Intracellular Calcium Antagonist Research Articles

Enhancement of superoxide production by interferon-γ on human neutrophils

Superoxide production of human neutrophils was enhanced within 10 min by the treatment of interferon-γ, when phorbol 12-myristate 13-acetate (PMA) was used as a stimulant. The enhancement of superoxide production by interferon-γ occurred only under the presence of calcium, and that it was inhibited by 1- (5-isoquinolinesulfonyl) -2-methylpiperazine (H-7), an inhibitor of protein kinase-C and 8- (N, N-diethylamino) -octyl- (3, 4, 5-trimethoxy) benzoate hydrochloride (TMB-8), an intracellular calcium antagonist. Quin 2 (acetoxy-methylester) fluorescent revealed that intracellular calcium arose immediately after the addition of interferon-γ.These data suggest that the enhancement of superoxide production by interferon-γ is closely related to protein kinase-C activation followed by the arising of intracellular calcium.

Antiulcer activity of the calcium antagonist propyl-methylenedioxyindene. IV. Effects on gastric lesions in rats induced by cold-restraint stress and thyrotropin-releasing hormone

Propyl-methylenedioxyindene (pr-MDI; 30 mg/kg, i.p.), an intracellular calcium antagonist, significantly reduced the number and size of erosions per stomach induced by cold-restraint stress by 69% and 86%, respectively. Our previous findings indicate that the antiulcer activity of pr-MDI is highly correlated with its inhibitory effect on gastric motor activity. Since central TRH is suggested as the brain mediator responsible for cold-restraint stress gastric ulcers in rats, the inhibitory action of pr-MDI was evaluated in the TRH-induced gastric lesion model. Pr-MDI (30 mg/kg) did not reduce the gastric erosions induced by intracisternal administration of 100ng RX77368, a stable thyrotropin-releasing hormone (TRH) analogue, even though it abolished the RX77368-induced stimulation of gastric emptying, gastric acidity, and acid output. Since pr-MDI (30 mg/kg, i.p.) significantly inhibited the stimulation of gastric motility by both cold-restraint stress and TRH, but only cold-restraint stress-induced gastric erosions were effectively reduced by the drug, the present findings suggest a possible dissociation between the ulcerogenic mechanisms of cold-restraint stress and intracisternal administration of TRH.

Examination of the Potential Antiilcer Activity of the Calcium Antagonist Propyl-Methylenedioxyindene

Since propyl-methylenedioxyindene (pr-MDI) exhibits significant protective effects against stress-induced ulcers in rats at subcardiovascular doses (10-30 mg/kg, i.p.), the aim of the present study was to explore the effect of this intracellular calcium antagonist on cysteamine-induced duodenal ulcers at the same low doses. Duodenal ulcers were induced in rats with a single dose of cysteamine (425 mg/kg, s.c.), which produced an 80% ulcer incidence within 24 h without affecting gastric acid concentration. Administration of pr-MDI (10 and 30 mg/kg, i.p.) at 0, 6 and 12 h post-cysteamine did not afford protection against ulceration. On the other hand, atropine (10 mg/kg, s.c., administered at 0, 6 and 12 h post-cysteamine) resulted in a 69% inhibition of ulceration, and the antacid Maalox (2 ml, administered p.o. at 0, 2, 4, 6 and 12 h post-cysteamine) completely prevented ulceration. The failure of pr-MDI to protect against duodenal ulceration is discussed in relation to its pharmacological mechanism of action and the pathogenetic mechanism of action of cysteamine.

Mechanism of angiotensin II stimulation of Na-K-Cl cotransport of vascular smooth muscle cells

