Entry Blocker Nifedipine Research Articles

Effect of Antidepressant Doxepin on Ca^(2+) Homeostasis and Viability in PC3 Human Prostate Cancer Cells

The effect of the antidepressant doxepin on cytosolic Ca²⁺ concentrations ([Ca²⁺](i)) and viability in PC3 human prostate cancer cells was explored. The Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺](i). Doxepin at concentrations of 500-1000 μM induced a [Ca²⁺](i) rise in a concentration-dependent manner. The response was reduced partly by removing Ca²⁺. Doxepin-evoked Ca²⁺ entry was suppressed by Ca²⁺ entry blockers (nifedipine, econazole, SK&F96365), and protein kinase C (PKC) modulators. In the absence of extracellular Ca²⁺, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) partly inhibit doxepin-induced [Ca²⁺](i) rise. Incubation with doxepin nearly inhibited thapsigargin or BHQ-induced [Ca²⁺](i) rise. Inhibition of phospholipase C (PLC) with U73122 failed to alter doxepin-induced [Ca²⁺](i) rise. At concentrations of 200-250 μM, doxepin killed cells in a concentration-dependent manner. This cytotoxic effect was not reversed by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'- tetraacetic acid/acetoxy methyl (BAPTA/AM). Annexin V/PI staining data implied that doxepin (200 and 250 μM) did not induce apoptosis. Collectively, in PC3 cells, doxepin induced a [Ca²⁺](i) rise by evoking PLC-independent Ca²⁺ release from stores including the endoplasmic reticulum and Ca²⁺ entry via PKC-sensitive store-operated Ca²⁺ channels. Doxepin caused cell death that was independent of [Ca²⁺](i) rises.

Microcirculation in rat soleus muscle after eccentric exercise: the effect of nifedipine

This paper explores the role of the calcium entry blocker nifedipine in the explanation of eccentric exercise-induced fibre damage by changes in skeletal muscle microcirculation. Eccentric exercise (EE) was induced by indirect stimulation of rat soleus muscle in its lengthening phase during cycling. Muscle damage was assessed by histology, electron microscopy and muscle tension 48 h later. Diameters of arterioles and venules, their response to dilator and constrictor stimuli and pattern of capillary flow were measured in epiiluminated muscles using intravital microscopy. Tetanic tension developed by EE muscles was lower (8.60 +/- 1.02, means +/- SEM, n = 8 N g(-1) wet weight compared to 12.25 +/- 0.56 in controls, P < 0.01). Electron microscopy showed changes similar to those in muscles exposed to EE by downhill running (Z line streaming, disruption of sarcolemma, swollen tubules). A total of 16% of muscle fibres were damaged, and fibre areas and interstitial space were enlarged. Capillary red blood cell flow showed tendency to a greater intermittency. Large venules were narrower, but arterioles and smaller venules had diameters similar to control muscles. Vessel dilatation to topically applied 10(-4) M adenosine was attenuated. Daily administration of calcium entry blocker nifedipine by gavage (2 mg/kg/day in two equal doses) removed the narrowing of venules, restored the dilator response of all vessels to adenosine and increased capillary:fibre ratio. The percentage of damaged fibres decreased to 4.7 and the size of the interstitial space and fibre areas was normalized. Thus muscle damage caused by eccentric exercise was attenuated by nifedipine due to its beneficial effect on muscle microcirculation, which was impaired by eccentric exercise.

Effects of nifedipine, verapamil and diltiazem on serum biochemical parameters and aortic composition of atherosclerotic chickens

Calcium appears to be involved in many of the cellular events, which are thought to be important in atherogenesis. In this study, we examine the effects of three calcium entry blockers (nifedipine, verapamil, and diltiazem at clinical and higher doses) on serum biochemical parameters and aortic calcium, cholesterol and triglyceride concentrations of atherosclerotic egg-fed chickens. All egg-fed chickens (treated and non-treated) showed an increase in serum total cholesterol, LDL-cholesterol and triglycerides without significant effect when calcium entry blockers were used. Increased HDL values were observed in clinical and high-dose nifedipine and clinical dose verapamil groups. The high-dose diltiazem group presented increased zinc values with respect to the clinical dose diltiazem and control groups. The sodium concentrations were significantly decreased in all the groups of animals treated with calcium entry blockers at high-doses and nifedipine at clinical doses. Measurements of aortic calcium concentration showed a significant decrease in the high-dose nifedipine and verapamil groups. Calcium channel blockers had a tendency to decrease total cholesterol in aortas. The values were statistically significant for the high-dose verapamil, and nifedipine groups. Only nifedipine showed a significant decrease for this parameter at clinical dosages. Triglyceride concentrations in aortas were significantly low in animals fed an atherogenic diet and treated with calcium channel blockers, without differences between drugs or dosages used in the experiment. In addition, the chicken atherosclerosis model has proved itself useful and very suitable for in vivo drug intervention studies.

