calcimycin and calmidazolium

Secretoneurin enhances the adhesion and transendothelial migration properties of monocytes and is a part of the peptide family encoded by the secretogranin II gene. The expression of the secretogranin II gene is upregulated in senescent endothelium. The present study was designed to examine the effects of secretoneurin on endothelium-dependent responsiveness. Isometric tension was measured in rings (with or without endothelium) of porcine coronary arteries. Secretoneurin did not induce contraction of quiescent or contracted rings. In preparations contracted by U-46619, relaxation was observed with high concentrations of the peptide. This relaxation was endothelium dependent and reduced by the nitric oxide synthase inhibitor N(ω)-nitro-l-arginine methyl ester (l-NAME). It was abolished when the preparations were incubated with l-NAME in combination with the cyclooxygenase inhibitor indomethacin. The relaxation was not affected by the combination of 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34) and 6,12,19,20,25,26-hexahydro-5,27:13,18:21,24-trietheno-11,7-etheno-7H-dibenzo[b,m][1,5,12,16]tetraazacyclotricosine-5,13-diiumditrifluoroacetate hydrate (UCL 1684), which abrogates endothelium-dependent hyperpolarizations. These results indicate that secretoneurin acutely induces relaxation through the activation of endothelial nitric oxide synthase (eNOS) and cyclooxygenase, with nitric oxide playing the dominant role. Prolonged (24 h) incubation with physiological concentrations of secretoneurin enhanced the relaxations to bradykinin and to the calcium ionophore A-23187, but this difference was not observed in preparations incubated with l-NAME or the calmodulin antagonist calmidazolium. Under these conditions, the relaxation to sodium nitroprusside remained unchanged. Incubation with secretoneurin significantly augmented the expression of eNOS and calmodulin as well as the dimerization of eNOS in cultures of porcine coronary arterial endothelial cells. These observations suggest that secretoneurin not only acutely causes but also, upon prolonged exposure, enhances endothelium-dependent relaxations.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Alkanes; Animals; Bradykinin; Calcimycin; Cardiovascular Agents; Coronary Vessels; Endothelium, Vascular; Imidazoles; Indomethacin; Neuropeptides; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Nitroprusside; Pyrazoles; Quinolinium Compounds; Secretogranin II; Swine; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents

Our previous studies showed that the prothoracicotropic hormone (PTTH) stimulated extracellular signal-regulated kinase (ERK) phosphorylation in prothoracic glands of Bombyx mori both in vitro and in vivo. In the present study, the signaling pathway by which PTTH activates ERK phosphorylation was further investigated using PTTH, second messenger analogs, and various inhibitors. ERK phosphorylation induced by PTTH was partially reduced in Ca(2+)-free medium. The calmodulin antagonist, calmidazolium, partially inhibited both PTTH-stimulated ERK phosphorylation and ecdysteroidogenesis, indicating the involvement of calmodulin. When the prothoracic glands were treated with agents that directly elevate the intracellular Ca(2+) concentration [either A23187, thapsigargin, or the protein kinase C (PKC) activator, phorbol 12-myristate acetate (PMA)], a great increase in ERK phosphorylation was observed. In addition, it was found that PTTH-stimulated ecdysteroidogenesis was greatly attenuated by treatment with PKC inhibitors (either calphostin C or chelerythrine C). However, PTTH-stimulated ERK phosphorylation was not attenuated by the above PKC inhibitors, indicating that PKC is not involved in PTTH-stimulated ERK phosphorylation. A potent and specific inhibitor of insulin receptor tyrosine kinase, HNMPA-(AM)(3), greatly inhibited the ability of PTTH to activate ERK phosphorylation and stimulate ecdysteroidogenesis. However, genistein, another tyrosine kinase inhibitor, did not inhibit PTTH-stimulated ERK phosphorylation, although it did markedly attenuate the ability of A23187 to activate ERK phosphorylation. From these results, it is suggested that PTTH-stimulated ERK phosphorylation is only partially Ca(2+)- and calmodulin-dependent and that HNMPA-(AM)(3)-sensitive receptor tyrosine kinase is involved in activation of ERK phosphorylation by PTTH.

Topics: Animals; Antibodies; Antibody Specificity; Bombyx; Calcimycin; Calcium; Calcium Signaling; Calmodulin; Ecdysteroids; Extracellular Signal-Regulated MAP Kinases; Genistein; Imidazoles; Insect Hormones; MAP Kinase Signaling System; Naphthalenes; Organophosphonates; Phosphorylation; Protein Kinase C; Receptor Protein-Tyrosine Kinases

When skeletal muscle is stretched or injured, myogenic satellite cells are activated to enter the cell cycle. This process depends on nitric oxide (NO) production by NO synthase (NOS), matrix metalloproteinase activation, release of hepatocyte growth factor (HGF) from the extracellular matrix, and presentation of HGF to the c-met receptor as demonstrated by a primary culture and in vivo assays. We now add evidence that calcium-calmodulin is involved in the satellite cell activation cascade in vitro. Conditioned medium from cultures that were treated with a calcium ionophore (A23187, ionomycin) for 2 h activated cultured satellite cells and contained active HGF, similar to the effect of mechanical stretch or NO donor treatments. The response was abolished by addition of calmodulin inhibitors (calmidazolium, W-13, W-12) or a NOS inhibitor N(G)-nitro-l-arginine methyl ester hydrochloride but not by its less inactive enantiomer N(G)-nitro-d-arginine methyl ester hydrochloride. Satellite cells were also shown to express functional calmodulin protein having a calcium-binding activity at 12 h postplating, which is the time at which the calcium ionophore was added in this study and the stretch treatment was applied in our previous experiments. Therefore, results from these experiments provide an additional insight that calcium-calmodulin mediates HGF release from the matrix and that this step in the activation pathway is upstream from NO synthesis.

Topics: Animals; Calcimycin; Calcium; Calmodulin; Cell Proliferation; Cells, Cultured; Culture Media, Conditioned; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hepatocyte Growth Factor; Imidazoles; Ionophores; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Rats; Rats, Sprague-Dawley; Satellite Cells, Skeletal Muscle; Signal Transduction; Sulfonamides

Neural tube (NT) closure is a multifactorial process that involves yet unresolved molecular mechanisms. It had been shown previously that high levels of nitric oxide (NO) block the process of NT closure in the chick embryo by inhibiting methionine synthase (MS). The MS inhibition and its effect on NT closure could be alleviated by folic acid, suggesting the involvement of the folate-methionine pathway in the process. Here we test the hypothesis that endogenous nitric oxide synthase (NOS) activity regulates the MS activity required in the process of NT closure. The experiments described here reveal that NOS activity per se, is indeed critical for NT closure in the chick embryo. Inhibition of NOS activity with either 2,4-diamino-6-hydroxypyrimidine (DAHP), which blocks biosynthesis of the NOS co-factor tetrahydrobiopterin (BH4), or with calmidazolium, which blocks calcium-calmodulin binding to NOS, resulted in reduced MS activity and consequently ablated NT closure. Addition of BH4 or the calcium ionophore A23187 restored NOS and MS activities, resulting in NT closure. The results described here imply that NOS and MS activities can serve as functional markers in this developmental process as they are essential in the process of NT closure.

Topics: 5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; Animals; Biopterins; Calcimycin; Calmodulin; Chick Embryo; Enzyme Inhibitors; Fatty Acids; Fluorescein; Fluorescent Dyes; Imidazoles; Indicators and Reagents; Neural Tube Defects; Nitric Oxide Donors; Nitric Oxide Synthase Type I; Nitroprusside; Vitamin B 12

Because the activity of the sodium pump (Na-K-ATPase) influences the secretion of aldosterone, we determined how extracellular potassium (K(o)) and calcium affect sodium pump activity in rat adrenal glomerulosa cells. Sodium pump activity was measured as ouabain-sensitive (86)Rb uptake in freshly dispersed cells containing 20 mM sodium as measured with sodium-binding benzofluran isophthalate. Increasing K(o) from 4 to 10 mM in the presence of 1.8 mM extracellular calcium (Ca(o)) stimulated sodium pump activity up to 165% and increased intracellular free calcium as measured with fura 2. Increasing K(o) from 4 to 10 mM in the absence of Ca(o) stimulated the sodium pump approximately 30% and did not increase intracellular free calcium concentration ([Ca(2+)](i)). In some experiments, addition of 1.8 mM Ca(o) in the presence of 4 mM K(o) increased [Ca(2+)](i) above the levels observed in the absence of Ca(o) and stimulated the sodium pump up to 100%. Ca-dependent stimulation of the sodium pump by K(o) and Ca(o) was inhibited by isradipine (10 microM), a blocker of L- and T-type calcium channels, by compound 48/80 (40 microg/ml) and calmidizolium (10 microM), which inhibits calmodulin (CaM), and by KN-62 (10 microM), which blocks some forms of Ca/CaM kinase II (CaMKII). Staurosporine (1 microM), which effectively blocks most forms of protein kinase C, had no effect. In the presence of A-23187, a calcium ionophore, the addition of 0.1 mM Ca(o) increased [Ca(2+)](i) to the level observed in the presence of 10 mM K(o) and 1.8 mM Ca(o) and stimulated the sodium pump 100%. Ca-dependent stimulation by A-23187 and 0.1 mM Ca(o) was not reduced by isradipine but was blocked by KN-62. Thus, under the conditions that K(o) stimulates aldosterone secretion, it stimulates the sodium pump by two mechanisms: direct binding to the pump and by increasing calcium influx, which is dependent on Ca(o). The resulting increase in [Ca(2+)](i) may stimulate the sodium pump by activating CaM and/or CaMKII.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Aldosterone; Animals; Calcimycin; Calcium; Calcium Channels; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Cells, Cultured; Enzyme Inhibitors; Extracellular Space; Female; Imidazoles; Intracellular Fluid; Ionophores; Isradipine; Potassium; Rats; Rats, Sprague-Dawley; Rubidium Radioisotopes; Sodium-Potassium-Exchanging ATPase; Staurosporine; Zona Glomerulosa