Previous studies from this laboratory have demonstrated that Na-K-Cl cotransport of vascular smooth muscle cells is inhibited by hormones that increase intracellular adenosine 3',5'-cyclic monophosphate (cAMP) levels (e.g., catecholamines) and is stimulated by hormones that increase intracellular guanosine 3',5'-cyclic monophosphate (cGMP) levels (e.g., atrial natriuretic peptides). Others have suggested that calcium may also modulate Na-K-Cl cotransport of vascular smooth muscle cells. The goal of the present study was to characterize the mechanism of angiotensin II stimulation of Na-K-Cl cotransport of early passage cultured vascular smooth muscle cells. We found that when vascular smooth muscle cells were treated with angiotensin II or a calcium ionophore, Na-K-Cl cotransport was markedly enhanced above basal levels. We found that when calcium influx was blocked with the calcium chelator EDTA or with three different chemical types of calcium-channel blockers, the stimulatory effects of angiotensin II on Na-K-Cl cotransport were markedly inhibited. Furthermore, when intracellular calcium mobilization was blocked with high concentrations of the calcium chelator quin2 or with the intracellular calcium antagonist 8-(diethyl-amino)octyl 3,4,5-trimethoxybenzoate (TMB-8), the stimulatory effects of angiotensin II on Na-K-Cl cotransport were also substantially inhibited. These results suggest that both calcium influx via receptor-operated calcium channels and intracellular calcium mobilization may play a role in stimulation of Na-K-Cl cotransport of vascular smooth muscle cells in response to angiotensin II.

Role of Calcium in Phytochrome-Controlled Nyctinastic Movements of Albizzia lophantha Leaflets

The involvement of Ca(2+) on phytochrome-controlled nyctinastic closure in Albizzia lophantha has been studied by testing the effect of the calcium ionophore 6S-[6alpha(2S(*),3S(*)),8beta(R(*)),9beta,11alpha]-5- methyl-amino)-2-[[3,9,11-trimethyl-8-[-1-methyl-2-oxo-2-(1H-pyrrol-2-yl) ethyl]-1,7-dioxaspiro[5.5]-undec-2yl] methyl]-4-benzoxazolecarboxylic acid (A23187) and the intracellular calcium antagonist 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8). An external supply of Ca(2+) or calcium ionophore A23187 to the Albizzia leaflets emulates the effect of red light irradiation and counteracts the inhibitory effect of far red light. The intracellular calcium antagonist TMB-8 supplied to Albizzia leaflets inhibits the effect of red light, but had no effect on far red irradiated plants. This suggests a dependence between phytochrome action and intracellular free Ca(2+). We suggest that calcium acts as a phytochrome messenger on control of ion fluxes that drive turgor changes in pulvinular motor cells.

Collateral Blood Flow Following Acute Coronary Artery Occlusion

The effects of a new intracellular calcium antagonist, KT-362 (150 and 300 micrograms/kg per min), on hemodynamics and collateral function (retrograde pressure and flow, radioactive microspheres) distal to an acute coronary artery occlusion were studied in anesthetized dogs and compared with the effects of the structurally related classical calcium channel blocker, diltiazem (15 and 30 micrograms/kg per min), and a saline-treated control group. In the saline series, there were no changes in systemic hemodynamics or coronary collateral blood flow over the 90-min ischemic period. KT-362 reduced mean aortic pressure, heart rate, and dP/dt whereas diltiazem only decreased aortic blood pressure. When blood pressure was controlled by a distal aortic cuff, heart rate was significantly reduced in both groups and dP/dt was reduced in the KT-362 series and increased in diltiazem-treated dogs. In both drug-treated groups, retrograde pressure and flow were significantly increased only when aortic pressure was controlled. Regional myocardial tissue blood flow in the nonischemic or ischemic region did not change significantly after KT-362 treatment despite its hypotensive actions, and in the presence of a constant aortic pressure, transmural collateral blood flow and the ischemic/nonischemic blood flow ratio tended to increase. In contrast, diltiazem treatment resulted in a significant decrease in the ischemic/nonischemic blood flow ratio in the absence of blood pressure control. In the presence of constant aortic pressure, blood flow to the nonischemic area was markedly increased by diltiazem whereas subendocardial blood flow was significantly increased in the ischemic area.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduction of Myocardial Ischemia-Reperfusion Injury by KT-362, a New Intracellular Calcium Antagonist in Anesthetized Dogs

