-
[show abstract]
[hide abstract]
ABSTRACT: Genetic variability in the ADD1 (Gly460Trp) and ADD2 (C1797T) subunits of the cytoskeleton protein adducin plays a role in the pathogenesis of hypertension, possibly via changes in intracellular cation concentrations. ADD2 1797CC homozygous men have decreased erythrocyte count and hematocrit. We investigated possible association between intra-erythrocyte cations and the adducin polymorphisms. In 259 subjects (mean age 47.7 years), we measured intra-erythrocyte Na(+) [iNa], K(+) [iK] and Mg(2+) [iMg], serum cations and adducin genotypes. Genotype frequencies (ADD1: GlyGly 61.5%, Trp 38.5%; ADD2: CC 80.4%, T 19.6%) complied with Hardy-Weinberg proportions. In men, ADD2 CC homozygotes (n=100) compared to T-carriers (n=23) had slightly lower iK (85.8 versus 87.5 mmol/l cells; P=0.107), higher iMg (1.92 versus 1.80 mmol/l cells; P=0.012), but similar iNa (6.86 versus 6.88 mmol/l cells; P=0.93). In men, iK, iMg and iNa did not differ according to ADD1 genotypes. In men, iK (R(2)=0.128) increased with age and serum Na(+), but decreased with serum total calcium and the daily intake of alcohol. iMg (R(2)=0.087) decreased with age, but increased with serum total calcium. After adjustment for these covariates (P<or=0.04 for all), findings in men for iK (CC versus T: 85.8 versus 87.3 mmol/l; P=0.14) and iMg (1.91 versus 1.82 mmol/l; P=0.03) remained consistent. In 136 women, none of the phenotype-genotype relations reached significance. Changes in intra-erythrocyte cations in ADD2 1797CC homozygous men might lead to osmotic fragility of erythrocytes, but to what extent they reflect systemic changes or are possibly involved in blood pressure regulation remains unknown.
Journal of Human Hypertension 06/2007; 21(5):387-92. · 2.80 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Myofibroblasts and transforming growth factor-beta 1 (TGF-beta 1) are key elements of cardiac tissue fibrosis development. The aim of this study was to determine whether the ability of TGF-beta 1 to affect the contractile activity of cardiac fibroblasts depends on their differentiation into myofibroblasts. Cardiac fibroblasts (from male adult Wistar rats) from passage 2 were therefore cultured to confluency and incubated on a hydrated collagen gel, both with and without TGF-beta 1 (0, 20, 40, 100, 200, 400 or 600 pmol/l), for 1, 2 and 3 days in a Dulbecco's Modified Eagle's Medium (DMEM) without fetal bovine serum (FBS). Growing cultures of cardiac fibroblasts were obtained by incubating second-passage fibroblasts in DMEM with 10% FBS with or without TGF-beta 1 (0 to 600 pmol/l) for 6 days. These fibroblasts were then further incubated on the collagen gel for 1, 2 and 3 days in DMEM without FBS. TGF-beta 1 dose-dependently increased the contraction of collagen gel mediated by cardiac fibroblasts, either added directly to the gel or after growing of the cardiac fibroblasts in the presence of TGF-beta 1 for 6 days, reaching a maximal effect at 100 pmol/l TGF-beta 1. In both culturing conditions, TGF-beta 1 also stimulated the [3H]-thymidine incorporation and the total protein content in the cardiac fibroblasts in the collagen gel lattice. TGF-beta 1 dose-dependently induced an increase in alpha-smooth muscle actin, a marker of myofibroblasts, in both culturing conditions. The TGF-beta 1-induced reduction of area of the collagen gel was negatively correlated to the TGF-beta 1-evoked appearance of alpha-smooth muscle actin in the collagen gel matrix. TGF-beta 1 increased the contractile activity of adult rat cardiac fibroblasts and their ability to differentiate into myofibroblasts. Because contractile activity was correlated with differentiation, the influence of TGF-beta 1 on cardiac fibroblast-induced collagen gel contraction may depend on the promotion of myofibroblast differentiation.
