous; Male; Middle Aged; Neoplasms; Reference Standards; United States; World Health Organization

Topics: Blood Pressure; Calcium Channel Blockers; Chronic Disease; Dihydropyridines; Diltiazem; Dose-Response Relationship, Drug; Drug Therapy, Combination; Female; Humans; Hypertension; Male; Middle Aged; Nifedipine; Stroke Volume

We studied the effects after single doses of niguldipine (0.3, 0.6, and 0.9 mg intravenously; 8 and 16 mg orally) and nifedipine (2 mg intravenously; 20 mg orally) in healthy male volunteers in randomized placebo-controlled experiments. Total peripheral resistance (TPR), heart rate-corrected electromechanical systole (QS2c), and preejection period (PEPc) were assessed noninvasively. Both drugs induced a similar pronounced decreased in TRP, indicating peripheral vasodilation, followed by increasing heart rate and cardiac output, a decrease in diastolic blood pressure, and a shortening of the PEPc. QS2c was unchanged after niguldipine. The prolongation of QS2c after oral nifedipine is suggestive of a negative inotropic effect. We conclude that the vasodilatory effects of dihydropyridines may (as for nifedipine) or may not (as for niguldipine) be associated with changes that are suggestive of negative inotropic effects, and that this difference is detectable by noninvasive methods in healthy subjects.

Topics: Administration, Oral; Adult; Blood Pressure; Calcium Channel Blockers; Cardiac Output; Dihydropyridines; Dose-Response Relationship, Drug; Heart Rate; Humans; Infusions, Intravenous; Male; Nifedipine

Glioma-initiating cells (GIC) are considered the underlying cause of recurrences of aggressive glioblastomas, replenishing the tumor population and undermining the efficacy of conventional chemotherapy. Here we report the discovery that inhibiting T-type voltage-gated Ca

Topics: Amino Acids; Animals; Biological Transport; Brain Neoplasms; Calcium Channel Blockers; Calcium Channels, T-Type; Cell Death; Cell Line, Tumor; Dihydropyridines; Glioma; Humans; Mice; Mycotoxins; Neoplastic Stem Cells; Potassium Channels, Calcium-Activated; Proteomics; Sodium; Unfolded Protein Response

The α1-adrenergic receptors (α1-ARs), which belong to a G protein-coupled receptor family, consist of three highly homologous subtypes known as α1A-ARs, α1B-ARs, and α1D-ARs. Our previous findings suggested that α1A-ARs are an important target for imipramine and electroconvulsive therapy. The current study sought to evaluate whether S-(+)-niguldipine and B8805-033, two selective antagonists of α1A-ARs, can evoke antidepressant-like effects in the forced swim test in rats. Both compounds were administered at three time points (24, 5, and 1 h before testing), and the effects of three doses (2, 5, and 10 mg/kg) of each compound were investigated. S-(+)-Niguldipine produced no antidepressant-like effects other than a 14% reduction in immobility time at the highest dose. Although B8805-033 at a dose of 2 mg/kg did not influence the rats' behavior, higher B8805-033 doses (5 and 10 mg/kg) produced significant reductions in immobility time (approximately 42 and 44% vs. controls, respectively; P<0.01). However, this effect was abolished by the concomitant administration of WAY100135, a serotonin receptor antagonist, suggesting that the observed antidepressant-like effects of B8805-033 are unrelated to α1A-ARs. Nevertheless, given the current dearth of selective α1A-AR agonists, the question of whether this particular subtype could be involved in antidepressant therapy mechanisms remains unresolved.

Topics: Adrenergic alpha-1 Receptor Antagonists; Animals; Antidepressive Agents; Dihydropyridines; Dioxins; Disease Models, Animal; Dose-Response Relationship, Drug; Male; Piperazines; Pyrimidinones; Rats; Rats, Wistar; Receptors, Adrenergic, alpha-1; Swimming; Time Factors

In whole cell patch-clamp recordings, we characterized the L-type Ca(2+) currents in bovine adrenal zona fasciculata (AZF) cells and explored their role, along with the role of T-type channels, in ACTH- and angiotensin II (ANG II)-stimulated cortisol secretion. Two distinct dihydropyridine-sensitive L-type currents were identified, both of which were activated at relatively hyperpolarized potentials. One activated with rapid kinetics and, in conjunction with Northern blotting and PCR, was determined to be Cav1.3. The other, expressed in approximately one-half of AZF cells, activated with extremely slow voltage-dependent kinetics and combined properties not previously reported for an L-type Ca(2+) channel. The T-type Ca(2+) channel antagonist 3,5-dichloro-N-[1-(2,2-dimethyl-tetrahydro-pyran-4-ylmethyl)-4-fluoro-piperidin-4-ylmethyl]-benzamide (TTA-P2) inhibited Cav3.2 current in these cells, as well as ACTH- and ANG II-stimulated cortisol secretion, at concentrations that did not affect L-type currents. In contrast, nifedipine specifically inhibited L-type currents and cortisol secretion, but less effectively than TTA-P2. Diphenylbutylpiperidine Ca(2+) antagonists, including pimozide, penfluridol, and fluspirilene, and the dihydropyridine niguldipine blocked Cav3.2 and L-type currents and inhibited ACTH-stimulated cortisol secretion with similar potency. This study shows that bovine AZF cells express three Ca(2+) channels, the voltage-dependent gating and kinetics of which could orchestrate complex mechanisms linking peptide hormone receptors to cortisol secretion through action potentials or sustained depolarization. The function of the novel, slowly activating L-type channel is of particular interest in this respect. Regardless, the well-correlated selective inhibition of T- and L-type currents and ACTH- and ANG II-stimulated cortisol secretion by TTA-P2 and nifedipine establish the critical importance of these channels in AZF cell physiology.

Topics: Action Potentials; Adrenocorticotropic Hormone; Angiotensin II; Animals; Benzamides; Calcium; Calcium Channel Blockers; Calcium Channels, L-Type; Calcium Channels, T-Type; Cattle; Cyclic AMP; Dihydropyridines; Fluspirilene; Gene Expression; Hydrocortisone; Microelectrodes; Nifedipine; Patch-Clamp Techniques; Penfluridol; Pimozide; Piperidines; Protein Isoforms; Single-Cell Analysis; Zona Fasciculata

Premature ejaculation is one of the most common male sexual dysfunctions. Current pharmacological treatments involve reduction in penile sensitivity by local anesthetics or increase of ejaculatory threshold by selective serotonin reuptake inhibitors. α1-Adrenoceptors (α1-ARs) and L-type calcium channels are expressed in the smooth muscles of the male reproductive tract, and their activations play an important role in the physiological events involved in the seminal emission phase of ejaculation.. To evaluate if the inhibition of the contractility of the vas deferens and seminal vesicle by α1-AR antagonism or the L-type calcium channel blockade can delay ejaculation.. The effects of the α1-AR antagonist tamsulosin and of the L-type calcium channel blockers, nifedipine and (S)-(+)-niguldipine, on contractions induced by norepinephrine in the rat vas deferens and seminal vesicles in vitro and on the ejaculation latency of male rats in behavioral mating tests were evaluated.. Tension development of vas deferens and seminal vesicles in response to norepinephrine in vitro and behavioral mating parameters were quantified.. Tension development of vas deferens and seminal vesicle to α1-AR activation was significantly inhibited by tamsulosin, nifedipine, and (S)-(+)-niguldipine. Tamsulosin displayed insurmountable antagonism of contractions induced by norepinephrine in the rat vas deferens and seminal vesicle. Ejaculation latency of male rats was not modified by tamsulosin, nifedipine, or (S)-(+)-niguldipine; however, both the number and weight of the seminal plugs recovered from female rats mated with male rats treated with tamsulosin were significantly reduced.. Seminal emission impairment by inhibition of vas deferens or seminal vesicle contractility by L-type calcium channel blockade or α1-AR antagonism is not able to delay the ejaculation.

Topics: Adrenergic alpha-1 Receptor Antagonists; Animals; Calcium Channel Blockers; Calcium Channels, L-Type; Dihydropyridines; Dose-Response Relationship, Drug; Ejaculation; Male; Muscle Contraction; Muscle, Smooth; Nifedipine; Norepinephrine; Rats; Rats, Wistar; Receptors, Adrenergic, alpha-1; Seminal Vesicles; Sulfonamides; Tamsulosin; Vas Deferens

Canine atrial cardiomyocytes display a constitutively active, acetylcholine-regulated, time-dependent K+ current (IKH) that contributes to atrial repolarization and atrial tachycardia-induced atrial-fibrillation promotion. Adrenergic stimulation favors atrial arrhythmogenesis but its effects on IKH are poorly understood.. Adrenergic modulation of IKH was studied in isolated canine atrial cardiomyocytes with whole-cell patch-clamping, and action-potential consequences were assessed in multicellular preparations with fine-tipped microelectrodes. Isoproterenol increased IKH in a concentration-dependent manner (maximum 103+/-22% increase), an effect mimicked by forskolin and 8-bromo-cyclic AMP. Isoproterenol effects were prevented by propranolol and the selective beta1-adrenoceptor blocker CGP-20712A, but not the beta2-blocker ICI-118551. Isoproterenol enhancement was prevented by pipette-administered protein kinase A (PKA) inhibitor peptide or by superfusion of H89 (PKA blocker). Phenylephrine decreased IKH in a reversible, concentration-dependent way. This effect was blocked by the alpha-antagonist prazosin and the selective alpha1A-blocker niguldipine, but not the alpha1B-blocker chloroethylclonidine or the alpha1D inhibitor BMY-7378. Phenylephrine effects were prevented by the phospholipase C (PLC) inhibitor U73122 and the protein kinase C (PKC) inhibitor bisindolylmaleimide. The PKC-activating phorbol ester PDD (but not its inactive analogue alpha-PDD) mimicked phenylephrine effects. Action potential recordings in the presence and absence of the selective IKH blocker tertiapin indicated a functional role of alpha- and beta-adrenergic actions on IKH. Adrenergic regulation of cholinergic agonist-induced K+ current paralleled that of IKH.. IKH is under dual regulation by the adrenergic system: beta1-adrenergic stimulation enhances IKH via cAMP-dependent PKA pathways, whereas alpha1A-adrenergic stimulation inhibits IKH via PLC-mediated PKC activation. Modulation of constitutive acetylcholine-regulated K+ current is a novel potential mechanism for adrenergic control of atrial repolarization.

Topics: Acetylcholine; Action Potentials; Adrenergic Agents; Adrenergic alpha-Agonists; Adrenergic alpha-Antagonists; Animals; Bee Venoms; Clonidine; Cyclic AMP-Dependent Protein Kinases; Dihydropyridines; Dogs; Dose-Response Relationship, Drug; Estrenes; Heart Atria; Imidazoles; Indoles; Isoproterenol; Isoquinolines; Maleimides; Myocytes, Cardiac; Patch-Clamp Techniques; Phenylephrine; Phorbol 12,13-Dibutyrate; Piperazines; Potassium Channels; Prazosin; Propanolamines; Propranolol; Protein Kinase C; Pyrrolidinones; Receptors, Adrenergic, alpha-1; Receptors, Adrenergic, beta-1; Sulfonamides; Type C Phospholipases

Efflux of cytotoxic agents mediated by P-glycoprotein is believed to be an important mechanism of multidrug resistance, which remains a serious limitation to successful chemotherapy in cancers such as metastatic breast cancer. A series of 4-aryl-1,4-dihydropyridines and corresponding aromatized 4-arylpyridines have been synthesized based on structure modifications of niguldipine to enhance multidrug resistance reversal activity, while minimizing calcium channel binding. Thirty new compounds were characterized. [(3)H]Vinblastine accumulation studies indicated that at a concentration level of 3 muM, 15 of 18 4-aryl-1,4-dihydropyridines and all 4-arylpyridines can successfully restore intracellular accumulation of vinblastine in a resistant human breast adenocarcinoma cell line, MCF-7/adr, which overexpresses P-glycoprotein. The most potent compounds led to an approximately 15-fold increase of vinblastine accumulation. All of the test compounds that significantly increased vinblastine accumulation in MCF/adr cells were able to substantially reduce IC(50) values of daunomycin and increase its cytotoxicity in MCF-7/adr-resistant cells, confirming the results of the vinblastine accumulation studies. Calcium channel binding assays for these newly synthesized compounds were conducted using rat cerebral cortex membrane. All but eight compounds demonstrated negligible calcium channel binding over the concentration range from 15 to 2500 nM. The results demonstrate that the newly synthesized series of 1,4-dihydropyridines and pyridines represent P-glycoprotein modulators with negligible calcium channel blocking activity.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Binding, Competitive; Calcium Channel Blockers; Calcium Channels; Cell Line, Tumor; Cerebral Cortex; Daunorubicin; Dihydropyridines; Humans; In Vitro Techniques; Pyridines; Rats; Structure-Activity Relationship; Vinblastine

There is evidence that some calcium (Ca(2+)) channel inhibitors enhance the protective activity of antiepileptic drugs. Since clinical trials have not provided consistent data on this issue, the objective of this study was to evaluate the interaction of a dihydropyridine, niguldipine, with conventional antiepileptics in amygdala-kindled rats. Niguldipine (at 7.5 but not at 5 mg/kg) displayed a significant anticonvulsant effect, as regards seizure and afterdischarge durations in amygdala-kindled convulsions in rats, a model of complex partial seizures. No protective effect was observed when niguldipine (5 mg/kg) was combined with antiepileptics at subeffective doses, i.e. valproate (75 mg/kg), diphenylhydantoin (40 mg/kg), or clonazepam (0.003 mg/kg). Unexpectedly, the combined treatment of niguldipine (5 mg/kg) with carbamazepine (20 mg/kg) or phenobarbital (20 mg/kg) resulted in a proconvulsive action. BAY k-8644 (an L-type Ca(2+) channel activator) did not modify the protective activity of niguldipine (7.5 mg/kg) or the opposite action of this dihydropyridine (5 mg/kg) in combinations with carbamazepine or phenobarbital. A pharmacokinetic interaction is not probable since niguldipine did not affect the free plasma levels of the antiepileptics. These data indicate that the opposite actions of niguldipine alone or combined with carbamazepine (or phenobarbital) were not associated with Ca(2+) channel blockade. The present results may argue against the use of niguldipine as an adjuvant antiepileptic or for cardiovascular reasons in patients with complex partial seizures.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Amygdala; Animals; Anticonvulsants; Calcium Channel Agonists; Calcium Channel Blockers; Carbamazepine; Dihydropyridines; Drug Combinations; Injections, Intraperitoneal; Kindling, Neurologic; Male; Phenobarbital; Rats; Rats, Wistar; Seizures

Human P-glycoprotein (P-gp), an integral membrane transport protein, is responsible for the efflux of various drugs, including cytostatics from cancer cells leading to multidrug resistance. P-gp is composed of two homologous half domains, each carrying one nucleotide binding site. The drug extrusion is ATP-dependent and can be inhibited by chemosensitizers, such as the dihydropyridine derivative dexniguldipine-HCl, through direct interaction with P-gp. To evaluate the mechanism(s) of chemosensitization and identify the binding sites of dexniguldipine-HCl, a tritium-labeled azido analog of dexniguldipine, [(3)H]B9209-005, was used as a photoaffinity probe. Using the multidrug resistant T-lymphoblastoid cell line CCRF-ADR5000, two proteins were specifically labeled in membranes by [(3)H]B9209-005. These proteins were identified by immunoprecipitation such as P-gp and its N-terminal fragment. The membranes were solubilized and the labeled P-gp proteins first isolated by lectin-chromatography and then digested with trypsin. SDS-polyacrylamide gel electrophoresisanalysis of the digest revealed a major radioactive 7-kDa fragment. The tryptic fragments were separated by high-performance liquid chromatography and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The MS results, corroborated by MALDI-MS of peptides after one step of Edman analysis, identified the radioactive 7-kDa band as the dexniguldipine-bound, tryptic P-gp peptide, 468-527. This sequence region is flanked by the Walker motifs A and B of the N-terminal ATP-binding cassette suggesting direct interaction of the chemosensitizer with the nucleotide binding site is involved in the mechanism of chemosensitization.

Topics: Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; Azides; Binding Sites; Chromatography, High Pressure Liquid; Dihydropyridines; Humans; Mass Spectrometry; Peptide Fragments; Photoaffinity Labels; Tritium; Trypsin; Tumor Cells, Cultured

Although agonist binding in adrenergic receptors is fairly well understood and involves residues located in transmembrane domains 3 through 6, there are few residues reported that are involved in antagonist binding. In fact, a major docking site for antagonists has never been reported in any G-protein coupled receptor. It has been speculated that antagonist binding is quite diverse depending upon the chemical structure of the antagonist, which can be quite different from agonists. We now report the identification of two phenylalanine residues in transmembrane domain 7 of the alpha(1a)-adrenergic receptor (Phe-312 and Phe-308) that are a major site of antagonist affinity. Mutation of either Phe-308 or Phe-312 resulted in significant losses of affinity (4-1200-fold) for the antagonists prazosin, WB4101, BMY7378, (+) niguldipine, and 5-methylurapidil, with no changes in affinity for phenethylamine-type agonists such as epinephrine, methoxamine, or phenylephrine. Interestingly, both residues are involved in the binding of all imidazoline-type agonists such as oxymetazoline, cirazoline, and clonidine, confirming previous evidence that this class of ligand binds differently than phenethylamine-type agonists and may be more antagonist-like, which may explain their partial agonist properties. In modeling these interactions with previous mutagenesis studies and using the current backbone structure of rhodopsin, we conclude that antagonist binding is docked higher in the pocket closer to the extracellular surface than agonist binding and appears skewed toward transmembrane domain 7.