The effects of a new intracellular calcium antagonist, KT-362 (5-[3-[[2-(3,4-dimethoxyphenyl)-ethyl]amino]-1-oxopropyl]- 2,3,4,5,-tetrahydro-1,5-benzothiazepine fumarate) on myocardial infarct size following a 90-min occlusion and 3-h reperfusion of the left anterior descending coronary artery (LAD) were determined in anesthetized dogs. Regional myocardial blood flow was measured by radioactive microsphere technique, and infarct size was determined using triphenyltetrazolium chloride histochemical stain. Vehicle or KT-362 (300 micrograms/kg/min for 20 min followed by 150 micrograms/kg/min for 80 min) was administered intravenously (i.v.) 10 min prior to coronary occlusion and continued throughout the occlusion period in separate experimental groups. KT-362 produced a reduction in heart rate (HR) and the HR-systolic pressure product. Mean arterial pressure (MAP) was reduced during occlusion and early reperfusion in the KT-362-treated group, and segment function (% segment shortening) was improved during the first hour of reperfusion. There were no differences in collateral blood flow between the two groups. However, at 3 h postreperfusion, ischemic zone subendocardial blood flow in the KT-362-treated group was significantly greater than in the vehicle-treated group, resulting in an increase in endo/epi. There were no differences in ventricular mass, mass of the area at risk, or percentage of the left ventricle at risk. As compared with the control group, KT-362 produced a marked reduction in myocardial infarct size expressed as a percentage of the area at risk infarcted, percentage of the left ventricle infarcted, and absolute weight of infarcted tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduction of Myocardial Ischemia-Reperfusion Injury by KT-362, a New Intracellular Calcium Antagonist in Anesthetized Dogs

Summary: The effects of a new intracellular calcium antagonist, KT-362 (5-[3-[[2-(3,4-dimethoxyphenyl)-ethyl]amino]-1-oxopropyl]-2,3,4,5,-tetrahydro-1,5-ben-zothiazepine fumarate) on myocardial infarct size following a 90-min occlusion and 3-h reperfusion of the left anterior descending coronary artery (LAD) were determined in anesthetized dogs. Regional myocardial blood flow was measured by radioactive microsphere technique, and infarct size was determined using triphenyltetrazolium chloride histochemical stain. Vehicle or KT-362 (300 μg/kg/min for 20 min followed by 150 μg/kg/min for 80 min) was administered intravenously (i.v.) 10 min prior to coronary occlusion and continued throughout the occlusion period in separate experimental groups. KT-362 produced a reduction in heart rate (HR) and the HR-systolic pressure product. Mean arterial pressure (MAP) was reduced during occlusion and early reperfusion in the KT-362-treated group, and segment function (% segment shortening) was improved during the first hour of reperfusion. There were no differences in collateral blood flow between the two groups. However, at 3 h postreperfusion, ischemic zone subendocardial blood flow in the KT-362-treated group was significantly greater than in the vehicle-treated group, resulting in an increase in endo/epi. There were no differences in ventricular mass, mass of the area at risk, or percentage of the left ventricle at risk. As compared with the control group, KT-362 produced a marked reduction in myocardial infarct size expressed as a percentage of the area at risk infarcted, percentage of the left ventricle infarcted, and absolute weight of infarcted tissue. This beneficial effect was most likely due to a decrease in myocardial energy requirements during occlusion as a result of inhibition of intracellular calcium release.

Effect of KT-362, a Putative Intracellular Calcium Antagonist, on Norepinephrine-Induced Contractions and Inositol Monophosphate Accumulation in Canine Femoral Artery

The purpose of the present study was to determine if the intracellular calcium antagonist, KT-362, inhibits norepinephrine- (NE) induced contractions and inositol monophosphate (IP) accumulation in canine femoral arteries preincubated with [3H]inositol. Norepinephrine dose-dependently increased contractile tension and produced a parallel stimulation of inositol phospholipid hydrolysis as measured by IP accumulation. This stimulation was inhibited by the selective alpha 1 adrenoceptor antagonist, prazosin (0.1 microM), indicating that the NE-induced stimulation of inositol phospholipid hydrolysis is coupled to alpha 1 adrenoceptor activation in canine femoral artery. Pretreatment with nitroglycerin (100 microM), 8-Br cyclic guanosine monophosphate (cGMP) (1 microM), or diltiazem (40 microM) inhibited contractile responses produced by NE; however, these agents had no significant effect on NE-induced IP accumulation. In contrast, pretreatment with KT-362 (10-100 microM) greatly inhibited both the NE-induced contractions and IP accumulation. KT-362 also produced a marked inhibition of NE-induced contractions in normal as well as zero calcium buffer, whereas diltiazem (40 microM) had no effect in zero calcium buffer. These results indicate that the mechanism of action of KT-362 differs from diltiazem, nitroglycerin, and 8-Br cGMP, and these data suggest that one of the mechanisms by which KT-362 antagonizes NE-induced vasoconstrictor responses is by decreasing inositol phospholipid hydrolysis in canine femoral artery.

