Overproduction of cyclo-oxygenase-2 (COX-2) is involved in the resistance to apoptosis in vascular smooth muscle cells from diabetic patients: a link between inflammation and apoptosis.

AIMS/HYPOTHESIS: Inflammation is a common feature in cardiovascular diseases, including diabetes mellitus. In addition to the well-known inflammatory role of cyclo-oxygenase-2 (COX-2), this protein has also been implicated in apoptosis resistance in tumour cells. Vascular smooth muscle cells (VSMC) from diabetic patients are also resistant to apoptosis because of an increased abundance of B cell lymphoma 2 protein (BCL2). In this work, we investigated whether overproduction of COX-2 was involved in the resistance to apoptosis in VSMC from diabetic patients. METHODS: VSMC were obtained from internal mammary arteries from patients who had undergone coronary artery bypass graft surgery. Apoptosis was measured by DNA fragmentation, BCL2 degradation and cytochrome c release. RESULTS: Apoptosis induced by C-reactive protein in cells from non-diabetic patients was mediated by COX-2. VSMC from diabetic patients showed higher basal levels of COX-2 compared with those from non-diabetic patients. Transfection of VSMC from non-diabetic patients with a plasmid containing COX-2 (also known as PTGS2) increased basal production of COX-2 and BCL2 and mimicked the resistance to apoptosis that occurs in diabetic patients. We also found a significant correlation (R = 0.846, p = 0.016) between COX-2 and BCL2 production in arterial rings from diabetic patients measured by confocal microscopy. However, inhibition of COX-2 production by small interfering RNA proved unable to reverse BCL2 production in diabetic VSMC. CONCLUSIONS/INTERPRETATION: These results suggest a link between inflammation (COX-2) and apoptosis resistance (BCL2) in the arteries of diabetic patients. This relationship is not causative and the common production of these two proteins may be co-regulated by shared regulatory elements in diabetes.

Study of garlic extracts and fractions on cholesterol plasma levels and vascular reactivity in cholesterol-fed rats.

Garlic is known for its pharmacologic and nutritional properties. In previous studies, garlic elicited a reduction in plasma levels of lipids by inhibiting hepatic cholesterol synthesis. The aim of this study was to investigate in an in vivo model the effects of garlic extract and some fractions on cholesterol levels and vascular reactivity in cholesterol-fed rats. Rats were fed a cholesterol-enriched diet for 16 wk and were divided into 10 groups as follows: control and hypercholesterolemic diet groups, 4 groups fed frozen garlic fractions and 4 groups fed raw garlic fractions with different doses. Blood samples were obtained to analyze HDL and LDL cholesterol levels. After treatment, rats were killed. The heart, liver and kidneys were weighed; the aorta was isolated, mounted in organ chambers and vascular reactivity was tested. Plasma concentration of cholesterol was 58 mg/dL (100%) at the beginning of the study and increased to 102 mg/dL (153%; hypercholesterolemic group) at the end of the treatment. Plasma total cholesterol decreased in all groups treated with garlic; moreover, this effect was higher in rats fed raw garlic fractions and extracts. LDL decreased significantly with respect to the hypercholesterolemic group in all groups treated with garlic fractions and extracts (P: < 0.01); however, an increase in HDL was found in those treated with frozen fractions and extracts. The liver:body weight ratio decreased in all treated groups. The relaxing effect of acetylcholine (ACh) was enhanced in arteries contracted with noradrenaline (NE). These data suggest that garlic fractions could prevent diet-induced hypercholesterolemia and vascular alterations in the endothelium-dependent relaxation associated with atherosclerosis.

Growth inhibitory activity of indapamide on vascular smooth muscle cells.