Topics: Adrenergic alpha-1 Receptor Antagonists; Adrenergic alpha-Antagonists; Amino Acid Sequence; Animals; Cell Membrane; Clonidine; Conserved Sequence; Cricetinae; Dihydropyridines; Dioxanes; Humans; Imidazoles; Models, Molecular; Molecular Sequence Data; Oxymetazoline; Phenylalanine; Piperazines; Prazosin; Protein Structure, Secondary; Rats; Receptors, Adrenergic, alpha-1; Structure-Activity Relationship

Various studies have shown that stressful manipulations in rats and mice lower the convulsant potency of GABA-related, but also some GABA-unrelated convulsants. The mechanism of this anticonvulsive effect of stress is still unknown.. We tested the possible involvement of alpha2-adrenoceptors in the previously observed anticonvulsive effect of swim stress.. The mice were, prior to exposure to swim stress and the IV infusion of picrotoxin, pre-treated with clonidine (an alpha2-adrenoceptor agonist), yohimbine (a non-selective alpha2-adrenoceptor antagonist), idazoxan (a selective alpha2-adrenoceptor antagonist), or niguldipine (an alpha1-adrenoceptor antagonist), and the latency to the onset of two convulsant signs was registered.. In control unstressed animals clonidine (0.1 and 1 mg/kg IP), yohimbine (2 mg/kg IP) and idazoxan (1 mg/kg IP) failed to affect the doses of picrotoxin needed to produce convulsant signs, while niguldipine (5 mg/kg IP) prolonged the latency, i.e. it enhanced the doses of picrotoxin producing running/bouncing clonus and tonic hindlimb extension. In swim stressed mice clonidine enhanced, while idazoxan decreased doses of picrotoxin needed to produce two convulsive signs. Yohimbine decreased the dose of convulsant needed to produce tonic hindlimb extension, while niguldipine enhanced doses of picrotoxin needed to produce both symptoms.. The results demonstrate the alpha2-adrenoceptor agonist-induced potentiation and alpha2-adrenoceptor antagonist-induced diminution of the anticonvulsive effect of stress. Additionally, they show the anticonvulsive effect of niguldipine in unstressed and stressed animals. Hence, the results suggest that alpha2-adrenoceptors are involved in the anticonvulsive effect of swim stress in mice.

Topics: Adrenergic Agonists; Adrenergic alpha-2 Receptor Agonists; Adrenergic alpha-2 Receptor Antagonists; Adrenergic Antagonists; Animals; Convulsants; Dihydropyridines; GABA-A Receptor Antagonists; Idazoxan; Infusions, Intravenous; Injections, Intraperitoneal; Male; Mice; Mice, Inbred CBA; Picrotoxin; Receptors, Adrenergic, alpha-2; Seizures; Stress, Physiological; Swimming

Recent results show that the intracellular uptake pattern of idarubicin (IDA) in multidrug-resistant (MDR) cells is nearly identical to that seen in the drug-sensitive parent cell line, whereas the MDR cells have minimal daunorubicin (DNR) uptake compared with the drug-sensitive parent cells. It is known that the major metabolite of IDA, idarubicinol (IDA-OL), has nearly the same cytotoxicity as IDA, while the cytotoxicity of daunorubicinol (DNR-OL) is about 1/30th of that of DNR. We examined the effect of the MDR modifiers verapamil and dexniguldipine on the efflux of IDA, DNR and their hydroxylated metabolites IDA-OL and DNR-OL in blast populations of acute myeloid leukemia (AML), in the MDR-negative cell line CEM-CCRF and in their MDR-positive counterpart (CEM-VBL). All patients with relapsed or persistent AML had been pretreated with IDA and cytosine arabinoside. The efflux of the anthracyclines was estimated by flow cytometry. A total of 36 patients with AML were investigated; 18 out of 36 AML blast populations showed an efflux of DNR, DNR-OL and IDA-OL. The efflux of DNR, DNR-OL and particularly IDA-OL could be reversed by 10 microM verapamil or 1 microM dexniguldipine. For IDA we found an effusion of 40 +/- 11% in all blast populations which could not be significantly inhibited by the modulators. Similar results for IDA were found in the MDR-positive cell line (CEM-VBL 100) and in their MDR-negative counterpart (CEM-CCRF). The incubation of CEM-CCRF cells with DNR, DNR-OL, IDA-OL and especially IDA led to MDR induction as determined by reverse transcription/polymerase chain reaction analysis with MDR-specific primer and by cellular efflux studies. We conclude that the outcome of chemotherapy with idarubicin is influenced by MDR, although IDA is not essentially MDR-dependent itself, but because IDA-OL is actively involved in multidrug resistance. Further investigations should consider the question of whether the combination of IDA and MDR modifiers can enhance the serum level of the active metabolite IDA-OL and can reverse the MDR pattern in cells treated with IDA.

Topics: Antineoplastic Agents; Blast Crisis; Calcium Channel Blockers; Daunorubicin; Dihydropyridines; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Humans; Idarubicin; Leukemia, Myeloid, Acute; Tumor Cells, Cultured; Verapamil

P-glycoprotein (P-gp) is a 170 kDa ATPase which can transport a wide range of natural product cytotoxic drugs out of cells, thus conferring the multidrug resistance (MDR) phenotype.. In this paper we used the 1,4-dihydropyridine (1,4-DHP) MDR-reversing agent dexniguldipine (DN), and a derivative with a quaternary nitrogen which is permanently charged, N-methyl-DN, to explore the sidedness of block of [3H]-vinblastine transport by P-gp.. In cytotoxicity assays, 1 microM DN sensitized MCF7 ADR cells, causing a 13-fold decrease in the EC50 of vinblastine from 400 +/- 80 nM to 30 +/- 25 nM. In marked contrast, N-methyl-DN was without effect. In intact MCF7 ADR cells, DN reversed the [3H]vinblastine uptake deficit with an EC50 of 445 +/- 100 nM, again, N-methyl-DN was inactive. In photoaffinity labelling studies using the arylazide [3H]-B9209-005 in whole cells, DN potently inhibited incorporation of the photoaffinity label into P-gp whilst N-methyl-DN was without effect. However, in photoaffinity labelling studies in membrane fragments, both DN and N-methyl-DN potently inhibited [3H]-B9209-005 photoaffinity labelling of P-gp. Furthermore, in membrane fragments [3H]-vinblastine binding to P-glycoprotein was potently inhibited by both N-methyl-DN (Ki 10.7 +/- 4.9 nM) and DN (Ki 11.2 +/- 3.8 nM), and both N-methyl-DN and DN blocked ATP-dependent [3H]-vinblastine transport into inside-out vesicles. Thus, in intact cells the permanently charged 1,4-dihydropyridine, N-methyl-DN is unable to reverse the MDR phenotype or photoaffinity labelling of P-gp. However, in cell fragments and inside-out vesicles, N-methyl-DN binds avidly to P-gp and this binding blocks [3H]-vinblastine transport.. These data are consistent with the hypothesis that 1,4-DHPs block [3H]-vinblastine binding, and thereby transport by P-gp, by acting at a domain accessible only from the cytoplasm.

Topics: Affinity Labels; Animals; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP-Binding Cassette Transporters; Azides; Binding Sites; Cell Division; Cell Membrane; Cells, Cultured; Dihydropyridines; Drug Interactions; Structure-Activity Relationship; Synaptic Vesicles; Vinblastine

Influence of JTH-601 [N-(3-hydroxy-6-methoxy-2,4,5-trimethylbenzyl)-N-methyl-2-(4-hydroxy-2-isopropyl-5-methylphenoxy)ethylamine hemifumarate], a selective alpha1-adrenoceptor antagonist, on alpha1-mediated positive inotropic effect (PIE) was studied in isolated rabbit papillary muscle (1 Hz at 37 degrees C). JTH-601 (0.1-10 microM) shifted the concentration-response curve (CRC) for PIE of phenylephrine mediated by alpha1-adrenoceptor (with timolol at 1 microM) to the right and downward. In the presence of 100 nM WB 4101, an alpha1A antagonist, the shift to the right disappeared and JTH-601 (1-3 microM) shifted CRC for phenylephrine downward. The antagonistic action of JTH-601 was unchanged by 100 nM (+)-niguldipine, another alpha1A antagonist. Following pretreatment with 10 microM chloroethylclonidine, an alpha1B antagonist, the shift of CRC for phenylephrine to the right disappeared and JTH-601 (3-10 microM) shifted CRC downward. Antagonistic action of JTH-601 (3 microM) was unaltered by 100 nM BMY 7378, an alpha1D antagonist. JTH-601 (10 microM) had no effect on beta-mediated PIE of isoproterenol. These results indicate that JTH-601 exerts an inhibitory action on alpha1-mediated PIE through antagonism of alpha1A- and/or alpha1B-adrenoceptors in rabbit ventricular myocardium. As an alpha1 antagonist, JTH-601 is much less potent in rabbit ventricular muscle than in smooth muscle.

Topics: Adrenergic alpha-Antagonists; Adrenergic beta-Agonists; Animals; Cresols; Depression, Chemical; Dihydropyridines; Dioxanes; In Vitro Techniques; Isoproterenol; Male; Myocardial Contraction; Papillary Muscles; Piperazines; Rabbits; Receptors, Adrenergic, alpha-1

A convenient functional assay of the multidrug resistance (MDR) pump is useful for the diagnosis of MDR-1 cancers and the quantitative determination of the potency of inhibitors of the pump. Calcein-AM, a substrate of the MDR pump, was used to determine the concentration of SDZ PSC833 needed to completely inhibit the pump in CEM/VLB100 drug-resistant cells. The initial rates (in percent) for calcein retention by these MDR-1 cells were used to calculate values for the percent initial efflux of calcein-AM through the MDR pump in the presence of the inhibitors PSC833, cyclosporinA, and dexniguldipine. The percent efflux values at 250 and 60 nM calcein-AM were used to calculate the required concentration of each inhibitor to produce half-inhibition (I50) of initial efflux through the pump. These results are consistent with a noncompetitive inhibition of the MDR pump by each of the three inhibitors.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Cyclosporine; Cyclosporins; Dihydropyridines; Drug Resistance, Neoplasm; Fluoresceins; Half-Life; Humans; Inhibitory Concentration 50; Kinetics; Leukemia; Lymphocytes; Molecular Weight; Tumor Cells, Cultured

The human U373 MG astrocytoma cell line has been widely used as a model system for the investigation of astrocyte function. The aim of this study was to establish which alpha1-adrenoceptors are present on these cells. The specific binding of [3H]prazosin to membranes of U373 MG cells (Bmax 32+/-3 fmol mg(-1) protein, Kd 0.27+/-0.03 nM) was inhibited in a monophasic manner by alpha1-antagonists that have different affinities for alpha1A-, alpha1B- and alpha1D-adrenoceptors. Estimates for pKi values were: prazosin 9.69+/-0.06, 5-methylurapidil 7.10+/-0.21; (+)-niguldipine 7.06+/-0.26; WB 4101 8.26+/-0.16; and BMY 7378 6.60+/-0.21. The specific binding of [3H]prazosin was reduced to low levels by pretreatment of cells with 10 microM chloroethylclonidine for 15 min. In the presence of 30 mM LiCl, 100 microM noradrenaline stimulated [3H]inositol phosphate accumulation by 2.1+/-0.1-fold of basal after 30-min incubation. The EC50 for the accumulation of [3H]IP1, the major product detected (85+/-2% of total [3H]IP1 + [3H]IP2 + [3H]IP3), was 0.38+/-0.05 microM. Noradrenaline-induced [3H]IP1 accumulation was also inhibited by alpha1-antagonists. Estimates for pKi values were: 5-methylurapidil 6.95+/-0.01; WB 4101 8.31+/-0.07; and BMY 7378 6.71+/-0.28. The accumulation of [3H]IP1 in response to 100 microM noradrenaline was not significantly affected by raising the extracellular Ca2+ concentration from 1.3 to 4 mM. Noradrenaline (100 microM) also produced an increase in intracellular Ca2+ (mean peak 86+/-5 nM above basal). Pretreatment with chloroethylclonidine (10 microM, 15 min) abolished noradrenaline-induced [3H]IP1 accumulation and Ca2+ mobilisation. Activation of the alpha1B-adrenoceptors by 10 microM phenylephrine increased [3H]thymidine uptake to 140+/-5% of control uptake. Taken together, these results indicate that U373 MG cells express a single class of alpha1-adrenoceptors, the alpha1B-subtype, which are coupled to phosphoinositide hydrolysis and calcium mobilisation, and which mediate a mitogenic response to alpha1-agonists.

Topics: Adrenergic alpha-Antagonists; Astrocytoma; Calcium; Cell Membrane; Clonidine; Dihydropyridines; Dioxanes; Dose-Response Relationship, Drug; Humans; Inositol Phosphates; Norepinephrine; Phenylephrine; Piperazines; Prazosin; Receptors, Adrenergic, alpha-1; Thymidine; Tumor Cells, Cultured

1. The effects of alpha1 adrenoceptor blocking agents doxazosin, indoramin, 5-methylurapidil, niguldipine, WB-4101 and chloroethylclonidine (CEC) on the force of contraction (Fc), velocity of contraction (+dF/dt) and relaxation (-dF/dt) of guinea pig papillary muscles were studied. 2. All examined substances were applied in a wide concentration range (0.01-30.0 microM) for at least 30 min at each concentration. Only alpha1a blockers [i.e., niguldipine (0.01-0.3 microM), 5-methylurapidil (1-30 microM) and WB-4101 (1-30 microM)] showed a concentration-dependent negative inotropic action. 3. This effect was significantly attenuated in the presence of glibenclamide (1 microM) and almost completely abolished by 1,3-dipropyl-8-p-sulfophenylxanthine (1 microM), an antagonist of adenosine receptors with a slight selectivity for the A1 subtype. 4. Pretreatment with dibenamine, an irreversible blocker of alpha1 adrenoceptors (0.6 microM for 40 min), abolished this effect, whereas pretreatment with CEC, an irreversible blocker of alpha1b adrenoceptors (1 microM for 20 min), and pertussis toxin (10 microg/kg IP, 4 to 5 days before experiments) diminished it. 5. The alpha1a adrenoceptor blocking agents in the presence of the unblocked alpha1b adrenoceptor trigger the negative inotropic action, which seems to include adenosine receptor stimulation and activation of ATP-sensitive K+ channels (K[ATP]) through an inhibitory G protein.

Topics: Adenosine; Adrenergic alpha-Antagonists; Animals; Depression, Chemical; Dihydropyridines; Dioxanes; Female; Guinea Pigs; In Vitro Techniques; Male; Myocardial Contraction; Piperazines; Potassium Channels; Receptors, Adrenergic, alpha-1

We have used a sensitive bioassay of calcium-mediated volume changes in mammalian absorptive intestinal epithelial cells to screen extracts of the skin of the amphibian Xenopus laevis for the presence of factors affecting ion transport. A 66-residue peptide, purified using reversed-phase high performance liquid chromatography techniques, caused isotonic volume reduction of guinea pig jejunal villus cells in suspension. This volume reduction required extracellular Ca2+ and was prevented by the dihydropyridine-sensitive Ca2+ channel blocker niguldipine. Structural analysis demonstrated the presence of eight cysteines and a primary structure homologous to that of the neurotoxin/cytotoxin family found in the venom of certain poisonous snakes. The structure of the peptide was identical to that of xenoxin-1 purified from dorsal gland secretions of X. laevis (Kolbe, M., Huber A., Cordier, P., Rasmussen, U., Bouchon, B., Jaquinod, M., Blasak, R., Detot, E., and Kreil, G. (1993) J. Biol. Chem. 268, 16458-16464). Xenoxin-1 (10 nM) caused volume changes that required extracellular Ca2+ and were comparable in magnitude and direction to changes caused by BayK-8644 (100 nM), a dihydropyridine-sensitive Ca2+ channel agonist. The initial rate of dihydropyridine-sensitive 45Ca2+ influx was substantially increased by xenoxin-1. Staurosporine (10 nM) prevented volume changes caused by ATP (250 microM) but had no effect on volume changes caused by BayK-8644 or xenoxin-1. We conclude that xenoxin-1 directly activated dihydropyridine-sensitive Ca2+ channels in villus cells and that a mammalian homologue to xenoxin-1 may exist.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Adenosine Triphosphate; Amino Acid Sequence; Amphibian Venoms; Animals; Calcium; Calcium Channel Agonists; Calcium Channel Blockers; Cells, Cultured; Dihydropyridines; Guinea Pigs; Jejunum; Molecular Sequence Data; Neurotoxins; Peptides; Skin; Staurosporine; Xenopus laevis

The newly designed pyridine derivative B9309-068 and a series of structurally different compounds were tested for their ability to modulate rhodamine 123 (RHO) efflux from CD56+ hematopoietic cells in the presence of either 10% fetal calf serum or undiluted human AB serum. Furthermore, efflux modulation was investigated on CD34+ blast populations obtained from four patients with relapsed state AML. Target cells were specified throughout by labeling with peridinine chlorophyll protein (PerCP)-conjugated monoclonal antibodies, allowing clear differentiation from RHO emission spectrum by flow cytometry. In the presence of low serum each compound efficiently modulated RHO efflux without significant differences in the range of final concentrations (1.0-3.0 microM). At 0.1 microM, however, RHO efflux was differentially modulated following the series GF120918 approximately B9309-068 > PSC 833 > DNIG approximately DVER. With CD56+ cells in the presence of undiluted human AB serum at a final modulator concentration of 0.1 microM, all chemosensitizers tested were found to be inefficient. At final concentrations of 0.3 microM or higher, distinct RHO efflux modulation was found with the following efficacies: B9309-068 approximately GF120918 > PSC 833 >> DVER approximately DNIG. The efficacies seen in undiluted human AB serum at 3.0 microM were comparable to those seen on CD56+ cells at final modulator concentrations of 0.1 microM in low serum. Our results identify the pyridine derivative B9309-068 as a promising compound for modulating P-glycoprotein-mediated drug resistance under conditions resembling the clinical setting. Nonetheless, modulation potencies of a series of structurally very different chemosensitizers was revealed to be substantially diminished at high serum concentrations in vitro.