The effects of TMB-8 on the shape changes of vascular endothelial cells resulting from exposure to various inflammatory agents

Pre-treatment of the endothelial cells lining the guinea pig inferior vena cava with 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8) (10 microM) in vitro significantly reduced the shape changes resulting from subsequent exposure to platelet activating factor (PAF) (0.1 microM), calcium ionophore A23187 (10 microM), histamine (300 microM), bradykinin (2 microM), prostaglandin E2 (PGE2) (30 microM), or leukotrienes (LT) C4, D4 or E4 (1 microM). Since TMB-8 is an intracellular calcium antagonist, this provides evidence to support the suggestion that these inflammatory agents increase the concentration of intracellular calcium which brings about a contraction of the actin-myosin complex resulting in endothelial cell shape changes, and the formation of interendothelial cell gaps.

P-375 - Effects of calcium entry blocker and TMB-8, intracellular calcium antagonist, on angiotensin II - and arg-vasopressin-induced renal vasoconstriction in dogs

P-375 - Effects of calcium entry blocker and TMB-8, intracellular calcium antagonist, on angiotensin II - and arg-vasopressin-induced renal vasoconstriction in dogs

Role of cell calcium in alpha-1 adrenergic receptor control of arachidonic acid release from brown adipocytes

Exposure of brown fat cells to phenylephrine, an agonist alpha-1 adrenergic receptors, activates a phospholipase A 2 which releases arachidonic acid. Since receptor activation of phospholipase A 2 requires calcium, experiments were undertaken to define more precisely the role played by calcium in the regulation of enzyme activity. In this study, adipocytes were loaded with the fluorescent calcium chelator quin2 in order to buffer intracellular calcium and block receptor stimulated changes in its concentration. When quin2 loaded adipocytes were incubated in buffer containing 0.10 mM clacium, the ability of phenylephrine to stimulate release of arachidonic acid was severely reduced. At an intracellular quin2 concentration of 6.6 nM stimulated arachidonic acid release was inhibited by more than 50% and at 13 mM it was completely blocked. In contrast, phenylephrine stimulation of inositol phosphate accumulation was unaffected by quin2. Quin2 also did not affect the liberation of arachidonic acid in response to exogenous phospholipase C, A23187 or forskolin. The intracellular calcium antagonist TMB-8 also ihibited phenylephrine-stimulation of arachidonic acid release and this effect was reversed by ionomycin. Basal phospholipase A 2 activity was increased by introduction of high calcium concentrations into cells rendered permeable with digitonin, but phenylephrine still caused a further increase in enzyme activity. These findings show a selective inhibition of phenylephrine activation of phospholipase A 2 by either the chelation of intracellular calcium with quin2 or by the calcium antagonist TMB-8 and suggest an essential role for intracellular calcium in alpha adrenergic stimulation of enzyme activity. However, because phenylephrine still stimulates enzymes activity in cells rendered permeable with digitonin, we suggest that the action of phenylephrine cannot be attributed solely to changes in intracellular calcium.

Hypoxic contractile response in isolated human pulmonary artery: role of calcium ion.

The mechanism for hypoxic pulmonary vasoconstriction (HPVC) was investigated in human pulmonary arterial strips. Hypoxia in the presence of histamine (10(-6) M) caused marked pulmonary arterial contraction, which was reversed by O2. The hypoxic contraction in the presence of histamine was inhibited by diphenhydramine, but not by cimetidine. The hypoxic histamine-mediated contraction was attenuated but still present in the absence of extracellular Ca2+, or by the inhibitors of voltage-dependent Ca2+ influx. However, it was inhibited significantly by a further depletion of intracellular Ca2+, or by HA 1004, an intracellular calcium antagonist. A low concentration (10(-7) M) of a calcium ionophore, A23187, enhanced the hypoxic contraction in the presence of histamine, whereas procaine completely inhibited it. W-7, a calmodulin inhibitor, significantly decreased the hypoxic histamine-mediated contraction, but 12-O-tetradecanoylphorbol-13-acetate (TPA), a C-kinase promotor, had no effect. The hypoxic contractile response was also observed in the presence of both A23187 and KCl instead of histamine, but the hypoxia-induced contraction with KCl alone was much smaller than that. These results indicate that hypoxia in the presence of certain other vasoactive agents has a potent contractile effect on the human pulmonary artery and that the response is dependent on Ca2+. Enhancement of both Ca2+ influx and Ca2+ release from intracellular storage sites by hypoxia, which interacts with calmodulin, were suggested to be involved in the mechanism of HPVC.