Methods and Findings in Experimental and Clinical Pharmacology 04/2003; 25(2):79-86. · 0.93 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The purpose of the present investigation was to describe the relative impact of genes and environment on the variance of the plasma constituents of the renin angiotensin system. We ascertained 56 male and 80 female adult same-sex twin pairs from the Flemish population. Plasma renin activity (PRA), the concentration of angiotensinogen (AGT) and angiotensin-converting enzyme (ACE) were measured, and path analysis was applied, after transformation toward normality. For PRA and AGT significant heritability was only detected in the male subgroup, with heritability estimates of 66% and 90%, respectively. Angiotensin-converting enzyme concentration was determined by additive genes for 43% of its variance, by shared environmental influences for 42%, and by specific environmental influences for 15%. The high heritability found for AGT is compatible with the results of earlier studies linking the M235T polymorphism of the angiotensinogen gene to plasma AGT levels. For PRA, we are the first to show significant heritability. Our results regarding ACE confirm the findings in other populations.
Journal of Human Hypertension 07/2002; 16(6):417-22. · 2.80 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The aim of the present study was to investigate whether collagen gel contraction can be induced by cardiac fibroblasts in serum-free conditions. Cardiac fibroblasts (from normal male adult rats) from passage 2 were cultured to confluency and added to a hydrated collagen gel in Dulbecco's Modified Eagle's Medium with or without fetal bovine serum for 1, 2, 3 or 7 days. Control gels containing adult rat cardiac fibroblasts showed a significant amount of contraction after 2 days of incubation in a serum-free medium, causing a contraction to 47% of the area at the start of the incubation. Increasing the percent of fetal bovine serum induced further stimulation of the collagen gel contraction by cardiac fibroblasts. Optimal conditions for collagen gel contraction by adult fibroblasts were obtained with 100,000 cells incubated for 1 (up to 3) days. The presence of insulin-transferrin-selenium in the medium caused a more pronounced stimulation of the collagen gel contraction by cardiac fibroblasts, while sodium azide inhibited this contraction.
Methods and Findings in Experimental and Clinical Pharmacology 10/2001; 23(7):377-82. · 0.93 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The primary objective of this randomised, placebo- controlled, double-blind, crossover study, was to evaluate and compare the longer term effects of the angiotensin II type 1 receptor antagonist losartan and the converting enzyme inhibitor enalapril on 24-h ambulatory blood pressure (BP). After a 4-week placebo run-in period, nine patients with essential hypertension entered the double-blind phase of the study, which consisted of three 6-week periods during which patients were treated with placebo, enalapril 20 mg o.d. or losartan 50 mg o.d. Losartan and enalapril, taken between 07.00 and 08.00, reduced ambulatory BP throughout the 24-h period. Average night time BP was reduced from 133/85 mm Hg on placebo to 124/78 mm Hg on enalapril and to 126/77 mm Hg on losartan. Daytime BP averaged 157/101 mm Hg on placebo, and was significantly lower during enalapril (142/91 mm Hg) than during losartan treatment (147/95 mm Hg). Clinic BP, measured 2 to 4 hours after drug intake, was reduced to the same extent by both drugs. The losartan-induced BP changes were significantly related to those obtained with enalapril (0.63 < r < 0.93). Ambulatory BP monitoring was repeated after 4 weeks of combined therapy in six patients. The BP lowering effect of the combination was not significantly better than that achieved with enalapril alone. In conclusion, losartan 50 mg o.d. and enalapril 20 mg o.d. lower BP to approximately the same extent, except for a more pronounced effect of enalapril on daytime ambulatory BP. The current study does not provide convincing evidence that addition of losartan to enalapril in the doses used further reduces BP.
Journal of Human Hypertension 03/2001; 15(3):161-7. · 2.80 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: An intracardiac aldosterone system which responds to short- and long-term physiological stimuli has been described. This cardiac generated aldosterone has possibly autocrine or paracrine actions. Normal cardiac tissue contains mineralocorticoid receptors (MR) and cardiac high affinity MR are localized in cardiac myocytes and endothelial cells. Data concerning the presence of MR in cardiac fibroblasts are, however, controversial. MR are not specific for aldosterone but they also bind glucocorticoids. Cardiac fibroblasts however contain the enzyme 11beta-hydroxy-steroid dehydrogenase II which converts these glucocorticoids to inactive metabolites. Discordant findings on the in vitro effect of aldosterone on the collagen synthesis in cardiac fibroblasts are reported and can at least partly attributed to the presence of various fibroblasts phenotypes. During chronic aldosterone infusion in uninephrectomized rats on a high-salt diet, a marked accumulation of interstitial and to a lesser extent perivascular collagen occurs in the heart in both ventricles. This cardiac fibrosis in this aldosteronism model is prevented by spironolactone. This effect of aldosterone is crucially dependent on the salt status of the rat. Indeed, rats on a restricted salt intake infused with aldosterone had no cardiac fibrosis above control levels. During the continuous infusion of aldosterone in the rat the appearance of fibrosis was delayed and starts 4 weeks after the beginning of the infusion which argues against a direct effect of aldosterone. The mechanism of aldosterone-salt induced cardiac fibrosis possibly involves angiotensin II acting through upregulated AT1 receptors and the cardiac AT1 receptor is the target for aldosterone. An accumulation of collagen in the heart has also been found in patients with adrenal adenomas and during chronic activation of the renin-angiotensin-aldosterone system such as in surgically induced unilateral renal ischemia, unilateral renal artery banding or renovascular hypertension. Spironolactone prevents aortic collagen accumulation in spontaneously hypertensive rats. In patients with stable chronic heart failure spironolactone treatment in addition to diuretics and angiotensin-converting enzyme (ACE) inhibition reduced circulating levels of procollagen type III N-terminal aminopeptide. Also, in the Randomized Aldactone Evaluation Study spironolactone coadministered with conventional therapy of ACE inhibitors, loop diuretics and digitalis in patients with symptomatic heart failure defined as NYHA classes III-IV reduces total mortality by 30%.