Topics: Acridines; Antigens, CD34; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP-Binding Cassette Transporters; Blood Proteins; Calcium Channel Blockers; CD56 Antigen; Cyclosporins; Dihydropyridines; Dose-Response Relationship, Drug; Flow Cytometry; Fluorescent Dyes; Gene Expression; Humans; Isoquinolines; Leukemia, Myeloid, Acute; Leukocytes, Mononuclear; Morpholines; Multidrug Resistance-Associated Proteins; Pyrimidines; Rhodamine 123; Rhodamines; Tetrahydroisoquinolines; Verapamil

P-glycoprotein(P-gp)- related resistance is one of the major obstacles in treating leukemia patients. Therefore, it is of clinical interest to find new potential modulators and compare their P-gp-modulating efficacy. The present analysis investigated the influence of P-gp modulators, such as verapamil, tamoxifen, droloxifene E, droloxifene Z, SDZ PSC 833 (PSC 833) and dexniguldipine in a leukemic T-cell line (CCRF-CEM) and its P-gp-resistant counterparts (CCRF-CEM/ACT400 and CCRF-CEM/VCR1000). P-gp expression was assessed with an immunocytological technique using the monoclonal antibody 4E3.16. It was characterized as the percentage of P-gp positive cells and also expressed as a D value by using the Kolmogorov Smirnov statistic. The efficacy of P-gp modulators was determined with the rhodamine-123 accumulation test and the MTT test. An in vitro modulator concentration between 0.1 microM and 3 microM was determined, where no genuine antiproliferative effect was apparent. The modulators PSC 833 and dexniguldipine were the significant (p

Topics: Antibodies, Monoclonal; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP-Binding Cassette Transporters; Cell Division; Cyclosporins; Dihydropyridines; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Humans; Leukemia, T-Cell; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Tamoxifen; Tumor Cells, Cultured; Vault Ribonucleoprotein Particles; Verapamil

In heart failure, homologous downregulation of beta-adrenoceptors contributes to impaired adrenergic responsiveness of the myocardium. We evaluated alpha 1-adrenoceptors (alpha 1-AR) in a sparsely innervated and a densely innervated peripheral artery in an experimental model of left ventricular dysfunction post-myocardial infarction.. [3H]Prazosin binding was determined in arterial segments of Wistar-Kyoto rats (WKY), and of Wistar rats 5 weeks after myocardial infarction (MI) or sham operation (SHAM).. In the thoracic aorta (TAO) of WKY, specific prazosin binding was: (i) prevented by the irreversible alpha 1B-AR and relatively selective alpha 1D-AR antagonist, chloroethylclonidine (CEC); (ii) displaced with low affinity (pKi 6.25) by the alpha 1A-AR selective ligand, (+)-niguldipine; and (iii) displaced with both high (pKi 10.4) and low (pKi 7.37) affinity by the alpha 1D-AR antagonist, BMY 7378. In mesenteric small arteries (MSA) of WKY, prazosin binding was: (i) reduced 50% by CEC; (ii) displaced in a biphasic fashion by (+)-niguldipine (pKi 8.60 and pKi 6.22); and (iii) displaced by BMY 7378 with low affinity only (pKi 6.86). Also in TAO of SHAM. prazosin binding was prevented by CEC, but neither 30 nM (+)-niguldipine nor 1 nM BMY 7378 affected it. In MSA of SHAM, prazosin binding was virtually abolished in the presence of 30 nM (+)-niguldipine and was not reduced by 1 nM BMY 7378. In TAO and MSA of MI, compared to SHAM, the density of binding sites tended to be increased rather than decreased and neither the affinity for the ligand nor the effects of alpha 1-AR subtype selective tools were significantly modified.. These findings indicate that: (i) radioligand binding can be applied in intact arterial segments to quantify and characterize alpha 1-AR; (ii) although differences seem to exist between rat strains, alpha 1B-AR and alpha 1D-AR predominate in rat thoracic aorta and alpha 1A-AR and alpha 1B-AR in mesenteric small arteries; and (iii) alpha 1-AR density is not reduced in the poorly innervated aorta and the densely innervated mesenteric small arteries of rats with heart failure due to myocardial infarction.

Topics: Adrenergic alpha-Antagonists; Animals; Aorta; Calcium Channel Blockers; Dihydropyridines; In Vitro Techniques; Male; Mesenteric Arteries; Myocardial Infarction; Piperazines; Prazosin; Protein Binding; Radioligand Assay; Rats; Rats, Inbred WKY; Rats, Wistar; Receptors, Adrenergic, alpha-1; Ventricular Dysfunction, Left

We report the effects of two new dihydropyridine derivatives, isradipine (4-(4'-benzofurazanyl)-1,4-dihydro-2,6-dimethyl-3,5-pyridinedic arboxylic acid methylisopropylester) and niguldipine (1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinecarboxylic acid 3-(4,4-diphenyl-1-piperidinyl)-propyl methyl ester hydrochloride), and of dantrolene (1-[(5-[p-nitrophenyl]furfurylidene)-amino]hydantoin sodium, an inhibitor of Ca2+ release from intracellular stores) on the protective efficacy of antiepileptic drugs against maximal electroshock-induced seizures. It was shown that dantrolene (5-20 mg/kg), isradipine (5-10 mg/kg) and niguldipine (up to 2.5 mg/kg) did not influence the electroconvulsive threshold in mice, although a higher dose of niguldipine (5 mg/kg) significantly elevated it. Dantrolene (10-20 mg/kg) and isradipine (1 mg/kg) did not affect the anticonvulsive activity of conventional antiepileptic drugs. In contrast, niguldipine (2.5-5 mg/kg) impaired the protective action of carbamazepine and phenobarbital. No effect of niguldipine (2.5-5 mg/kg) was observed upon the anticonvulsive efficacy of diphenylhydantoin and valproate. BAY k-8644 (methyl-1,4-dihydro-2,6-dimethyl-5-nitro-4- [(2-trifluoromethyl)-phenyl]-pyridine-5-carboxylate, an L-type Ca2+ channel agonist) did not reverse the action of niguldipine alone or the niguldipine-induced impairment of the anticonvulsive action of carbamazepine and phenobarbital. Niguldipine did not influence the free plasma levels of carbamazepine and phenobarbital, so a pharmacokinetic interaction is not probable. The results suggest that in contrast to the anticonvulsive activity of niguldipine against electroconvulsions, this Ca2+ channel inhibitor significantly weakened the protective action of both carbamazepine and phenobarbital. These effects do not seem to result from the blockade of voltage-dependent Ca2+ channels. Isradipine and dantrolene did not have a modulatory action on the threshold for electroconvulsions or on the anticonvulsive activity of antiepileptic drugs. It may be concluded that the use of niguldipine, isradipine, and dantrolene in epileptic patients seems questionable.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Anticonvulsants; Calcium Channel Agonists; Calcium Channel Blockers; Carbamazepine; Dantrolene; Dihydropyridines; Disease Models, Animal; Drug Interactions; Electroshock; Epilepsy; Female; Isradipine; Lethal Dose 50; Mice; Motor Activity; Phenobarbital; Phenytoin; Random Allocation; Seizures; Valproic Acid

1. Alterations in the cardiac alpha 1-adrenoceptor and its subtypes in hypothyroid rats were studied by radioligand binding assays and reverse transcription-polymerase chain reaction (RT-PCR). Hypothyroidism was created by feeding rats with 0.2% 2-thiouracil solution instead of daily drinking water for 20 days. 2. The density of cardiac alpha 1-adrenoceptors (Bmax) was increased from 67.5 +/- 4.3 fmol/mg in control rats to 81.1 +/- 7.2 fmol/mg (P < 0.05) in hypothyroid rats. 3. Compared with control rats, in hypothyroid rats the percentages of high-affinity sites for (+)-niguldipine and 5-methylurapidil were increased from 13.8 +/- 5.6 and 31.9 +/- 6.3%, respectively, to 24.9 +/- 7.3 and 45.5 +/- 2.4%, respectively (both P < 0.05), while those for BMY7378 were decreased from 37.2 +/- 8.9 to 23.8 +/- 8.4% (P < 0.05), respectively. The percentage of high-affinity sites for WB4101 was not significantly different in control and hypothyroid rats (43.3 +/- 9.1 and 39.4 +/- 3.6%, respectively). 4. Reverse transcription-PCR experiments revealed that the steady state levels of mRNA for alpha 1A- and alpha 1B-adrenoceptors were increased, while those for alpha 1D-adrenoceptor were decreased in the hearts of hypothyroid rats. 5. The concentration-contraction response curves for noradrenaline in the presence of a beta-adrenoceptor antagonist in control and hypothyroid rats showed that the maximal response was reduced from 344 +/- 58 to 200 +/- 23 mg, respectively (P < 0.05). 6. The data suggest that in hypothyroid rats the total number of cardiac alpha 1-adrenoceptors is increased. The change is subtype-selective, with levels of alpha 1A- and alpha 1B-adrenoceptors being increased and levels of alpha 1D-adrenoceptors being reduced. Furthermore, the positive inotropic response mediated by alpha 1-adrenoceptors is reduced in hypothyroid rats.

Topics: Adrenergic alpha-Antagonists; Animals; Atrial Function; Binding, Competitive; Dihydropyridines; Dioxanes; DNA Primers; Heart Atria; Hypothyroidism; Male; Myocardial Contraction; Norepinephrine; Phenethylamines; Piperazines; Polymerase Chain Reaction; Protein Binding; Radioligand Assay; Rats; Receptors, Adrenergic, alpha; RNA, Messenger; Tetralones; Thiouracil

The effects of nifedipine, niguldipine, nimodipine and nitrendipine on the high K+-induced intracellular Ca2+ ([Ca2+]i) transient in dibutyryl cAMP-differentiated neuroblastoma x glioma hybrid NG 108-15 cells were studied by using the fluorescent Ca2+ indicator fura-2. It was observed that nifedipine at the concentration of 50 microM inhibited the high K+-induced [Ca2+]i transient by about 60%; niguldipine at the concentration of 10 microM caused a reduction of about 65% in the high K+-induced calcium signal and a further increase in the concentration up to 50 microM did not result in a significant further reduction in the high K+-induced calcium signal. However, on the other hand, nimodipine and nitrendipine at 50 microM inhibited almost completely the high K+-induced [Ca2+]i transient. Consequently, it was demonstrated in the present study that nimodipine and nitrendipine inhibit both L- and N-type calcium channels and thus seem to be unique among the dihydropyridines examined in their effects on calcium channels in dibutyryl cAMP-differentiated neuroblastoma x glioma hybrid NG 108-15 cells, whereas nifedipine and niguldipine appear to block mainly L-type calcium channels.

Topics: Animals; Bucladesine; Calcium; Calcium Channel Blockers; Calcium Channels; Cell Differentiation; Dihydropyridines; Evoked Potentials; Glioma; Hybrid Cells; Kinetics; Mice; Neuroblastoma; Nimodipine; Nitrendipine; Potassium; Rats

Dexniguldipine hydrochloride (B859-35, a dihydropyridine with antitumor and multidrug resistance-reverting activity) inhibits both the DNA cleavage and religation reactions of purified human DNA topoisomerase I at concentrations >1 microM, whereas at concentrations <1 microM it inhibits selectively the religation step and stabilizes the covalent topoisomerase I-DNA intermediate in a similar fashion as camptothecin. Inhibition of religation by camptothecin can be overcome by increasing the concentration of the DNA substrate in the religation reaction, indicating a competitive type of inhibition. In contrast, dexniguldipine hydrochloride decreases rate constants of topoisomerase I-mediated DNA religation independently of the concentration of the DNA substrate, suggesting a noncompetitive mechanism of inhibition, which is different from that of camptothecin.

Topics: Antineoplastic Agents; Dihydropyridines; DNA Replication; DNA Topoisomerases, Type I; Enzyme Stability; Humans; Kinetics; Recombinant Proteins; Topoisomerase I Inhibitors

The thioether phospholipid ilmofosine (BM 41 440) is a new anti-cancer drug presently undergoing phase II clinical trials. Because resistance to anti-tumour drugs is a major problem in cancer treatment, we investigated the resistance of different cell lines to this compound. Here we report that the multidrug-resistant cell lines MCF7/ADR, CCRFNCR1000, CCRF/ADR500, CEM/VLB100 and HeLa cell lines transfected with a wild-type and mutated (gly/val185) multidrug resistance 1 gene (MDR1) are cross-resistant to ilmofosine compared with the sensitive parental cell lines. In CEMNM-1 cells, in which the resistance is associated with an altered topoisomerase II gene, no cross-resistance to ilmofosine was observed. Ilmofosine is not capable of modulating multidrug resistance and neither does it reduce the labelling of the P-glycoprotein (P-gp) by azidopine nor alter ATPase activity significantly. The resistance to ilmofosine in multidrug-resistant CCRF/VCR1000 cells cannot be reversed by the potent multidrug resistance modifier dexniguldipine-HCI (B8509-035). A tenfold excess of ilmofosine does not prevent the MDR-modulating effect of dexniguldipine-HCl. Treatment of cells with ilmofosine does not alter the levels of MDR1 mRNA. Long-term treatment of an ilmofosine-resistant Meth A subline with the drug does not induce multidrug resistance, indicating that ilmofosine does not increase the level of P-gp. Determination of the MDR2 mRNA levels in the cells revealed that the resistance pattern to ilmofosine is not correlated with the expression of this gene. It is concluded, therefore, that multidrug-resistant cells are cross-resistant to ilmofosine and that the compound is not a substrate of Pgp. No association between the expression of the MDR2-encoded P-gp and resistance to ilmofosine was observed. It is supposed that MDR1-associated alterations in membrane lipids cause resistance to ilmofosine.

Topics: Antibiotics, Antineoplastic; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Division; Cell Transformation, Neoplastic; Dihydropyridines; Dose-Response Relationship, Drug; Doxorubicin; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Genes, MDR; HeLa Cells; Humans; Phospholipid Ethers; Time Factors; Tumor Cells, Cultured

1. It was examined by means of BMY 7378, a selective antagonist of alpha 1D-adrenoceptors, whether alpha 1D-adrenoceptors contribute to the regulation of myocardial contractility and hydrolysis of phosphoinositide (PI) in rabbit ventricular muscle. 2. BMY 7378 had a biphasic antagonistic action on the positive inotropic effect (PIE) of phenylephrine depending on the concentration. BMY 7378 at 1-10 nM shifted the concentration-response curve (CRC) for the PIE of phenylephrine to the right and downward and at 100 nM to 1 microM it antagonized the PIE in a competitive manner, the slope of Schild plot being 0.93 and the pA2 being 7.17 +/- 0.09. 3. The inhibitory action of BMY 7378 at 1-10 nM is ascribed to the selective action on alpha 1-adrenoceptors because the PIE of neither isoprenaline nor endothelin-3 and angiotensin II was affected by this compound over this concentration range. 4. In the presence of 100 nM WB 4101, the antagonistic action of BMY 7378 at 1-10 nM remained unchanged but the antagonistic action of BMY 7378 at 100-300 nM disappeared. The antagonistic action of BMY 7378 at 1 nM was unaffected by 100 nM (+)-niguldipine. 5. Following pretreatment with chloroethyldonidine, BMY 7378 at 1 nM inhibited the maximal response to phenylephrine but the pD2 value for phenylephrine was increased in the presence of BMY 7378. The CRC for phenylephrine was shifted to the left in the presence of 10-100 nM BMY 7378 but it was shifted to the right by BMY 7378 at 300 nM. 6. Stimulation of PI hydrolysis induced by phenylephrine was not affected by BMY 7378 up to 10 nM but it was reduced significantly by BMY 7378 at higher concentrations (100 nM to 1 microM). 7. BMY 7378 inhibited the [3H]prazosin specific binding to the rabbit ventricular membrane fraction in a monophasic manner with a pKi value of 7.53 +/- 0.09. 8 The results indicate that in rabbit ventricular muscle, BMY 7378 at 1-10 nM suppressed the maximal response to phenylephrine (probably mediated by alpha 1D-adrenoceptors) and at 10-100 nM it inhibited the negative inotropic effect of phenylephrine, the mechanisms of which remain to be characterized. At higher concentrations (100 nM to 1 microM) BMY 7378 antagonized the functional and biochemical response via a presumed interaction mainly with the alpha 1D-adrenoceptor and partially with the alpha 1A-adrenoceptor.

Topics: Adrenergic alpha-Antagonists; Animals; Binding Sites; Calcium Channel Blockers; Dihydropyridines; Dioxanes; Dose-Response Relationship, Drug; Heart; Heart Ventricles; Male; Myocardial Contraction; Myocardium; Papillary Muscles; Piperazines; Prazosin; Rabbits; Receptors, Adrenergic, alpha-1

The pharmacokinetics of oral dexniguldipine, a new multidrug-resistance-modifying agent under clinical evaluation, and its pyridine metabolite M-1 were determined in plasma, tumor, and renal tissue in Wag/Rij rats bearing a multidrug-resistant CC531 colon adenocarcinoma tumor under the renal capsule. The pharmacokinetics were studied in four experiments. After a single administration of dexniguldipine (30 mg/kg), tumors and kidneys were collected after 5 (experiment 1), 24 (experiment 2), and 48 h (experiment 3). In the fourth experiment, dexniguldipine was given once daily for 3 consecutive days at a dose of 30 mg/kg. In all experiments, plasma samples were collected at regular intervals. The concentrations of dexniguldipine and M-1 could be determined in plasma in most of the rats at up to 32 h after drug administration. The area under the curve (AUC) of dexniguldipine and M-1 varied by a factor of 2-6 in the four experiments. High tumor-tissue concentrations of dexniguldipine were observed. The concentrations were highest in the multiple-dose experiment (2014 +/- 1005 ng/g tissue). High degrees of correlation (> 0.8) were established between the concentrations of dexniguldipine measured in plasma and tumor as well as renal tissue. Overall, tumor-tissue concentrations of M-1 comprised one-third of the dexniguldipine concentrations measured.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Colonic Neoplasms; Dihydropyridines; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Humans; Kidney; Neoplasm Transplantation; Pyridines; Rats; Rats, Inbred Strains

A photoreactive dihydropyridine (DHP), BZDC-DHP (2,6-dimethyl-4-(2-(trifluoromethyl)-phenyl)-1,4-dihydropyridine-3,5- dicarboxylic acid (2-[3-(4-benzoylphenyl)propionylamino]ethyl) ester ethyl ester), and its tritiated derivative were synthesized as novel probes for human p-glycoprotein (p-gp). (-)-[3H]BZDC-DHP specifically photolabeled p-gp in membranes of multidrug-resistant CCRF-ADR5000 cells. In reversible labeling experiments a saturable, vinblastine-sensitive and high-affinity (Kd = 16.3 nM, Bmax = 58 pmol/mg of protein, k(+1) = 0.031 nM-1 min-1, k(-1) = 0.172 min-1) binding component was present in CCRF-ADR5000 membranes but absent in the sensitive parent cell line. Binding was inhibited by cytotoxics and known chemosensitizers with a p-gp characteristic pharmacological profile. For eight chemosensitizers tested, the potency for binding inhibition correlated (r > 0.94) with the potency for drug transport inhibition (measured using rhodamine 123 accumulation). The DHP niguldipine and a structurally related pyrimidine stereoselectively stimulated reversible (-)-[3H]BZDC-DHP binding, suggesting that more than one DHP molecule can bind to p-gp at the same time. Our data demonstrate that DHPs label multiple chemosensitizer domains on p-gp, distinct from the vinblastine interaction site. (-)-[3H]BZDC-DHP represents a valuable tool to characterize the molecular organization of chemosensitizer binding domains on p-gp by both reversible binding and photoinduced covalent modification. It provides a novel simple screening assay for p-gp active drugs.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Benzophenones; Binding Sites; Biological Transport; Calcium Channels; Dihydropyridines; Drug Resistance, Multiple; Humans; Models, Biological; Molecular Probes; Photosensitizing Agents; Vinblastine

The chemosensitizing potency of dexniguldipine hydrochloride (B8509-035) on epidoxorubicin was assessed in a multidrug-resistant (MDR) tumour model, the intrinsic MDR rat colon carcinoma CC531. In vitro in the sulphorhodamine B cell-viability assay the cytotoxicity of epidoxorubicin was increased approximately 15-fold by co-incubation with 50 ng/ml dexniguldipine. In vivo concentrations of dexniguldipine 5 h after a single oral dose of 30 mg/kg were 72 (+/- 19 SD) ng/ml in plasma and 925 (+/- 495 SD) ng/g in tumour tissue. Levels of the metabolite of dexniguldipine, M-1, which has the same chemosensitizing potential, were 26 (+/- 6 SD) ng/ml and 289 (+/- 127 SD) ng/g respectively. The efficacy of treatment with 6 mg/kg epidoxorubicin applied intravenously combined with 30 mg kg-1 day-1 dexniguldipine administered orally for 3 days prior to epidoxorubicin injection was evaluated on tumours grown under the renal capsule. Dexniguldipine alone did not show antitumour effects in vivo. Dexniguldipine modestly, but consistently, potentiated the tumour-growth-inhibiting effect of epidoxorubicin, reaching statistical significance in two out of four experiments. In conclusion, these experiments show that dexniguldipine has potency as an MDR reverter in vitro and in vivo in this solid MDR tumour model.