Ca2+ requirement of prostanoid but not of superoxide production by rat Kupffer cells.

The release of the prostanoids prostaglandin D2 (PGD2), prostaglandin E2 (PGE2) and thromboxane induced by zymosan and phorbol ester in cultured rat Kupffer cells was found to depend on the extracellular concentration of Ca2+ to some extent. Prostanoid formation following the addition of the calcium ionophore A 23187 was totally inhibited when calcium ions were withdrawn from the medium whereas the prostanoid synthesis from added arachidonic acid was independent of Ca2+. A half-maximal rate of PGE2 release by cells treated with zymosan, phorbol ester or A23187 was obtained at 0.6-0.7 microM free extracellular Ca2+ and greater than or equal to 100 microM free Ca2+ was required to stimulate PGE2 formation maximally. The calmodulin antagonist R24571 partially inhibited the release of PGE2 elicited by zymosan and A23187 but not by phorbol ester or arachidonic acid. Verapamil and nifedipine, two calcium channel blockers, had no effect on the formation of PGE2 irrespective of the stimulus. TMB 8 [3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester] an intracellular calcium antagonist, inhibited the synthesis of PGE2 induced by zymosan and phorbol ester. The superoxide formation following the addition of zymosan and phorbol ester was not influenced by removal of calcium ions from the medium or by addition of the various calcium antagonists. The data presented here suggest that Ca2+-dependent reactions are involved in the synthesis of prostanoids induced by zymosan and phorbol ester and that both extracellular Ca2+ and mobilization of Ca2+ from intracellular stores are needed to induce maximally the production of prostanoids in cultured rat Kupffer cells.

Differential role of extra- and intracellular calcium in the release of EDRF and prostacyclin from cultured endothelial cells.