Amphotericin B remains the agent of choice for treatment of severe fungal infections. Its use is hindered by adverse effects, including infusion-related fever, chills, and hypotension, as well as nephrotoxicity with secondary anemia, hypokalemia, and hypomagnesemia. Amphotericin B-induced transcription and expression of interleukin (IL)-1beta by human monocytes is believed to be involved in mediating infusion-related adverse effects. It is shown here that agents that increase intracellular calcium [Ca++]i (A23187 and thapsigargin) in human monocytic cells also induce IL-1beta expression. Furthermore, amphotericin B-induced IL-1beta expression is attenuated by the calmodulin antagonist calmidazolium. Amphotericin B 5.41 microM increases [Ca++]i by up to 300 nM in these cells. In the presence of a nominal calcium buffer or EGTA, amphotericin B-induced IL-1beta expression is attenuated. Thus, amphotericin B acts as an ionophore to increase [Ca++]i and activates calmodulin-mediated expression of IL-1beta in human monocytes.

Topics: Amphotericin B; Calcimycin; Calcium; Calmodulin; Cell Line; Enzyme Inhibitors; Gene Expression Regulation; Humans; Imidazoles; Interleukin-1; Kinetics; Monocytes; Thapsigargin

The tachyzoite of Toxoplasma gondii must successfully invade a host cell before it can replicate. Depletion of the Ca2+ in the external medium (EGTA) reduced tachyzoite invasion, suggesting that the initial tachyzoite-host cell interaction is Ca2+ dependent. The interaction of tachyzoites with host cells was also inhibited by Ca2+ channel blockers (verapamil) and calmodulin antagonists (trifluoperazine, calmidazolium). The calmodulin concentrated at the apical end of the tachyzoite could be involved in cytoskeleton movement and conoid extrusion. Invasion also depends on changes in tachyzoite cytosolic calcium. Depletion of Ca2+ with A23187+EGTA and release of Ca2+ from intratachyzoite pools (nuclear and perinuclear areas) inhibited invasion. In contrast, Ca-ionophore and thapsigargin which increase host cell cytosolic Ca2+, significantly decreased tachyzoite invasion. We therefore suggest that the effect of the drug is significantly different from the localized Ca2+ signal that is produced after parasite attachment to its host cell receptors and leads to its internalization into the host cell.

Topics: Animals; Calcimycin; Calcium; Calcium Channel Blockers; Calmodulin; Carcinoma, Squamous Cell; Cytosol; Epithelial Cells; Humans; Imidazoles; Ionophores; Toxoplasma; Trifluoperazine; Tumor Cells, Cultured; Verapamil

In cultured rat vascular smooth muscle cells, angiotensin II (Ang II) induced a rapid increase in mitogen-activated protein kinase (MAPK) activity through the Ang II type 1 receptor, which was insensitive to pertussis toxin but was abolished by the phospholipase C inhibitor, U73122. The Ang II-induced MAPK activation was not affected by the protein kinase C inhibitor, GF109203X, and was only partially impaired by pretreatment with a phorbol ester, whereas both treatments completely prevented MAPK activation by the phorbol ester. Intracellular Ca2+ chelation by TMB-8, but not extracellular Ca2+ chelation or inhibition of Ca2+ influx, abolished Ang II-induced MAPK activation. The calmodulin inhibitor, calmidazolium, and the tyrosine kinase inhibitor, genistein, completely blocked MAPK activation by Ang II as well as by the Ca2+ ionophore A23187. Ang II caused a rapid increase in the binding of GTP to p21(ras), and this was inhibited by genistein, TMB-8, and calmidazolium but not by pertussis toxin or GF109203X. These data suggest that Ang II-induced MAPK activation through the Ang II type 1 receptor could be mediated by p21(ras)activation through a currently unidentified tyrosine kinase that lies downstream of Gq-coupled Ca2+/calmodulin signals.

Topics: Adenosine Triphosphate; Amino Acid Sequence; Angiotensin II; Animals; Aorta, Thoracic; Calcimycin; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; Cells, Cultured; Enzyme Activation; Enzyme Inhibitors; Estrenes; Gallic Acid; Genistein; GTP-Binding Proteins; Imidazoles; Indoles; Isoflavones; Kinetics; Maleimides; Molecular Sequence Data; Muscle, Smooth, Vascular; Peptides; Pertussis Toxin; Protein Kinase C; Proto-Oncogene Proteins p21(ras); Pyrrolidinones; Rats; Rats, Sprague-Dawley; Signal Transduction; Substrate Specificity; Type C Phospholipases; Virulence Factors, Bordetella

The possible role of intracellular Ca2+ level on Trypanosoma cruzi differentiation was explored. The addition to epimastigotes of a Triatoma infestans intestinal homogenate, which that triggers off the differentiation to the infective metacyclic form, induced a sudden rise in [Ca2+]i from the basal value, 94 +/- 28 to 584 +/- 43 nmole/liter. This increase was not affected by the presence of EGTA in the medium. Trypsin-treated intestinal homogenate did not alter the [Ca2+]i of epimastigotes. Calmodulin inhibitors (Calmidazolium, Trifluoperazine, and Chlorpromazine) blocked differentiation. Although the calcium ionophore ionomycin increased [Ca2+]i to 342 +/- 29 nmole/liter, it was unable to induce differentiation by itself. BAY K8644 and Methoxyverapamil (agonist and antagonist of Ca2+ channels, respectively) were unable to affect [Ca2+]i by themselves, or when added to stimulated parasites, and did not exert a stimulatory or inhibitory effect on morphogenesis. BAPTA/AM, a Ca2+ chelator, partially blocked the rise in [Ca2+]i and morphogenesis; this effect was reversed by ionomycin. The requirement of intracellular Ca2+ on epimastigote multiplication was also evaluated. The addition of EGTA to the culture medium led to a decrease in epimastigote multiplication till it practically ceased in the sixth passage. When such parasites were transferred to LIT they partially recovered the growth rate. Parasites from passages III, IV, and V in the Ca(2+)-depleted medium maintained their basal [Ca2+]i, but when treated with the intestinal homogenate, the rise in [Ca2+]i was abrogated. Accordingly, the differentiation percentages of such parasites dropped significantly compared with controls.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Calcimycin; Calcium; Calcium Channel Agonists; Calcium Channel Blockers; Chelating Agents; Chlorpromazine; Dopamine Antagonists; Egtazic Acid; Enzyme Inhibitors; Gallic Acid; Gallopamil; Imidazoles; Ionomycin; Ionophores; Morphogenesis; Triatoma; Trifluoperazine; Trypanosoma cruzi

The role of Ca2+ in the gravitropic perception and/or response mechanism of Coprinus cinereus was examined by treating stipes with inhibitors of Ca2+ transport and calmodulin. Inhibitors had no effect on gravity perception but significantly diminished gravitropism. It is concluded that, under the conditions tested, Ca2+ is not involved in gravity perception by Coprinus stipes, but does contribute to transduction of the gravitropic impulse. The results would be consistent with regulation of the gravitropic bending process requiring accumulation of Ca2+ within a membrane-bound compartment. Treatment of stipes with an actin inhibitor caused a significantly delayed response, a result not observed with the Ca2+ inhibitors. This suggests that cytoskeletal elements may be involved directly in perception of gravity by Coprinus stipes while Ca(2+)-mediated signal transduction may be involved in directing growth differentials.