Abnormal vascular smooth muscle cell proliferation has a fundamental role in the pathogenesis of vascular diseases. Indapamide is an oral diuretic antihypertensive drug effective for patients with mild or moderate essential hypertension. We now investigated the effects of indapamide on the growth of aortic vascular smooth muscle cells (A10 cell line). Indapamide inhibited cell proliferation as measured by the tetrazolium salt XTT (sodium 3'-[1-(phenylamino-carbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate) test. The increase in cell number was significantly reduced in the presence of indapamide 10(-6) and 5 x 10(-4) M (P < 0.05 n = 3 and P < 0.01, n = 3, respectively). Serum-induced DNA synthesis, determined as the incorporation of 5-bromo-2'-deoxyuridine (BrdU), was concentration-dependently inhibited by indapamide. BrdU incorporation was 47.2+/-1.6% (10% foetal calf serum). Indapamide treatment markedly prevented BrdU incorporation (37.2+/-2.1%, 29.2+/-4.8%, 15.0+/-1.8%, 8.7+/-2.1%) indapamide 10(-6), 10(-5), 5 x 10(-5) and 5 x 10(-4) M, respectively. Cell-cycle progression was also evaluated. Flow cytometry analysis of DNA content in synchronised cells revealed blocking of the serum-inducible cell-cycle progression by indapamide. This inhibition was abolished when the drug was added 2 h after serum repletion, indicating that indapamide must act at the early events of a cell cycle to be fully effective against DNA synthesis. In addition, serum-induced intracellular Ca2+ movements and also p44/p42 mitogen-activated protein kinase (MAPK) phosphorylation were studied in the presence or absence of indapamide. Indapamide 10(-5) and 5 x 10(-5) M decreased significantly cytosolic free calcium, and the p44/p42 mitogen-activated protein kinase phosphorylation (5 x 10(-5) M) stimulated by 10% foetal calf serum. In accordance with this finding, indapamide (5 x 10(-4) M) caused a 95% to 99% decrease in the early elevation of c-fos expression as evaluated by northern blot analysis of mRNA induced after serum addition. In conclusion, our results indicate that indapamide reduces vascular smooth muscle cell proliferation by a mechanism which involves a decrease in the intracellular Ca2+ movements that might link with the mitogen-activated protein kinase (MAPK) pathway, altering cell-cycle progression.

Effect of trandolapril on vascular responsiveness in cholesterol-fed rabbit-isolated arteries.

According to the World Health Organisation, cardiovascular disorders are one of the main causes of morbi/mortality in the western world. The effect of trandolapril (0.3 mg kg(-1) day(-1)), a non-sulphydryl angiotensin-converting enzyme (ACE) inhibitor, on the vascular responsiveness in aorta isolated from hypercholesterolemic rabbits was examined. Three groups of rabbits (n=30) were used: Group 0 (control group); Group 1 (hypercholesterolemic group, 0.5% (wt/wt) cholesterol-enriched diet) and Group 2 (hypercholesterolemic+trandolapril 0.3 mg kg(-1) day(-1)). After 18 weeks of treatment, the rabbits were killed and the thoracic aorta, proximal coronary and mesenteric (5th branch) arteries were isolated, cleaned off and mounted in an organ bath. Trandolapril had no significant effect on plasma cholesterol, high density lipoprotein (HDL) or low density lipoprotein (LDL). Despite the lack of effect of the drug on the above-mentioned parameters, treatment with trandolapril improved endothelium-dependent relaxation induced by acetylcholine in aortic and mesenteric rings from hypercholesterolemic rabbits treated with trandolapril. The relaxation induced by 10(-5) M acetylcholine were 65.0+/-4.0%; 24. 0+/-9.4% (P<0.01, n=10) and 51.3+/-7.0% (P<0.01, n=10) in aortic rings from Groups 0, 1 and 2, respectively, and 50.0+/-12.0%; 10. 1+/-10.0% (P<0.01, n=10); 61.0+/-9.7% (P<0.01, n=10) in small mesenteric rings from Groups 0, 1 and 2, respectively. In addition, trandolapril treatment improved the increase in serotonin-induced contraction in proximal coronary arteries with respect to the hypercholesterolemic group. On the other hand, we did not find any differences among the group in endothelium-independent relaxation induced by sodium nitroprusside. These results provide evidence that trandolapril restores endothelium-dependent relaxation in hypercholesterolemic rabbit-isolated arteries. These data suggest that trandolapril might have beneficial action in the prevention of vascular alteration involved in atherosclerosis.