Journal of Molecular and Cellular Cardiology 07/2000; 32(6):865-79. · 5.17 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To compare the effects of a highly beta1-selective adrenoceptor antagonist bisoprolol with those of atenolol and placebo on endurance exercise capacity in young, healthy male volunteers.
Twelve subjects randomly received oral placebo, atenolol (100 mg/day) or bisoprolol (10 mg/day) for 3 weeks, following a double-blind cross-over design.
At the end of each period, the subjects performed an endurance exercise test on the bicycle ergometer at 70% of maximal aerobic power. Cardiac output was measured by means of an automated CO2-rebreathing method. Venous blood was sampled before, during and after exercise.
Exercise duration was not significantly different between the two drugs tested. Total exercise duration was significantly reduced by bisoprolol (-19.4 +/- 6.7%, P< 0.01) (mean +/- SEM) and by atenolol (-29.8 +/- 6.6%, P< 0.001), compared with placebo. Atenolol and bisoprolol were equally effective in lowering resting plasma renin activity, heart rate and systolic blood pressure. Resting and exercise stroke volume were significantly increased by both drugs, so that cardiac output was not significantly affected. Both drugs induced significant decreases in plasma-free fatty acid concentrations during recovery and blunted the exercise-induced increase. There were no significant relationships between the reduction of exercise duration and the haemodynamic changes or the degree of impairment of the exercise-induced increase in free fatty acid release resulting from beta-blockade.
It is concluded that both drugs affect endurance exercise capacity in young, normotensive men, with a tendency to a smaller reduction during bisoprolol treatment. Haemodynamic variables are unlikely to be involved in the reduction of endurance exercise capacity. The role of the reduced availability of plasma free fatty acids remains unclear.
Journal of Hypertension 02/2000; 18(1):35-43. · 4.02 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A double blind, placebo-controlled, parallel study was conducted on the effect of a high daily oral calcium supplementation of 1 g elemental calcium, given twice daily for 16 weeks in normal male subjects, on plasma renin, aldosterone, kallikrein, cGMP, cAMP, and calciotropic hormones, intracellular calcium concentrations, and plasma total and ionized calcium. After a 1-month run-in period on a limited use of dairy products, the subjects (n = 32) were allocated to a placebo or a calcium group. Placebo or 1 g elemental calcium was administered twice daily in the morning and evening for 16 weeks. All subjects were investigated at baseline and after 1, 2, 4, 8, and 16 weeks of placebo or calcium administration. A decreased intraerythrocyte and intraplatelet Ca2+ concentration was observed in the calcium-treated subjects. Compared with the placebo group, an increase in the plasma renin activity (PRA) in the calcium group was observed after 4, 8, and 16 weeks of oral calcium administration. However, plasma aldosterone and urinary excretion of aldosterone, kallikrein, cGMP, and cAMP were not changed during calcium administration. Oral calcium supplementation in these men was also accompanied by a reduction in the plasma concentration of intact parathyroid hormone and 1,25-dihydroxyvitamin D3, and an increase in 24-h urinary calcium excretion, but no change in the plasma total Ca2+ concentration, serum ionized Ca2+ level, and plasma phosphate or 25-hydroxyvitamin D3. Our data show that the increase in PRA observed in men during oral calcium supplementation is accompanied by a reduction in the intracellular free and total Ca2+ concentration in platelets and erythrocytes and by a decrease in the plasma concentration of intact parathormone and 1,25-dihydroxyvitamin D3.