Topics: Adenocarcinoma; Animals; Antineoplastic Combined Chemotherapy Protocols; Biotransformation; Colonic Neoplasms; Dihydropyridines; Drug Resistance, Multiple; Drug Screening Assays, Antitumor; Drug Synergism; Epirubicin; Male; Neoplasm Transplantation; Rats; Rats, Inbred Strains; Tumor Cells, Cultured

Dexniguldipine hydrochloride (DNIG) is a potent antineoplastic agent with well-documented anti-(protein kinase C) activity and an ability to reverse multidrug resistance. Given the importance of protein kinase C (PKC) activity in proliferation and differentiation, we examined the effect of DNIG on several parameters of Friend erythroleukemia cell (FELC) activity. Particular attention was paid to proliferation, hexamethylene-bisacetamide-(HMBA)-induced differentiation, nuclear localization of protein kinase C, and nuclear protein phosphorylation. P-glycoprotein expression was also followed as an indicator of changes in multidrug resistance. At 2.5 microM, DNIG caused a significant decrease in the rate of FELC proliferation, while maintaining a cellular viability of greater than 80%, whether exposure to the drug was continuous over 96 h or took the form of a 6-h pulse/chase. DNA synthesis was decreased in cells exposed to DNIG for 20 h. Flow cytometry showed a marked increase in the percentage of cells in S phase of the cell cycle. Phosphorylation studies revealed decreased phosphorylation of two nuclear proteins (80 kDa and 47 kDa) following a 4-h exposure to the drug. HMBA-induced differentiation was significantly inhibited with continuous exposure to DNIG, and this effect appears to be a pre-commitment one, as 6-h pulse/chase exposures also resulted in inhibition of differentiation. Cells induced to differentiate with HMBA also demonstrated a decrease in the quantity of the 80-kDa phosphoprotein. Western blotting revealed that, even in the face of decreased phosphorylation, exposure to this PKC inhibitor resulted in an increase in the amount of nuclear PKC alpha. Finally, levels of P-glycoprotein were decreased in the presence of this drug. Our work identifies several effects of the PKC inhibitor DNIG on FELC and suggests several roles for PKC in regulating FELC proliferation and differentiation. Additionally, these results suggest that this PKC inhibitor may increase the effect of other chemotherapeutic drugs, particularly S-phase-specific ones, by increasing the length of S phase and decreasing multidrug resistance. The possibility of combination therapy with DNIG and other antineoplastic agents should be investigated further in light of these findings.

Topics: Acetamides; Animals; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Cycle; Cell Differentiation; Dihydropyridines; Friend murine leukemia virus; Leukemia, Erythroblastic, Acute; Mice; Mice, Inbred DBA; Nuclear Proteins; Phosphorylation; Protein Kinase C; Tumor Cells, Cultured

The interaction of cytostatics and chemosensitizers with the dexniguldipine binding site of P-glycoprotein was investigated in photoaffinity labeling experiments. A tritiated azidoderivative of the chemosensitizer dexniguldipine with dihydropyridine structure, [3H]B9209-005, was used to irreversibly label P-glycoprotein. The apparent affinity of cytostatics and chemosensitizers to this binding site was estimated from labeling experiments in the presence of increasing concentrations of compounds. From the cytostatics tested, the vinca alkaloids and taxol showed the highest affinity, anthracyclins possessed moderate affinity while methotrexate, ara C and camptothecin, cytostatics not involved in P-glycoprotein-mediated multidrug resistance, were almost inactive. The chemosensitizers GF 120918, cyclosporin A and SDZ PSC-833 inhibited photoincorporation with the highest potency. Steep dose-inhibition curves were obtained with the cyclic peptides and S9788, indicating that these compounds may bind allosterically to a separate binding site. Compounds with dihydropyridine structure with or without chemosensitizing potency were also tested and some structure-activity relationships could be derived from the data. Our data show that inhibition of photoaffinity labeling by [3H]B9209-005 is a valuable and reliable system for measuring the interaction with and potency of chemosensitizing compounds at P-glycoprotein. Furthermore, data obtained in this test system are well suited to investigate structure-activity relationships for chemosensitizers at P-glycoprotein. In addition cytostatics underlying P-glycoprotein-mediated multidrug resistance can be identified.

Topics: Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; Binding Sites; Cell Line; Dihydropyridines; Dose-Response Relationship, Drug; Drug Resistance, Multiple; Humans

A relatively simple electroanalytical procedure for the determination of niguldipine in biological samples is described. The technique involves adsorptive accumulation of the drug at the hanging mercury drop electrode (HMDE) followed by a differential-pulse polarographic determination of the preconcentrated species. The adsorptive stripping response is evaluated with respect to various experimental conditions, such as solvent composition and pH of the supporting electrolyte, accumulation potential and accumulation time. After a simple sample preparation, the method can be used for the determination of niguldipine in blood and urine. Interfering substances are simply removed by precipitation, adding a small amount of 5% ZnSO4 solution and ethanol to the urine or blood sample and centrifuging the mixture. A limit of detection of 6.7 ng per ml of urine and 41 ng per ml of blood is found with a mean recovery of 96% in urine and 71% in blood. The mean relative errors are 8.4% and 2.2%, respectively.

Topics: Adsorption; Calcium Channel Blockers; Dihydropyridines; Electrochemistry; Electrodes; Humans; Indicators and Reagents; Mercury; Reproducibility of Results; Sensitivity and Specificity; Sulfates; Zinc Compounds; Zinc Sulfate

Inhibition of protein kinase C (PKC) is discussed as a new approach for overcoming multidrug resistance (MDR) in cancer chemotherapy. For evaluation of this concept we applied the bisindolylmaleimide GF 109203X, which shows a highly selective inhibition of PKC isozymes alpha, beta 1, beta 2, gamma, delta and epsilon in vitro. The efficacy of this compound in modulation of MDR was examined using several P-glycoprotein (P-gp)-overexpressing cell lines including a MDR1-transfected HeLa clone, and was compared with the activities of dexniguldipine-HCI (DNIG) and dexverapamil-HC1 (DVER), both of which essentially act via binding to P-gp. As PKC alpha has been suggested to play a major role in P-gp-mediated MDR, cell lines exhibiting different expression levels of this PKC isozyme were chosen. On crude PKC preparations or in a cellular assay using a cfos(-711)CAT-transfected NIH 3T3 clone, the inhibitory qualities of the bisindolylmaleimide at submicromolar concentrations were demonstrated. At up 1 microM final concentrations of the PKC inhibitor GF 109203X, a concentration at which many PKC isozymes should be blocked substantially, no cytotoxic or MDR-reversing effects whatsoever were seen, as monitored by 72 h tetrazolium-based colorimetric MTT assays or a 90 min rhodamine 123 accumulation assay. Moreover, depletion of PKC alpha by phorbol ester in HeLa-MDR1 transfectants had no influence on rhodamine 123 accumulation after 24 or 48 h. MDR reversal activity of GF 109203X was seen at higher final drug concentrations, however. Remarkably, [3H]vinblastine-sulphate binding competition experiments using P-gp-containing crude membrane preparations demonstrated similar dose dependencies as found for MDR reversion by the three modulators, i.e. decreasing efficacy in the series dexniguldipine-HCl > dexverapamil-HCl > GF 109203X. Similar interaction with the P-gp in the micromolar concentration range was revealed by competition of GF 109203X with photoincorporation of [3H]azidopine into P-gp-containing crude membrane preparations. No significant effect of the PKC inhibitor on MDR1 expression was seen, which was examined by cDNA-PCR. Thus, the bisindolylmaleimide GF 109203X probably influences MDR mostly via direct binding to P-gp. Our work identifies the bisindolylmaleimide GF 109203X as a new type of drug interacting with P-gp directly, but does not support the concept of a major contribution of PKC to a P-gp-associated MDR, at least using the particular cellular mode

Topics: 3T3 Cells; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Azides; Dihydropyridines; Drug Resistance, Multiple; Enzyme Inhibitors; Gene Expression; Humans; Indoles; Maleimides; Mice; Phosphorylation; Protein Kinase C; Verapamil

Experiments were performed to determine the subtypes of alpha-adrenoceptors involved in the contraction induced by rilmenidine in isolated canine cutaneous veins. Rings of saphenous vein (without endothelium) were suspended for the recording of isometric force in physiological salt solution. All experiments were performed in the presence of propranolol (to antagonize beta-adrenoceptors), cocaine (to inhibit neuronal uptake) and hydrocortisone (to inhibit extraneuronal uptake). In the presence of rauwolscine (an alpha 2-adrenergic blocker), rilmenidine caused concentration-dependent contractions which were inhibited by prazosin (nonselective alpha 1-antagonist) and by (+)niguldipine (selective alpha 1A-adrenergic antagonist), but not by (-)niguldipine. After treatment with phenoxybenzamine (to alkylate alpha 1-adrenoceptors), rilmenidine evoked contractions of the canine saphenous vein which were antagonized competitively by rauwolscine. The combination of rauwolscine and prazosin did not abolish contractions evoked by the highest concentrations of rilmenidine. Although binding experiments using 3H-idazoxan suggested the existence of a nonadrenergic binding site (around 20% of the total binding), contractile studies failed to demonstrate their involvement in the increases in tension evoked by rilmenidine. These experiments suggest that the contractions evoked by rilmenidine in isolated canine veins are mediated by both alpha 1A- and alpha 2-adrenoceptors.

Topics: Adrenergic alpha-1 Receptor Agonists; Adrenergic alpha-2 Receptor Agonists; Adrenergic alpha-Agonists; Adrenergic alpha-Antagonists; Animals; Brimonidine Tartrate; Calcium Channel Blockers; Clonidine; Dihydropyridines; Dogs; Female; Idazoxan; Imidazoles; Imidazoline Receptors; In Vitro Techniques; Isotope Labeling; Male; Medetomidine; Muscle Contraction; Muscle, Smooth; Osmolar Concentration; Oxazoles; Phenoxybenzamine; Prazosin; Protein Binding; Quinoxalines; Radioligand Assay; Receptors, Adrenergic, alpha-1; Receptors, Adrenergic, alpha-2; Receptors, Drug; Rilmenidine; Saphenous Vein; Tritium; Yohimbine

The L-type Ca2+ current inhibition by the enantiomers of the dihydropyridine niguldipine was investigated at various holding potentials (-40 to -120 mV) and stimulus frequencies (0.1-1 Hz), using guinea-pig ventricular myocytes. Block of whole-cell current is both voltage- and concentration-dependent. (S)-Niguldipine is more potent than its (R)-enantiomer. However, the extent of enantioselectivity is rather small (< or = x 4.4). Importantly, this value does not increase when stimulus conditions favour the inactivated channel state, although this leads to more potent block. This is in contrast to our expectation based on modulated receptor hypothesis, and to the high enantioselectivity of niguldipine binding found in guinea-pig heart membranes (x 40). We conclude that the common modulated receptor hypothesis had to be refined to explain the effects of niguldipine enantiomers.

Topics: Animals; Calcium Channel Blockers; Calcium Channels; Dihydropyridines; Guinea Pigs; Patch-Clamp Techniques

1. The affinity of the alpha 1-adrenoceptor antagonist SB 216469 (also known as REC 15/2739) has been determined at native and cloned alpha 1-adrenoceptor subtypes by radioligand binding and at functional alpha 1-adrenoceptor subtypes in isolated tissues. 2. In radioligand binding studies with [3H]-prazosin, SB 216469 had a high affinity at the alpha 1A-adrenoceptors of the rat cerebral cortex and kidney (9.5-9.8) but a lower affinity at the alpha 1B-adrenoceptors of the rat spleen and liver (7.7-8.2). 3. At cloned rat alpha 1-adrenoceptor subtypes transiently expressed in COS-1 cells and also at cloned human alpha 1-adrenoceptor subtypes stably transfected in Rat-1 cells, SB 216469 exhibited a high affinity at the alpha 1a-adrenoceptors (9.6-10.4) with a significantly lower affinity at the alpha 1b-adrenoceptor (8.0-8.4) and an intermediate affinity at the alpha 1d-adrenoceptor (8.7-9.2). 4. At functional alpha 1-adrenoceptors, SB 216469 had a similar pharmacological profile, with a high affinity at the alpha 1A-adrenoceptors of the rat vas deferens and anococcygeus muscle (pA2 = 9.5-10.0), a low affinity at the alpha 1B-adrenoceptors of the rat spleen (6.7) and guinea-pig aorta (8.0), and an intermediate affinity at the alpha 1D-adrenoceptors of the rat aorta (8.8). 5. Several recent studies have concluded that the alpha 1-adrenoceptor present in the human prostate has the pharmacological characteristics of the alpha 1A-adrenoceptor subtype. However, the affinity of SB 216469 at human prostatic alpha 1-adrenoceptors (pA2 = 8.1) determined in isolated tissue strips, was significantly lower than the values obtained at either the cloned alpha 1a-adrenoceptors (human, rat, bovine) or the native alpha 1A-adrenoceptors in radioligand binding and functional studies in the rat. 6. Our results with SB 216469, therefore, suggest that the alpha 1-adrenoceptor mediating contractile responses of the human prostate has properties which distinguish it from the cloned alpha 1a-adrenoceptor or native alpha 1A-adrenoceptor. Since it has previously been shown that the receptor is not the alpha 1B- or alpha 1D-adrenoceptor, the functional alpha 1-adrenoceptor of the human prostate may represent a novel receptor with properties which differ from any of the alpha 1-adrenoceptors currently defined by pharmacological means.

Topics: Adrenergic alpha-1 Receptor Antagonists; Adrenergic alpha-Antagonists; Animals; Cattle; Chromones; Dihydropyridines; Dioxanes; Dose-Response Relationship, Drug; Guinea Pigs; Humans; Male; Prazosin; Prostate; Radioligand Assay; Rats; Rats, Wistar

The aim of this study was to assess the contribution of alpha 1-adrenoceptor subtypes to noradrenaline (NA)-induced inositol phosphate formation in rat striatum. In cross-chopped slices and in the presence of 10 mM LiCl, NA stimulated the accumulation of [3H]inositol phosphates. After 60-min incubation with 100 microM NA, [3H]IP1 was the major product detected (82 +/- 3% of total [3H]inositol phosphates). Best-fit values for the concentration-response curve for NA-induced [3H]IP1 accumulation yielded an EC50 of 9.4 +/- 1.1 microM, maximum effect of 210 +/- 3% of basal, and Hill coefficient (nH) of 1.1 +/- 0.1. Pre-treatment of the slices for 30 min with the alkylating agent chloroethylclonidine (100 microM) failed to decrease significantly the response to 100 microM NA. Inhibition curves for four alpha 1-antagonists, (+)-niguldipine, prazosin, WB-4101 and 5-methylurapidil (5-MU), best-fit to a single-site model with pKi values of 9.4 ((+)-niguldipine), 9.2 (prazosin and WB-4101) and 8.8 (5-MU). The putative alpha 1 D-selective antagonist BMY 7378 reduced the response to NA only partially (30 +/- 3% inhibition at 1 microM: pKi 7.24). NA-induced [3H]IP1 accumulation was significantly reduced (to 20 +/- 9% of controls) by Ca2+ removal and increased as the extracellular Ca2+ concentration was raised from nominally zero (no added Ca2+) to 1 mM Ca2+. NA-induced [3H]IP1 accumulation was reduced by both the non-selective Ca2+ channel blocker Ni2+ (58 +/- 3% inhibition at 2 mM) and nimodipine, an antagonist of L-type voltage-operated Ca2+ channels (77 +/- 4% inhibition at 3 microM). Taken together these results indicate that NA-induced inositol phosphate formation in striatal slices is mediated by activation of alpha 1A-adrenoceptors coupled to Ca2+ entry and Ca2+ activation of phospholipase C.