1. The effects of extracellular Ca2+ on the release of endothelium-derived relaxing factor (EDRF) and prostacyclin (PGI2), and on the intracellular free calcium concentration [( Ca2+]i), were studied in cultured bovine aortic endothelial cells. 2. Receptor-mediated stimulation of endothelial cells with bradykinin (10 nM) elicited a transient release of EDRF (assayed by its stimulant effect on purified soluble guanylate cyclase) and of PGI2 (measured by radioimmunoassay for 6-keto prostaglandin F1 alpha). 3. Bradykinin (10 nM) also increased [Ca2+]i (measured with the fluorescent probe indo-1) from 125 +/- 11 nM to 631 +/- 59 nM, with the same time course as for autacoid release. 4. In Ca2+-free medium, [Ca2+]i was still increased by bradykinin but declined faster (within 1 min) to resting levels than in the presence of extracellular Ca2+. 5. PGI2 release was almost completely abolished in Ca2+-free medium. The intracellular calcium antagonist TMB-8 evoked a similar inhibition of PGI2 release. 6. In contrast, bradykinin-induced EDRF release was not significantly affected by TMB-8 but was completely abolished in Ca2+-free medium. 7. When endothelial cells were stimulated with the receptor-independent drug thimerosal (an inhibitor of the enzyme acyl-CoA-lysolecithin-acyl-transferase; 5 microM), a long-lasting release of EDRF (greater than 90 min) and PGI2 (greater than 20 min) was observed. 8. In contrast to bradykinin stimulation, thimerosal-induced autacoid release was associated with only a slight increase of [Ca2+]i to 201 +/- 13 nM after 40 min. 9. After removal of extracellular Ca2 + from thimerosal-stimulated endothelial cells, [Ca2+] was little affected during the observation time of 90s. EDRF release was completely abolished within 90s whereas PGI2 release was unchanged. 10. We conclude that EDRF production is directly controlled by extracellular Ca2+ during both receptor-dependent and independent stimulation. This effect of extracellular Ca2 + is not mediated by changes in [Ca2+]i. In contrast, PGI2 release is closely correlated to [Ca2+]i in bradykininstimulated endothelial cells. However, the results obtained during thimerosal stimulation indicate that there is not necessarily a tight coupling between the absolute level of [Ca2+]i and the amount of PGI2 released. 9. After removal of extracellular Ca2 + from thimerosal-stimulated endothelial cells, [Ca2+] was little affected during the observation time of 90s. EDRF release was completely abolished within 90s whereas PGI2 release was unchanged. 10. We conclude that EDRF production is directly controlled by extracellular Ca2+ during both receptor-dependent and independent stimulation. This effect of extracellular Ca2 + is not mediated by changes in [Ca2+]i. In contrast, PGI2 release is closely correlated to [Ca2+]i in bradykininstimulated endothelial cells. However, the results obtained during thimerosal stimulation indicate that there is not necessarily a tight coupling between the absolute level of [Ca2+]i and the amount of PGI2 released. 9. After removal of extracellular Ca2 + from thimerosal-stimulated endothelial cells, [Ca2+] was little affected during the observation time of 90s. EDRF release was completely abolished within 90s whereas PGI2 release was unchanged. 10. We conclude that EDRF production is directly controlled by extracellular Ca2+ during both receptor-dependent and independent stimulation. This effect of extracellular Ca2 + is not mediated by changes in [Ca2+]i. In contrast, PGI2 release is closely correlated to [Ca2+]i in bradykininstimulated endothelial cells. However, the results obtained during thimerosal stimulation indicate that there is not necessarily a tight coupling between the absolute level of [Ca2+]i and the amount of PGI2 released.

Protection of rat atrial myocardium against electrical, mechanical and structural aspects of injury caused by exposure in vitro to conditions of simulated ischaemia.

1. Rat isolated and superfused atria were exposed for varying periods to a solution simulating the composition of extracellular fluid during myocardial ischaemia (SI). 2. Atria subjected to SI showed a loss of systolic contractile tension, a rise in diastolic tension, a shortening of electrical refractory periods, a slowing of action potential conduction velocity and disruption of the mitochondrial ultrastructure. All these features were reversible when the muscle was returned to normal superfusate. 3. Atria pretreated with a superfusate containing a calcium channel antagonist, a calmodulin inhibitor or an intracellular calcium antagonist showed fewer features of the response to SI than did controls. 4. Atria pretreated with a superfusate containing various non-steroidal anti-inflammatory agents did not show identical responses to SI. Sulphinpyrazone protected against all features of the response to SI but ibuprofen, flurbiprofen and GP25671 (a metabolite of sulphinpyrazone) had little effect. Flufenamate, phenylbutazone and salicylate enhanced the responses to SI.

Role of Calcium in Prolactin-Stimulated c-mycGene Expression and Mitogenesis in NB2 Lymphoma Cells*

Receptor-activated transmembrane calcium flux has been implicated as a mediator of the actions of many growth factors and hormones. We examined the effects of PRL, calcium ionophores, and calcium antagonists on 45Ca2+ flux, c-myc gene expression, and DNA synthesis in the PRL-dependent rat Nb2 lymphoma cell line. PRL had no detectable effects on 45Ca2+ uptake or efflux, and the mitogenic effects of PRL could not be reproduced by the calcium ionophore A23187 alone or in combination with the tumor-promoting phorbol ester 12-O-tetra-decanoyl-phorbol-13 acetate (TPA). PRL, but not A23187 or TPA, stimulated c-myc gene expression in quiescent Nb2 cells. Exposure to PRL for brief periods (15 min to 4 h), followed by extensive washing, resulted in a time- and dose-dependent activation of DNA synthesis measured 16 h later. This activation was not blocked by addition of excess anti-PRL antiserum after the wash steps, indicating that the observed stimulation was not due to residual PRL. Despite the marked increase in DNA synthesis, removal of PRL after 4 h prevented mitosis, suggesting that PRL may be required throughout the cell cycle for Nb2 cell proliferation. Although continuous incubation with calcium antagonists resulted in a dose-dependent inhibition of PRL-stimulated DNA synthesis, activation of DNA synthesis by brief exposure to PRL was not inhibited by the presence of EGTA, calcium channel blockers (nifedipine, cobalt chloride), or calmodulin inhibitors (trifluoperazine, N-6-aminohexyl-5-chloronaphthalene sulfonamide). PRL-stimulated c-myc expression was attenuated, but not blocked, by the calcium channel antagonists. However, the putative intracellular calcium antagonist TMB-8 inhibited both c-myc expression and DNA synthesis in a dose-dependent manner (IC50 = 16 microM). Nb2 cells were sensitive to TMB-8 throughout G1 of the cell cycle, but inhibition of DNA synthesis was greatest when TMB-8 was present during the first 3 h of mitogen presentation, indicating a block in the transition from G0 to G1 of the cell cycle. The effect of TMB-8 suggests that release of a small intracellular calcium pool may mediate the early actions of PRL in Nb2 cells.