Actions of R‐ and S‐verapamil and nifedipine on rat vascular and intestinal smooth muscle

1 We have investigated the actions of the calcium entry blockers nifedipine, R-verapamil and S-verapamil in rat aorta, colon and vas deferens. 2 In aorta and colon, these agents produced concentration-dependent relaxations of KCl (80 mM)-induced contractions. In both tissues, the order of potency was nifedipine > S-verapamil > R-verapamil. However, nifedipine showed selectivity for aorta (potency ratio, colon/aorta: 4.36), S-verapamil showed no selectivity (0.62), but R-verapamil showed selectivity for colon (0.19). 3 In prostatic portions of rat vas deferens, nifedipine (10 microM) abolished the contraction to a single electrical stimulus, but R- and S-verapamil were without effect. In epididymal portions of rat vas deferens, R- and S-verapamil inhibited alpha1-adrenoceptor-mediated contractions to a single electrical stimulus at concentrations of 10 microM and above. 4 In conclusion, R-verapamil may prove useful as an intestinal selective calcium entry blocker in the treatment of intestinal disease with a hypermotility component, e.g. irritable bowel syndrome.

Signal transduction underlying carbachol-induced contraction of human urinary bladder.

The present study was designed to reexamine the muscarinic acetylcholine receptor subtype mediating carbachol-induced contraction of human urinary bladder and to investigate the underlying signal transduction. Based upon the nonselective tolterodine, the highly M(2)-selective (R)-4-[2-[3-(4-methoxy-benzoylamino)-benzyl]-piperidin-1-ylmethyl]piperidine-1-carboxylic acid amide (Ro-320-6206), and the highly M(3)-selective darifenacin and 3-(1-carbamoyl-1,1-diphenylmethyl)-1-(4-methoxyphenylethyl)pyrrolidine (APP), contraction occurs via M(3) receptors. The phospholipase C inhibitor 1-(6-[([17beta]-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl)-1H-pyrrole-2,5-dione (U 73,122) (1-10 microM) did not significantly affect carbachol-stimulated bladder contraction. The phospholipase D inhibitor butan-1-ol relative to its negative control butan-2-ol (0.3% each) caused small but detectable inhibition of carbachol-induced bladder contraction. The Ca(2+) entry blocker nifedipine (10-100 nM) strongly inhibited carbachol-induced bladder contraction. In contrast, 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole HCl (SK&F 96,365) (1-10 microM), an inhibitor of store-operated Ca(2+) channels, caused little inhibition. The protein kinase C inhibitor bisindolylmaleimide I (1-10 microM) did not significantly affected carbachol-induced bladder contraction. In contrast, trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide (Y 27,632) (1-10 microM), an inhibitor of rho-associated kinases, concentration dependently and effectively attenuated the carbachol responses. We conclude that carbachol-induced contraction of human urinary bladder via M(3) receptors largely depends on Ca(2+) entry through nifedipine-sensitive channels and activation of a rho kinase, whereas phospholipase D and store-operated Ca(2+) channels contribute only in a minor way. Surprisingly, phospholipase C or protein kinase C do not seem to be involved to a relevant extent.

Signal transduction underlying carbachol-induced contraction of rat urinary bladder. I. Phospholipases and Ca2+ sources.