American Journal of Hypertension 01/2000; 12(12 Pt 1-2):1217-24. · 3.18 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: In response to humoral and mechanical stimuli, the myocardium adapts to increased work load through hypertrophy of individual muscle cells. Myocardial hypertrophy is characterized by an increase in cell size in the absence of cell division and is accompanied by changes in gene expression. Angiotensin II (Ang II), the effector peptide of the renin-angiotensin system (RAS), regulates volume and electrolyte homeostasis and is involved in cardiac and vascular growth in rats. In this review, the role of RAS in myocyte protein synthesis (myocyte hypertrophy) and in induction of gene expression will be discussed in rat cardiomyocytes in culture. Traditional RAS can be considered as a system in which circulating Ang II is delivered to target tissues or cells. However, a local RAS has also been described in cardiac cells and evidence has been accumulated for autocrine and/or paracrine pathways by which biological actions of Ang II can be mediated. These actions of Ang II are primarily mediated through Ang II receptors subtype I (AT1-R). When evaluating the effects of Ang II in situ, both changes in circulating levels and local production have to be taken into account. Contrasting results have been found concerning the in vitro effect of Ang II on the protein synthesis in cardiac myocytes and can be at least partly be attributed to methodological problems such as assay of de novo protein synthesis and isolation and separation procedure of cardiac myocytes. The Ang II-induced hypertrophic effect also depends on the existence of nonmyocytes in a cardiocyte culture. In rat cardiocytes, AngII also causes induction of many immediately-early genes (c-fos, c-jun, jun-B, Egr-1 and c-myc) and induces also late markers of cardiac hypertrophy (skeletal alpha-actin and atrial natriuretic peptide expression) and growth factors (TGF-beta 1 gene expression). In vivo AngII via AT1-R, causes not only ventricular hypertrophy but also a shift to the fetal phenotype of the myocardium. Angiotensin-converting enzyme inhibitors and AngII receptor antagonists of the subtype I not only induce the regression but also prevent the development of cardiac hypertrophy in experimental rat models.
Methods and Findings in Experimental and Clinical Pharmacology 07/1999; 21(5):363-74. · 0.93 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: In response to humoral and mechanical stimuli, the myocardium adapts to increased work load through hypertrophy of individual muscle cells. Myocardial hypertrophy is characterized by an increase in cell size in the absence of cell division and is accompanied by changes in gene expression. Angiotensin II (ANG II), the effector peptide of the renin-angiotensin system (RAS), regulates volume and electrolyte homeostasis and is involved in cardiac and vascular growth in rats. In this review, the role of RAS on the myocyte protein synthesis (myocyte hypertrophy) and on the induction of gene expression will be discussed in rat cardiomyocytes in culture. The traditional RAS can be considered as a system in which circulating ANG II is delivered to target tissues or cells. However, a local RAS has also been described in cardiac cells and evidence has been accumulated for autocrine and/or paracrine pathways by which biological actions of ANG II can be mediated. These actions of ANG II are primarily mediated through ANG II receptors of the subtype I (AT1-R). When evaluating the effects of ANG II in situ, both changes in circulating levels and local production have to be taken into account. Discrepant findings on the in vitro effect of ANG II on the protein synthesis in cardiac myocytes are described and can be at least partly be attributed to methodological problems such as assay of the de novo protein synthesis, isolation and the separation procedure of cardiac myocytes. The ANG II-induced hypertrophic effect also depends on the existence of non-myocytes in a cardiocyte culture. In rat cardiocytes ANG II also causes induction of many immediately-early genes (c-fos, c-jun, jun-B, Egr-1 and c-myc) and induces also late markers of cardiac hypertrophy (skeletal alpha-actin and atrial natriuretic peptide expression) and growth factors (TGF-beta1 gene expression). In vivo ANG II via AT1-R, causes not only ventricular hypertrophy, independently of blood pressure, but also a shift to the fetal phenotype of the myocardium. Angiotensin-converting enzyme inhibitors and ANG II receptor antagonists of the subtype I not only induce the regression, but also prevent the development of cardiac hypertrophy in experimental rat models.