Topics: Adrenergic alpha-1 Receptor Agonists; Adrenergic alpha-1 Receptor Antagonists; Adrenergic alpha-Agonists; Adrenergic alpha-Antagonists; Alkylating Agents; Animals; Calcium; Calcium Channel Blockers; Clonidine; Dihydropyridines; Dioxanes; In Vitro Techniques; Inositol Phosphates; Male; Neostriatum; Norepinephrine; Piperazines; Rats; Rats, Wistar; Receptors, Adrenergic, alpha-1

The newly synthesized dihydropyridine derivative B859-35 was previously shown in vitro to be highly effective in reversing multidrug resistance (MDR) of P-glycoprotein positive tumor cell lines, such as the adriamycin (ADR) resistant erythroleukemia F4-6RADR cells. In the current study B859-35 was investigated for its efficiency in reversing MDR in an in vivo tumor model for preclinical testing of MDR-modulators. F4-6RADR cells were injected into the right flank of nude mice while the parent cells were injected into the left flank. The animals were treated i.p. with ADR (9.0 mg/kg body weight) combined with B859-35 (5, 10, or 25 mg/kg) or, for comparison and validation, with verapamil (VRP) (75 mg/kg). The effects of ADR and the MDR-modulator combination were evaluated by histological morphometry of the tumors. While ADR alone was shown to be ineffective in resistant cells, the combinations of ADR + B859-35 as well as of ADR + VRP were highly active in reducing the number of viable cells in the resistant tumor nodule by 67 +/- 9% or by 53 +/- 11% of controls. This model provides evidence that even in vivo, MDR modulators can be effective in reversing drug resistance. In addition, it presents a potentially useful and rapid preclinical system for in vivo studies on the modification of drug resistance.

Topics: Animals; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; Calcium Channel Blockers; Dihydropyridines; Drug Resistance, Neoplasm; Humans; Immunohistochemistry; Male; Mice; Mice, Nude; Neoplasm Transplantation; Tumor Cells, Cultured; Verapamil

Topics: Adrenergic alpha-1 Receptor Antagonists; Adrenergic alpha-Antagonists; Calcium Channel Blockers; Cell Line; Cloning, Molecular; Dihydropyridines; Humans; Receptors, Adrenergic, alpha-1

alpha-Adrenergic stimulation is known to play a role in cardiac arrhythmogenesis and to modulate a variety of cardiac K+ currents. The effects of alpha-adrenergic stimulation on Cl- currents are largely unknown. Many cardiac cell types show a volume-sensitive Cl- current induced by cell swelling (ICl.swell). The present experiments were designed to assess the potential alpha-adrenergic modulation of ICl.swell in rabbit atrial myocytes. ICl.swell was induced with the use of a hypotonic superfusate, under conditions designed to prevent currents carried by K+, Na+, and Ca2+ ions. A basal Cl- current (ICl.b) was observed under isotonic conditions in 128 of 150 cells (85%), had the same dependency on [Cl-]o as ICl.swell, and was reduced by cell shrinkage induced by hypertonic superfusion, suggesting that ICl.b is carried by the same volume-sensitive Cl- conductance as ICl.swell. Phenylephrine produced a concentration-dependent and near-complete inhibition of ICl.b and ICl.swell, with EC50 values of 86 +/- 5 and 72 +/- 7 (mean +/- SEM) mumol/L, respectively, at +20 mV. Norepinephrine (administered in the presence of 1 mumol/L propranolol) also inhibited ICl.b and ICl.swell, with EC50 values of 2.6 +/- 0.1 and 2.8 +/- 0.4 mumol/L, respectively. The concentration-response curve for phenylephrine was shifted significantly (P < .001) to the right by the alpha 1-adrenoceptor antagonist prazosin and by the alpha 1A-receptor antagonists (+)-niguldipine and 5-methylurapidil but was unaltered by the alpha 1B-receptor antagonist chloroethylclonidine (100 mumol/L). Inhibition of protein kinase C (PKC) with staurosporine, H-7, or 18-hour preincubation with the phorbol ester 4 beta-phorbol 12-myristate 13-acetate (PMA, 500 nmol/L) blocked the effects of phenylephrine on ICl.swell, and the highly selective PKC inhibitor bisindolylmaleimide blocked the effects of norepinephrine on ICl.swell and ICl.b. Both PMA and 1-oleoyl-2-acetylglycerol inhibited ICl.swell in a concentration-dependent fashion. In blinded studies, the phorbol ester phorbol 12,13-didecanoate (PDD) reduced ICl.swell by 91 +/- 3%; its inactive analogue 4 alpha-PDD had no effect (mean change, 3 +/- 1%). Preincubation with pertussis toxin (PTX) prevented the actions of phenylephrine on ICl.swell, indicating a role for a PTX-sensitive guanine nucleotide-binding (G) protein. We conclude that alpha-adrenergic agonists inhibit volume-sensitive Cl- currents in rabbit atrial cells by interacting with an alpha 1A-adren

Topics: Action Potentials; Adrenergic alpha-Antagonists; Analysis of Variance; Animals; Antihypertensive Agents; Atrial Function; Autonomic Nervous System; Calcium Channel Blockers; Chloride Channels; Dihydropyridines; Electrophysiology; Heart Atria; In Vitro Techniques; Ion Channels; Norepinephrine; Phenylephrine; Piperazines; Prazosin; Protein Kinase C; Rabbits; Receptors, Adrenergic, alpha; Stimulation, Chemical

Plasma membranes were prepared from the P-glycoprotein expressing human breast cancer cell line MCF-7 ADR. [3H]Vinblastine bound to these membranes saturably with a Bmax of 24 pmol/mg of protein and KD of 23 nM. In contrast, membranes from the parent cells MCF-7 WT, which do not express P-glycoprotein, did not bind [3H]vinblastine with high affinity. Cytotoxics known to be transported by P-glycoprotein inhibited the binding of [3H]vinblastine, as did multidrug reversing agents including the 1,4-dihydropyridine, dexniguldipine-HCl (Ki, 15 nM). In dissociation kinetic experiments, dexniguldipine-HCl accelerated the dissociation of [3H]vinblastine from P-glycoprotein, indicating a negative heterotropic allosteric mechanism of action through a drug binding site distinct from that of vinblastine. Other 1,4-dihydropyridines tested also accelerated [3H]vinblastine dissociation from P-glycoprotein, however, multidrug reversing drugs of different chemical classes, including quinidine, verapamil and cyclosporin A did not. These results suggest that P-glycoprotein of MCF-7 ADR cell membranes possesses at least two drug acceptor sites which are allosterically coupled: receptor site-1 which binds vinca alkaloids, and receptor site-2 which binds 1,4-dihydropyridines such as dexniguldipine-HCl, which had the highest affinity of the tested derivatives.

Topics: Allosteric Regulation; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Membrane; Dihydropyridines; Drug Resistance, Multiple; Drug Synergism; Humans; Ions; Kinetics; Nucleotides; Protein Binding; Tritium; Tumor Cells, Cultured; Vinblastine

P-glycoprotein is an energy-dependent drug extrusion pump for a variety of anticancer drugs and is involved in the development of multidrug resistance in cancer. Dexniguldipine-HCl is a potent chemosensitizer for P-glycoprotein-mediated multidrug resistance in vitro, and clinical phase I/II trials are underway. To investigate the mechanisms of chemosensitization and to identify the binding sites for dexniguldipine-HCl on target proteins involved in chemosensitization, [3H]B9209-005, an azido derivative of dexniguldipine-HCl, was synthesized and used as a photoaffinity ligand. In two models of multidrug resistance reversal, i.e., sensitization to vincristine and modulation of rhodamine-123 uptake, B9209-005 and dexniguldipine-HCl showed identical biological activities. Photoaffinity labeling experiments with [3H]B9209-005 in cell membranes from multidrug-resistant CCRF ADR-5000 cells, in comparison with labeling experiments with [3H]azidopine (an established photoaffinity ligand for P-glycoprotein), showed that [3H]B9209-005 labeled two proteins, with apparent molecular masses of 170 and 95 kDa. The pharmacological specificity of labeling was demonstrated by inhibition of photoincorporation by several cytostatic drugs transported by P-glycoprotein, as well as by chemosensitizers. Immunoprecipitation of the labeled proteins with the P-glycoprotein-specific monoclonal antibody C 219 and with a site-directed polyclonal antibody to the amino-terminal sequence of P-glycoprotein (amino acids 389-406) identified these proteins as intact P-glycoprotein and the amino-terminal fragment thereof. No specific labeling was obtained in the drug-sensitive parent cell line CCRF-CEM, which is devoid of significant P-glycoprotein expression. Maximal labeling of 17 pmol of the 170-kDa protein/mg of crude membrane protein was obtained. The affinity of [3H]B9209-005 for binding to and photoincorporation into P-glycoprotein was 5-fold greater than that of [3H]azidopine, and photoincorporation of [3H]B9209-005 showed a different photoincorporation pattern, compared with [3H]azidopine, in that the latter compound was incorporated specifically into the carboxyl-terminal 55-kDa fragment of P-glycoprotein. In contrast to [3H]azidopine, no specific labeling of this fragment was obtained with [3H]B9209-005, indicating different binding sites for or different photoincorporation of the two dihydropyridine ligands. Because B9209-005 carries the photoreactive azido group in the dihydropyri

Topics: Affinity Labels; ATP Binding Cassette Transporter, Subfamily B, Member 1; Azides; Cell Line; Dihydropyridines; Drug Resistance, Multiple; Drug Synergism; Humans; Photochemistry

1. The present study characterizes and classifies alpha 1-adrenoceptor-mediated vasoconstriction in the isolated perfused kidney of rat using quantitative receptor pharmacology and compares the results to radioligand binding studies (made in cloned alpha 1-adrenoceptor subtypes, native alpha 1A-adrenoceptors in submaxillary gland of rat, and alpha 1A-adrenoceptors in several other tissues of rat). 2. Concentration-effect curves to noradrenaline in the presence of 5-methyl-urapidil were biphasic, indicating alpha 1-adrenoceptor heterogeneity. The alpha 1-adrenoceptor subtype mediating the first phase (low affinity for 5-methyl-urapidil) could not be 'isolated' for detailed pharmacological characterization but was defined by a sensitivity to inhibition by chloroethylclonidine and an inability of methoxamine to activate the site. Additionally, vasoconstriction mediated by this alpha 1-adrenoceptor subtype or subtypes was abolished by nitrendipine (1 microM), thereby allowing characterization of the second, high affinity site for 5-methyl-urapidil. 3. The following antagonists interacted competitively with noradrenaline at the alpha 1-adrenoceptor for which 5-methyl-urapidil exhibits high affinity (pKB value): WB 4101 (10.3) > prazosin (9.5) approximately HV 723 (9.3) approximately 5-methyl-urapidil (9.2) > phenotolamine (8.6) > spiperone (pA2 = 8.1) approximately oxymetazoline (7.9). In contrast, insurmountable antagonism was seen with S(+)- and R(-)-niguldipine, the S(+)-isomer being approximately 30 fold more potent than the R(-)-isomer. Receptor protection experiments indicated that S(+)-niguldipine interacted directly with alpha 1-adrenoceptors. Dehydroniguldipine acted as a competitive antagonist (pKB = 9.0). Thus, the results with antagonists define the alpha 1-adrenoceptor as an alpha 1A-adrenoceptor. 4. An agonist 'fingerprint' was constructed in the presence of nitrendipine to define further the alpha 1A-adrenoceptor. The following order and relativity of agonist potency was obtained: cirazoline (1) approximately adrenaline (2) > noradrenaline (5) > phenylephrine (23) approximately amidephrine (31) > methoxamine (71) >> isoprenaline (1456) approximately dopamine (2210). 5. A high correlative association was shown between the affinity of antagonists obtained functionally in the isolated perfused kidney of rat and pKi values obtained from binding experiments with the cloned bovine alpha 1C-adrenoceptor (R2 = 0.85), native alpha 1A-adrenoceptors in sub

Topics: Adrenergic alpha-Agonists; Adrenergic alpha-Antagonists; Animals; Autoradiography; Binding, Competitive; Calcium Channel Blockers; Cattle; Cricetinae; Dihydropyridines; Dose-Response Relationship, Drug; Kidney; Kinetics; Male; Perfusion; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic, alpha-1; Reference Values; Regression Analysis; Stereoisomerism; Submandibular Gland; Vasoconstriction

We have cloned the human alpha 1d-adrenergic receptor (AR) and compared the pharmacological properties of the three recombinant human alpha 1-AR subtypes in SK-N-MC cells. SK-N-MC cells natively express a mixture of alpha 1-AR subtypes, and the use of an inducible expression system allowed us to directly compare the recombinant and native subtypes without concern for cell-specific processing or microenvironment. The human alpha 1d-AR was expressed from a cDNA/gene fusion construct cloned from human SK-N-MC cell cDNA and human genomic libraries. This receptor is deduced to contain 572 amino acids with 98% identity to the rat alpha 1d-AR in the transmembrane domains and, when expressed in human embryonic kidney 293 cells, has alpha 1-AR binding properties similar to those of the rat alpha 1d-AR. Norepinephrine increased inositol phosphate formation and mobilized intracellular Ca2+ in transfected 293 cells. Reverse transcription-polymerase chain reaction analysis of the three cloned human subtypes (alpha 1a, alpha 1b, and alpha 1d) in mRNA from SK-N-MC cells, which natively express alpha 1A- and alpha 1B-like pharmacology, showed abundant alpha 1a and alpha 1d but fewer alpha 1b transcripts. The three human clones were expressed in SK-N-MC cells using isopropyl-beta-D-thiogalactoside-inducible vectors. Upon induction, alpha 1-AR density was increased with the recombinant subtype comprising 67-80% of total alpha 1-ARs. Inhibition curves for (+)-niguldipine and 5-methylurapidil fit best to a two-site model in uninduced cells, indicating significant receptor heterogeneity. Isopropyl-beta-D-thiogalactoside induction altered the potencies of both compounds, causing most inhibition curves to fit best to a one-site model. (+)-Niguldipine was 100-fold more potent at the alpha 1a-AR than at alpha 1b- or alpha 1d-ARs, whereas 5-methylurapidil had similar potencies at alpha 1a- and alpha 1d-ARs and about 10-fold lower affinity at the alpha 1b-AR. We conclude that the complex alpha 1A- and alpha 1B-like pharmacology observed in native SK-N-MC cells is due to expression of all three subtypes in different proportions, independently of cell-specific processing or environmental factors, and that the alpha 1a-AR cDNA encodes the pharmacologically defined alpha 1A subtype.

Topics: Adrenergic alpha-Antagonists; Animals; Base Sequence; Cell Line; Cloning, Molecular; Dihydropyridines; DNA Primers; DNA, Complementary; Gene Expression; Humans; Isopropyl Thiogalactoside; Molecular Sequence Data; Phenethylamines; Piperazines; Polymerase Chain Reaction; Rats; Receptors, Adrenergic, alpha-1; Recombinant Proteins; Restriction Mapping; Tetralones; Transfection

It has previously been shown that dexniguldipine-HCl (B8509-035) is a potent chemosensitizer in multidrug resistant cells [Hofmann et al., J Cancer Res Clin Oncol 118: 361-366, 1992]. It is shown here that dexniguldipine-HCl causes a dose-dependent reduction of the labeling of the P-glycoprotein by azidopine, indicating a competition of dexniguldipine-HCl with the photoaffinity label for the multidrug resistance gene 1 (MDR-1) product. Exposure to dexniguldipine-HCl results in a dose-dependent accumulation of rhodamine 123 in MDR-1 overexpressing cells. In the presence of 1 microM dexniguldipine-HCl, rhodamine 123 accumulated in multidrug resistant cells to similar levels as in the sensitive parental cell lines. At this concentration, dexniguldipine-HCl enhances the cytotoxicities of Adriamycin and vincristine. The resistance modulating factors (RMF), i.e. IC50 drug/IC50 drug + modulator, were found to be proportional to the expression of MDR-1, ranging from 8 to 42 for Adriamycin and from 16 to 63 for vincristine. Transfection with the MDR-1 gene was found to be sufficient to sensitize cells to the modulation by dexniguldipine-HCl. The compound does not affect the expression of the MDR-1 gene. Dexniguldipine-HCl has no effect on a multidrug resistant phenotype caused by a mutation of topoisomerase II. It is concluded that dexniguldipine-HCl modulates multidrug resistance by direct interaction with the P-glycoprotein.

Topics: Affinity Labels; ATP Binding Cassette Transporter, Subfamily B, Member 1; Azides; Cell Line; Cell Survival; Dihydropyridines; DNA Topoisomerases, Type II; Dose-Response Relationship, Drug; Doxorubicin; Drug Interactions; Drug Resistance, Multiple; Humans; Rhodamine 123; Rhodamines; Transfection; Vincristine

A variety of agents are capable of overcoming P-glycoprotein-mediated multidrug resistance (MDR) in vitro. However, the clinical potential of these compounds is often limited due to high plasma protein binding. We compared the efficacy of several MDR-reversing compounds in serum-free culture medium and under serum conditions by means of a functional assay. Using flow cytometry the efflux of the fluorescent dye rhodamine 123 (Rh123) was measured from normal peripheral blood CD8+ T-lymphocytes which express low levels of P-glycoprotein. Inhibition of Rh123 efflux by R-verapamil, dexnigludipine-HCl, cyclosporin A, SDZ PSC833 and the protein kinase C (PKC) inhibitor CGP 41251 was determined in serum-free medium and in serum at concentrations from 0.1 to 50 mumol/l. With the exception of SDZ PSC833 all MDR modulators showed an insufficient or suboptimal modulation of P-glycoprotein under serum conditions at concentrations achievable in vivo. The highest potency under serum conditions demonstrated SDZ PSC833: even at a concentration of 0.5 mumol/l a sufficient inhibitory effect was observed. Subsequently this approach was applied to patients suffering from B-cell chronic lymphocytic leukaemia (B-CLL; n = 3) and acute myeloid leukaemia (AML; n = 2) which were positive in the Rh123 efflux assay. As for normal CD8+ T-lymphocytes, much higher drug concentrations were required under serum conditions to effectively inhibit Rh123 efflux from the leukaemic cells. Thus the interpretation of results of clinical 'modulator' trials should consider the decreased bioavailability of MDR-reversing agents.