We have reexamined the muscarinic receptor subtype mediating carbachol-induced contraction of rat urinary bladder and investigated the role of phospholipase (PL)C, D, and A2 and of intra- and extracellular Ca2+ sources in this effect. Based on the nonsubtype-selective tolterodine, the highly M2 receptor-selective (R)-4-[2-[3-(4-methoxy-benzoylamino)-benzyl]-piperidin-1-ylmethyl]-piperidine-1-carboxylic acid amide (Ro-320-6206), and the highly M3 receptor-selective darifenacin and 3-(1-carbamoyl-1,1-diphenylmethyl)-1-(4-methoxyphenylethyl)pyrrolidine (APP), contraction occurs via M3 receptors. Carbachol stimulated inositol phosphate formation in rat bladder slices, and this was abolished by the phospholipase C inhibitor 1-(6-[([17beta]-3-methoxyestra-1,3,5[10]-trien-17-yl)-amino]hexyl)-1H-pyrrole-2,5-dione (U 73,122; 10 microM). Nevertheless, U 73,122 (1-10 microM) did not significantly affect carbachol-stimulated bladder contraction. Carbachol had only little effect on PLD activity in bladder slices, but the PLD inhibitor butan-1-ol, relative to its negative control butan-2-ol (0.3% each), caused detectable inhibition of carbachol-induced bladder contraction. The cytosolic PLA2 inhibitor arachidonyltrifluoromethyl ketone weakly inhibited carbachol-induced contraction at a concentration of 300 microM, but the cyclooxygenase inhibitor indomethacin (1-10 microM) remained without effect. The Ca2+ entry blocker nifedipine (10-100 nM) almost completely inhibited carbachol-induced bladder contraction. In contrast, 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole HCl (SKF 96,365; 10 microM), an inhibitor of store-operated Ca2+ channels, caused little inhibition. We conclude that carbachol-induced contraction of rat bladder largely depends on Ca2+ entry through nifedipine-sensitive channels and, perhaps, PLD, PLA2, and store-operated Ca2+ channels, whereas cyclooxygenase and, surprisingly, also PLC are not involved to a relevant extent.

Endothelial factors and microvascular hypertensive disease.

This paper discusses the role of endothelial dysfunction in human hypertension, especially in relation to small resistance artery structure, as well as the effects of anti-hypertensive drugs on endothelial function of small arteries in human and experimental hypertension. A significant impairment of endothelial function was observed in human essential hypertension as well as in secondary forms of hypertension. No correlation was observed with vascular structure. In animal models of genetic hypertension there is substantial evidence for a beneficial effect of anti-hypertensive treatment with angiotensin converting enzyme (ACE) inhibitors, calcium entry blockers and angiotensin II receptor blockers on endothelial function in small resistance arteries. A significant improvement in endothelial dysfunction may be observed in hypertensive patients after prolonged treatment with ACE inhibitors (cilazapril, lisinopril), calcium entry blockers (nifedipine), and angiotensin II receptor blockers (losartan), while atenolol and hydrochlorotiazide proved to be ineffective in this regard despite similar blood pressure reductions. We conclude that: (i) the development of hypertension is usually associated with the presence of endothelial dysfunction in small resistance arteries of essential hypertensive patients; (ii) vascular structure does not seem to be the major determinant of endothelial function, at least in subcutaneous small resistance arteries; (iii) anti-hypertensive therapy with ACE inhibitors, angiotensin II receptor blockers and calcium entry blockers may improve endothelial function; (iv) a decrease in blood pressure seems to be necessary but not sufficient to obtain a beneficial effect on the endothelium in humans.

Neuropeptide Y enhances potassium excretion by mechanisms distinct from those controlling sodium excretion

Neuropeptide Y (NPY) is an established modulator of renal function. Although NPY reduces renal blood flow and does not alter glomerular filtration rate, it enhances diuresis and natriuresis. Although initial studies on natriuresis did not detect kaliuresis, we now report that a retrospective analysis of previous studies regarding natriuresis demonstrates NPY-induced kaliuresis under several experimental conditions. Kaliuresis was observed despite a marked reduction in urinary potassium concentrations, which may explain why it has not been noted in some initial studies. In a direct comparison of NPY-induced kaliuresis and natriuresis, both effects were slow in onset (requiring >45 min to develop fully) and blocked by the cyclooxygenase inhibitor indomethacin. While natriuresis occurred solely via a Y5 receptor, kaliuresis involved a Y1 receptor and an additional receptor subtype, possibly Y2. The L-type Ca2+ entry blocker nifedipine abolished natriuresis but did not inhibit kaliuresis. A combination of experiments with the bradykinin B2 receptor antagonist icatibant, the angiotensin II receptor antagonist losartan, and the converting enzyme inhibitor ramiprilat revealed that NPY-induced natriuresis involves bradykinin while kaliuresis involves angiotensin II. We conclude that NPY-induced kaliuresis is much less pronounced than natriuresis and is mediated by distinct mechanisms.