Journal of Molecular and Cellular Cardiology 06/1999; 31(5):949-70. · 5.17 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To evaluate the role of protein kinase C (PKC) and intracellular calcium and particularly Ca(2+)-uptake in the initiation of lymphocyte mitogenesis, the proliferation of human peripheral blood mononuclear cells (PBMC) was investigated during calcium entry blockade with nifedipine (an L-type calcium channel blocker) and mibefradil (an L- and T-type calcium channel blocker with a higher selectivity for T-type channels). The rate of [3H]-thymidine, [3H]-uridine and [3H]-leucine incorporation into control and concanavalin A-stimulated PBMC cultured for 3 days in the presence or absence of the calcium channel blockers nifedipine or mibefradil (1, 10 or 50 microM) is assayed. Nifedipine and mibefradil concentration-dependently reduced cell number and [3H]-thymidine incorporation or de novo DNA synthesis in control and concanavalin A-stimulated PBMC, as well as de novo RNA and protein synthesis. The proliferative response of nifedipine- or mibefradil-treated cells was restored by addition of phorbol-12-myristate-13-acetate (PMA), an exogenous PKC activator. Our data show that PBMC treated with the Ca2+ channel blockers nifedipine or mibefradil are still capable of proliferating in response to PMA. However, in PKC-depleted cells, the proliferative response of PBMC was suppressed.
Methods and Findings in Experimental and Clinical Pharmacology 06/1999; 21(4):253-9. · 0.93 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To evaluate the role of intracellular calcium and particularly Ca2+-uptake in the initiation of lymphocyte mitogenesis, the effect of mibefradil, which blocks both L- and T-type calcium channels with a more selective blockade of T-type channels, on the proliferation of human peripheral blood mononuclear cells (PBMCs) is compared with the effect of nifedipine, which blocks only the L-type calcium channel. The rate of [3H]thymidine incorporation into control and concanavalin A-stimulated PBMCs in the presence or absence of the calcium channel blockers mibefradil or nifedipine (1, 10, or 50 microM), and of the intracellular calcium antagonist 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (TMB-8; 1, 10, 25, or 50 microM) was assayed in the cells cultured for 3 days. The cellular cytotoxicity and the cell number in growing cultures also was determined in mibefradil- or nifedipine-treated control or stimulated cells. Restoration of the proliferative response in mibefradil- or nifedipine-treated cells was investigated by addition of exogenous interleukin-2. Interleukin-2-receptor expression in the cells was monitored by using anti-activated T-cell antigen (Tac) antibody, and the interleukin-2 production in the cell supernatants of the cultures was determined by an enzyme-amplified sensitive immunoassay. Mibefradil and nifedipine concentration-dependently reduced the cell number and the [3H]thymidine incorporation or the de novo DNA synthesis in control and concanavalin A-stimulated human PBMCs. Mibefradil exhibited a more pronounced inhibition of the proliferation of human PBMCs than did nifedipine. The inhibitory effect of mibefradil or nifedipine on DNA synthesis was dependent on the timing of treatment with the drugs. The inhibitory effect of mibefradil or nifedipine on the lymphoproliferative response was nearly abolished if the drugs were added 20 h after cell stimulation. A markedly reduced inhibitory effect was found when mibefradil or nifedipine was added 1-7 h after cell stimulation. However, regardless of time of addition, TMB-8 caused a persistent inhibition of the proliferation of human PBMCs. The inhibitory effect of mibefradil or nifedipine on the proliferation of human PBMCs is nearly abolished by addition of the calcium channel activator Bay K 8644. The proliferative response of mibefradil- or nifedipine-treated cells is restored by addition of exogenous interleukin-2. The normal expression of interleukin-2 receptors was preserved, whereas the interleukin-2 production was blocked in the presence of mibefradil or nifedipine. Our data show that mibefradil has a more pronounced inhibitory effect on the proliferation of human PBMCs than nifedipine and that this inhibitory effect on DNA synthesis is dependent on the timing of treatment with both drugs.