Topics: Calcimycin; Calcium; Calcium Channel Blockers; Chelating Agents; Coprinus; Cytochalasin B; Cytoskeleton; Egtazic Acid; Enzyme Inhibitors; Gravitropism; Gravity Sensing; Imidazoles; Ionophores; Signal Transduction; Verapamil

In the present study, we investigated the effect of interferon-gamma (IFN-gamma) on cellular calcium ion concentration [Ca2+]i and inositol 1,4,5-trisphosphate (Ins 1,4,5-P3) formation in the human renal carcinoma cell line CaKi-1. We also examined the possible role of a Ca(2+)-dependent mechanism during IFN-gamma-induced intercellular adhesion molecule 1 (ICAM-1) antigen expression. IFN-gamma caused a rapid concentration-dependent rise in [Ca2+]i, which was partly inhibited by diltiazem, a calcium channel blocker, TMB-8, an inhibitor of intracellular calcium redistribution, and in calcium-free medium. IFN-gamma caused a fourfold increase in Ins 1,4,5-P3 formation. The induction of ICAM-1 antigen expression was synergistically enhanced by 4-bromocalcium ionophore A23187. Finally, the calcium antagonists diltiazem. TMB-8 and EGTA, as well as two potent inhibitors of Ca(2+)-dependent kinases, calmidazolium (R24571) and W7, had no or only a minor inhibitory effect on IFN-gamma induction. Our data suggest that IFN-gamma increases [Ca2+]i in CaKi-1 cells by stimulating influx of Ca2+ and release of Ca2+ from intracellular stores, probably via Ins 1,4,5-P3 formation. IFN-gamma signal transduction in our model may not be limited to an increase in [Ca2+]i and Ins 1,4,5-P3, since IFN-gamma-induced ICAM-1 antigen expression was abrogated to a minor degree by calcium antagonists and not coupled to Ins 1,4,5-P3 formation.

Topics: Antigens; Calcimycin; Calcium; Carcinoma, Renal Cell; Cell Adhesion Molecules; Cell Survival; Culture Media; Cyclic AMP-Dependent Protein Kinase Type II; Cyclic AMP-Dependent Protein Kinases; Humans; Imidazoles; Inositol 1,4,5-Trisphosphate; Intercellular Adhesion Molecule-1; Interferon-gamma; Kidney Neoplasms; Sulfonamides; Tumor Cells, Cultured

Intercellular adhesion molecule-1 (ICAM-1) is an important cell surface adhesion receptor of the immune system. Its cell surface expression on a wide variety of cells, including cancer cells, is regulated by various proinflammatory cytokines. In the present study, we investigated the role of calcium (Ca2+) and calmodulin (CaM) in the retinoic acid and gamma-interferon (IFN-gamma) signaling in the human neuroblastoma cell line SK-N-SH for up-regulating ICAM-1 expression. A 24-h incubation in the presence of Ca(2+)-mobilizing agents (A23187 and thapsigargin) resulted in the induction of ICAM-1 expression. Both Ca(2+)-mobilizing agents stimulated ICAM-1 expression additively to IFN-gamma but not to retinoic acid, suggesting that IFN-gamma does not use Ca2+ to stimulate ICAM-1, whereas retinoic acid might use it in part. As a second messenger, Ca2+ can be coupled with calmodulin. Using calmodulin inhibitors (W7 and calmidazolium), we found that retinoic acid-stimulated, A23187-stimulated, and thapsigargin-stimulated but not FIN-gamma-stimulated ICAM-1 were inhibited. Calmodulin signaling elicited by retinoic acid was an early event occurring within the first h of retinoic acid treatment, providing evidence that they may both be coupled to regulate gene expression. Using a novel CaM kinase II inhibitor, KN-62, we demonstrated that retinoic acid stimulated ICAM-1 expression in a CaM kinase II-dependent fashion. The mechanisms whereby CaM kinase II mediates retinoic acid activity on ICAM-1 expression remain to be elucidated.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Calcimycin; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Cell Adhesion Molecules; Enzyme Activation; Humans; Imidazoles; Intercellular Adhesion Molecule-1; Interferon-gamma; Isoquinolines; Neuroblastoma; Piperazines; Protein Kinase C; Sulfonamides; Terpenes; Thapsigargin; Tretinoin; Tumor Cells, Cultured; Up-Regulation

Cytoplasmic dynein is a microtubule-binding protein which is considered to serve as a motor for retrograde organelle movement. In cultured fibroblasts, cytoplasmic dynein localizes primarily to lysosomes, membranous organelles whose movement and distribution in the cytoplasm have been shown to be dependent on the integrity of the microtubule cytoskeleton. We have recently identified conditions which lead to an apparent dissociation of dynein from lysosomes in vivo, indicating that alterations in membrane binding may be involved in the regulation of retrograde organelle movement (Lin, S. X. H., and C. A. Collins. 1993. J. Cell Sci. 105:579-588). Both brief serum withdrawal and low extracellular calcium levels induced this alteration, and the effect was reversed upon addition of serum or additional calcium. Here we demonstrate that the phosphorylation state of the dynein molecule is correlated with changes in its intracellular distribution in normal rat kidney fibroblasts. Dynein heavy chain phosphorylation level increased during serum starvation, and decreased back to control levels upon subsequent addition of serum. We found that okadaic acid, a phosphoprotein phosphatase inhibitor, mimicked the effects of serum starvation on both phosphorylation and the intracellular redistribution of dynein from a membrane-associated pool to one that was more soluble, with similar dose dependence for both phenomena. Cell fractionation by differential detergent extraction revealed that a higher proportion of dynein was present in a soluble pool after serum starvation than was found in comparable fractions from control cells. Our data indicate that cytoplasmic dynein is phosphorylated in vivo, and changes in phosphorylation state may be involved in a regulatory mechanism affecting the distribution of this protein among intracellular compartments.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenosine Triphosphate; Alkaloids; Animals; Bucladesine; Calcimycin; Calmodulin; Cell Line; Colforsin; Culture Media; Cytoplasm; Dyneins; Edetic Acid; Ethers, Cyclic; Imidazoles; Kidney; Kinetics; Macromolecular Substances; Okadaic Acid; Phosphates; Phosphoprotein Phosphatases; Phosphorus Radioisotopes; Phosphorylation; Rats; Staurosporine; Sulfonamides; Tetradecanoylphorbol Acetate

Recent reports indicate that calmodulin inhibitors (CIs) can modify cadmium toxicity in rodents. For instance, pretreatment with CIs prevents cadmium-induced testicular damage in mice and substantially reduces such damage in rats, the latter effect coinciding with significant alterations in cadmium distribution. Although the basis of these effects is unclear, it is frequently observed that metal-binding proteins such as metallothionein (MT) are involved in acquired tolerance to cadmium and this could be true of tolerance induced by CIs. Thus, we examined the effects of various CIs on MT gene expression. Treatment of WF/NCr rats with known CIs, including trifluoperazine (TPZ), N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W7), calmidazolium (CMZ), chlorpromazine (CPZ), and the calcium ionophore, A23187, increased hepatic MT gene expression, as assessed by mRNA levels, in all cases. Furthermore, hepatic MT protein levels were 40 to 180 micrograms MT/g wet wt (g ww) in rats treated with CIs or A23187 compared to control levels of 10 micrograms MT/g ww. Treatment with CPZ and CMZ did not increase renal MT protein after exposure, although increases in renal MT mRNA were observed. However, the CIs TPZ and W7 and the calcium ionophore increased both renal MT protein and MT mRNA levels. In contrast, no increases in testicular MT mRNA or the testicular cadmium binding protein (TCBP) levels were seen with any of the treatments. Treatment with CIs or A23187 produced increases in zinc levels in the liver, but not in the kidneys or testes. These results indicate that CIs, such as TPZ, W7, CMZ, and CPZ, as well as the calcium ionophore A23187, have a marked stimulatory effect on hepatic and renal MT gene expression and that calcium regulatory pathways may play an important role in this induction of MT.

Topics: Animals; Base Sequence; Cadmium; Cadmium Chloride; Calcimycin; Calmodulin; Chlorides; Chlorpromazine; Gene Expression Regulation; Imidazoles; Kidney; Liver; Male; Metallothionein; Molecular Sequence Data; Rats; Rats, Wistar; RNA, Messenger; Sulfonamides; Testis; Trifluoperazine

Washed human erythrocytes were basically incubated in phosphate-buffered saline at 37 degrees C with different concentrations (1-5 mumol l-1) of the divalent cationophore A23187. This ionophore induced a decrease by about 75% of ATP content and energy charge (EC) and a concomitant increase in ADP and AMP contents in a dose-dependent fashion such that an inverse ATP/ADP relationship developed. EGTA at 1 mumol l-1 annihilated the effect of A23187 on energy status. When glucose was added to the basic incubation medium A23187 inclusion resulted in an elevated lactate production concomitantly with a partial restoration of EC. Introduction of Mg2+ to basic incubation medium containing glucose and the ionophore resulted in a sharp increase in lactate production with an energy state that was maintained at a control level. It is concluded that the low EC of erythrocytes obtained by this ionophore is the result of runaway ATPases dissociated from glycolysis. Since human erythrocytes are devoid of organelles other than the plasma membrane it is concluded that the ionophoric effect is exerted in the plasma membrane.