Effects of the selective cyclooxygenase-2 inhibitor nimesulide on vascular contractions in endothelium-denuded rat aorta

We have examined the effects of the selective cyclooxygenase-2 inhibitor nimesulide and the non-selective cyclooxygenase inhibitor indomethacin on vascular responsiveness of endothelium-denuded rat aorta. Isometric contractions were obtained to the α-adrenoceptor agonists phenylephrine (full agonist) and clonidine (partial agonist relative to phenylephrine) and to endothelin-1 and KCl. Maximum contractile responses to the partial agonist clonidine were significantly reduced by nimesulide (10 μM) and by indomethacin (10 μM) to 60.8±8.5% ( n=8) and 69.0±9.6% ( n=12) of control, respectively, as compared with the effects of vehicle (99.0±5.8%; n=17). The inhibitors had lesser effects against contractions to phenylephrine: nimesulide had no significant effect, whereas indomethacin caused a small but significant reduction in the maximum contraction to phenylephrine to 90.3±5.0% ( n=12) of control (vehicle: 108.0±5.2%, n=15 nimesulide: 111.8±5.9%, n=5). Neither nimesulide nor indomethacin had any effect on contractions to endothelin-1 or KCl. These actions differed from the effects of the Ca 2+ entry blocker nifedipine, which significantly reduced contractions to clonidine and KCl to a similar extent. The maximum contraction to clonidine was also significantly reduced by the thromboxane receptor antagonist SQ 29548 (1 μM) to 83.4±6.4% of control ( n=7) (vehicle 115.5±7.5%, n=7). It is concluded that the cyclooxygenase inhibitors nimesulide or indomethacin reduce vascular responsiveness to α-adrenoceptor agonists in endothelium-denuded rat aorta, presumably by preventing the formation of vasoconstrictor prostaglandins in aortic smooth muscle by cyclooxygenase-2. This reduced vascular responsiveness was most clearly seen with the partial agonist clonidine.

Regulation of red blood cell filterability by Ca2+ influx and cAMP-mediated signaling pathways.

To investigate the mechanism of the regulation of human red blood cell deformability, we examined the deformability under mechanical stress. Washed human red blood cells were rapidly injected through a fine needle, and their filterability was measured using a nickel mesh filter. The decrease in filterability showed a V-shaped curve depending on the extracellular Ca2+ concentration; the maximum decrease was achieved at approximately 50 muM. The decreased filterability was accompanied by no change in cell morphology and cell volume, indicating that the decrease in filterability can be ascribed to alterations of the membrane properties. Ca2+ entry blockers (nifedipine and felodipine) inhibited the impairment of filterability under mechanical stress. Prostaglandins E1 and E2, epinephrine, and pentoxifylline, which are thought to modulate the intracellular adenosine 3',5'-cyclic monophosphate (cAMP) level of red blood cells, improved or worsened the impaired filterability according to their expected actions on the cAMP level of the cells. These results strongly suggest that the membrane properties regulating red blood cell deformability are affected by the signal transduction system, including Ca(2+)-dependent and cAMP-mediated signaling pathways.

Ca2+ channel blockers verapamil and nifedipine inhibit apoptosis induced by 25-hydroxycholesterol in human aortic smooth muscle cells

We have characterized the death of human aortic smooth muscle cells induced by 25-hydroxycholesterol, an oxidation product of cholesterol. Chromatin condensation characteristic of apoptosis was observed by enzymatic (TUNEL) staining of chromatin, and by electron microscopy. Fourteen percent of cells treated with 5 microg/ml of 25-hydroxycholesterol for 24 h displayed chromatin degradation as determined by positive TUNEL staining. Addition of TNF alpha (10 ng/ml) and IFN gamma (20 ng/ml) increased the proportion of TUNEL positive cells to 30%, whereas the cytokines alone were without effect. After 48 h, 40% of the cells treated with 5 microg/ml of 25-hydroxycholesterol were TUNEL positive, and 21% of the cells displayed chromatin condensation. Oligonucleosomal DNA fragmentation typical of apoptosis was demonstrated by agarose gel electrophoresis. Furthermore, activation of the ICE-like protease caspase 3 (CPP32) was observed in cells treated with 25-hydroxycholesterol. Addition of the Ca2+ entry blockers verapamil or nifedipine to the culture medium inhibited apoptosis by more than 70% and reduced cytotoxicity, while removal of Ca2+ from culture medium reduced apoptosis by 42%. Within a few minutes after addition, 25-hydroxycholesterol induced intracellular Ca2+ oscillations with a frequency of approximately 0.3-0.4 min(-1). Thus it appears that Ca2+ influx through plasma membrane channels is an important signal in oxysterol-induced apoptosis. Addition of TNF alpha and IFN gamma enhanced cytotoxicity and resulted in a higher proportion of apoptotic cells, suggesting that inflammatory cytokines can increase the cytotoxicity of lipid oxidation products.

