piperidines and alpha-beta-methyleneadenosine-5--triphosphate

Nicotinamide phosphoribosyltransferase (NAMPT) is a crucial enzyme in the biosynthesis of intracellular NAD+. NAMPT inhibitors have potent anticancer activity in several preclinical models by depleting NAD+ and ATP levels. Recently, we demonstrated that CD73 enables the utilization of extracellular NAD+/nicotinamide mononucleotide (NMN) by converting them to Nicotinamide riboside (NR), which can cross the plasmamembrane and fuel intracellular NAD+ biosynthesis in human cells. These processes are herein confirmed to also occur in a human ovarian carcinoma cell line (OVCAR-3), by means of CD73 or NRK1 specific silencing. Next, we investigated the anti-tumor activity of the simultaneous inhibition of NAMPT (with FK866) and CD73 (with α, β-methylene adenosine 5'-diphosphate, APCP), in an in vivo human ovarian carcinoma model. Interestingly, the combined therapy was found to significantly decrease intratumor NAD+, NMN and ATP levels, compared with single treatments. In addition, the concentration of these nucleotides in ascitic exudates was more remarkably reduced in animals treated with both FK866 and APCP compared with single treatments. Importantly, tumors treated with FK866 in combination with APCP contained a statistically significant lower proportion of Ki67 positive proliferating cells and a higher percentage of necrotic area. Finally, a slight but significant increase in animal survival in response to the combined therapy, compared to the single agents, could be demonstrated. Our results indicate that the pharmacological inhibition of CD73 enzymatic activity could be considered as a means to potentiate the anti-cancer effects of NAMPT inhibitors.

Topics: 5'-Nucleotidase; Acrylamides; Adenosine Triphosphate; Animals; Cell Line, Tumor; Cytokines; Female; GPI-Linked Proteins; Humans; Mice; Mice, Nude; NAD; Niacinamide; Nicotinamide Mononucleotide; Nicotinamide Phosphoribosyltransferase; Ovarian Neoplasms; Piperidines; Pyridinium Compounds; RNA Interference; RNA, Small Interfering

The purpose of this study was to examine the pharmacologic plasticity of cholinergic, non-adrenergic non-cholinergic (NANC), and purinergic contractions in neurogenic bladder strips from spinal cord injured (SCI) rats. Bladder strips were harvested from female rats three to four weeks after T(9)-T(10) spinal cord transection. The strips were electrically stimulated using two experimental protocols to compare the contribution of muscarinic and NANC/purinergic contractions in the presence and the absence of carbachol or muscarine. The endpoints of the study were: (1) percent NANC contraction that was unmasked by the muscarinic antagonist 4-DAMP, and (2) P2X purinergic contraction that was evoked by α,β-methylene ATP. NANC contraction accounted for 78.5% of the neurally evoked contraction in SCI bladders. When SCI bladder strips were treated with carbachol (10 μM) prior to 4-DAMP (500 nM), the percent NANC contraction decreased dramatically to only 13.1% of the neurally evoked contraction (P=0.041). This was accompanied by a substantial decrease in α,β-methylene ATP evoked P2X contraction, and desensitization of purinergic receptors (the ratio of subsequent over initial P2X contraction decreased from 97.2% to 42.1%, P=0.0017). Sequential activation of the cholinergic receptors with carbachol (or with muscarine in neurally intact bladders) and unmasking of the NANC response with 4-DAMP switched the neurally evoked bladder contraction from predominantly NANC to predominantly cholinergic. We conclude that activation of muscarinic receptors (with carbachol or muscarine) blocks NANC and purinergic contractions in neurally intact or in SCI rat bladders. The carbachol-induced inhibition of the NANC contraction is expressed more in SCI bladders compared to neurally intact bladders. Along with receptor plasticity, this change in bladder function may involve P2X-independent mechanisms.

Topics: Acetylcholine; Adenosine Triphosphate; Animals; Carbachol; Cholinergic Agonists; Female; Muscarinic Antagonists; Muscle Contraction; Muscle, Smooth; Neuronal Plasticity; Piperidines; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Receptors, Purinergic; Spinal Cord Injuries; Urinary Bladder