Topics: Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Calcimycin; Calcium; Energy Metabolism; Erythrocytes; Glucose; Humans; Imidazoles; Ion Pumps

1. The role of calmodulin in endothelium-dependent relaxations in the canine coronary artery, was investigated by use of the inhibitor of calmodulin, calmidazolium. 2. The endothelium-dependent relaxations to adenosine diphosphate (ADP) and nebivolol, a beta-adrenoceptor antagonist, in control solution, and to bradykinin in high potassium solution (to inhibit endothelium-dependent hyperpolarization), were abolished by nitro-L-arginine (30 microM), an inhibitor of nitro oxide-synthase. Calmidazolium (10 microM) did not inhibit these relaxations. 3. Calmidazolium did not affect the endothelium-independent relaxations to SIN-1, an exogenous donor of nitric oxide (NO). 4. The relaxations to bradykinin and to the calcium ionophore A23187 in control solution were inhibited to a small extent by calmidazolium (10 microM). 5. Bradykinin and A23187 induced relaxations in the presence of nitro-L-arginine (30 microM) that were abolished by calmidazolium (10 microM) but not affected by glibenclamide (10 microM), an inhibitor of ATP-sensitive K+ channels. 6. The endothelium-independent relaxations to lemakalim, an ATP-sensitive K+ channel opener, were not affected by calmidazolium (10 microM) but were inhibited by glibenclamide (10 microM). 7. These results suggest that calmidazolium does not inhibit the endothelium-dependent relaxations due to endothelium-derived NO in the canine coronary artery but inhibits either the production of endothelium-derived hyperpolarizing factor (EDHF) from endothelial cells or its effects on vascular smooth muscle cells. Furthermore these results suggest that EDHF contributes to endothelium-dependent relaxations in the canine coronary artery.

Topics: Adenosine Diphosphate; Adrenergic beta-Antagonists; Animals; Arginine; Benzopyrans; Bradykinin; Calcimycin; Calmodulin; Coronary Vessels; Cromakalim; Dogs; Endothelium, Vascular; Ethanolamines; Imidazoles; In Vitro Techniques; Muscle Relaxation; Muscle, Smooth, Vascular; Nebivolol; Nitric Oxide; Nitroarginine; Pyrroles; Vasodilator Agents

1. The effects of the calmodulin antagonists, calmidazolium and fendiline were investigated on endothelium-dependent hyperpolarization in the canine coronary artery. The membrane potential of vascular smooth muscle cells was measured with the microelectrode technique. 2. Smooth muscle cells of the canine coronary artery had a resting membrane potential of -50 mV. Bradykinin and the Ca(2+)-ionophore, A23187, induced concentration- and endothelium-dependent hyperpolarization. The hyperpolarization induced by a supramaximal concentration of bradykinin (10(-6) M) reached approximately 20 mV. 3. Calmidazolium (10(-5) M) and fendiline (10(-4) M) inhibited hyperpolarization induced by bradykinin and A23187. By contrast, calmidazolium did not affect the hyperpolarization induced by lemakalim, an opener of ATP-sensitive K(+)-channels. 4. These observations suggest that calmodulin is involved in the generation of endothelium-dependent membrane hyperpolarization of vascular smooth muscle.

Topics: Animals; Benzopyrans; Bradykinin; Calcimycin; Calmodulin; Coronary Vessels; Cromakalim; Dogs; Endothelium, Vascular; Female; Fendiline; Imidazoles; Male; Membrane Potentials; Muscle, Smooth, Vascular; Potassium Channels; Pyrroles

Agents like carbachol and cholecystokinin (CCK), that activate chief cell phosphoinositidase C, thereby increasing cell calcium concentration, increase cAMP levels in cholera toxin-treated, but not control, gastric chief cells. In the present study, we found that phorbol esters, like PMA, that activate protein kinase C, also cause augmentation of chief cell cAMP levels. The maximal effect with PMA (100 nM) was about 50% of the maximal response with CCK (10 nM) or carbachol (100 microM). Because protein kinase C is a calcium-dependent enzyme, we examined the effect of modulating cell calcium levels with the ionophore A23187. The ionophore alone caused a dose-dependent augmentation of cAMP levels. Adding 100 nM PMA caused an additive response, such that a maximal cAMP response, equal to that seen with 100 microM carbachol, was observed with 30 nM A23187. Carbachol-, A23187-, and PMA-induced augmentation of cAMP levels was progressively reduced by increasing concentrations of calmidazolium, a calmodulin inhibitor. Combination of phorbol esters that activate protein kinase C with ionophores that increase cell calcium mimics the actions of CCK and carbachol on cAMP levels in cholera toxin-treated chief cells.

Topics: Animals; Calcimycin; Calmodulin; Carbachol; Cholera Toxin; Gastric Mucosa; Guinea Pigs; Imidazoles; In Vitro Techniques; Kinetics; Male; Phorbol 12,13-Dibutyrate; Phorbols; Signal Transduction; Sincalide; Tetradecanoylphorbol Acetate

Theca cells were collected from the second largest preovulatory follicle. Chelation of extracellular calcium with EGTA attenuated LH (10 ng)-induced androstenedione production by theca cells, and this effect was more pronounced in calcium-deficient than in calcium-replete incubation medium. Incubation of theca cells with steroidogenic agonists in the presence of the calcium channel blocker verapamil (100 microM) suppressed androstenedione production stimulated by LH (a 57% decrease), the adenylate cyclase activator forskolin (a 59% decrease) and the cyclic adenosine monophosphate (cAMP) analog 8-bromo-cAMP (a 61% decrease). Furthermore, 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), a putative inhibitor of intracellular calcium mobilization, suppressed LH-induced androstenedione production in a dose-dependent fashion. The calmodulin inhibitors trifluoperazine (100 microM) and R24571 (50 microM) inhibited androstenedione production stimulated by hormonal (LH) and non-hormonal (forskolin, 8-bromo-cAMP) agonists (decreases ranging from 76 to 98%). While increasing the intracellular calcium ion concentrations with the calcium ionophore A23187 did not affect basal concentrations of androstenedione, treatment of LH-stimulated cells with the ionophore caused dose-dependent inhibition of androstenedione production; these effects were enhanced by coincubation with phorbol 12-myristate 13-acetate (a known activator of protein kinase C). We conclude that the mobilization of calcium is critical for agonist-stimulated steroidogenesis in hen theca cells, apparently requiring the interaction of calcium with its binding protein, calmodulin. Furthermore, increased cytosolic calcium concentrations may be involved in the suppression of androstenedione production, possibly as a result of an interaction with protein kinase C.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Androstenedione; Animals; Calcimycin; Calcium; Calmodulin; Chickens; Colforsin; Dose-Response Relationship, Drug; Female; Imidazoles; Luteinizing Hormone; Theca Cells; Trifluoperazine; Verapamil

The effects of TMB-8 and calmidazolium were investigated on mast cell responses believed to be mediated by protein kinase C, i.e. histamine release induced by TPA (tetradecanoyl-phorbol-acetate) in combination with sub-threshold concentrations of the ionophore A23187 and with antigen. Inhibition with both drugs was found in the same concentration range as observed earlier and could be counteracted by glucose, indicating an impaired oxidative energy production. Hence, the test drugs do not reveal protein kinase C selectivity.

Topics: Animals; Calcimycin; Calcium Channel Blockers; Calmodulin; Gallic Acid; Histamine Release; Imidazoles; In Vitro Techniques; Male; Mast Cells; Phorbol Esters; Rats; Rats, Inbred Strains

The role of calcium influx in dexamethasone-induced fragmentation of DNA was studied in the glucocorticoid-sensitive human lymphoid line of T cell derivation (CEM-C7). Reduction of calcium content in the medium or the use of EGTA increased DNA fragmentation and appeared to slightly enhance the effect of dexamethasone. Incubation of isolated nuclei in the presence of high concentrations of calcium did not bring about significant DNA fragmentation. Calmidazolium, an antagonist of calmodulin dependent reactions did not reduce the sensitivity of CEM-C7 cells to dexamethasone nor did it modify the response to dexamethasone of the resistant CEM-C1 line. It appears that in contrast to rodent thymocytes, massive calcium influx is not per se responsible for the initiation of directed cell killing (apoptosis).