Effects of mercuric chloride and methyl mercury on cholinergic neuromuscular transmission in the guinea-pig ileum.

The effects of mercuric chloride (HgCl2) and methyl mercury (MeHg) were examined on basal mechanical activity and electrically-induced neurogenic cholinergic contractions (twitch contractions) in longitudinal muscle-myenteric plexus strips from guinea-pig distal ileum. Both compounds at 0.33 microM slightly enhanced the amplitude of twitch contractions in approximately 50% preparations. This effect was probably due to facilitation of acetylcholine (ACh) release since 0.1 and 1 microM mercurials increased electrically-evoked tritium outflow from [3H]choline preloaded muscle layer with attached myenteric plexus. Conversely, higher mercury concentrations inhibited twitch contractions (HgCl2 IC50 = 21.3 +/- 6.4 microM; MeHg IC50 = 45.1 +/- 5.5 microM), as well as contractions to exogenous ACh (0.1 microM) in resting preparations, and concomitantly increased the basal tone. The former effects possibly reflected an antimuscarinic activity of mercury, while the latter was related to alterations of calcium homeostasis in the effector cells. Indeed, the effect of HgCl2 on basal tone was antagonized by the Ca2+ entry blocker nifedipine (3, 10, 30 nM), indicating Hg-induced facilitation of Ca2+ influx through voltage-dependent channels. On the whole, our results suggest that cholinergic neuromuscular transmission and Ca(2+)-dependent mechanisms underlying smooth muscle contractility are targets for mercury toxicity in the intestine.

Oedema formation with the vasodilators nifedipine and diazoxide

To determine whether the common side effect of ankle oedema with arteriolar vasodilators such as the calcium entry blocker (CEB) nifedipine and the potassium channel opener (PCO) diazoxide is the direct result of peripheral vasodilation or merely a consequence of renal sodium retention. In 12 healthy sitting volunteers we studied for 3 h the effects of 20 mg nifedipine, 150 mg diazoxide intravenously, 25 mg captopril and placebo on oedema formation and sodium excretion. Foot swelling was determined with a new accurate device (coefficient of variation 0.30%), which uses Archimedes principle to measure water displacement induced by immersion of the foot. Blood pressures were recorded with a Hawksley random-zero sphygmomanometer. All of the active drugs decreased diastolic blood pressure (captopril by 9 +/- 2%, nifedipine by 4 +/- 3% and diazoxide by 2 +/- 2%, compared with an increase of 5 +/- 2% with placebo). Foot volume increased acutely after administration of nifedipine (by 2.6 +/- 0.4%), whereas it remained stable with placebo and the other drugs. Administration of captopril and nifedipine induced increases of fractional sodium excretion (by 20 +/- 9% and 40 +/- 20%, respectively) in contrast to the decreases with placebo and diazoxide (by 13 +/- 11% and 24 +/- 10%, respectively). Only administration of nifedipine induced significant, albeit small, increases in haemoglobin and serum albumin levels. Administration of nifedipine increased foot volume and natriuresis simultaneously, thereby supporting the hypothesis that development of ankle oedema with CEB is a local phenomenon at the site of vasodilation. The absence of a similar increase in foot volume with diazoxide administration should be interpreted with caution because of the rather minor effect of this dose of diazoxide on blood pressure. However, it could be indicative of a different mechanism of oedema formation with PCO.