Topics: Acute Disease; Antimetabolites, Antineoplastic; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; CD8-Positive T-Lymphocytes; Cyclosporine; Dihydropyridines; Dose-Response Relationship, Drug; Drug Resistance, Multiple; Flow Cytometry; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Leukemia, Myeloid; Rhodamine 123; Rhodamines; Verapamil

Digital imaging fluorescence microscopy was used to investigate the effect of the B subunit of cholera toxin on calcium homeostasis in neuroblastoma N18 cells. The B subunit, which binds specifically to ganglioside GM1 in the outer leaflet of the cell membrane, was found to induce a sustained increase of intracellular calcium concentration ([Ca2+]i). The increase in [Ca2+]i was not observed in the absence of extracellular calcium, or in the presence of the calcium chelator EGTA, and was blocked by nickel. The B subunit was also found to induce an influx of manganese ions, as indicated by a quench of the intracellular fura-2 fluorescence. These data suggest that the B subunit induces an increase in calcium influx in N18 cells. Potassium-induced depolarization also stimulated manganese influx; however, after the onset of depolarization-induced influx, the B subunit had no further effect. This occlusion suggests involvement of voltage-dependent calcium channels. Treatment with BayK8644, a dihydropyridine agonist selective for L-type calcium channels, induced manganese influx that was not altered by the B subunit and apparently blocked the effect of the B subunit itself. Furthermore, the dihydropyridine L-type channel antagonists niguldipine or nicardipine completely inhibited B subunit-induced manganese influx. Thus, the B subunit-induced manganese influx is likely due to activation of an L-type voltage-dependent calcium channel. Spontaneous influx of manganese ions was also inhibited by nicardipine or niguldipine and by exogenous gangliosides. Ganglioside GM1 was more potent than GM3, but globoside had no significant effect. The modulation of L-type calcium channels by endogenous ganglioside GM1 has important implications for its role in neural development, differentiation, and regeneration and also for its potential function in the electrical excitability of neurons.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Calcium; Calcium Channel Blockers; Calcium Channels; Cell Line; Cholera Toxin; Dihydropyridines; Egtazic Acid; Fura-2; G(M1) Ganglioside; G(M3) Ganglioside; Manganese; Mice; Microscopy, Fluorescence; Neuroblastoma; Nicardipine; Nickel; Potassium; Tumor Cells, Cultured

The low affinity binding sites identified in crude membranes from different excitable tissues with the dihydropyridine (DHP) calcium (Ca2+) channel ligands have confused researches in the field of Ca2+ channels as they can represent low affinity state(s) of the DHP receptor, or they can be labelled with DHP-type Ca2+ channel ligands. The aim of this communication was to provide more evidence for the existence of separate DHP binding sites on the surface of cultured green monkey renal cells (GMRC). The saturation ligand binding experiments with [3H]-nitrendipine (NTP) and photoaffinity labelling studies with (-)-[3H]-azidopine (AZI) were performed in order to identify and further characterize the DHP receptor on cultured GMRC. Specific high affinity sites identified on GMRC with [3H]-NTP (Bmax = 0.78 +/- 0.03 pmol/mg protein and KD = 0.06 +/- 0.1 nmol/l in native cells) and photolabelled with AZI represent DHP receptor on L-type Ca2+ channels. The low affinity binding sites photolabelled with AZI on GMRC (9.84 +/- 2.4 pmol/mg protein and KD = 3.21 +/- 1.25 nmol/l in native cells) were significantly increased after preincubation of GMRC with low concentrations of DHPs nitrendipine and nisoldipine. Preincubation of GMRC with Ca2+ channel agonist (-)BAYK 8644 significantly reduced specific photolabelling with AZI on GMRC and increased low affinity labelling. Preincubation of (+)BAYK 8644 was without any effect. Niguldipine (DHP with the voluminous substituent on the port side of the DHP ring) partially inhibited specific photolabelling with AZI on GMRC and also partially reduced the maximal number of low affinity binding sites labelled with AZI. Our results support the hypothesis of separate subsites in the region of DHP receptor of GMRC and the existence of the "marginal" photolabelling of specific DHP binding sites identified on Ca2+ channels.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Affinity Labels; Animals; Azides; Binding Sites; Calcium Channels; Calcium Channels, L-Type; Cells, Cultured; Chlorocebus aethiops; Dihydropyridines; Kidney; Kinetics; Muscle Proteins; Nisoldipine; Nitrendipine; Propanolamines; Receptors, Adrenergic, beta

In the clinical therapy of cancer, resistance to many cytostatic drugs is a major cause of treatment failure. Among other mechanisms, the expression and pumping activity of P-glycoprotein (PGP) in the membrane of resistant cancer cells is responsible for the reduced uptake of cytostatics. The blockade or inhibition of PGP activity by chemosensitisers seems to be a tenable way to restore sensitivity to antineoplastic drugs and therapeutic efficacy. In the present work the influence of the new chemosensitiser dexniguldipine on rhodamine-123 accumulation in multidrug-resistant leukaemia cells was investigated. Dexniguldipine increases cellular rhodamine-123 accumulation dose-dependently.pEC50 values (-log concentration of drug showing a half maximal effect) in accumulation studies are dependent on pH of the test system and are in the range of 6.5 (pH 7.2) to 7.2 (pH 8.0) for dexniguldipine. In comparison with other chemosensitisers such as SDZ PSC 833, cyclosporin A, verapamil, dipyridamole, quinidine and amiodarone, dexniguldipine is the most potent drug in this test system. In addition to equilibrium measurements of rhodamine-123 accumulation, efflux of rhodamine-123 was analysed in the absence and presence of chemosensitisers. A clear dose-dependency was seen and, moreover, a dramatic decrease in efflux rates was achieved in the presence of chemosensitisers. The described system can be used to investigate PGP-mediated drug transport on a pharmacological and biochemical basis.

Topics: Antineoplastic Agents; Cyclosporins; Dihydropyridines; Dose-Response Relationship, Drug; Drug Resistance, Multiple; Humans; Hydrogen-Ion Concentration; Leukemia; Rhodamine 123; Rhodamines; Tumor Cells, Cultured

Cell membranes were prepared from the multidrug resistant, P-glycoprotein expressing human lymphoblastoid cell line CCRF-ADR 5000. The P-glycoprotein of these membranes possessed high affinity binding sites for [3H]vinblastine, with a Kd of 8 +/- 2 nM and Bmax of 17 +/- 8 pmol/mg of protein. The binding of [3H]vinblastine to P-glycoprotein was not ATP-dependent, and was inhibited by cytotoxic drugs with the following potency order; vincristine > doxorubicin > etoposide. The 1,4-dihydropyridine and multidrug resistance reversing agent, dexniguldipine-HCl, inhibited binding with a Ki value of 37 nM. The multidrug resistance reversing agent cyclosporin A, and the cytotoxics doxorubicin and etoposide did not alter the kinetics of [3H]vinblastine dissociation from P-glycoprotein; however, the 1,4-dihydropyridines dexniguldipine-HCl and nicardipine accelerated dissociation of [3H]vinblastine. These data suggest that P-glycoprotein possesses at least two allosterically coupled drug acceptor sites; receptor site 1 which binds vinblastine, doxorubucin, etoposide and cyclosporin A, and receptor site 2 which binds dexniguldipine-HCl and other 1,4-dihydropyridines.

Topics: Allosteric Regulation; ATP Binding Cassette Transporter, Subfamily B, Member 1; Binding, Competitive; Calcium Channel Blockers; Cell Division; Cyclosporine; Dihydropyridines; Doxorubicin; Drug Resistance, Multiple; Etoposide; Humans; Kinetics; Mathematics; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Tritium; Tumor Cells, Cultured; Vinblastine; Vincristine

Recent evidences from molecular biology, radioreceptor binding and functional studies indicate that the alpha 1-adrenoceptor population is heterogeneous and can at least be divided into two subclasses named alpha 1A and alpha 1B. The present study was designed to obtain, a selective enrichment of rat brain cortical membranes with each subtype of alpha 1-adrenoceptor using alkylating agents. [3H]prazosin binding to rat cortical membranes was saturable and of high affinity (KD = 0.11 +/- 0.02 nM; Bmax = 132.5 +/- 7.2 fmol/mg protein). All ligands competed for specific [3H]prazosin binding in a statistically significant biphasic manner (%Rhigh = 30-40%; %Rlow = 60-70%). These sites meet generally accepted and recently described pharmacologic criteria for their identification as the alpha 1A- and alpha 1B-adrenoceptors. After pretreatment of membranes with benextramine (1 microM) in the presence of clonidine (1 microM), the antagonists, WB4101, (+)-niguldipine and phentolamine, displaced the radioligand with an inhibition curve steeper than in control membranes and with Ki values that agree with those obtained for the low affinity site present in control membranes. On the other hand, after pretreatment with chloroethylclonidine (10 microM) in the presence of WB4101 (1 nM), Hill coefficients for the displacement of the radioligand by WB4101, (+)-niguldipine, and phentolamine, were also increased, but in contrast to the situation described above, the Ki values agree with those obtained for the high affinity site present in control membranes. In conclusion, this method of partial alkylation of receptors could be a valuable tool for separately studying the pharmacological characteristics of the alpha 1-adrenoceptor subtypes in native membranes of cerebral tissue.

Topics: Adrenergic alpha-Antagonists; Alkylating Agents; Alkylation; Animals; Binding Sites; Cerebral Cortex; Clonidine; Cystamine; Dihydropyridines; Dioxanes; Phenoxybenzamine; Phentolamine; Prazosin; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, Adrenergic, alpha

The alpha 1-adrenoceptors present in liver membranes from rats, hamsters and mice were characterized using [3H]prazosin. In the liver membranes from the three species a relatively large number of receptors was observed (500-900 fmol/mg of protein) and the affinities for [3H]prazosin were very similar (0.2-0.3 nM). Membrane preincubation with 10 microM chloroethylclonidine markedly decreased [3H]prazosin binding and higher concentrations essentially abolished specific binding of this radioligand. Binding competition experiments indicated the following orders of potency: a) for agonists: oxymetazoline > epinephrine > or = norepinephrine >> methoxamine and b) for antagonists: prazosin > WB 4101 > or = phentolamine = benoxathian > 5-methyl urapidil. The affinity for (+)niguldipine was also low but there was variation between the three species. Total RNA obtained from the liver of these species hybridized with the alpha 1B-adrenergic cDNA probe. The data suggest that these receptors correspond to the alpha 1B subtype.

Topics: Adrenergic alpha-Agonists; Adrenergic alpha-Antagonists; Animals; Binding Sites; Binding, Competitive; Cell Membrane; Clonidine; Cricetinae; Dihydropyridines; Liver; Male; Mesocricetus; Mice; Prazosin; Rats; Rats, Wistar; Receptors, Adrenergic, alpha; Species Specificity

The dihydropyridine, dexniguldipine hydrochloride (B859-35), has shown therapeutic activity in experimentally induced neuroendocrine hamster lung tumors and demonstrated antiproliferative effects in a mammary cancer cell line via inhibition of Ca2+ calmodulin. Studies in NIH 3T3 fibroblasts have provided evidence that dexniguldipine may also inhibit protein kinase C (PKC). In this study, we have tested the hypothesis that dexniguldipine may inhibit the proliferation of lung cancer cells in response to autocrine or exogenous activation of PKC. Using a panel of human lung cancer cell lines, we show that dexniguldipine is a potent inhibitor of mitogenic signal transduction pathways dependent on PKC activation in several small-cell and non-small-cell lung cancer cell lines while it failed to inhibit cyclic-AMP-dependent cell proliferation.

Topics: Adenocarcinoma; Antineoplastic Agents; Carcinoid Tumor; Carcinoma, Adenosquamous; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Cell Division; Dihydropyridines; Humans; Lung Neoplasms; Protein Kinase C; Tumor Cells, Cultured

A series of five potential modulators of resistance were tested for their relative ability, as compared with verapamil, to sensitize CEM lymphoblastoid leukemia drug-resistant tumor sublines expressing either the classic or the atypical multidrug-resistance (MDR) phenotype to vinblastine or teniposide. Maximal non-cytotoxic concentrations of each modulator were tested and sensitization induces (SIs) were derived by comparing the drug concentration required to inhibit growth by 50% in their presence or absence. Like verapamil (10 microM) itself, three of the other modulators tested, namely, S9788 (4 microM), flunarizine (20 microM) and quinidine (30 microM), resulted in 2- to 3-fold sensitization of vinblastine against the parental CEM cells, and comparable effects were noted in the CEM/VM-1 cells, which were not cross-resistant to vinblastine. In contrast, cyclosporin A (0.5 microM) and B859-35 (2 microM) did not enhance vinblastine growth inhibition in these lines. However, the greatest sensitization with all the modulators was noted in the classic MDR VBL1000 cells, with SIs ranging from 40- to 350-fold, except for cyclosporin A, which proved ineffective at the concentration tested (SI, 2.6). The greatest extent of differential sensitization of these VBL1000 tumor cells occurred with quinidine or B859-35, which proved significantly more effective than verapamil alone. Combinations of modulators resulted in additive effects, with B859-35 plus cyclosporin A proving superior to B859-35 plus verapamil. In contrast, none of these compounds proved effective as a sensitizer to teniposide. The growth-inhibitory effects of this drug were not modified significantly in either the 92-fold teniposide-resistant VM-1 cells or in the parental cells. Addition of verapamil itself also failed to modulate teniposide growth inhibition in the VBL1000 cells, which express significant cross-resistance to this drug (36-fold). However, SI values of 3- to 5-fold were obtained using quinidine or B859-35. These results serve (a) to emphasise the need to monitor the effects of modulators not only on drug-resistant cells but also on their drug-sensitive counterparts so as to ensure differential sensitization such that normal sensitive tissues are not likely to be adversely influenced and (b) to highlight the observation that the extent of modulation differs depending not only on the antitumor drug used but also on the mechanism of drug resistance expressed. This in vitro model system

Topics: Cyclosporine; Dihydropyridines; Dose-Response Relationship, Drug; Drug Resistance; Drug Screening Assays, Antitumor; Flunarizine; Humans; Leukemia, T-Cell; Piperidines; Quinidine; Teniposide; Triazines; Tumor Cells, Cultured; Verapamil; Vinblastine

The influence of a newly developed alpha 1-adrenoceptor antagonist, HV723 (alpha-ethyl-3,4,5-trimethoxy-alpha-(3-((2-(2-methoxy-phenoxy)ethyl)-ami no)- propyl) benzeneacetonitrile fumarate), on the positive inotropic effect and acceleration of phosphoinositide hydrolysis induced by phenylephrine via alpha 1-adrenoceptors in the presence of bupranolol (0.3 microM) was studied in the rabbit ventricular muscle: (1) HV723 at low concentrations (1-100 pM) attenuated the maximal inotropic response by 15-20% without altering the pD2 value for and [3H]inositol monophosphate accumulation induced by phenylephrine. The inhibitory action of HV723 (1-100 pM) showed a close resemblance to that of a selective alpha 1A antagonist, (+)-niguldipine. (2) HV723 (> or = 1 nM) shifted the concentration-response curve for phenylephrine to the right and downwards in association with a partial inhibition (42.5%) of [3H]inositol monophosphate accumulation. The IC50 values of HV723 for the inhibition of the inotropic response and phosphoinositide hydrolysis were approximately equal to the Kilow value determined by displacement of [3H]prazosin-specific binding. The mode of the inhibitory action of HV723 (> or = 1 nM) resembled that of another alpha 1A antagonist, WB 4101. These results indicate that HV723 shows a differential antagonistic action on the alpha 1A-mediated responses depending on the concentration: HV723 (1-100 pM) selectively inhibits the (+)-niguldipine-sensitive subclass to lead to a decrease in the maximal inotropic response with no change in phosphoinositide hydrolysis; HV723 (> or = 1 nM) may antagonize the WB 4101-sensitive subtype coupled to acceleration of phosphoinositide hydrolysis.

Topics: Acetonitriles; Adrenergic alpha-Antagonists; Animals; Binding, Competitive; Bupranolol; Calcium Channel Blockers; Dihydropyridines; Heart Ventricles; Hydrolysis; In Vitro Techniques; Male; Myocardial Contraction; Myocardium; Phenylephrine; Phosphatidylinositols; Prazosin; Rabbits; Receptors, Adrenergic, alpha

We have studied the role of alpha 1A- and alpha 1B-adrenoceptors in noradrenaline- and methoxamine-stimulated inositol phosphate accumulation in rat renal cortical slices. [3H]Prazosin binding studies with and without inactivation of alpha 1B-adrenoceptors by chloroethylclonidine treatment suggested that noradrenaline lacks relevant selectivity for alpha 1-adrenoceptor subtypes. Both agonists stimulated [3H]inositol phosphate accumulation with similar maximal effects. The alpha 1A-selective antagonists 5-methyl-urapidil and (+)-niguldipine inhibited inositol phosphate formation by both agonists with shallow biphasic curves but the high affinity component was only 15%-31% and 38%-41%, respectively. The irreversible alpha 1B-selective antagonist chloroethylclonidine inhibited inositol phosphate generation by both agonists by 54%-57%. In contrast to our previous data in rat cerebral cortical slices, we conclude that in rat renal cortex both alpha 1A- and alpha 1B-adrenoceptors are involved in noradrenaline- and methoxamine-stimulated inositol phosphate generation.

Topics: Adrenergic alpha-Antagonists; Animals; Dihydropyridines; Inositol Phosphates; Kidney Cortex; Kinetics; Male; Methoxamine; Norepinephrine; Piperazines; Rats; Rats, Wistar; Receptors, Adrenergic, alpha; Substrate Specificity; Tritium

Modulators for the reversal of multidrug resistance such as R-verapamil and B8509-035, a dihydropyridine, effectively overcome multidrug resistance in vitro and are currently undergoing clinical trial. One problem with their use is the application protocol; the question as to whether they should be given by continuous administration or in sequential doses in combination with the cytotoxic drugs has to be addressed. Therefore, we examined the influence of the exposure time and the sequence of modulator administration on the active transport of the fluorescent dye rhodamine 123 (R123), a substrate for the P-glycoprotein, in the resistant lymphoblastoid cell line VCR1000 and the parental nonresistant cell line CCRF-CEM. Our results demonstrate the importance of coadministration of R-verapamil and the cytotoxic agent for the modulation of multidrug resistance, whereas the exposure sequence does not seem to be such an essential parameter in the case of B8509-035. This observation should be considered for the further design of clinical studies.