Topics: Biological Transport; Calcimycin; Calcium; Calmodulin; Cell Division; Cell Nucleus; Cell Survival; Dexamethasone; DNA Damage; Humans; Imidazoles; In Vitro Techniques; Lymphocytes; Tumor Cells, Cultured

GH produces an acute but transient insulin-like response in adipocytes that have been deprived of GH for at least 3 h. The insulin-like response is followed by a period of refractoriness during which a second insulin-like response to GH cannot be elicited. These studies were undertaken to evaluate the role of calcium in the insulin-like response and refractoriness. Methionyl human GH (hGH) (100 ng/ml) increased the incorporation of D-[3-3H] glucose into lipid by 50-100% in fat cells that preincubated for 3 h without hormone and usually by less than 10% in fat cells that were made refractory by exposure to 100 ng/ml of hGH in the first hour of incubation. Insulin (100 microU/ml) increased lipogenesis by 3-5-fold whether fat cells were sensitive or refractory to GH. To determine whether calcium plays a role in either the insulin-like response to GH or the refractory phenomenon, we examined the effects of trifluoroperazine (20 or 50 microM) and calmidazolium (1.0 microM) which block calmodulin, as well as verapamil (30 microM), which blocks calcium channels. These agents did not interfere with stimulation of incorporation of D-[3-3H]glucose into lipid by GH (100 ng/ml) or insulin (100 microU/ml) but restored sensitivity to the insulin-like effects of GH in otherwise refractory cells. When freshly isolated, and hence refractory, cells were incubated for 1 h in calcium-free medium that contained 0.5 mM EGTA, GH stimulated lipogenesis by 30% (P less than 0.001) even though the response to insulin was markedly decreased in the absence of calcium. Conversely, when added to sensitive cells in the presence of normal extracellular Ca2+ concentrations during the 4th h of incubation, the calcium ionophore, A23187 (1.0 microM) produced an apparent refractoriness to the insulin-like effect of GH but had no effect on the response to insulin. The data indicate that maintenance of refractoriness to the insulin-like action of GH depends upon a calcium-calmodulin-sensitive process and suggest that, in producing refractoriness, GH may increase availability of intracellular calcium perhaps by increasing the rate of calcium entry into adipocytes. Since changes in responsiveness to GH were not paralleled by changes in responsiveness to insulin, it is suggested that the calcium dependent process responsible for refractoriness must lie at an early step in the signaling pathway.

Topics: Adipose Tissue; Animals; Calcimycin; Calcium; Calmodulin; Egtazic Acid; Glucose; Growth Hormone; Imidazoles; Insulin; Male; Rats; Signal Transduction; Trifluoperazine; Verapamil

Treatment of human erythrocytes with ionophore A23187 (10 mumol.l-1) and Ca2+ (0.05-0.5 mmol.l-1) or Sr2+ (0.2-1 mmol.l-1) in results in a concentration-dependent acceleration of the transmembrane reorientation (flip) of the lipid probes lysophosphatidylcholine and palmitoylcarnitine to the inner membrane leaflet after their primary insertion into the outer leaflet. Mg2+, Mn2+, Zn2+ and La3+ do not accelerate flip. Ca2(+)-induced flip acceleration depends also on the ionophore concentration. It is reversed by removal of Ca2+ with EDTA. A causal role of Ca2(+)-induced membrane protein degradation and decrease of the polyphosphoinositide level in flip acceleration could be excluded. Likewise, calmodulin-dependent processes are probably not involved since the calmodulin antagonist calmidazolium (2-10 mumol.l-1) does not suppress but even enhances the Ca2(+)-induced flip acceleration. The same is true for the Ca2+ antagonist flunarizine. These drugs do not alter flip rate in the absence of Ca2+. At high Ca2+ (1-5 mmol.l-1) an initial flip acceleration is followed by flip normalization. High concentrations of Mn2+ and Mg2+ slow down flip rates. The selective acceleration of flip by Ca2+ and Sr2+ is discussed to be due to a local detachment of the membrane skeleton from the bilayer, whereas the unselective slow down of flip by divalent cations might be due to a stabilization of the membrane bilayer by the cations. After loading of cells with Ca2+ (but not with Mn2+) the inner membrane leaflet phospholipid phosphatidylserine becomes rapidly exposed to the outer membrane surface, as detectable by its accessibility to phospholipase A2 (5 min).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Biological Transport; Calcimycin; Calcium; Calpain; Cations, Divalent; Erythrocyte Membrane; Flunarizine; Humans; Imidazoles; Kinetics; Lipid Bilayers; Phospholipids

A study was made of calcium metabolism during germ tube formation in Sporothrix schenckii yeast cells. A net efflux of calcium was observed very early in the transformation process and remained constant thereafter. The efflux of calcium in yeast cells induced to form germ tubes was twice that observed in yeast cells not induced to form germ tubes. Two peaks of calcium uptake were observed in germ tube forming yeast cells at 30 and 300 minutes following inoculation, while non-induced yeast cells, a continuous increase in uptake was observed which ultimately reached higher values than the ones obtained in germ tube forming cells. Substances which affect calcium metabolism in other cells such as cobalt ions, ionophore A23187 and compound R24571 were observed to inhibit germ tube formation and calcium uptake. In addition, ionophore A23187 was found to increase calcium efflux to approximately twice the control values. The inhibition of germ tube formation brought about by substances which inhibit calcium uptake or increase efflux suggests that the intracellular calcium concentration in these cells must be precisely regulated for the yeast to mycelium transition to occur.

Topics: Calcimycin; Calcium; Calmodulin; Cobalt; Enzyme Inhibitors; Imidazoles; Sporothrix

A 40-50% reduction in soybean [Glycine max (L.) Merr. cv. Century 84] hypocotyl elongation occurred 24 h after application of mechanical stress. Exogenous Ca2+ at 10 mM inhibited growth by 28% if applied with the Ca2+ ionophore A23187 to the zone of maximum hypocotyl elongation. La3+ was even more inhibitory than Ca2+, especially above 5 mM. Treatment with ethyleneglycol-bis-(beta-aminoethylether)-N, N, N', N'-tetraacetic acid (EGTA) alone had no effect on growth of non-stressed seedlings at the concentrations used but negated stress-induced growth reduction by 36% at 4 mM when compared to non-treated, stressed controls. Treatment with EDTA was ineffective in negating stress-induced growth inhibition. Calmodulin antagonists calmidazolium, chlorpromazine, and 48/80 also negated stress-induced growth reduction by 23, 50, and 35%, respectively.

Topics: Calcimycin; Calcium; Calmodulin; Cations, Divalent; Chelating Agents; Chlorpromazine; Darkness; Dimethyl Sulfoxide; Edetic Acid; Egtazic Acid; Enzyme Inhibitors; Glycine max; Hypocotyl; Imidazoles; Ionophores; Lanthanum; Physical Stimulation; Stress, Mechanical

A yeast-mycelium (Y-M) transition of Candida albicans (3153A) was induced by 1.5 mM CaCl2.2H2O in defined liquid medium, pH 7, at 25 degrees C. Germ tube formation was detected after approximately 8 h and peaks of maximum germination occurred at approximately 20 h in all experimental treatments. Non-toxic concentrations of the calmodulin inhibitor R24571 almost completely suppressed germ tube formation whereas trifluoperazine (TFP) and the Ca2+ ionophore A23187 were only about half as effective. Further Ca2+ addition failed to reverse the inhibitory effect of R24571 and induced only about 10% of the cells inhibited by TFP or A23187 to germinate.

Topics: Calcimycin; Calcium; Calmodulin; Candida albicans; Hydrogen-Ion Concentration; Imidazoles; Morphogenesis; Trifluoperazine

Calcium ionophore A23187 (0.3-0.4 microM) elevated cellular angiotensin-converting enzyme activity (ACE) 2-7-fold after 48 h incubation with bovine pulmonary artery endothelial cells in culture. Cycloheximide (0.1 micrograms/ml) blocked the elevation in ACE produced by A23187. The increase in ACE was inhibited by 0.2 mM EGTA, 50 microM verapamil and 50 microM nifedipine, and was not associated with changes in cellular cAMP. Melittin, a phospholipase A2 activator, or addition of exogenous arachidonic acid failed to reproduce the elevation, and indomethacin only partially blocked the A23187 effect. The elevation of ACE was also inhibited by the calcium-calmodulin inhibitor, calmidazolium. Thus, we postulate that the ionophore A23187 elevates ACE in endothelial cells through a calcium-dependent mechanism other than phospholipase A2 activation. The elevation depends on new protein synthesis and involves calcium-calmodulin-dependent cellular mechanisms.

Topics: Animals; Calcimycin; Cattle; Cells, Cultured; Cyclic AMP; Cycloheximide; Egtazic Acid; Endothelium, Vascular; Imidazoles; Nifedipine; Peptidyl-Dipeptidase A; Time Factors; Verapamil

Topics: Adolescent; Albuterol; Asthma; Calcimycin; Child; Child, Preschool; Cromolyn Sodium; Enzyme Inhibitors; Granulocytes; Humans; Imidazoles; In Vitro Techniques; Membrane Potentials; N-Formylmethionine Leucyl-Phenylalanine; Pancreatic Elastase; Superoxides; Tetradecanoylphorbol Acetate

The role of the plasma membrane in the regulation of lens fiber cell cytosolic Ca2+ concentration has been examined using a vesicular preparation derived from calf lenses. Calcium accumulation by these vesicles was ATP dependent, and was releasable by the ionophore A23187, indicating that calcium was transported into a vesicular space. Calcium accumulation was stimulated by Ca2+ (K1/2 = 0.08 microM Ca2+) potassium (maximally at 50 mM K+), and cAMP-dependent protein kinase; it was inhibited by both vanadate (IC50 = 5 microM) and the calmodulin inhibitor R24571 (IC50 = 5 microM), indicating that this pump was plasma-membrane derived and likely calmodulin dependent. Valinomycin, in the presence of K+, stimulated calcium uptake, suggesting that the calcium pump either countertransports K+, or is regulated in an electrogenic fashion. Inhibition of calcium uptake by selenite and p-chloromercuribenzoate demonstrates the presence of an essential -SH group(s) in this enzyme. Calcium release from calcium-filled lens vesicles was enhanced by Na+, demonstrating that these vesicles also contain a Na:Ca exchange carrier. p-Chloromercuribenzoate and p-chloromercuribenzoate sulfonic acid also promoted calcium release from calcium-filled vesicles, suggesting that this release, like calcium uptake, is in part mediated by a cysteine-containing protein. We conclude that lens fiber cell cytosolic Ca2+ concentration could be regulated by a number of plasma membrane processes. The sensitivity of both calcium uptake and release to -SH reagents has implications in lens cataract formation, where oxidation of lens proteins has been proposed to account for the elevated cytosolic Ca2+ in this condition.