Body fat as a predictor of the antihypertensive effect of nifedipine

The aim of this study was to investigate the relationship between the blood pressure response and body fat in patients treated with the calcium entry blocker nifedipine. Forty untreated subjects with moderate hypertension completed the study. Height, weight, body mass index (BMI) and body fat (measured by near infrared spectrophotometry using Futrex 5000) were measured before the start of the study. Based on blood pressure measurements before and 30 min after one capsule of 10 mg nifedipine, the patients were allocated to treatment with 10 mg nifedipine daily in the responder group (reduction in diastolic blood pressure > or = 10 mmHg) and with 20 mg nifedipine daily in the non-responder group. Finally, the blood pressure was measured after 1 week. Twelve patients were classified as responders and 28 patients as non-responders. The responder/non-responder groups were comparable with respect to the blood pressure readings initially and to age, whereas the non-responders had higher weight (P < 0.01) and BMI (P < 0.01), more body fat (P < 0.01) and were smaller in height (P < 0.01). After 1 week no additional antihypertensive effect was observed in either group. The blood pressure reduction was negatively correlated to the body fat mass in kilogrammes (P < 0.05 for systolic and P < 0.01 for diastolic blood pressure) in the responder group but not in the non-responder group, whereas no significant correlations were found to BMI in either group. Our data indicate that the body fat can be used to predict the antihypertensive effect of nifedipine.

Calcium and the Endothelin-1 and α1-Adrenergic Stimulated Phosphatidylinositol Cycle in Cultured Rat Cardiomyocytes

Cultured neonatal rat cardiac myocytes have been utilized as a model for the study of the effect of variations in cytoplasmic free Ca2+ on the activity of phospholipase C, a key enzyme in agonist-stimulated signal transduction through the phosphoinositide pathway. Cells prelabelled with [3H]inositol were exposed to various agents in an attempt to modulate the cytoplasmic free Ca2+ concentration and the formation of [3H]inositolphosphates (15-30 min) in the presence of Li+ was taken as a measure of phospholipase C activity. Not the basal but the endothelin-1 (10-8 M) induced [3H]inositolphosphate production (15 min) was stimulated 1.54- and 1.43-fold by A23187 (10-8 μM external Ca2+) and 50 mM K+ (1.3 mM external Ca2+) treatment of cells, respectively. The phenylephrine (10-4 M)induced response was also stimulated (1.35-fold) by A23187, however it was 43% inhibited by high K+. Ouabain (10-8 μM) treatment of cells did not affect either or agonist stimulated phosphoinositide turnover. On the other hand, total removal of external free Ca2+ by addition of 50 μM ethylene glycol bis (β-aminoethyl ether) (N,N,N′,N′-tetraacetic acid strongly inhibited (75%) the endothelin-1 induced but not the basal phospholipase C activity. Endothelin-1 binding to its receptor was shown not to be inhibited by the absence of external Ca2+ while resynthesis of [3H]phosphatidylinositol 4,5-bisphosphate was not rate-limiting under this condition. The lack of external Ca2+ eventually resulted in total standstill of the ET-1 induced PtdIns turnover after 30 min. Although not always as predicted, effects on basal and agonist-activated phospholipase C were observed too when cells were treated with low Ca2+ medium, Ca2+ entry blocker nifedipine (1 μM) or Ca2+-channel agonist Bay K8644 (1 μM) but most of these effects were only seen after 90 min incubation. Fluorometric (fura-2) measurements showed that total removal of external free Ca2+ for a short period decreased, while short exposure to high K+ increased cytoplasmic free Ca2+ but neither Ca2+ free buffer or nifedipine nor Bay K8644 had any effect. Furthermore, in saponin-permeabilized cardiomyocytes we could demonstrate that basal as well as GTPγS (30 μM) stimulated phospholipase C activity was strongly activated by free Ca2+ in the concentration range of 0.1-10 μM. We conclude that in the intact cardiomyocyte the signalling pathway through phospholipase C/phosphatidylinositol 4,5-bisphosphate, stimulated by agonist-receptor interaction that activates GTP-binding proteins as does GTPγS, is likely be a Ca2+ dependent process.

Actions of abscisic acid and the analogue SD217595 on calcium mediated activity of rat vas deferens smooth muscle

1. 1. K +-induced and Ca 2+-induced contractures of rat prostatic and epididymal vas deferens smooth muscle were used to screen ABA and its analogue SD217595 for Ca 2+ modulatory activity. 2. 2. The Ca 2+ agonist BAY K8644 significantly enhanced Ca 2+-induced contractures while the Ca 2+ entry blocker nifedipine strongly inhibited such contractures in both types of vas deferens preparation over the whole Ca 2+ concentration range employed. 3. 3. At 10 −7 mol 1 −1 ABA had no significant effect on K +-induced or Ca 2+-induced contractures nor did it change the phasic/tonic force ratio in the preparations. 4. 4. The ABA analogue SD217595 strongly inhibited Ca 2+-induced contractures over the whole Ca 2+ concentration range employed in both prostatic and epididymal vas deferens. 5. 5. It is concluded that ABA is without significant Ca 2+ modulatory activity in this smooth muscle preparation but the ABA analogue SD217595 possesses strong Ca 2+ entry blocking ability.