In the present study, the plasticity of the non-adrenergic non-cholinergic (NANC) response was investigated. Isolated rat bladder strips were electrically stimulated and the evoked contractions were isometrically recorded. The NANC part of the contractions were unmasked by applying 500 nM 4-DAMP, a potent muscarinic antagonist. Treatment of the bladder strips with 10 microM carbachol (a cholinergic agonist) increased the muscle tone but did not alter the neurally evoked contractions. However, carbachol decreased: (1) the NANC response from 74.6% to 33.3% of control and (2) the purinergic contractile response to alpha,beta-methylene ATP (alpha,beta-mATP) (10 microM) from 97.0% to 43.4% (p<0.05). Treatment with the cholinesterase inhibitor eserine (10 microM) also significantly decreased the NANC response to 21.1% (p<0.0001). The purinergic receptor antagonist suramin (100 microM) did not affect the neurally evoked contractions, however; subsequent addition of 4-DAMP decreased the contractions to 31%. Activation of the smooth muscle cholinergic receptors (with carbachol or eserine) and purinergic receptors (with alpha,beta-mATP) decreased the NANC contractions and the direct contractile response to alpha,beta-mATP. When the electrically evoked contractions were facilitated by the L-type Ca2+ channel activator, Bay-K 8644 the subsequent application of 4-DAMP did not unmask inhibited NANC contractions. We conclude that activation of muscarinic receptors by cholinergic agonist, carbachol or by endogenous acetylcholine (ACh) induce a cascade of events that leads to diminished purinergic response and consequently an inhibition of the bladder NANC response.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Adenosine Triphosphate; Animals; Antineoplastic Agents; Atropine; Calcium Channel Agonists; Carbachol; Cholinergic Agonists; Cholinesterase Inhibitors; Electric Stimulation; Female; In Vitro Techniques; Muscarinic Antagonists; Muscle Contraction; Muscle Relaxation; Muscle, Smooth; Physostigmine; Piperidines; Rats; Rats, Sprague-Dawley; Receptors, Cholinergic; Suramin; Urinary Bladder

In cyclophosphamide-induced cystitis in the rat, detrusor function is impaired and the expression and effects of muscarinic receptors altered. Whether or not the neuronal transmission may be affected by cystitis was presently investigated. Responses of urinary strip preparations from control and cyclophosphamide-pretreated rats to electrical field stimulation and to agonists were assessed in the absence and presence of muscarinic, adrenergic and purinergic receptor antagonists. Generally, atropine reduced contractions, but in contrast to controls, it also reduced responses to low electrical field stimulation intensity (1-5 Hz) in inflamed preparations. In both types, purinoceptor desensitization with alpha,beta-methylene adenosine-5'-triphosphate (alpha,beta-meATP) caused further reductions at low frequencies (<10 Hz). The muscarinic receptor antagonists atropine, 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) ('M(1)/M(3)/M(5)-selective'), methoctramine ('M(2)-selective') and pirenzepine ('M(1)-selective') antagonized the tonic component of the electrical field stimulation-evoked contractile response more potently than the phasic component. 4-DAMP inhibited the tonic contractions in controls more potently than methoctramine and pirenzepine. In inflamed preparations, the muscarinic receptor antagonism on the phasic component of the electrical field stimulation-evoked contraction was decreased and the pirenzepine and 4-DAMP antagonism on the tonic component was much less efficient than in controls. In contrast to controls, methoctramine increased -- instead of decreased -- the tonic responses at high frequencies. While contractions to carbachol and ATP were the same in inflamed and in control strips when related to a reference potassium response, isoprenaline-induced relaxations were smaller in inflamed strips. Thus, in cystitis substantial changes of the efferent functional responses occur. While postjunctional beta-adrenoceptor-mediated relaxations are reduced, effects by prejunctional inhibitory muscarinic receptors may be increased.

Topics: Adenosine Triphosphate; Adrenergic alpha-Antagonists; Adrenergic beta-Antagonists; Animals; Cyclophosphamide; Cystitis; Diamines; Electric Stimulation; In Vitro Techniques; Male; Muscarinic Antagonists; Muscle Contraction; Parasympatholytics; Phentolamine; Piperidines; Pirenzepine; Propranolol; Purinergic P2 Receptor Antagonists; Rats; Rats, Sprague-Dawley; Suramin; Sympatholytics; Urinary Bladder

To evaluate the effect of St. John's wort (SJW), an effective and safe herbal antidepressant, on rat bladder contractility. Recent data have suggested a strong association between depression and urinary incontinence.. Strips were cut from the bladder body and placed in organ baths containing Krebs solution. Contractions were induced by electrical field stimulation (EFS) and, in some experiments, by exogenous alpha,beta (alpha,beta)-methylene adenosine triphosphate.. St. John's wort was significantly more active in inhibiting the EFS-induced contractions than the alpha,beta-methylene adenosine triphosphate-induced contractions, suggesting both a presynaptic site of action and a direct inhibition of bladder smooth muscle. The inhibitory effect of SJW on EFS-induced contractions was unaffected by methysergide, haloperidol, phentolamine plus propranolol (antagonists that block the action of the neurotransmitters 5-hydroxytriptamine, dopamine, and noradrenaline on their own receptors), the L-type calcium channel antagonist verapamil, capsazepine (which blocks the vanilloid receptor), or cannabinoid CB1 receptor antagonist SR141716A. However, the opioid receptor antagonist naloxone significantly reduced the inhibitory effect of SJW on EFS-induced contractions. Among the chemical constituents of SJW tested, hyperforin and, to a lesser extent, the flavonoid kaempferol showed inhibitory effects.. The results of our study demonstrated that SJW inhibits excitatory transmission of the rat urinary bladder and also directly inhibits smooth muscle contractility. The inhibitory effect on excitatory transmission could involve, at least in part, opioid receptors. SJW may be evaluated for its possible use in treating urinary incontinence in depressed patients.