Topics: 4-Chloromercuribenzenesulfonate; Adenosine Triphosphate; Animals; Calcimycin; Calcium; Calmodulin; Carrier Proteins; Cattle; Cell Membrane; Chloromercuribenzoates; Cyclic AMP; Cytosol; Imidazoles; Lens, Crystalline; Membrane Proteins; p-Chloromercuribenzoic Acid; Potassium; Protein Kinases; Sodium-Calcium Exchanger; Sodium-Potassium-Exchanging ATPase; Valinomycin; Vanadates

We have studied the effect of a calcium ionophore, A23187, and the purported calmodulin inhibitors, calmidazolium and chlorpromazine, on direct intercellular communication between smooth muscle cells in the myometrium of delivering rats. The extent of cell-to-cell coupling was determined by exposing one portion of small strips of longitudinal myometrium to 2-[3H] deoxy-D-glucose (2-DG) and determining the distribution and apparent diffusion coefficient (Da) for this tracer after a 5-h period for diffusion. The distribution and Da for 2-DG were significantly (p less than 0.05) reduced by exposure to A23187 in Krebs-Ringer solution with 2.5 mM Ca++, partially reduced in Krebs solution with A23187 and low Ca++ (1-10 microM), but the drug had no effect when used with Ca++-free solutions with [ethylenebis (oxyethylene-nitrilo)] tetraacetic acid (EGTA). The calmodulin inhibitors blocked the effects of A23187 in a dose-dependent fashion, and at higher concentrations, the extent of 2-DG diffusion was not different from that in control tissues. Surprisingly, however, a dose-dependent reduction in coupling was also observed in tissues exposed to the calmodulin inhibitors alone. Structural studies failed to reveal any change in the area of gap junctions between the myometrial cells following the above treatments, suggesting that the reduced exchange of 2-DG resulted from a decrease in the permeability of gap junctions between the muscle fibers.

Topics: Animals; Calcimycin; Calmodulin; Cell Communication; Chlorpromazine; Female; Imidazoles; Intercellular Junctions; Labor, Obstetric; Myometrium; Pregnancy; Rats

A study was performed to determine the possible role of calmodulin (CaM) in regulating gonadotropin-releasing hormone (GnRH)-induced luteinizing hormone (LH) release from the bovine pituitary using three structurally unrelated calmodulin antagonists. Primary calf anterior pituitary cell cultures (3 X 10(5)/well) were treated with either LH secretogogue (GnRH, 100 ng/ml; A23187, 2.5 microM; theophylline, 1 mM; prostaglandin E2, 1 microM; estradiol, 25 ng/ml; or KCl, 25 mM; final concentrations) or secretogogue plus CaM inhibitor in 1 ml Hanks' balanced salt solution plus 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid for 6 h. Significant (P less than 0.01) inhibition of GnRH- and A23187-stimulated LH release was obtained with calmidazolium (CMZ; 1-10 nM) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7; 1-10 microM). Both CMZ (10 nM) and W-7 (10 microM) significantly reduced (P less than 0.05) LH release by all of the other agents tested as well. In contrast, trifluoperazine (TFP; 0.1-100 microM) had no effect against most of the secretogogues tested. These results suggest that GnRH-stimulated LH release is in part a Ca2+-CaM-dependent process and may implicate a common CaM-dependent mechanism for LH release in general. The data also demonstrate a marked dichotomy in response between the phenothiazine, TFP, and other CaM antagonists in the calf pituitary.

Topics: Animals; Calcimycin; Calmodulin; Cattle; Dinoprostone; Female; Gonadotropin-Releasing Hormone; Imidazoles; Luteinizing Hormone; Male; Pituitary Gland, Anterior; Prostaglandins E; Sulfonamides; Trifluoperazine

The roles of protein kinase C, calcium and calmodulin in mediating insulin-stimulated lipogenesis by rat adipocytes were investigated using the protein kinase C activator, phorbol myristate acetate (PMA); the protein kinase C inhibitors, H7 and polymixin B; the calcium ionophore, A23187; the calcium channel blocker, verapamil; and the calmodulin inhibitor, calmidazolium. PMA caused a concentration-dependent, parallel left shift of the insulin-lipogenesis dose response curve. Both PMA- and insulin-stimulated lipogenesis were inhibited by H7 and polymixin B. A23187 enhanced the stimulatory action of both insulin and PMA was not inhibited by H7. The stimulatory effects of insulin and PMA were inhibited by verapamil and calmidazolium. These data indicate that insulin receptor-lipogenesis coupling in rat adipocytes is mediated by protein kinase C-elicited calcium influx and activation of calmodulin.

Topics: Adipose Tissue; Animals; Calcimycin; Calcium; Calcium Channel Blockers; Cells, Cultured; Glucose; Imidazoles; Insulin; Kinetics; Lipids; Male; Polymyxin B; Protein Kinase C; Rats; Tetradecanoylphorbol Acetate; Verapamil

The role of the calcium messenger system in the regulation of ion absorption across the teleost intestine was studied using pharmacological intervention. Radiochloride transport was independent of external Ca2+ over the range 10 microM to 2.5 mM. Treatment with the Ca2+ ionophore A23187 (to hyperpolarization of the apical membrane potential of intestinal epithelial cells. The Ca2+-calmodulin antagonists trifluoperazine (TFP) and calmidazolium (R24571) produced opposite effects, i.e., stimulation of Cl- absorption and cellular depolarization. Treatment with TFP or R24571 will block or override the inhibitory action of A23187. These data suggest a regulatory role for Ca2+ in the control of intestinal NaCl absorption and mediation via calmodulin.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Calcimycin; Calcium; Calmodulin; Female; Fishes; Imidazoles; Intestinal Absorption; Ion Channels; Male; Microscopy, Electron; Trifluoperazine

8-Bromo cyclic AMP (cAMP) (10(-4) M) inhibits Na absorption in isolated chicken enterocytes as has been reported previously. Direct measurements of intracellular pH (pHi) using 5,6-carboxyfluorescein diacetate showed that both 8-bromo cAMP and the diuretic amiloride (10(-3) M) stimulated a persistent decrease in pHi of approximately 0.1 pH units, effects that were Na dependent and were not additive when cells were stimulated with both agents. These results suggest inhibition of an amiloride-sensitive Na/H exchange by cAMP. Direct measurements of intracellular Ca [Ca]i were also made using quin 2. 8-Bromo cAMP (10(-4) M) stimulated an immediate and persistent (greater than 10 min) increase in [Ca]i of approximately 20 nM, an effect that was not dependent on extracellular Ca. Pretreatment of cells with the specific calmodulin inhibitor calmidazolium (10(-7) M) and the intracellular Ca-buffering agent MAPTAM blocked cAMP's effects on pH and Na uptake, but did not interfere with amiloride's effects. An increase in [Ca]i stimulated by the Ca ionophore A23187 (10(-6) M) was sufficient by itself to decrease pHi and inhibit amiloride-sensitive Na influx in isolated enterocytes. We conclude that cAMP stimulates the release of endogenous Ca in isolated enterocytes. This increase in [Ca]i appears to be essential for inhibition of amiloride-sensitive Na-H exchange by this cyclic nucleotide.