Effect of somatostatin on resistance and on capacitance rabbit isolated arteries.

The effects of somatostatin, a tetradecapeptide isolated from hypothalamus extracts, were studied on the vascular reactivity of aorta and mesenteric arteries isolated from rabbits. We also investigated whether or not Ca(2+) movements were implicated in these effects. Rabbit aorta and mesenteric (fifth branch) arteries were isolated, cleaned off, and mounted in an organ bath containing Godfraind solution or physiological saline solution (PSS), respectively. Somatostatin (10(-8)-10(-4) M) produced a concentration-dependent inhibition of the contractile responses induced by high K(+) (80 mM) or noradrenaline (10(-6) M in aorta or 10(-4) M in mesenteric arteries) in both arteries studied. The inhibitory effect of somatostatin was greater in mesenteric resistance vessels (IC(50) 3.1+/-2.3x10(-5) M, and 5.2+/-4.8x10(-8) M with KCl and noradrenaline, respectively). Contractile responses produced by the addition of Ca(2+) (1-5 mM) to Ca(2+)-free high K(+) solution were also concentration dependently inhibited by somatostatin in aorta. Furthermore, somatostatin decreased noradrenaline-induced contraction attributed to intracellular Ca(2+) release in aorta, and inhibited 45Ca(2+) uptake stimulated by high K(+) or by noradrenaline. However, it did not modify 45Ca(2+) uptake in resting mesenteric resistance arteries. Taken together, these results suggest that somatostatin exerts an inhibitory effect on vascular contractions induced by some stimulating agents in different arteries isolated from rabbits, being more potent in mesenteric arteries.

L-Citrulline, the by-product of nitric oxide synthesis, decreases vascular smooth muscle cell proliferation.

Endothelium injury plays an important role in atherosclerosis. Damage to the endothelium results in vascular smooth muscle cell proliferation. Natriuretic peptides present a potent antimitogenic action, mediating their biological effects via the binding of guanylate cyclase-linked atrial natriuretic peptide (ANP) receptor and the production of cyclic GMP. In a previous study, we demonstrated that L-citrulline, the by-product of nitric oxide synthesis, could relax rabbit aortic rings by stimulating the guanylate cyclase-linked ANP receptor. In this work, we investigated the effect of L-citrulline on vascular smooth muscle cell proliferation. L-Citrulline (10(-8) M) significantly decreased rat aortic (A10 cell line) vascular smooth muscle proliferation. The percentage of inhibition exerted by L-citrulline on days 3, 5, and 7 of the proliferation curve was 20.0 +/- 0.5%, 37.5 +/- 8.3%, and 28. 5 +/- 7.2%, respectively. In addition, L-citrulline also inhibited serum-induced DNA synthesis, measured as 5-bromo-2'-deoxyuridine incorporation. 5-Bromo-2'-deoxyuridine incorporation into nuclei of vehicle-treated cells was 40.5 +/- 2.4%, whereas in L-citrulline-treated cells the percentage decreased to 36.0 +/- 4.1%, 29.1 +/- 2.0% (P <.01, n = 4), 30.5 +/- 2.4% (P <.05, n = 4), and 23.1 +/- 0.5% (P <.001, n = 4) for 10(-10), 10(-9), 10(-8), and 10(-7) M, respectively. Zaprinast, a phosphodiesterase type V inhibitor, enhanced 5-bromo-2'-deoxyuridine incorporation in serum-stimulated cells. Moreover, L-citrulline inhibition of serum-stimulated DNA synthesis was abolished by HS-142-1 (10(-5) M), an ANP receptor antagonist. In another group of experiments, L-citrulline was shown to increase intracellular cyclic GMP levels from 2.1 +/- 0.2 pmol of cGMP/mg protein to 4.1 +/- 0.1 for L-citrulline (10(-8) M) (P <.001, n = 3). These findings suggest that L-citrulline decreases vascular smooth muscle cell proliferation in the A10 cell line by acting on DNA synthesis by mechanisms that involve the ANP receptor.

Vasodilatory effect in rat aorta of eriodictyol obtained from Satureja obovata.