Journal of Cardiovascular Pharmacology 05/1999; 33(4):595-604. · 2.29 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The renin-angiotensin-aldosterone system has emerged as a potential candidate for the accumulation of collagen in cardiac fibroblasts. The traditional renin-angiotensin-aldosterone system can be considered a system in which circulating angiotensin II or aldosterone is delivered to target tissue or cells. However, an independent local renin-angiotensin system has also been described in cardiac cells and evidence has been accumulated for autocrine and/or paracrine pathways by which biological actions of angiotensin II can be mediated. These actions of angiotensin II are primarily mediated through angiotensin II receptors of the subtype I (AT1). When evaluating the effects of angiotensin II in situ, changes in circulating levels and local production both have to be taken into account. Functional angiotensin II receptors have been documented in cardiac fibroblasts although the presence of aldosterone receptors in cardiac fibroblasts is obscure, and the expression of mRNA for mineralocorticoid receptors in cardiac fibroblasts has been described. In vitro, angiotensin II increased cardiac fibroblast-mediated collagen synthesis and mRNA levels of collagen type I, type III, pro-alpha 1 (I) collagen, pro-alpha 1 (III) collagen and fibronectin, and inhibited matrix metalloproteinase I activity. The ability of angiotensin II to induce collagen synthesis and expression of collagen in cardiac fibroblasts may be mediated by an increase in transforming growth factor-beta 1 in an autocrine/paracrine fashion. The angiotensin II-stimulated secretion and expression of collagen was completely abolished by AT1 receptor antagonism, but not affected by AT2 receptor antagonism. The discordant findings that have been reported concerning the in vitro effect of aldosterone on collagen synthesis in cardiac fibroblasts can at least partly be attributed to differences in methodology such as the use of the total population or a sub-population of cardiac fibroblasts. In vivo, chronic infusion of angiotensin II or aldosterone increased the collagen volume fraction in the ventricles. Angiotensin-converting enzyme (ACE) inhibition and AT1 receptor antagonism, but not AT2 receptor antagonism, reduced collagen deposition in the myocardium in spontaneously hypertensive rats. The cardioprotective mechanism of action of ACE inhibitors can be attributed to local blockade of the formation of angiotensin II, to the degradation of bradykinin or to the release of nitric oxide and/or eicosanoids. Angiotensin-converting enzyme inhibitors also reduced collagen deposition in rat myocardium following myocardial infarction suggesting that collagen deposition may in part result from mechanisms other than through AT1 receptors. However, further research is necessary to unravel the various mechanisms involved in the action of angiotensin-converting enzyme inhibitors or of AT1 receptor antagonists on collagen deposition in the myocardium.
Methods and Findings in Experimental and Clinical Pharmacology 05/1999; 21(3):215-27. · 0.93 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A double-blind, placebo-controlled parallel study was conducted on the effect of mibefradil, both an L- and T-type Ca2+-channel blocker with a more selective blockade of T-type channels, administered once daily for 1 week to normal male subjects, on blood pressure, intracellular cationic concentrations, sodium-proton exchange rate and 3H-thymidine incorporation in peripheral blood mononuclear cells (PBMC).
After a 1-week run-in period on placebo, the subjects (n = 40) were allocated to a placebo or a mibefradil group. Placebo or 50 mg mibefradil was administered once daily in the morning for 1 week. All subjects were investigated at baseline and after 1 week of placebo or mibefradil administration. Standing or recumbent blood pressure and heart rate of subjects in the mibefradil group was decreased (P < 0.05 or less) compared with that of subjects in the placebo group.
Decreased (P < 0.001) intracellular free Ca2+ concentration and reduced (P < 0.001) 3H-thymidine incorporation in the PBMC were observed in the mibefradil-treated subjects. The intracellular sodium, potassium or magnesium concentration as well as the sodium-proton exchange rate were not changed during mibefradil administration.
The blood pressure lowering action of mibefradil in men is accompanied by a decrease in intracellular free Ca2+ concentration. Mibefradil also reduced the 3H-thymidine incorporation or de novo DNA synthesis in PBMC by modulating the calcium homeostasis.