Topics: Alprostadil; Amiloride; Aminoquinolines; Animals; Buffers; Butyrates; Calcimycin; Calcium; Carrier Proteins; Chelating Agents; Chickens; Cyclic AMP; Glycine; Hydrogen-Ion Concentration; Imidazoles; In Vitro Techniques; Intestine, Small; Nigericin; Sodium-Hydrogen Exchangers; Valinomycin

Human amnion prostaglandin E2 (PGE2) synthesis increases with the onset of labour, and this synthesis is Ca2+-dependent. To understand better the mechanism of Ca2+-stimulated PGE2 biosynthesis, studies were performed to identify the presence of the intracellular Ca2+-mediator, calmodulin, in human amnion and to examine its role in PGE2 synthesis. Calmodulin-like activity was identified by the ability of the microsomal and cytosolic fractions of the 105,000g centrifugation of amnion homogenate to stimulate cyclic AMP-dependent phosphodiesterase activity. Cytosolic fractions consistently stimulated phosphodiesterase activity more than microsomal fractions (P less than 0.001) in paired samples from term human amnions. This activity was calcium-dependent. The cytosolic and microsomal factors increased the Vmax but not the Km of phosphodiesterase. There were no differences in these parameters with the onset of labour. The distribution of calmodulin-like activity between microsomes and cytosol was similar to the distribution of calmodulin mass as determined by radioimmunoassay. Three structurally different inhibitors of calmodulin activity, calmidazolium, trifluoperazine and W7, were tested for their ability to inhibit cytosolic factor-stimulated phosphodiesterase activity and to inhibit PGE2 output from dispersed amnion cells obtained before the onset of labour at term (cesarean section cells) or after spontaneous labour and vaginal delivery (spontaneous labour cells). The 50% inhibitory concentrations of the calmodulin antagonists in the phosphodiesterase assay were: trifluoperazine (6.7 microM), calmidazolium (0.11 microM), and W7 (24 microM). Trifluoperazine inhibited both basal and calcium ionophore (A23187)-stimulated PGE2 output from cesarean section cells and spontaneous labour amnion cells. Calmidazolium inhibited basal PGE2 output in cesarean section cells and spontaneous labour cells, but had no effect on A23187-stimulated output. W7 inhibited only the ionophore-stimulated PGE2 output in cesarean section amnion cells. The rank order of inhibition of both phosphodiesterase activation and basal PGE2 output was: calmidazolium greater than trifluoperazine greater than W7. These results suggest that human amnion contains calmodulin and that its distribution, concentration and activity remain unchanged with the onset of labour. The data suggest, although not conclusively, that calmodulin may, in part, play a role in amnion cell PGE2 production. Further inv

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Amnion; Calcimycin; Calcium; Calmodulin; Cytosol; Dinoprostone; Female; Humans; Imidazoles; Microsomes; Pregnancy; Prostaglandins E; Sulfonamides; Trifluoperazine

Calcium ionophore (A23187)-stimulated prostaglandin (PG) E2 and I2 (measured as 6-keto PGF1 alpha) release from cultured rabbit coronary microvessel endothelial (RCME) cells in a time- and dose-dependent manner. A23187-stimulated PG release was reduced by the calcium channel blockers nifedipine, verapamil and diltiazem and by the intracellular calcium blocker, 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate. A23187-stimulated PG release was significantly reduced by lowering the calcium concentration in the buffer to concentrations of 0.2 mM or less. A23187-stimulated 45Ca uptake was not inhibited by nifedipine (0.5 microM), diltiazem (10 micron) or verapamil (50 microM) although these same concentrations of calcium channel blockers significantly inhibited A23187-stimulated PG release. However, these concentrations of calcium blockers did inhibit K+ (10 mM)-valinomycin (5 microM)-stimulated 45Ca uptake, indicating that, although RCME cells probably have voltage-dependent calcium channels and although calcium influx via these channels is blocked by the calcium channel blockers, voltage-dependent calcium influx plays little or no role in A23187-stimulated 45Ca influx and PG release. KCl-valinomycin significantly stimulated PG release, but this increase was not significantly affected by either nifedipine (0.5 microM) or diltiazem (10 microM) despite complete inhibition of KCl-valinomycin-stimulated 45Ca influx. Verapamil (50 microM) exhibited a small but significant suppression of KCl-valinomycin-stimulated PG release. These observations most likely indicate that calcium influx by voltage-dependent calcium channels plays little or no role in the events leading to either A23187- or KCl-valinomycin-stimulated PG release. The calmodulin antagonists, trifluoperazine and calmidazolium, also reduced A23187-stimulated PG release. In vitro studies of porcine pancreatic phospholipase (PL) A2 activity suggested that the inhibitory actions of trifluoperazine, but not the calcium antagonists, may be mediated by direct inhibitory actions on PLA2. Studies with [3H]arachidonic acid (AA)- and [14C]stearic acid-prelabeled RCME cells suggested that A23187 stimulated both PLA2 and PLC activity, leading to the release of AA. Studies with exogenous AA indicated that reducing calcium availability by reducing buffer calcium concentrations resulted in an enhanced conversion of exogenous AA to PGs. RCME cells incubated in nominally calcium-free buffer exhibited a decreased rate of AA

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Calcium; Calcium Channel Blockers; Calmodulin; Cells, Cultured; Coronary Vessels; Endothelium; Imidazoles; Kinetics; Microcirculation; Prostaglandins; Rabbits; Trifluoperazine; Valinomycin

Calmidazolium in macromolecular concentrations inhibited the electric and contractile activity of smooth muscle cells (SMC). The concentrations causing a 50% inhibition of oscillations on the action potential (AP) plate were equal to 1 X 10(-6) microM, AP amplitude was 3 X 10(-6) microM and contraction amplitude was 1 X 10(-6) microM. Calcium ionophore A 23187/8 X 10(-7) microM, added to the normal Krebs solution, decreased rapid AP components amplitude and increased the contraction power of the isolated SMC strip by 62 +/- 9%. A 23187, though to a lesser extent, increased smooth muscle contractions during the action of calmidazolium. With combined use of A 23187 and calmidazolium, rapid AP components were depressed to a greater extent than each of them taken separately. The data obtained point to the presence of calmodulin or similar protein in SMC of the calcium channels.

Topics: Action Potentials; Animals; Calcimycin; Calmodulin; Depression, Chemical; Dose-Response Relationship, Drug; Drug Interactions; Guinea Pigs; Imidazoles; In Vitro Techniques; Membrane Potentials; Muscle Contraction; Muscle, Smooth

The effects of selected calmodulin-antagonists, i.e. calmidazolium (R24571), trifluoperazine, cis- and transflupenthixol, chlorpromazine, and imipramine, on rat mast cells and on mast cell histamine release were investigated. The drugs induced histamine release, apparently by cytotoxic effects, with a rank order of potency in accordance with their lipid solubility and with maximal release at calmidazolium (5 mumol/l), trifluoperazine (40 mumol/l), cis- and trans-flupenthixol (50 mumol/l), chlorpromazine (100 mumol/l), and imipramine (500 mumol/l). Inhibition of the histamine release induced by antigen, compound 48/80, and the ionophore A23187 was only observed with some of the drugs tested, with maximal inhibition at calmidazolium (2 mumol/l), chlorpromazine (25-50 mumol/l), and imipramine (100-250 mumol/l). The concentration-response curve for histamine release induced by calmidazolium was significantly shifted to the right by antigen (i.e. horse serum) in the medium and the addition of antigen was capable of immediately stopping the release induced by calmidazolium, indicating binding of calmidazolium by antigen. Similar effects on the actions of calmidazolium were observed with phosphatidylserine. The inhibition by calmidazolium of the histamine release induced by antigen, compound 48/80, and the ionophore A23187 was significantly counteracted by glucose in the medium. The findings do not confirm an involvement of calmodulin in the histamine release process in rat mast cells. The ability of calmidazolium to bind to proteins and phospholipids in the medium indicates multiple cellular targets for calmidazolium, and the observations with glucose suggest an impaired mitochondrial function to be of major significance.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Calcimycin; Calmodulin; Chlorpromazine; Flupenthixol; Glucose; Histamine Release; Imidazoles; Imipramine; In Vitro Techniques; Male; Mast Cells; p-Methoxy-N-methylphenethylamine; Rats; Rats, Inbred Strains; Trifluoperazine

By using the calmodulin antagonists, calmidazolium and N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7), the hypothesis was investigated as to whether calmodulin is involved in the sequence of events leading to the endothelium-dependent vascular smooth muscle relaxation. Endothelium-dependent relaxations were studied on two different preparations, the rabbit aorta and the pulmonary artery of the guinea pig. Relaxations were produced in the precontracted rings (noradrenaline 3 X 10(-6) mol/l) in response to acetylcholine, 10(-8) to 10(-6) mol/l (aorta), histamine, 3 X 10(-8) to 1 X 10(-6) mol/l (pulmonary artery) or the calcium ionophore A 23187, 1 X 10(-8) to 3 X 10(-7) mol/l (aorta and pulmonary artery). In the presence of calmidazolium and W-7 the endothelium-dependent relaxation was inhibited in a dose dependent manner. This inhibition was seen in a concentration range that coincides with calmodulin inhibition. The half maximal concentrations of calmidazolium for the inhibition of the relaxation of the aorta induced by acetylcholine and A 23187 were 3 X 10(-6) mol/l and 1.4 X 10(-6) mol/l and that of W-7 were 3.1 X 10(-5) and 3.6 X 10(-5) mol/l, respectively. Complete inhibition was obtained both for acetylcholine-and for A 23187-induced relaxations by preincubation with 1 X 10(-5) mol/l calmidazolium or 1 X 10(-4) mol/l W-7. The half maximal concentrations of calmidazolium for the inhibition of the relaxation of the pulmonary artery in response to histamine and A 23187 were 2.7 X 10(-6) mol/l and 3 X 10(-6) mol/l and complete inhibition was achieved at 1 X 10(-5) mol/l.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acetylcholine; Animals; Calcimycin; Calmodulin; Endothelium; Female; Guinea Pigs; Histamine; Imidazoles; In Vitro Techniques; Male; Nitroprusside; Norepinephrine; Rabbits; Sulfonamides; Vasodilation