The vasodilator effect of eriodictyol (5,7,3',4'-tetrahydroxyflavanone), isolated previously from the medicinal plant Satureja obovata Lag., was studied in rat thoracic aorta rings. Eriodictyol relaxed in a concentration-dependent manner the noradrenaline (10(-6) M) and KCl (80 mM) induced contractions. The relaxant effect was more potent in noradrenaline precontracted preparations (IC50 = 6.11 +/- 0.2 x 10(-5) M) than in those precontracted with KCl (IC50 = 2.96 +/- 0.1 x 10(-4) M). Eriodictyol produced weakly concentration-dependent inhibition of the phasic component induced by KCl and noradrenaline while the inhibition of the tonic phase of these contractions was more pronounced. These effects were endothelium independent. In addition, eriodictyol (10(-5) and 5 x 10(-5) M) inhibited CaCl2 cumulative concentration response curves. Eriodictyol weakly inhibited the release of calcium from the sarcoplasmic reticulum and its contribution to the relaxant effect seems to be slight. We have also observed the relaxant effect of eriodictyol on phorbol-12-myristate-13-acetate (PMA) (10(-7) M) induced contractions both in normal calcium (IC50 = 4.69 +/- 0.3 x 10(-5) M) and calcium-free medium (IC50 = 3.74 +/- 0.4 x 10(-5) M). Finally we studied the effects on protein kinase C (PKC) activity. This flavonoid did not show any activity. These results suggest that the vasodilator effect of eriodictyol in rat thoracic aorta could be partially related to the inhibition of calcium influx or other enzymatic protein subsequent to activation of PKC related to the activation of contractile proteins like myosin light chain kinase (MLCK).

Study of Calcium Dobesilate in Diabetic Rats.

According to the World Health Organization (WHO) 74% of diabetic patients die of vascular complications. Previous reports have shown that endothelium-dependent relaxation of diabetic vasculature is more sensitive to free radical-induced injury. Calcium dobesilate (DOBE) has been successfully used in the treatment of diabetic retinopathy. The aims of this study were to investigate the in vivo and ex vitro effects of DOBE on both contractile and relaxing responses in isolated diabetic rat aorta. Four groups of rats were used: Wistar rats (Group 0); spontaneously diabetic rats (BB/wor rats) (Group 1); BB/wor rats treated with DOBE 50 mg/kg/day (Group 2); and BB/wor rats treated with 500 mg/kg/day (Group 3). At 180 days after the development of diabetes, the animals were killed and the thoracic aorta were isolated, cleaned off, and mounted in an organ chamber. Two groups of experiments were carried out. In the first group (in vitro), incubation with DOBE 10(-4) in aortic rings isolated from BB/wor rats decreased the contraction induced by noradrenaline (NA) 10(-6) M (1.21 +/- 0.11 g vs 0.67 +/- 0.01 g P < 0.01, n = 8 in diabetic rings with or without the presence of DOBE 10(-4) M, respectively), and this decrease was prevented by propranolol 10(-6) M (1.20 +/- 0.6 g). DOBE 10(-5) and 10(-4) M increased the endothelium-dependent relaxation induced by ACh in BB/wor rats [the maximal relaxation with ACh 10(-5) M was 50.0 +/- 5.1 vs 72.0 +/- 11.0 (p < 0.05, n = 8) and 69.0 +/- 7.8 (p < 0.05, n = 8) in BB/wor rats and after the incubation with DOBE 10(-5) and 10(-4) M, respectively], however, incubation with DOBE did not modify the endothelium-independent relaxation in these rats. In the second part of the study (ex vitro), we found an increase in the endothelium-dependent relaxation in arteries from diabetic rats treated with DOBE (Groups 2) compared with Group 1 (BB/wor rats) although we did not find any improvement in the endothelium-independent relaxation. Thus, in spontaneously diabetic rats, DOBE restored endothelium-dependent, but not independent, relaxation to normal and also decreased the contractile responses induced by NA through a mechanism that involves beta-adrenergic receptors.

In vitro effects of calcium dobesilate on the responsiveness of spontaneously diabetic rat aorta.