European Journal of Clinical Pharmacology 02/1999; 54(12):911-5. · 2.85 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To evaluate the role of intracellular calcium and particularly Ca2+ uptake in the initiation of lymphocyte mitogenesis, the effect of mibefradil--which blocks both L- and T-type calcium channels with a more selective blockade of T-type channels--on the proliferation of human peripheral blood mononuclear cells (PBMC) is compared with the effect of nifedipine, which blocks only the L-type calcium channel. The rate of 3H-thymidine, 3H-uridine, and 3H-leucine incorporation into control and concanavalin A-stimulated PBMC in the presence or absence of the calcium channel blockers mibefradil or nifedipine (1, 10, or 50 micromol/L), and of the intracellular calcium antagonist TMB-8 or the calmodulin antagonist W-7 (1, 10, 25, or 50 micromol/L) was assayed in cells cultured for 3 days. The cellular cytotoxicity and the cell number in growing cultures was also determined in mibefradil- or nifedipine-treated control or stimulated cells. Mibefradil and nifedipine reduced the cell number and the 3H-thymidine, 3H-uridine, or 3H-leucine incorporation or the de novo DNA, RNA, or protein synthesis in control and concanavalin A-stimulated human PBMC in a concentration-dependent manner. Mibefradil exhibited a more pronounced inhibition than nifedipine. The inhibitory effect of mibefradil or nifedipine on DNA synthesis was dependent upon the timing of treatment with the drugs. The inhibitory effect of mibefradil or nifedipine on the lymphoproliferative response was nearly abolished if the drugs were added 20 h after cell stimulation. A markedly reduced inhibitory effect was found when mibefradil or nifedipine were added 1 to 7 h after cell stimulation. However, regardless of time of addition, TMB-8 and W-7 caused a persistent inhibition of the proliferation of human PBMC. Our data show that mibefradil had a more pronounced inhibitory effect on the proliferation of human PBMC than nifedipine and that this inhibitory effect on de novo DNA synthesis was dependent upon the timing of treatment with both drugs. Mibefradil and nifedipine also reduce RNA and protein synthesis in human PBMC. Therefore, administration of these calcium channel blockers to inhibit cellular proliferation might be most beneficial at anatomic sites where cellular proliferation is not already an active process, while being ineffective in the presence of ongoing active proliferation, as suggested by some prospective studies.
American Journal of Hypertension 01/1999; 11(12):1461-8. · 3.18 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To determine whether phosphodiesterase (PDE) is involved in the degradation of cGMP in human erythrocytes, we studied the cell cGMP content in the presence of different PDE inhibitors: zaprinast and dipyridamole, specific inhibitors of cGMP-binding, cGMP-specific PDE (cG-BPDE); vinpocetine, a specific inhibitor of Ca2+, calmodulin-dependent phosphodiesterase (CaM-PDE); an unspecific inhibitor, 3-isobutyl-1-methylxanthine (IBMX). IBMX, zaprinast, and dipyridamole at 30 microM did not affect the intracellular cGMP content. However, vinpocetine at this concentration increased the cGMP content by 102 +/- 14% (p < 0.05). The effect of vinpocetine was dose-dependent, reached the maximal level after 1 min of incubation and flattened at the same level. Ca2+ (10 microM) in the presence of the Ca(2+)-ionophore, A23187 (5 microM), decreased the cGMP content (-23% +/- 4; p < 0.05), which can be explained by the CaM-PDE activation. The Ca(2+)-induced decrease in cGMP was completely inhibited by the CaM antagonist, W-7 (100 microM). These data suggest that erythrocytes contain Ca2+, CaM-PDE.
Methods and Findings in Experimental and Clinical Pharmacology 06/1998; 20(5):387-93. · 0.93 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To determine whether protein kinase C is necessary for the calcium activation of the Na+/H+ exchange in human erythrocytes by studying activation by calcium of erythrocyte Na+/H+ exchange in control cells, in protein kinase C-depleted cells after downregulation of protein kinase C with phorbol-12-myristate-13-acetate and in cells that had been treated beforehand with phorbol-12-myristate-13-acetate with and without the calpain inhibitor E-64d.
Erythrocyte Na+/H+ exchange was measured by determining the initial rates of the influx of Na+ into Na+-depleted, acid loaded cells. The effects of various concentrations (0-1 mmol/l) of CaCl2 and the effects of 1 mmol/l CaCl2 on activation of the intracellular pH and on the external Na+ activation of Na+/H+ exchange were studied. The effects of 1 mmol/l CaCl2 on Na+/H+ exchange in control cells and cells that had been incubated beforehand with and without 1 micromol/l phorbol-12-myristate-13-acetate and with E-64d and 1 micromol/l phorbol-12-myristate-13-acetate for 1, 2, 3 and 24 h were also investigated.
Addition of Ca2+ to a concentration in the range 0-1 mmol/l in the presence of calcimycin resulted in stimulation of Na+/H+ exchange: 1 mmol/l CaCl2 increased (P< 0.001) the erythrocyte Na+/H+ exchange by 74%. Calcium increased the maximum rate for activations by intracellular pH and by external Na+ of Na+/H+ exchange, whereas it did not affect the Michaelis-Menten constants for activation by intracellular H+ and external Na+. However, calcium did not activate the Na+/H+ exchange in protein kinase C downregulated erythrocytes and administration of the calpain inhibitor E-64d could not prevent this inactivation.
Our data indicate that protein kinase C is necessary for the activation by calcium of the erythrocyte Na+/H+ exchange.