These studies were designed to test the hypothesis that changes in intracellular Ca2+ levels and activation of the calcium ion- and phospholipid-dependent protein kinase C were required for the induction of macrophage tumoricidal activity by interferon-gamma (IFN-gamma). Phenothiazines and R24571, known antagonists of calcium-binding proteins and therefore nonspecific inhibitors of protein kinase C, blocked in a dose-dependent manner the induction of macrophage cytocidal activity by either natural or recombinant IFN-gamma. Macrophages depleted of intracellular Ca2+ by chelation with Quin 2, were also unresponsive to IFN-gamma. These treatments effected neither the binding of IFN-gamma to its cell surface receptor nor the normal intracellular processing of IFN-gamma. Activators of protein kinase C (such as phorbol esters) and Ca2+ ionophores when added alone did not effect the activation state of the macrophage population. However, macrophages exposed to both drugs in combination were elevated into the primed activation state such that in the presence of a second signal (lipopolysaccharide or heat killed Listeria monocytogenes), the cells were triggered to express full levels of tumoricidal activity. The capacity of phorbol esters to induce cellular activation correlated with their ability to bind and to activate protein kinase C. No synergistic effect was observed between IFN-gamma and protein kinase C activators and/or Ca2+ ionophores, indicating that the drugs could only prime and could not trigger macrophages for tumor cell killing. These results thus support the concept that protein kinase C activation and mobilization of intracellular Ca2+ are essential steps in the pathway of IFN-gamma-dependent induction of non-specific tumoricidal activity in macrophages.

Topics: Aminoquinolines; Calcimycin; Calcium; Cytotoxicity, Immunologic; Imidazoles; Interferon-gamma; Macrophage Activation; Macrophages; Neoplasms, Experimental; Protein Kinase C; Tetradecanoylphorbol Acetate; Trifluoperazine

We have studied the effect of angiotensin II and bradykinin on prostaglandin output and vascular tone during extracellular calcium depletion and administration of calcium antagonists and calmodulin inhibitors to elucidate the mechanism of action in the isolated rat kidney perfused with Tyrode's solution. Administration of angiotensin II (0.028-0.28 nmol) or bradykinin (0.28-2.8 nmol) enhanced the output of prostaglandin E2 and 6-keto-prostaglandin F1 alpha in a dose-dependent manner. Angiotensin II, but not bradykinin, produced renal vasoconstriction. Omission of calcium from the medium or infusion of calcium entry blockers, diltiazem (60 microM), or nimodipine (47 microM), failed to alter prostaglandin output elicited by angiotensin II or bradykinin; however, the effect of angiotensin II to produce renal vasoconstriction was inhibited. If calcium was omitted from the medium, the intracellular calcium antagonists, 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (23 microM), dantrolene sodium (31 microM), or ryanodine (2 microM), attenuated prostaglandin output caused by angiotensin II but not bradykinin. Calmodulin inhibitors, trifluoperazine (2 microM), napthalene sulfonamide hydrochloride (2 microM), or calmidazolium (2 microM), diminished prostaglandin output elicited by angiotensin II, but not that caused by bradykinin. Trifluoperazine, but not naphthalene sulfonamide or calmidazolium, attenuated the renal vasoconstrictor effect of angiotensin II. Prostaglandin output induced by angiotensin II and bradykinin were inhibited by mepacrine and indomethacin, whereas, the prostaglandin output caused by exogenous arachidonic acid (33 nmol) was abolished by indomethacin but was unaltered by mepacrine, calcium antagonists, and calmodulin inhibitors. From these data, we conclude that angiotensin II produces renal vasoconstriction by a mechanism dependent on extracellular calcium but not calmodulin, whereas angiotensin II-induced prostaglandin output depends on intracellular calcium and calmodulin. In contrast, bradykinin appears to stimulate prostaglandin synthesis by a calcium/calmodulin-independent mechanism.

Topics: 6-Ketoprostaglandin F1 alpha; Angiotensin II; Animals; Bradykinin; Calcimycin; Calcium; Calcium Channel Blockers; Calmodulin; Dantrolene; Diltiazem; Dinoprostone; Gallic Acid; Imidazoles; Indomethacin; Kidney; Male; Nicotinic Acids; Nimodipine; Prostaglandins E; Quinacrine; Rats; Rats, Inbred Strains; Ryanodine; Stimulation, Chemical; Sulfonamides; Trifluoperazine; Vasoconstriction

Intercellular gap-junctional communication was measured using [14C]citrulline incorporation in co-cultures of argininosuccinate lyase-deficient human fibroblasts and argininosuccinate synthetase-deficient Chinese Hamster V79 cells. As previously shown, in this system junctional communication is completely inhibited by the tumor promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). In the absence of extracellular calcium, TPA inhibition was less pronounced. However, synergism with calcium ionophore A23187 could not be demonstrated. Chlorpromazine, trifluoperazine and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester partially antagonised the effect of TPA. No antagonism was demonstrable between calmidazolium and TPA. Treatment of co-cultures with exogenous phospholipase C or 1-oleoyl-2-acetylglycerol (OAG) resulted in communication inhibition, suggesting that protein kinase C activation is involved in the mechanism of phorbol ester-mediated communication inhibition. However co-cultures which had been made refractory to TPA by prolonged exposure to high concentrations remained sensitive to inhibition by phospholipase C and OAG. These results suggest either that diacylglycerol can produce other effects independent of protein kinase C activation, or that refractoriness to phorbol esters is not simply due to a decrease in the amount of protein kinase C.

Topics: Animals; Calcimycin; Cell Communication; Cells, Cultured; Chlorpromazine; Cricetinae; Cricetulus; Diglycerides; Gallic Acid; Humans; Imidazoles; Phorbols; Tetradecanoylphorbol Acetate; Type C Phospholipases

The involvement of the calcium-calmodulin system in the gonadotropic regulation of granulosa cell steroidogenesis during follicular development was assessed by comparing the effects of agents known to alter calcium metabolism or calmodulin activity on gonadotropin-stimulated progesterone production by rat granulosa cells at different stages of cellular differentiation. Granulosa cells from pregnant mare's serum gonadotropin-injected rats (PMSG-cells) could be stimulated to produce steroids by both follicle-stimulating hormone (FSH) and luteinizing hormone (LH), whereas those from 17 beta-estradiol-pretreated animals (E2-cells) only responded to to FSH. Although PMSG-cells demonstrated greater capacities for adenosine 3': 5'-cyclic monophosphate (cyclic AMP) and progesterone production, their sensitivity to the various agents tested was similar to that of the E2-cells. [Ethylene-bis(oxyethylene-nitrilo)] tetraacetic acid (EGTA) markedly reduced both basal and FSH-stimulated progesterone production by both cell preparations. Verapamil, an agent which inhibits cellular uptake of calcium, attenuated cyclic AMP and progesterone production by both cell groups at similar concentrations. Calcium ionophore A23187, a stimulator of cellular calcium uptake, significantly stimulated progesterone production by both E2-cells and PMSG-cells. While FSH-stimulated progesterone production by both cell groups was lowered by the calmodulin inhibitor, 1-[bis(p-chlorophenyl)methyl]-3-[2,4-dichloro-beta-(2,4-dichlorobenzy loxy) phenethyl] imidazolium chloride (R24571), cyclic AMP production by PMSG-cells in response to both FSH and LH was significantly reduced by R24571 and trifluoperazine (TFP), another calmodulin inhibitor. These findings indicate that the calcium-calmodulin system is involved in the regulation of granulosa cell steroidogenesis by both FSH and LH. Its participation in the gonadotropic regulation is independent of the stage of follicular maturation and cellular differentiation.

Topics: Animals; Calcimycin; Calcium; Calmodulin; Cell Differentiation; Estradiol; Female; Gonadotropins, Equine; Granulosa Cells; Imidazoles; Rats; Rats, Inbred Strains; Steroids

Carbamylcholine-stimulated catecholamine release from adrenal chromaffin cells was completely inhibited by pretreatment of the cells for 10 min with 1 microM calmidazolium. Catecholamine release due to 55 mM K+ and ionophore A23187 was also inhibited by calmidazolium but less effectively than release due to carbamylcholine. Inhibition of release appeared to be due to an effect of calmidazolium on a step distal to Ca2+ entry, since the carbamylcholine-stimulated rise in the concentration of intracellular free calcium, monitored using quin-2, was unaffected by calmidazolium. The possibility was considered that calmidazolium inhibited secretion through an effect on protein kinase C rather than calmodulin. However, the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), had no demonstrable effect on catecholamine release, arguing against a significant role for protein kinase C in secretion from adrenal chromaffin cells. These results give further support to the notion that calmodulin plays a role in the secretory process in chromaffin cells.

Topics: Adrenal Medulla; Animals; Calcimycin; Carbachol; Catecholamines; Cattle; Chromaffin Granules; Enzyme Inhibitors; Imidazoles; Kinetics; Phorbols; Potassium; Tetradecanoylphorbol Acetate