We tested the effect of calcium dobesilate (DOBE) in aorta from spontaneously diabetic (BB/wor) rats. The contraction induced by 10(-6) M noradrenaline (NA) in BB/wor rats was smaller than that induced in control rats (1.21+/-0.11 vs 0.82+/-0.02 g, P<0.01, n=8, respectively) in arteries with intact endothelium. Incubation with DOBE (10(-4) M) impaired the contractions induced by NA in BB/wor rats (1.21+/-0.11 vs 0.67+/-0.01 g, P<0.01, n=8). The effect of DOBE was reversed by 10(-6) M propranolol (0.67+/-0.01 vs 1.20+/-0.60g, P<0.001, n=8, with 10(-4) M DOBE and 10(-4)M DOBE plus 10(-6) M propranolol, respectively). DOBE increased the endothelium-dependent relaxation in arteries from diabetic rats. These findings suggest that DOBE might improve vascular reactivity in BB/wor rats.

Calcium dobesilate increases endothelium-dependent relaxation in endothelium-injured rabbit aorta.

Calcium dobesilate (DOBE) is an orally administered angioprotective agent which is used in some vascular diseases such as diabetic retinopathy, although its mechanism of action is not yet fully understood. The aim of this work was to correlate previous rising single quote, left (low)in vitro' findings carried out in our laboratory with an rising single quote, left (low)ex vivo' model of endothelium-injury by overdose of vitamin D2. Male New Zealand White rabbits were used. The study was divided into two protocols. Protocol 1: 10 days of treatment; and Protocol 2: 30 days of treatment. Rabbits in each group were treated with vitamin D2 (200"000 IU day-1) for the first 2 days and two groups were subsequently treated with DOBE at different doses (50 mg kg-1 per day or 500 mg kg-1 per day). The concentration-response curve induced by NA (10(-8)-10(-4) M) in aorta arteries was shifted downwards in the groups treated with DOBE (in both Protocol 1 and 2), whereas only in Protocol 2 (30 days of treatment) was this curve affected in the hypervitaminic group. The endothelium-dependent relaxation induced by ACh (10(-8)-10(-5) M) decreased in the hypervitaminic groups (in both Protocol 1 and 2) but only in Protocol 2 (30 days of treatment) was the endothelium-dependent relaxation restored to normal (control, untreated group) in both DOBE-treated groups. The endothelium-independent relaxation induced by sodium nitroprusside (SNP) (10(-8)-10(-4) M) decreased in the hypervitaminic groups only in Protocol 1. We did not find differences in the DOBE-treated groups in any protocol compared with the control (untreated) group. These findings show evidence that DOBE restored endothelial functionality in endothelium-injured rabbit aorta only after 30 days of treatment. (c) 1998 The Italian Pharmacological Society.

Relaxant effects of L-citrulline in rabbit vascular smooth muscle.

1. Vascular endothelium plays a pivotal role in the control of vascular tone through the release of vasoactive factors such as EDRF (NO). 2. The aim of this study was to investigate whether the addition of exogenous L-citrulline, the byproduct of the NO-synthesis, could relax vascular smooth muscle. 3. L-citrulline relaxed both endothelium-denuded and endothelium-intact rabbit aortic rings precontracted with noradrenaline 10(-6) M (maximum relaxations induced by L-citrulline 10(-8) M were 74.1+/-5.2% vs 51.3+/-2.8% in endothelium-denuded and endothelium-intact arteries, respectively). 4. This relaxant effect was enhanced by zaprinast (a phosphodiesterase type 5 inhibitor) and inhibited by HS-142-1 (a particulate guanylate cyclase inhibitor) and by apamin (a K(Ca)-channel blocker). 5. L-citrulline (10(-13)-10(-8) M) increased cGMP levels in aortic rings (maximum value with L-citrulline 10(-8) M was 0.165+/-0.010 pmol cGMP mg(-1) of tissue vs 0.038+/-0.009 pmol mg(-1) of tissue in basal). 6. L-citrulline as well as NO were released from endothelial cells in culture stimulated with ACh. The values were 6.50+/-0.50 microM vs 2.30+/-0.20 microM (stimulated with ACh and basal respectively) for L-citrulline and 4.22+/-0.10 microM vs 0.87+/-0.26 microM (stimulated with ACh and basal respectively) for NO. 7. These results suggest that L-citrulline could be released together with NO from endothelium and may have actions complementary to those of NO in the control of vascular smooth muscle relaxation.