Journal of Hypertension 04/1998; 16(3):305-10. · 4.02 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To investigate the interrelationship between erythrocyte Na+/H+ exchange rate and free cytosolic Ca2+ concentration, the effect of the (non)selective protein kinase C inhibitors staurosporine, Ro 31-8220 and CGP 41251 (1 micromol/L) and of the protein kinase C activator phorbol-12-myristate-13-acetate (PMA, 1 micromol/L) was studied in vitro on these variables. PKC depleted erythrocytes were obtained after 24 h PMA down-regulation of the cells and intracellular Ca2+ clamping was obtained using quin-2 AM and fluo-3 AM. PMA increased (P < .05) the erythrocyte Na+/H+ exchange activity and this rise was accompanied by an increase in the free cytosolic Ca2+ concentration. When staurosporine and Ro 31-8220 were added to erythrocytes in suspension, a decrease in free cytosolic Ca2+ concentration was also found, whereas no significant change was observed after CGP 41251 administration. The Na+/H+ exchange rate was decreased in the 24 h PMA down-regulated erythrocytes as well as in Ca2+-clamped cells. Addition of Ca2+ in a concentration range of 0 to 1 mmol/L in the presence of calcimycin resulted (P < .001) in a stimulation of Na+/H+ exchange by 74%. Calcium increased the Vmax for cellular pHi or external Na+ activation of Na+/H+ exchange, whereas it did not affect the Km for H+(i) or external Na+ activation. However, in PKC down-regulated cells, calcium did not activate the Na+/H+ exchange in erythrocytes and the calpain inhibitor E-64d did not prevent this inactivation. Our data show a concomitant increase in free cytosolic Ca2+ concentration and Na+/H+-exchange rate upon protein kinase C activation and a corresponding decrease in both variables upon PKC inhibition, indicating a Ca2+ requirement for protein kinase C activation of Na+/H+ exchange.
American Journal of Hypertension 01/1998; 11(1 Pt 1):81-7. · 3.18 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The proliferation response of human peripheral blood mononuclear cells (PBMC) to concanavalin A (con A) was tested in a medium with or without addition of fetal calf serum (FCS) or a serum substitute. The time profile of the proliferative response of PBMC to con A was different in cells cultured in a medium supplemented with or without FCS. This different pattern occurred for the con A-induced DNA-synthesis as well as for the RNA- or protein-synthesis. The peak proliferation rate and the stimulation index of proliferation of human PBMC to con A was also higher when the cells were cultured in a medium with 10% FCS compared to cells cultured in a FCS-free medium. Substitution of the fetal calf serum by a serum substitute even induced a profound inhibition of the de novo synthesis of DNA in human PBMC. The results indicate that a lymphocyte proliferation response to con A can still be obtained in a culture medium where no FCS was added, although a weaker stimulation occurred in comparison to a culture medium with FCS. However, addition of a serum substitute caused a marked inhibition of the lymphocyte proliferation rate.
Clinica Chimica Acta 09/1997; 264(1):91-101. · 2.54 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: To determine the effects of angiotensin II (AII) (1-8) on cytosolic free calcium concentrations in the absence and in the presence of the selective angiotensin subtype 1 (AT1) receptor antagonist losartan and of the selective angiotensin subtype 2-receptor antagonist P-186 in human peripheral blood mononuclear cells (PBMC). We also assessed the effect of the AII analogues AII (2-8), AII (3-8) and AII (4-8) on the cytosolic free-calcium concentration in human PBMC.
The cytosolic free-calcium concentration was assayed in human peripheral blood mononuclear cells by measuring the fluorescence of fura-2 entrapped by these cells.
Administration of AII caused a concentration-dependent increase in the cytosolic free-calcium concentration in human peripheral blood mononuclear cells with a half-maximal increase at 5 x 10(-8) mol/l. Also administration of the heptapeptide AII (2-8) increased the intracellular free-calcium concentration in human PBMC, whereas AII (3-8) and AII (4-8) had no effect. The AII (1-8)-induced rise in cytosolic free-calcium concentration was blocked completely by losartan but not by P-186.
Our data demonstrate that the effects of AII on the cytosolic free-calcium concentration in human PBMC are AT1 receptor-mediated since they were abolished by the specific AII AT1 receptor antagonist losartan but not by the specific angiotensin subtype 2 receptor antagonist P-186.
Journal of Hypertension 09/1997; 15(8):871-6. · 4.02 Impact Factor