Topics: Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Differentiation; Dihydropyridines; Dose-Response Relationship, Drug; Drug Interactions; Drug Resistance; Flow Cytometry; Humans; Membrane Glycoproteins; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Rhodamines; Tumor Cells, Cultured; Verapamil

Dexniguldipine-HCl (DNIG)--a prospective clinical modulator of p170-glycoprotein (pgp170)-mediated multidrug resistance (MDR)--was evaluated in a drug-accumulation assay in MDR murine leukemia cell strain F4-6RADR expressing pgp170. The compound elevated low accumulation of either doxorubicin (DOX), daunorubicin (DNR), or mitoxantrone (MITO) in resistant F4-6RADR cells to the very levels observed in drug-sensitive F4-6 precursor cells. In parallel with the increase in DNR content (F4-6RADR, solvent: 303 +/- 27 pmol/mg protein; DNIG (3.3 mumol/l): 1,067 +/- 174 pmol/mg protein; F4-6P, solvent: 948 +/- 110 pmol/mg protein; n = 8-9, SEM), the amount of DNR tightly bound to the acid precipitate pellet obtained from F4-6RADR (i.e., protein, DNA, RNA) increased 3.9-times to the levels observed in sensitive F4-6 cells. The main pyridine metabolite of DNIG displayed similar activity. Concentration-response analysis revealed that DNIG and R,S-verapamil (VER) induced 100% reversal of the DNR accumulation shortage associated with the MDR phenotype but DNIG was 8 times more potent than VER (50% inhibitory concentration (IC50), 0.73 vs 5.4 mumol/l). In keeping with the accumulation assay, DNIG was about 10 times more potent than VER in sensitizing F4-6RADR cells to the cytostatic and cytotoxic effects of DNR in proliferation assays. In conclusion, DNIG is a potent in vitro modulator, improving (a) the accumulation of anthracycline-like cytostatics, (b) drug access to cellular binding sites, and (c) the cytostatic action of DNR in F4-6RADR leukemia cells of the MDR phenotype.

Topics: Animals; Cell Division; Daunorubicin; Dihydropyridines; Dose-Response Relationship, Drug; Drug Resistance; Friend murine leukemia virus; Leukemia, Erythroblastic, Acute; Mice; Tumor Cells, Cultured; Verapamil

It has been shown previously that verapamil and other calcium antagonists and calmodulin inhibitors can reverse multidrug resistance. We compared the potency of the dihydropyridine derivatives (4R)-3-[3-(4,4-diphenyl-1-piperadinyl)-propyl]-5-methyl-1,4-dihydr o-2,6- dimethyl-4-(3-nitrophenyl)-pyridine-3,5-dicarboxylate-hydrochloride (B859-35), a metabolite of B859-35, niguldipine and (R)-nitrendipine to that of (RS)-verapamil in reversing multidrug resistance. The accumulation of the fluorescent dye rhodamine 123, which is transported by the P-glycoprotein, was determined by a flow cytometer. Multidrug-resistant human HeLa KB-8-5 and Walker rat carcinoma cells were incubated in the presence and in absence of the drugs indicated above. We found that 0.1 microM B859-35 increases the accumulation of rhodamine 123 in multidrug-resistant KB-8-5 and Walker cells more effectively than 1 microM (RS)-verapamil. In sensitive KB-3-1 cells addition of the drugs had no significant influence on the accumulation of rhodamine 123. IN KB-8-5 cells, 10 nM Adriamycin caused a reduction of cell growth to 85% compared to untreated controls (= 100%). If 1 microM B859-35, B859-35 metabolite, niguldipine, verapamil or (R)-nitrendipine was added to 10 nM Adriamycin, growth reduction compared with untreated controls increased to 12%, 11%, 23%, 63%, and 82% respectively. The effect of 0.1 microM B859-35 was a reduction in proliferation to 38%, that of 0.1 microM verapamil to 72%. These data illustrate that B859-35, a compound with antitumor activity in several tumors, is at least ten times more potent than racemic verapamil in reversing multidrug resistance.

Topics: Animals; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; Calcium Channel Blockers; Cell Division; Dihydropyridines; Drug Resistance; Humans; Membrane Glycoproteins; Nitrendipine; Rats; Rhodamines; Verapamil

The (-)-isomer of verapamil is 10-fold more potent as a calcium antagonist than the (+)-isomer. However, both enantiomers are equally effective in increasing cellular accumulation of anticancer drugs [Gruber et al., Int J Cancer 41: 224-226, 1988]. In addition to verapamil, there exists a wide variety of stereoisomers with phenylalkylamines and dihydropyridine structures which markedly differ in their potency as calcium antagonists. We have tested these drugs for their ability to increase intracellular accumulation of [3H]vinblastine ([3H]VBL) in a doxorubicin-resistant cell line (F4-6RADR) derived from the Friend mouse leukemia cell line (F4-6P) and in COS-7 monkey kidney cells. Both cell types express substantial amounts of multidrug resistance gene 1 mRNA and P-glycoprotein as revealed by RNA and immuno blot analysis. The enantiomers with phenylalkylamine structures [(+/-)-verapamil; (+/-)-devapamil; (+/-)-emopamil)] and with dihydropyridine structures [(+/-)-isradipine; (+/-)-nimodipine; (+/-)-felodipine; (+/-)-nitrendipine; (+/-)-niguldipine] increased [3H]VBL accumulation in both cell lines at micromolar concentrations. Although the stereoisomers of these drugs differ markedly in their potency as calcium channel blockers they were about equally effective in increasing VBL levels in the cells. There was no substantial difference in the potencies of the phenylalkylamine drugs in affecting cellular [3H]VBL transport. Major potency differences, however, were observed in the dihydropyridine drug series with the niguldipine isomers as the most effective drugs. Moreover, the niguldipine enantiomers were equally as effective in reversing VBL resistance in F4-6RADR cells as were the verapamil enantiomers. Since (-)-niguldipine (B859-35) displays a 45-fold lower affinity for calcium channel binding sites than (+)-niguldipine, but is equally potent in inhibiting drug transport by P-glycoprotein and in reversing drug resistance, it may be, in addition to (+)-verapamil, another useful candidate drug for the treatment of multidrug resistance in cancer patients.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Binding, Competitive; Biological Transport; Calcium Channel Blockers; Cell Line; Dihydropyridines; Drug Resistance; Haplorhini; Immunoblotting; Membrane Glycoproteins; Mice; RNA, Messenger; Stereoisomerism; Verapamil; Vinblastine

The influence of the alpha 1A-adrenoceptor subtype-selective antagonist (+)-niguldipine on the alpha 1-mediated positive inotropic effect was assessed in the isolated rabbit ventricular myocardium. (+)-Niguldipine displaced the specific binding of [3H]prazosin to a membrane fraction derived from rabbit ventricular muscle with high (Ki = 64.6 pmol/l; RH = 23%) and low (Ki = 7.08 nmol/l) affinity. (+)-Niguldipine displaced specific [3H]CGP-121177 binding only at very high concentrations (Ki = 118 nmol/l). (+)-Niguldipine at 0.1 pmol/l and higher shifted the concentration-response curve for the alpha 1-mediated positive inotropic effect downwards, but at higher concentrations (up to 10 and 100 nmol/l) it did not cause a further shift of the curve. (+)-Niguldipine (1-100 nmol/l) did not affect the beta-mediated positive inotropic effect and the basal force of concentration. (-)-Niguldipine also showed a selective inhibitory action on the alpha 1-adrenoceptor-mediated positive inotropic effect, but its affinity and potency were approximately 1-2 log units lower than those of (+)-niguldipine. The present results indicate that the alpha 1A-adrenoceptor subtype is involved in the alpha 1-mediated positive inotropic effect. (+)-Niguldipine (or (-)-niguldipine with lower affinity) is able to antagonize selectively the cardiac alpha 1A-adrenoceptor-mediated positive inotropic effect. The magnitude of the alpha 1A-mediated inotropic effect, however, may be much less than that mediated by the alpha 1B-subtype in the rabbit ventricular myocardium.

Topics: Adrenergic alpha-Antagonists; Adrenergic beta-Antagonists; Animals; Binding Sites; Calcium Channel Blockers; Dihydropyridines; Dose-Response Relationship, Drug; Heart; Male; Myocardial Contraction; Prazosin; Propanolamines; Rabbits; Receptors, Adrenergic, alpha; Receptors, Adrenergic, beta; Stimulation, Chemical

Radioligand binding studies were undertaken in renal membranes of normotensive and hypertensive rats in order to test the hypothesis that there are alterations in renal alpha 1-adrenergic subtypes of genetic hypertensive animals. The highly selective competitive compound, (+)-niguldipine, was used to distinguish high-affinity (alpha 1a) from low-affinity (alpha 1b) sites, after initial studies demonstrated that this compound had greater selectivity than 5-methylurapidil in distinguishing alpha 1a and alpha 1b sites in rat renal membranes. In contrast to the significant difference in the blood pressure of the spontaneously hypertensive rats (delta BP = 74 mm Hg), there was no difference in the renal alpha 1-adrenergic receptor density. Membranes from the whole kidneys of spontaneously hypertensive rats (SHRs) possessed 31% alpha 1a and 69% alpha 1b sites with -log(Ki) values of 10.0 +/- 0.3 and 7.1 +/- 0.1, respectively, for (+)-niguldipine. However, these values were not different from those obtained from renal membranes of the normotensive Wistar-Kyoto (WKY) rats. These results indicate that in spite of the elevated blood pressure during the established phase of hypertension, the number, the affinity, and the ratio of the alpha 1a and alpha 1b appear not to be responsible for the manifestation of hypertension during this phase.

Topics: Animals; Dihydropyridines; Hypertension; In Vitro Techniques; Kidney; Kidney Medulla; Male; Membranes; Piperazines; Prazosin; Radioligand Assay; Rats; Rats, Inbred SHR; Rats, Inbred Strains; Rats, Inbred WKY; Receptors, Adrenergic, alpha

The whole-cell tight seal recording technique was used to investigate the effects of niguldipine, a novel dihydropyridine, on Ca2+ currents in guinea pig atrial cells. Ca2+ currents were separated into T-type and L-type components by an appropriate voltage protocol. Extracellular application of 1 microM (+/-)-niguldipine (NIG) resulted in a pronounced blockade of both T-type (to 20 +/- 10% of control, n = 5) and L-type Ca2+ currents (to 28 +/- 12% of control, n = 5). Current to voltage relationships clearly showed that both Ca2+ currents were blocked over the whole voltage range examined (-60 to +40 mV). The inhibitory effect of niguldipine on T-type Ca2+ currents was found to be voltage-dependent, i.e. prolonged hyperpolarization to -90 mV led to a partial and transient removal of NIG block. The IC50 for T-type Ca2+ current inhibition by (+/-)-NIG was determined as 0.18 microM. NIG action is stereospecific. (+)-niguldipine was found to be more potent than (-)-niguldipine in blocking both Ca2+ currents. This study demonstrates the Ca2+ antagonistic action of the dihydropyridine NIG, which may not discriminate between T- and L-type Ca2+ channels.

Topics: Animals; Calcium; Calcium Channel Blockers; Dihydropyridines; Guinea Pigs; Heart; In Vitro Techniques; Myocardium

The selective antagonists (+)-niguldipine and 5-methylurapidil (5-MU) were used to more clearly identify the alpha 1-adrenergic receptor subtypes involved in second messenger responses in slice and culture preparations of rat brain. The alpha 1-adrenergic receptor activating [3H]inositol phosphate (InsP) formation in neocortical and hippocampal slices appeared to have mixed characteristics. Although the low potency of (+)-niguldipine indicated involvement of the alpha 1B subtype, 5-MU had an alpha 1A-like potency at this subtype. (+)-Niguldipine did not inhibit the alpha 1 receptor-mediated potentiation of the cAMP response to either isoproterenol or adenosine in cortical slices, even at high concentrations. 5-MU inhibited both cAMP responses, although this inhibition appeared non-competitive. Thus, these receptors are clearly different from those mediating InsP formation. In primary glial cultures, (+)-niguldipine also had a low potency in blocking norepinephrine-stimulated [3H]InsP formation, consistent with involvement of the alpha 1B subtype. However, both 5-MU and WB 4101 had high potencies in blocking this response, suggesting involvement of the alpha 1A subtype. Inactivation of the alpha 1B subtype by pretreatment of cultures with chloroethylclonidine did not increase the potencies of any of these antagonists. The inhibition by 5-MU and WB 4101 was competitive in both control and chloroethylclonidine-pretreated cultures, whereas the inhibition by (+)-niguldipine was primarily noncompetitive. The use of these more selective antagonists shows that the current alpha 1A/alpha 1B subclassification scheme is inadequate to identify the receptors mediating these responses. None of the responses were blocked by (+)-niguldipine with the high potency expected at the alpha 1A subtype, although all InsP responses were blocked by 5-MU with a relatively high (alpha 1A-like) potency. In addition, very low affinity and noncompetitive effects of (+)-niguldipine were observed. These data raise the possibility of additional subtypes of alpha 1-adrenergic receptors or as yet unidentified functional interactions between known subtypes.

Topics: Adrenergic alpha-Antagonists; Alkylation; Animals; Brain; Calcium Channel Blockers; Cells, Cultured; Cyclic AMP; Dihydropyridines; Dioxanes; Female; Inositol Phosphates; Male; Piperazines; Pregnancy; Rats; Rats, Inbred Strains; Receptors, Adrenergic, alpha; Second Messenger Systems; Tritium

(+)-Niguldipine inhibited specific 125I-BE 2254 binding more potently in membrane preparations from rat tissues enriched in the alpha 1A subtype (hippocampus and vas deferens) than those with the alpha 1B subtype (liver and spleen). Inhibition curves for (+)-niguldipine were better fit by a two-site model in most tissues, although Kl values for each site varied markedly between tissues. The potency of this lipophilic drug was highly dependent on tissue concentration, probably accounting for most of this variability. Pretreatment of membranes with chloroethylclonidine (CEC) to inactivate the alpha 1B subtype did not completely eliminate the low affinity sites for (+)-niguldipine, particularly in heart. Saturation analysis showed that (+)-niguldipine competitively inhibited both alpha 1A and alpha 1B subtypes. However, substantial non-competitive inhibition was also observed in several tissues. Analysis of inhibition curves for 5-methylurapidil gave similar proportions of alpha 1A and alpha 1B receptor sites as were calculated for (+)-niguldipine in various tissues. Although (+)-niguldipine and 5-methylurapidil revealed variable proportions of low affinity sites in CEC-pretreated hippocampus and heart, this was not observed with inhibition curves for WB 4101 and phentolamine. These results are generally consistent with the previously defined alpha 1A and alpha 1B subtypes. 5-Methylurapidil currently appears to be the best antagonist for discriminating these subtypes; (+)-niguldipine shows similar selectivity but is complicated by a high lipophilicity. However, the persistence of low affinity sites for 5-methylurapidil and (+)-niguldipine after CEC pretreatment and the noncompetitive effects of (+)-niguldipine in some tissues raise the possibility of an additional subtype(s) of alpha 1-adrenergic receptors in rat tissues.

Topics: Adrenergic alpha-Antagonists; Animals; Binding Sites; Binding, Competitive; Calcium Channel Blockers; Clonidine; Dihydropyridines; Dioxanes; Kinetics; Male; Membranes; Phentolamine; Piperazines; Rats; Rats, Inbred Strains; Receptors, Adrenergic, alpha; Time Factors

In rat ventricular myocytes, the effects of alpha adrenoceptor stimulation on outward currents were studied by means of the whole cell voltage-clamp technique. Phenylephrine (30 microM) in the presence of propranolol (1 microM) to block beta adrenoceptors reduced voltage-activated transient outward current. Both components of transient outward current were affected, i.e., peak current (Ipeak) was reduced by 25.3 +/- 1.8%, the outward current at the end of a clamp step (Ilate) was reduced by 39.1 +/- 3.5% (n = 5; holding potential -40 mV, clamp step to +20 mV). In order to describe the alpha-1 adrenoceptor subtypes involved in this action, the effect of phenylephrine was also investigated after pretreatment of the cells with various antagonists. Pretreatment with prazosin (0.3 microM) abolished completely the phenylephrine effect. The alpha-1A adrenoceptor subtype-selective antagonists 5-methylurapidil and (+)-niguldipine (0.1 microM each) and the irreversible alpha-1B adrenoceptor subtype antagonist chloroethyl-clonidine (100 microM) blocked the phenylephrine effect on Ipeak, but merely attenuated the effect on Ilate, whereas pretreatment with a combination of chloroethylclonidine and (+)-niguldipine suppressed the phenylephrine-induced effect on both outward current components just like prazosin did. In conclusion, stimulation of both adrenoceptor subtypes is required for reduction of Ipeak, but stimulation of either alpha-1A or alpha-1B subtype is sufficient for reduction of Ilate. Therefore, stimulation of both alpha-1 adrenoceptor subtypes contributes to the phenylephrine-induced reduction in transient outward currents of isolated rat myocytes.

Topics: Adrenergic alpha-Antagonists; Animals; Clonidine; Dihydropyridines; Electrophysiology; Heart; Membrane Potentials; Myocardium; Phenylephrine; Piperazines; Propranolol; Rats; Receptors, Adrenergic, alpha; Stimulation, Chemical

The dihydropyridine derivative B859-35 inhibits phospholipid- and calcium-dependent protein kinase C (PKC) in cell-free extracts from NIH3T3 cells. Inhibition is competitive with regard to phosphatidylserine. At 1 microM phosphatidylserine, half-maximal inhibition (IC50) is obtained at approximately 2.5 microM B859-35. 12-O-Tetradecanoylphorbol-13-acetate (TPA)-dependent activation of the Na+/H+ antiporter was used to determine whether the enzyme is also affected in intact cells. The activity of the antiporter was monitored by following the dimethylamiloride-sensitive cytosolic alkalinization. It is demonstrated that B859-35 depresses the TPA-induced alkalinization with an IC50 of 5 microM, indicating that PKC in intact cells and the enzyme in cell-free extracts are equally sensitive to the drug. TPA-induced expression of the c-fos gene was used as an additional marker for intracellular PKC activity. Activation of c-fos expression was determined by measuring chloramphenicol acetyltransferase (CAT) activity in cells transfected with a c-fosCAT construct in which the CAT gene is expressed under the control of the endogenous human c-fos promoter. The studies revealed that 2.5 microM B859-35, a concentration equivalent to the IC50 in cell-free extracts, significantly depresses TPA-induced c-fosCAT expression. B859-35 inhibited cellular proliferation of NIH3T3 cells with an IC50 of approximately 5 microM. This is close to the IC50 for the anti-PKC activity of B859-35. It is suggested that the inhibition of PKC contributes to the growth inhibition following exposure to B859-35.