Distortion of plant cell plate formation by the intracellular-calcium antagonist TMB-8

Caulonema tip cells ofFunaria deposit new oblique cross walls of specific morphology and placement by a highly defined reorientation mechanism. In the presence of the purported intracellular Ca2+ antagonist 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), these cross walls form in the proper place but exhibit a distorted morphology. Video microscopy indicates that the deformation takes place during the reorientation of the cell plate from a perpendicular to an oblique configuration. Electron micrographs of TMB-8 treated cells indicate a stabilization of phragmoplast microtubules and a greater amount of vesicles and membrane in the developing cell plate. TMB-8 treated cells also show intense chlortetracycline fluorescence from mitochondria, vesicles and endoplasmic reticulum as compared to untreated cells indicating that TMB-8 is blocking release of Ca2+ from intracellular stores. It is concluded that this may cause distortation of cross walls as they form by delaying vesicle fusion, stabilizing microtubules, and increasing the amount of new wall material in the developing cell plate.

Inositol trisphosphate stimulates the release of calcium from intact vacuoles isolated from Acer cells

On addition of inositol trisphosphate, intact vacuoles isolated from Acer pseudoplatanus cell suspension cultures release part of their calcium content. The process was specific, dose-dependent (IC 50 = 0.2μM) and was inhibited by an intracellular calcium antagonist. The calcium efflux elicited by inositol trisphosphate increased with the age of the cell suspension cultures, the maximum effect being obtained when the cultures reached the stationary phase. It is suggested that vacuoles play a role as an endocellular calcium store that is responsive to inositol trisphosphate in plants.

The effects of an intracellular calcium antagonist HA 1077 on delayed cerebral vasospasm in dogs.

The effectiveness of calcium antagonists on a chronic cerebral vasospasm after an SAH is still under debate. Calcium channel blockers such as nimodipine, nifedipine etc. can dilate spastic arteries by intrathecal administration, but not by systemic (iv or po) use. HA 1077 is a novel and potent calcium antagonist vasodilator which is considered to act by employing different mechanisms from the usual calcium channel blockers since it inhibits 1. calcium ionophore A 23187 induced contraction in arterial strips and 2. phenylephrine induced contraction in calcium free media, suggesting that its site of action is in the intracellular space. HA 1077 is water soluble and relatively stable in light. In the present study, the efficacy of HA 1077 was evaluated on dogs by using the spiral arterial strips in vitro and by angiography in vivo. In the arterial strips from the control dogs, a 50% relaxation of KCl (15 mM) induced contraction was obtained by a 10(-6) M HA 1077 for the "intracranial" basilar and middle cerebral arteries, while a 10(-5) M was needed to obtain the same effect for the "extracranial" common carotid and vertebral arteries, indicating that HA 1077 is more effective for the intracranial arteries. A vasospasm was produced by the "two haemorrhage" model of Varsos et al. The average angiographic diameter of the basilar artery was reduced to 60% of the control on SAH day 7. Intravenous infusion of HA 1077 (0.5-3 mg/kg/30 min) significantly dilated the spastic basilar artery (up to 20-30%), for over 2 hours. A fall in the systemic BP remained less than 20% during this time.(ABSTRACT TRUNCATED AT 250 WORDS)