Calcium dobesilate: pharmacology and future approaches.

1. Calcium dobesilate (2,5-dihydroxybenzene sulfonate) is a drug commonly used in the treatment of diabetic retinopathy and chronic venous insufficiency. 2. The pharmacology of calcium dobesilate reveals its ability to decrease capillary permeability, as well as platelet aggregation and blood viscosity. 3. Furthermore, recent data show that calcium dobesilate increases endothelium-dependent relaxation owing to an increase in nitric oxide synthesis.

Calcium dobesilate increased endothelium-dependent relaxation in isolated rabbit aorta.

1. The aim of this study was to investigate the effects of calcium dobesilate on relaxant and contractile responses in the isolated rabbit aorta. 2. Calcium dobesilate (10(-6) and 10(-4) M) shifted the concentration-response curve induced by noradrenaline (10(-8)-10(-4) M) downward and to the right, the IC50 being 5.1 +/- 1.1 x 10(-7) M in the control and 7.5 +/- 1.2 x 10(-6) M and 3.1 +/- 1.9 x 10(-6) M in the presence of calcium dobesilate, 10(-6) M and 10(-4) M respectively. 3. Calcium dobesilate, 10(-5) M, increased the endothelium-dependent relaxation induced by acetylcholine (10(-8)-10(-5) M) but had no actions in the absence of endothelium.

Effects of calcium dobesilate on the synthesis of endothelium-dependent relaxing factors in rabbit isolated aorta.

1. Some cardiovascular disturbances which occur in diabetics are a consequence of alterations in vascular contractility as well as in endothelium-dependent relaxation. 2. Calcium dobesilate (DOBE) is a drug used in diabetic retinopathy and its mechanism of action is not yet understood. 3. The aim of this study was to investigate the effects of DOBE on synthesis and release of endothelium-dependent relaxing factor (EDRF) and endothelium-dependent hyperpolarizing factor (EDHF) in rabbit isolated aorta. 4. Endothelium-dependent relaxation induced by acetylcholine (ACh) (10(-8)-(10(-5) M) increased in the presence of DOBE 10(-5) M only when vascular endothelium was kept intact. 5. NG-nitro-L-arginine methyl ester (L-NAME; 10(-8)-10(-4) M progressively decreased the enhancing effect of DOBE on endothelium-dependent relaxation whereas it was progressively increased by L-Arg. 6. DOBE 10(-5) M increased in a non-significant manner endothelium-dependent relaxation induced by ACh when the arteries were incubated with both L-NAME 10(-4) M and indomethacin 10(-5) M. 7. DOBE (10(-6) M and 10(-5) M) was able to scavenge superoxide anion radicals generated by the hypoxanthine/xanthine oxidase reaction. 8. These results provide evidence that DOBE is able to affect the vascular disorders associated with diabetes mellitus since it enhances the synthesis of endothelium-dependent relaxing factors.

Action of elgodipine on atherosclerosis development, cell growth, and oncogene expression in vascular smooth muscle cells.

1. Elgodipine (ELG) had no significant effect on aortic content of cholesterol, plasma cholesterol, or triglycerides concentrations in cholesterol-fed rabbits. However, ELG administration produced a dose-dependent and significant reduction of calcium content of the aorta. 2. In vitro studies showed that ELG was able to inhibit vascular smooth muscle proliferation through a mechanism independent of the expression of the transcription factors c-fos and c-jun.

Action of probucol in arteries from normal and hypercholesterolaemic rabbits.