Topics: 1-Methyl-3-isobutylxanthine; 3T3 Cells; Animals; Base Sequence; Calcium Channel Blockers; Carrier Proteins; Cell Division; Chloramphenicol O-Acetyltransferase; Cytosol; Dihydropyridines; Enhancer Elements, Genetic; Gene Expression; Genes, fos; Humans; Hydrogen-Ion Concentration; Kinetics; Mice; Molecular Sequence Data; Phosphatidylserines; Protein Kinase C; Recombinant Fusion Proteins; Sodium-Hydrogen Exchangers; Tetradecanoylphorbol Acetate; Transcription, Genetic; Transfection

We have recently demonstrated that the dihydropyridine-derivative B859-35 has a selective chemotherapeutic effect on experimentally induced neuroendocrine lung tumors in hamsters. These tumors resembled human atypical lung carcinoids morphologically and expressed mammalian bombesin, calcitonin and neuron-specific enolase. In the hamster model, B859-35 had no antiproliferative effect on pulmonary adenomas of Clara cell origin. In this study, we have tested the antiproliferative effects of B859-35 and of the Ca(2+)-channel blocker Verapamil in vitro on three human lung cancer cell lines. The neuroendocrine cell line NCI-H727 is derived from a lung carcinoid and expresses mammalian bombesin and calcitonin. Two non-neuroendocrine cell lines are derived from peripheral pulmonary adenocarcinomas, with line NCI-H322 expressing features of Clara cells while line NCI-H358 expresses features of alveolar type II cells. B859-35 was a potent antiproliferative agent in the neuroendocrine line NCI-H727 at concentrations as low as 0.001 pM, while it inhibited cell proliferation in the two other cell lines at concentrations of 100 nM and above. Verapamil inhibited cell proliferation in the neuroendocrine line NCI-H727 at concentrations of 1 nM and above.

Topics: Adenocarcinoma; Antineoplastic Agents; Calcium Channel Blockers; Carcinoid Tumor; Cell Division; Dihydropyridines; Humans; Lung Neoplasms; Tumor Cells, Cultured; Verapamil

An automated non-chiral high-performance liquid chromatographic method is described for the determination of the new anti-proliferative agent B859-35 in serum. This method employs sample clean-up of 1 ml of biofluid by liquid-solid extraction with the AASP (Advanced Automatic Sample Preparation) system. First separation is achieved on a LiChrospher-60-RP-Select-B column. A fraction of this elute is then collected by solid-phase trapping. Thereafter, the final chromatogram is developed on a narrow-bore Hypers1-CPS column and quantified with ultraviolet detection at 230 nm. The limit of quantitation of the assay is 250 ng/ml. Linearity was proven in the range 0.25-100 ng/ml. Typical figures for precision at these concentrations are 7.4 and 3.3%, and for accuracy 8.0 and 1.3%, respectively. An application of this method to the study of pharmacokinetics of B859-35 in serum samples of cancer patients is given.

Topics: Antineoplastic Agents; Cell Division; Chromatography, High Pressure Liquid; Dihydropyridines; Humans; Reproducibility of Results; Spectrophotometry, Ultraviolet

It has previously been shown that B-859-35 ((-)-3-methyl-5- 3-(4,4-diphenyl-l-piperidinyl)-propyl-l,4-dihydro- 2,6-dimethyl-4-(3-nitrophenyl)-pyridine-3,5-dicarboxylate-hydrochloride) exerts a selective carcinostatic effect on some tumors. In order to evaluate whether the anti-cancer activity of B-859-35 can be modulated, we combined the new drug with several established anti-tumor drugs. A combination of B-859-35 with VP-16 (etoposide) in MDR(multi-drug-resistant-gene)-expressing Walker rat carcinoma cells shows synergism. A combination of B-859-35 with doxorubicin results in stronger synergism than verapamil/doxorubicin, especially at low concentrations of B-859-35. The resistance of mdrl(human multi-drug-resistance-gene)-expressing human HeLa KB-8-5 cells to doxorubicin can be reversed with non-toxic or weakly toxic concentrations of B-859-35 to the sensitivity of the parent KB-3-l cells. The finding that an anti-tumor drug is able to reverse multi-drug resistance makes B-859-35 an interesting drug for cancer treatment.

Topics: Actins; Animals; Antineoplastic Agents; Calcium Channel Blockers; Carcinoma 256, Walker; Cell Division; Cell Line; Dihydropyridines; DNA Topoisomerases, Type II; Doxorubicin; Drug Resistance; Drug Screening Assays, Antitumor; Drug Synergism; Etoposide; HeLa Cells; Humans; KB Cells; Mammary Neoplasms, Experimental; Molecular Structure; Rats

Topics: Antineoplastic Agents; Calmodulin; Dihydropyridines; Humans; Tumor Cells, Cultured

The effect of both isomers of niguldipine, a highly selective alpha 1-adrenoceptor antagonist and dihydropyridine calcium channel blocker, on noradrenaline-stimulated inositol phosphate (IP) accumulation and adenosine 3':5'-cyclic monophosphate (cyclic AMP) potentiation was examined. Both isomers inhibited noradrenaline-stimulated IP accumulation. (+)-Niguldipine was 100 fold more potent than (-)-niguldipine. Potentiation of beta-adrenoceptor-stimulated cyclic AMP by noradrenaline was only partially inhibited by both isomers. The dihydropyridine, israpidine, did not inhibit either second messenger response. This study provides further evidence that the alpha 1-adrenoceptors mediating IP accumulation and cyclic AMP potentiation are different.

Topics: Animals; Calcium Channel Blockers; Cerebral Cortex; Cyclic AMP; Dihydropyridines; In Vitro Techniques; Inositol Phosphates; Isoproterenol; Isradipine; Norepinephrine; Pyridines; Rats; Receptors, Adrenergic, alpha; Second Messenger Systems; Stereoisomerism

The chemotherapeutic effect of B859-35, the (-)-enantiomer of dihydropyrine 3-methyl-5-3-(4,4-diphenyl-1-piperidinyl)-propyl-1,4-dihydro-2,6-dimethy l-4- (3-nitrophenyl)-pyridine-3,5-dicarboxylate-hydrochloride (niguldipine), was tested on tumors induced in Syrian golden hamsters by N-nitrosodiethylamine (DEN). Peripheral pulmonary adenomas/adenocarcinomas were induced in hamsters maintained under ambient air conditions by multiple s.c. injections of DEN for 20 weeks. We have reproducibly shown that within this time interval lung adenomas develop in a significant number of the animals. The carcinogen treatment was discontinued at this point and one group of these hamsters was given B859-35 intragastrically 5 days/week for 20 weeks while the second group of such tumor-bearing hamsters were kept for an identical time interval without further treatment. Neuroendocrine lung tumors were induced in hamsters maintained in an atmosphere of 60% O2 by multiple s.c. injections of DEN for 8 weeks. We have reproducibly shown that within this short time interval neuroendocrine lung tumors develop in a significant number of the animals. The carcinogen treatment was discontinued at this point and the animals were returned to ambient air conditions. One group of these tumor-bearing hamsters was then given B859-35 intragastrically 5 days/week for 20 weeks while a second group of these hamsters was kept untreated for an identical time interval. A control group was given s.c. injections of saline for 20 weeks under ambient air conditions. A dramatic and selective anticarcinogenic effect of B859-35 was observed on the neuroendocrine lung tumors and nasal cavity tumors induced by DEN/hyperoxia while tumors of larynx/trachea were not affected. B859-35 had no effect on peripheral adenomas/adenocarcinomas, nasal cavity tumors, papillary polyps of larynx/trachea, or liver tumors induced by DEN under ambient air conditions.

Topics: Animals; Cricetinae; Diethylnitrosamine; Dihydropyridines; Laryngeal Neoplasms; Lung Neoplasms; Male; Mesocricetus; Nose Neoplasms; Tracheal Neoplasms

Niguldipine, a novel dihydropyridine derivative, was tested for its effects on the cytoplasmic free Ca2+ concentration of mouse thymocytes. In quin-2-loaded cells, a concentration-dependent rise of cytoplasmic Ca2+ can be detected, which requires extracellular Ca2+. The effect of niguldipine reaches a maximum after about 5 min; a similar time course has been observed, when using concanavalin A as a stimulus. Niguldipine provokes influx of Ca2+ into thymocytes, but not of Mn2+. Moreover, the effect of niguldipine exhibits some degree of stereospecificity, since (-)-niguldipine was more effective than its (+)-enantiomer. The action of niguldipine could be reversed by addition of bovine serum albumin, but not by addition of nitrendipine. None of several agents tested (e.g. felodipine, nitrendipine, trifluoperazine, cloxacepride, phenylephrine and ouabain) could mimic the effect of niguldipine at a concentration of 1 microM.

Topics: Animals; Calcium; Calcium Channel Blockers; Cells, Cultured; Dihydropyridines; Male; Mice; Serum Albumin; Stereoisomerism; Thymus Gland

1. Vascular smooth muscle cells were isolated from the portal vein and from pial vessels of the cow. They were voltage-clamped with a single patch electrode technique (whole cell recording) in order to analyse the effects of niguldipine on ionic membrane currents. Due to adsorption of niguldipine to plastic and glass, the effective concentrations are lower than the nominal concentrations by a factor of about 3. 2. Niguldipine reduced Ca-currents (ICa of the L-type, voltage operated) at nominal concentrations greater than 0.1 microM up to a complete block at 1 microM (50% block at 0.4 microM). Nominal concentrations between 50 and 200 nM facilitated ICa ('Ca-agonistic effect'). The Ca-agonistic effects of niguldipine showed modest use- but strong voltage-dependence. 3. Niguldipine increased the outward currents at nominal concentrations greater than 10 nM. The extra outward currents reversed at -85 mV, the result suggesting that niguldipine had increased potassium currents, IK. Maximal facilitation of IK by niguldipine was about 400% and was obtained at 1 microM, half-maximal facilitation was obtained with a nominal concentration of 20 nM. 4. Both reduction of ICa and facilitation of IK may contribute to vasodilatation by niguldipine. Due to its greater sensitivity, the effects on IK may dominate.

Topics: Animals; Calcium; Calcium Channel Blockers; Calcium Channels; Cattle; Cerebral Veins; Dihydropyridines; In Vitro Techniques; Membrane Potentials; Muscle, Smooth, Vascular; Portal Vein; Potassium Channels

The enantiomers of the 1,4-dihydropyridine (DHP) niguldipine (3-methyl-5-[3-(4,4-diphenyl-1-piperidinyl)-propyl]- 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-pyridine-3,5-dicarboxylate- hydrochloride) were investigated with respect to their interaction with 1,4-DHP receptors on L-type Ca2+ channels and alpha-adrenoceptors. The Ki values for niguldipine were dependent on the membrane protein concentrations in the radioligand binding assay. 'True' Ki values (at extrapolated 'zero' membrane protein) were determined with guinea-pig membranes for (+)-niguldipine and were found to be 85 pmol/l for the 1,4-DHP receptor of skeletal muscle, 140 pmol/l for that of brain and 45 pmol/l for that of heart. (-)-Niguldipine was approximately 40 times less potent. (+)-Niguldipine (Ki: 78 nmol/l) and (-)-niguldipine (Ki: 58 nmol/l) bound with approximately equal affinity to the alpha 1-adrenoceptors ('alpha 1B') in liver cell membranes. The (+)-niguldipine alpha 1-adrenoceptor inhibition data for rat brain cortex membranes were better fitted by a two-site model. The high-affinity component ('alpha 1A') had a Ki value of 52 pmol/l in competition experiments with [3H]prazosin. The low-affinity site (alpha 1B) had 200- to 600-fold less affinity. (-)-Niguldipine was greater than 40-fold less potent at alpha 1A- but was nearly equipotent to the (+)enantiomer at alpha 1B-sites. (+)-Niguldipine was the most selective compound for discriminating alpha 1A- from alpha 1B-adrenoceptors and is a novel prototype for 1,4-DHPs which bind with nearly equal affinity to skeletal muscle and brain or heart 1,4-DHP receptors.

Topics: Animals; Calcium Channel Blockers; Calcium Channels; Cerebral Cortex; Dihydropyridines; Guinea Pigs; In Vitro Techniques; Iodine Radioisotopes; Isradipine; Liver; Male; Muscles; Myocardium; Pyridines; Rats; Receptors, Adrenergic, alpha; Stereoisomerism

Niguldipine is a 1,4-dihydropyridine derivative that combines L-type Ca2+ channel-blocking effects and alpha 1-adrenolytic activity within a single molecule, exemplifying a novel approach in the treatment of hypertension. As niguldipine is a very hydrophobic compound, it (1) readily adsorbs to surfaces of the plastic-ware often used in radioligand binding assays and (2) partitions into the hydrophobic membrane compartments. Both phenomena decrease the actual free drug concentration in radioligand-binding assays and lead to gross underestimation of the affinity of niguldipine (and other hydrophobic ligands) for the 1,4-dihydropyridine binding domain of the L-type Ca2+ channel or for alpha 1A adrenoceptors, respectively. Partitioning of the hydrophobic molecules into the membrane phase leads to a dependence of the Ki value on "total receptor" concentration despite mathematic corrections of the experimentally determined IC50 values. The Ki dependence was mimicked by adding denatured membranes (devoid of high-affinity receptor-binding activity) to native membrane preparations. Loss to pipet tips and tubes was avoided by a special dilution protocol. Partitioning into the hydrophobic membrane compartments needed more elaborate correction procedures.

Topics: Adsorption; Animals; Buffers; Calcium Channel Blockers; Calcium Channels; Chemical Phenomena; Chemistry; Dihydropyridines; Guinea Pigs; Ligands; Osmolar Concentration; Rats; Rats, Inbred Strains; Receptors, Adrenergic, alpha; Research Design; Water

[3H]5-Methyl-urapidil, a potent antihypertensive derivative of urapidil, binds to alpha 1A-adrenoceptors in rat brain cortex membranes with a dissociation constant (KD) of 0.89 nM and a Bmax of 116 fmol/mg protein. The ligand does not bind to purified liver cell membranes (alpha 1B-adrenoceptors). [3H]5-Methyl-urapidil also labels 5-HT1A receptors in brain membranes (KD: 0.84 nM and Bmax: 235 fmol/mg protein). (+/-)-Niguldipine, a novel 1,4-dihydropyridine with Ca2+-antagonistic as well as alpha 1A-adrenoceptor blocking properties, is a competitive inhibitor of [3H]5-methyl-urapidil binding to alpha 1A-adrenoceptors. In contrast to those for prazosin, the Ki values for niguldipine were highly dependent on the membrane protein concentration, indicating partitioning of niguldipine into hydrophobic compartments unavailable for alpha-adrenoceptor interaction. The extrapolated, 'true' Ki values were as follows: (+/-)-niguldipine: 0.298 nM, (-)-niguldipine: 3.12 nM, (+)-niguldipine: 0.145 nM.

Topics: Animals; Calcium Channel Blockers; Cerebral Cortex; Dihydropyridines; In Vitro Techniques; Male; Membranes; Piperazines; Potassium; Rats; Rats, Inbred Strains; Receptors, Adrenergic, alpha; Receptors, Serotonin; Stereoisomerism

Radioligand binding studies suggest that alpha 1-adrenoceptor recognition sites are heterogeneous. Several adrenergic agents discriminate between two adrenoceptor binding sites designated alpha 1A and alpha 1B. In the present study we demonstrate for the first time that these two subtypes exist in the human brain. 5-Methyl-urapidil and (+)-niguldipine, which have previously been shown to be alpha 1A-selective, inhibited [3H]prazosin binding to cortical membranes in a biphasic manner. The irreversible alpha 1B-ligand, chloroethylclonidine, preferentially eliminated the binding sites with low affinity for (+)-niguldipine. In contrast, BE 2254 and unlabelled prazosin displaced the radioligand in a monophasic manner. The IC50 values for prazosin were not affected by pretreatment of the membranes with chloroethylclonidine. Our data on human brain membranes are in excellent agreement with recent findings in rat tissues and suggest that the alpha 1-adrenoceptor subtypes in human brain are similar to those in rat tissues.

Topics: Adrenergic alpha-Antagonists; Brain Chemistry; Calcium Channel Blockers; Cerebral Cortex; Dihydropyridines; Humans; In Vitro Techniques; Phenethylamines; Piperazines; Prazosin; Receptors, Adrenergic, alpha; Tetralones

B 844-39 is a new dihydropyridine calcium antagonist with a long-lasting antihypertensive action. Preliminary tests in chronically instrumented normotensive dogs revealed that B 844-39 (0.3 mg/kg p.o.) caused a marked decrease in blood pressure which was accompanied by a counterregulatory increase in heart rate. Both effects outlasted the 6-h observation period. There was no sign of cardiac depression in left ventricular positive dP/dtmax or sonomicrometrically evaluated subendocardial systolic shortening. B 844-39 was also tested in renal hypertensive dogs over a period of 12 days to investigate its potential in the long-term treatment of hypertension. A dosage of 0.3 mg/kg given orally twice a day led to a marked and persistent decrease in blood pressure, which was accompanied by a positive chronotropic effect and increases in plasma renin activity and angiotensin II. This initial counterregulatory response was blunted within several days of chronic treatment. After completion of the 12-day treatment period, the blood pressure reduction persisted for greater than 14 days. B 844-39 induced a marked and persistent reduction in blood pressure in hypertensive dogs, even with prolongation of the dosing interval to 24 h. The hypotensive effect of this drug was more pronounced in hypertensive than in normotensive animals.

Topics: Animals; Antihypertensive Agents; Blood Pressure; Calcium Channel Blockers; Dihydropyridines; Dogs; Female; Hypertension, Renal; Male; Renin