Topics: Acetylcholine; Adenosine Triphosphate; Animals; Anthracenes; Antidepressive Agents; Atropine; Bridged Bicyclo Compounds; Capsaicin; Depression; Electric Stimulation; Female; Haloperidol; Hypericum; Kaempferols; Male; Methysergide; Muscle Contraction; Muscle, Smooth; Naloxone; Perylene; Phentolamine; Phloroglucinol; Piperidines; Plant Extracts; Propranolol; Pyrazoles; Quercetin; Rats; Rats, Wistar; Rimonabant; Rutin; Terpenes; Tetrodotoxin; Urinary Bladder; Urinary Incontinence; Verapamil

1. The muscarinic acetylcholine receptors mediating the contractile response elicited to endogenous acetylcholine released by the selective P2X receptor agonist alpha,beta-methylene ATP (mATP) were investigated in guinea-pig ileum. 2. mATP (0.1 - 30 microM) elicited a concentration-dependent neurogenic contractile response inhibited by tetrodotoxin (TTX) and antagonized by the non-selective muscarinic receptor antagonist N-methylscopolamine (NMS). 3. The contractile response to mATP was pertussis toxin-insensitive, irreversibly antagonized by N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard), and unaffected by the muscarinic M(2)/M(4) receptor selective antagonist AF-DX 116 (1 microM). 4. When measured in the presence of histamine and isoproterenol after treatment with 4-DAMP mustard, mATP elicited a pertussis toxin-sensitive contractile response potently antagonized by AF-DX 116. 5. Collectively, our data suggest that endogenous acetylcholine released by mATP can elicit a direct contractile response through the muscarinic M(3) receptor and an indirect contractile response through the muscarinic M(2) receptor by antagonizing the relaxant effects of isoproterenol on histamine induced contraction.

Topics: Adenosine Triphosphate; Animals; Diphenylacetic Acids; Dose-Response Relationship, Drug; Guinea Pigs; Ileum; In Vitro Techniques; Male; Muscarinic Antagonists; Muscle Contraction; N-Methylscopolamine; Pertussis Toxin; Piperidines; Pirenzepine; Receptor, Muscarinic M2; Receptors, Muscarinic; Tetrodotoxin; Virulence Factors, Bordetella

This study investigated the cannabinoid receptor, known to inhibit neuronally-evoked contractions of the mouse isolated urinary bladder, in bladder sections isolated from mouse, rat, dog, pig non-human primate or human. The CB(1)-like pharmacology of the cannabinoid receptor in mouse isolated bladder observed previously was confirmed in this study by the rank order of agonist potencies: CP 55940>/=WIN 55212-2>HU 210>JWH 015>anandamide, the high affinity of the CB(1) selective antagonist, SR 141716A (apparent pK(B) 8.7), and the low affinity of the CB(2) antagonist, SR 144528 (apparent pK(B)<6.5). In these studies, SR 141716A (10-100 nM) significantly potentiated electrically-evoked contractions in this tissue by an undetermined mechanism. A similar rank order of agonist potencies was determined in rat isolated bladder sections (CP 55, 940> or =WIN 55212-2>JWH 015). In this tissue, the maximal inhibitory effect of all agonists was lower than in the mouse bladder. Indeed, the effects of both HU 210 and anandamide were too modest to quantify potency accurately. In the rat isolated bladder, SR 141716A (30 nM) or SR 144528 (100 nM), reversed the inhibitory effect of WIN 55212-2 (apparent pK(B) = 8.4 and 8.0, respectively) or JWH 015 (apparent pK(B) = 8.2 and 7.4, respectively). These findings may demonstrate pharmacological differences between the rat and mouse orthologues of the CB(1) receptor. Alternatively, they may be attributed to a mixed population of CB(1) and CB(2) receptors that jointly influence neurogenic contraction of the rat bladder, but cannot be differentiated without more selective ligands. WIN 55212-2 had no effect on electrically-evoked contractions of bladder sections isolated from dog, pig, cynomolgus monkey and human. These findings suggest that the effect of cannabinoid agonists to inhibit neurogenic contraction of the mouse and rat bladder is not conserved across all mammalian species.

Topics: Acetylcholine; Adenosine Triphosphate; Animals; Atropine; Benzoxazines; Calcium Channel Blockers; Carbachol; Cholinergic Agonists; Dogs; Dose-Response Relationship, Drug; Electric Stimulation; Electrophysiology; Humans; In Vitro Techniques; Macaca fascicularis; Male; Mice; Morpholines; Muscle Contraction; Naphthalenes; Piperidines; Pyrazoles; Rats; Receptor, Cannabinoid, CB2; Receptors, Cannabinoid; Receptors, Drug; Receptors, Muscarinic; Rimonabant; Species Specificity; Swine; Urinary Bladder