1. The effect of probucol on the vascular reactivity of different arteries isolated from rabbits was studied as well as its effects on the development of atherosclerosis in a cholesterol-fed rabbit model. 2. Probucol 10(-6)-5 x 10(-4)M produced a concentration-dependent inhibition of the contractile responses induced by KCI (80 mM), the sequence for the IC50 was: mesenteric artery (5th branch, 4.8 +/- 2.6 x 10(-5) M) > aorta (8.2 +/- 2.3 x 10(-5) M) > femoral artery (> 5 x 10(-4) M). The response to noradrenaline was: mesenteric artery (5th branch, 4.2 +/- 1.3 x 10(-5) M) > aorta (3.2 +/- 3.0 x 10(-4) M) > femoral (> 5 x 10(-4) M). 3. In the aorta, probucol (10(-5)-10(-4) M) shifted the concentration-response curves to Ca2+ downward and to the right. 4. Probucol at 5 x 10(-5) M and 5 x 10(-4) M showed a reduction in the 45Ca2+ uptake in resting, non-stimulated aortic rings as well as the uptake induced by both noradrenaline 10(-6) M and KCI 80 mM. 5. In experiments in vivo, probucol did not affect lipid profiles; however, drug-treatment significantly decreased the cholesterol content of aortic tissue and the extent of intimal surface covered with atherosclerotic lesions. 6. The vascular reactivity was recovered in femoral arteries from rabbits on the atherogenic diet plus probucol. 7. It is concluded that the effect of probucol in vascular smooth muscle can be attributed to an inhibition of Ca2+ entry through both potential- and receptor-operated pathways. Moreover our findings suggest that the effects of probucol on movement of calcium in vascular smooth muscle may play an important role in the mechanism of antiatherogenic properties of this drug.

Comparative study of elgodipine and nisoldipine on the contractile responses of various isolated blood vessels.

The effects of elgodipine, a new dihydropyridine derivative, were compared to those of nisoldipine on contractile responses in various isolated artery rings and on mechanical activity in portal vein segments. Arteries used were: rabbit aorta, mesenteric (fifth branch), femoral and basilar, and sheep coronary arteries. Elgodipine and nisoldipine (10(-16)-3 x 10(-6) M) produced a concentration-dependent inhibition of the contractile responses induced by high K+ (80 mM), 5-hydroxytryptamine (10(-5) M) or noradrenaline (10(-6) M or 10(-4) M) in all the arteries studied. The inhibitory effect of elgodipine was greater in mesenteric resistance vessels (IC50 = 8.0 +/- 2.1 x 10(-12) M and 2.0 +/- 0.5 x 10(-13) M for the depression of high K(+)- and agonist-induced contraction, respectively), and in coronary arteries (IC50 = 2.6 +/- 0.3 x 10(-10) M and 9.0 +/- 1.4 x 10(-8) M for the inhibition of high K(+)- and agonist-induced contraction, respectively). In addition, the action of elgodipine in peripheral resistance vessels and in the coronary artery was more prominent than in aorta or femoral arteries, and this tissue selectivity was more apparent for elgodipine than for nisoldipine. In rat portal vein elgodipine (IC50 = 6.5 +/- 0.9 x 10(-8) M) and nisoldipine (IC50 = 8.5 +/- 1.3 x 10(-8) M) reduced in a concentration-dependent manner the development of mechanical activity. Furthermore, contractile responses produced by the addition of Ca2+ (1-5 mM) to Ca(2+)-free high K+ solution were also concentration dependently inhibited by elgodipine. However, elgodipine did not modify noradrenaline-induced contractions attributed to intracellular Ca2+ release. The results of this study indicate that elgodipine has potent vasodilator properties and vascular selectivity. The mechanisms through which elgodipine relaxes vascular smooth fibres seem to be related to its ability to inhibit the entry of extracellular Ca2+ into the cell.

Effects of indapamide on atherosclerosis development in cholesterol-fed rabbits.