Arterial smooth muscle responses in adult and moderately aged spontaneously hypertensive rats.

In order to further clarify differences between spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats as well as the effects of ageing, vascular smooth muscle responses of mesenteric arterial rings and intracellular free calcium concentration ([Ca2+]i) in platelets and lymphocytes were studied in 20-week-old and 32-week-old animals. Arterial contractile responses induced by noradrenaline and potassium chloride were comparable in 20-week-old SHR and WKY rats, whereas at 32 weeks of age maximal contractile force generation to both of these agents was clearly lower in SHR. In both age groups the calcium entry blocker nifedipine was more effective in inhibiting potassium chloride-evoked responses in SHR than in WKY rats, and arterial relaxation responses by endothelium-dependent (acetylcholine) and endothelium-independent (nitroprusside, isoprenaline) mechanisms were more pronounced in WKY rats when compared with SHR. The ability of vascular smooth muscle cells to sequester calcium was evaluated by first depleting cellular calcium stores with maximal contractions to noradrenaline in calcium-free buffer, whereafter calcium was returned to the organ bath. After a 10 min. calcium loading period the arterial rings were rechallenged with noradrenaline. Both in 20-week-old and 32-week-old rats these responses were less marked in SHR than in WKY rats, suggesting reduced ability of smooth muscle cells to sequester calcium. In addition, platelets and lymphocytes were used as cell models to examine [Ca2+]i in the experimental groups by the fluorescent indicator quin-2. In these two cell types [Ca2+]i was higher in SHR than in WKY rats in both of the age groups studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Investigation into vanadate-induced potentiation of smooth muscle contractility in the rabbit isolated ileum

The action of sodium orthovanadate (Na 3VO 4) on spontaneous mechanical activity of the longitudinal muscle was investigated in isolated segments of rabbit distal ileum. Vanadate (0.3–1000 μM) concentration-dependently enhanced the amplitude of phasic contractions (pendular movements) and caused the muscle tone to slightly increase at the highest concentrations. Both these effects were mimicked by the Ca 2+ channel activator BAY K 8644 (10–1000 nM). Vanadate- and BAY K 8644-induced potentiation of mechanical activity was antagonized by the Ca 2+ entry blocker nifedipine (3 nM). In Ca 2+-free, K +-depolarized preparations, vanadate (100 μM) failed to contract the musculature, but potentiated the contractile response to applied calcium (CaCl 2: 30–300 μM). The action of vanadate was similar to that of BAY K 8644 (3 nM) and was antagonized by nifedipine (0.1 nM). These results suggest that extracellular calcium is required for vanadate-induced smooth muscle excitation which, at least in part, appears to arise from facilitation of calcium influx through voltage-dependent Ca 2+ channels.

Influence of the endothelium on Ca2+ dependency of angiotensin II-induced contractions of rat aortic rings.

Endothelium-dependent relaxation has been demonstrated to be involved in the regulation of vascular tone and extracellular Ca2+ was found to play a prominent role in this process. Since the dependency on extracellular Ca2+ appeared to differ considerably within the arterial tree, possibly as a consequence of vessel-related endothelium-dependent mechanisms, we investigated the effects of different compounds affecting Ca2+ entry (nifedipine, CoCl2) on angiotensin II-induced contractions of rat aortic rings with and without endothelium as well as the responses in a Ca(2+)-"free" solution. For this purpose, rat aortic rings were either undone from their endothelial layer by gentle mechanical rubbing or care was taken to keep the intima intact in case rings where endothelium were required. The presence of an intact endothelium was confirmed by acetylcholine-induced relaxation. A stronger responsiveness towards angiotensin I, both after a complete concentration-response curve and after a single maximal concentration of angiotensin II was observed in arterial segments without endothelium. The maximal contraction to a single concentration of angiotensin II (0.1 microM) in the rings without endothelium amounted to 75.8 +/- 3.8% of the preceding response to a supramaximal concentration of noradrenaline (= Emax). In rings without the endothelial layer, the contraction was 34.8 +/- 3.7% of Emax. This indicates an endothelium-induced relaxation in aortic rings with endothelium. After incubation with the Ca2+ entry blocker nifedipine (1 microM) both rings with and without endothelium were inhibited to the same extent, contractions amounted to 30.7 +/- 1.8% and 19.6 +/- 1.3% of Emax, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of high calcium diet on arterial smooth muscle function and electrolyte balance in mineralocorticoid‐salt hypertensive rats