We examined the effect of indapamide (IND) on the development of atherosclerosis lesions in rabbits maintained on a 1% (wt/wt) cholesterol-enriched diet for a 16-week period. IND was supplemented to the diet at three different levels to correspond to doses of 0.1, 0.3, and 1 mg/kg/day. Throughout the treatment, dietary consumption and body weight gains were comparable among the experimental and control groups. IND had no significant effect on plasma cholesterol, triglycerides, or phospholipids concentrations. Despite the lack of effect of the drug on these parameters, its administration produced a tendency toward a reduction in the aortic content of cholesterol and a dose-dependent and significant decrease in aortic damage. In the aortic arch, the extent of intimal aortic surface covered by grossly discernible atherosclerotic lesions was decreased by IND 1 mg/kg/day from 11.02 +/- 1.10 to 6.00 +/- 1.00% (p < 0.05) and from 9.72 +/- 1.39 to 5.37 +/- 1.20% (p < 0.05) in the remaining thoracic sections. In addition, the former dose also reduced the number of lesions per square centimeter from 3.69 +/- 0.68 to 1.72 +/- 0.53% (p < 0.05), and from 3.37 +/- 0.85 to 1.55 +/- 0.46% (p < 0.05) in aortic arch and in thoracic sections, respectively. The possibility that IND reduces the development of atherosclerotic lesions produced by diet-induced hypercholesterolemia through a mechanism involving its calcium antagonist and/or its antioxidant activity is discussed.

Effects of indapamide on contractile responses and 45Ca2+ movements in various isolated blood vessels.

The effects of indapamide on contractile responses in various isolated artery rings and on spontaneous mechanical activity in portal vein segments were investigated. Arteries used were: rabbit aorta, mesenteric (fifth branch), femoral and basilar, and sheep coronary arteries. 45Ca2+ uptake was also analysed in rabbit mesenteric arteries. Indapamide (10(-10)-3 x 10(-4) M) produced a concentration-dependent inhibition of the contractile responses induced by high K+ (80 mM), 5-hydroxytryptamine (10(-5) M), and noradrenaline (10(-6) or 10(-4) M) in all the arteries studied. The inhibitory effect was greater in mesenteric (fifth branch) the IC50 values being 9.2 +/- 3.0 x 10(-6) and 5.5 +/- 4.0 x 10(-8) M for depression of high K(+)- and agonist-induced contractions, respectively. Indapamide inhibited in a concentration-dependent manner the contractile responses elicited by the addition of Ca2+ (1-5 mM) to Ca(2+)-free high K+ solution as well as the spontaneous mechanical activity of rat portal vein. Indapamide also reduced the 45Ca2+ uptake in rabbit mesenteric arteries stimulated by noradrenaline (10(-4) M) or by high K+ (80 mM) without affecting the Ca2+ influx in resting tissues. Our results indicate that indapamide blocked both depolarization-and noradrenaline-induced Ca2+ influx while it did not modify passive Ca2+ entry. Peripheral resistance vessels were demonstrated to be the arteries most sensitive to indapamide vascular effects.

Effects of dotarizine on 45Ca2+ movements and contractile responses in vascular smooth muscle.

The inhibitory effects of dotarizine on 45Ca2+ movements and contractile responses were studied and compared, using the same parameters measured in rabbit aorta and basilar smooth muscle. Dotarizine 10(-9)-10(-5) M inhibited the contractile responses induced by high K+ (80 mM), noradrenaline (10(-6) M) or 5-hydroxytryptamine (5-HT, 10(-5) M). These effects were observed when dotarizine was added before or after the induced contractions and were more potent in basilar arteries than in aorta. Moreover, dotarizine at concentrations less than 10(-6) M did not modify the contractile response obtained in aorta rings. Contractile responses induced by the addition of Ca2+ to Ca(2+)-free high-K+ solution were also concentration dependently inhibited by dotarizine 10(-7)-10(-6) M in aorta and basilar arteries. Dotarizine also inhibited the contractile response induced by caffeine (20 mM) in aortic rings incubated in normal or in Ca(2+)-free medium. Dotarizine reduced the 45Ca(2+) uptake stimulated by high K+, noradrenaline or 5-HT even in the aorta or basilar artery, but the inhibition was greater in basilar arteries than in aorta. These results suggest that, in rabbit, dotarizine inhibits Ca2+ entry through Ca2+ channels, being more selective for the basilar artery, probably by acting on multiple sites to decrease the availability of intracellular free Ca2+ required for activation.