1. The effects of a high calcium diet (2.5%) on blood pressure, electrolyte balance, plasma and tissue atrial natriuretic peptide (ANP), cytosolic free Ca2+ concentration ([Ca2+]i), and arterial smooth muscle responses were studied in one-kidney deoxycorticosterone (DOC)-NaCl hypertensive Wistar rats. 2. Calcium supplementation for 8 weeks markedly attenuated the development of DOC-NaCl hypertension and the associated cardiac hypertrophy, and prevented the DOC-NaCl-induced sodium-volume retention as judged by reduced plasma Na+, and decreased plasma and ventricular ANP concentrations in high calcium-fed DOC-NaCl rats. However, calcium supplementation did not affect the DOC-NaCl-induced rise in platelet [Ca2+]i. 3. Smooth muscle contractions of isolated mesenteric arterial rings in response to depolarization by K+ (20-30 mM) were enhanced in DOC-NaCl-treated rats, this enhancement being abolished by concurrent oral calcium loading. The Ca2+ entry blocker nifedipine (10 nM) inhibited the contractions induced by K+ (30-125 mM) more effectively in DOC-NaCl rats than in controls, while the inhibition in calcium-loaded DOC-NaCl rats was significantly greater than in controls only with 30 mM K+. 4. The contractions of mesenteric arterial rings induced by omission of K+ from the organ baths were used to evaluate cell membrane permeability to ions. In chemically denervated rings the onset of the gradual rise in contractile force in K(+)-free medium occurred earlier, and the rate of the contraction was faster in DOC-NaCl-treated rats than in controls and high calcium-fed DOC-NaCl rats. Smooth muscle relaxation induced by 0.5 mM K+ upon K(+)-free contractions was clearly slower in DOC-NaCl rats than in controls and calcium-supplemented DOC-NaCl rats. 5. The functions of arterial smooth muscle Na+, Ca2+ exchange and Ca(2+)-ATPase were evaluated by the aortic contractions elicited by low Na+ medium, and the subsequent relaxation responses induced by Ca(2+)-free solution (in the presence of 5 mM caffeine, 1 microM nifedipine and 10 microM phentolamine). The rate of aortic low Na+ contractions (evaluating Ca2+ influx via Na+, Ca2+ exchange), as well as that of subsequent relaxations was slower in DOC-NaCl-treated rats than in controls, whether the relaxation was induced in normal (144.0 mM) or low (1.2 mM) organ bath Na+ concentration (reflecting Ca2+ extrusion by both Ca(2+)-ATPase and Na+, Ca2+ exchange, and by Ca(2+)-ATPase alone, respectively). However, in calcium-supplemented DOC-NaCl rats the aortic responses did not differ from control. The difference between the relaxation rate in normal and low Na+ concentration in each aortic ring,representing the contribution of Na+, Ca2+ exchange in these relaxations, was comparable in all groups.6. In conclusion, calcium supplementation clearly attenuated the development of hypertension, cardiac hypertrophy, and sodium retention induced by the DOC-NaCI treatment. However, the associated rise in platelet [Ca2+], was not prevented, suggesting that in this form of experimental hypertension increased dietary calcium does not lower blood pressure by reducing [Ca2+]i. The results from vascular responses in vitro suggest that in arterial smooth muscle the DOC-NaCl treatment increased contractile sensitivity to depolarization, voltage-dependent Ca2+ entry and cell membrane permeability to ions, and attenuated relaxation responses and vascular Na+, K+-ATPase function. The results further suggest reduced ability of the cell membrane to transport Ca2+ (possibly via Ca2+-ATPase) in DOC-NaCl hypertension. The high calcium diet opposed these alterations. The present results thus provide evidence that the antihypertensive effect of a high calcium diet in mineralocorticoid-salt hypertension is mediated by its beneficial effects on systemic sodium balance and arterial smooth muscle function.