Effects of dihydropyridines on tension and calcium-45 influx in isolated mesenteric resistance vessels from spontaneously hypertensive and normotensive rats

Contractile tension responses to norepinephrine and depolarizing potassium (80 mM K+), as well as calcium-45 influx stimulated by these agents, were studied in isolated mesenteric resistance vessels (each 100 microM internal diameter) from spontaneously hypertensive rats (SHRs) and from normotensive Wistar Kyoto rats (WKYs). Inhibitory effects of 2 dihydropyridine Ca++ antagonists, PN 200-110 (isradipine) and nisoldipine, on these parameters were also determined. Contractile responses to 80 mM K+ were inhibited by both Ca++ antagonists with the same potency and efficacy in SHR compared with WKY vessels (PN 200-110 IC50 = 2.8 +/- 1.3 X 10(-8) M in SHRs and 2.5 +/- 1.5 X 10(-8) M in WKYs; nisoldipine IC50 = 1.1 +/- 0.4 X 10(-8) M in SHRs and 1.2 +/- 0.9 X 10(-8) M in WKYs). However, contractile responses to norepinephrine (10(-4) M) were inhibited less potently by nisoldipine in SHR vessels (IC50 = 2.2 +/- 0.3 X 10(-9) M) compared with WKY vessels (IC50 = 1.6 +/- 0.6 X 10(-10) M). Similarly, PN 200-110 tended to be less (but not significantly less) potent in SHR vessels (IC50 = 3.3 +/- 1.8 X 10(-8) M) than in WKY vessels (IC50 = 3.4 +/- 0.9 X 10(-9) M); its efficacy was significantly depressed in the SHR vessels (by approximately 20%). When norepinephrine-stimulated calcium-45 influx was determined in the presence of these Ca++ antagonists, a similar profile emerged with respect to a comparison of SHR and WKY vessels. These results support a previously hypothesized alteration in receptor-activated Ca++ influx pathways in SHR mesenteric resistance vessels.     

Effect of calcium antagonists on aortae isolated from normotensive and hypertensive rats: relaxation and calcium influx blockade

Verapamil and diltiazem were equally potent (ie, similar EC50s) in relaxing potassium-contracted aortas of spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. The mechanical EC50s produced approximately 50% calcium influx blockade, suggesting a causal link between relaxation and calcium influx blockade. Nitrendipine was about 250 times more potent in relaxing aortic smooth muscle in SHR than in WKY rats (EC50s in -log [M] were 14.10 +/- 0.30 and 11.70 +/- 0.54, respectively). This difference was not affected by endothelial denudation, and was present when nitrendipine was used by preincubation rather than during established potassium chloride contractions. In spite of the different relaxant potency of nitrendipine in SHR and WKY rats, both strains showed similar EC50s for calcium influx blockade for this compound (9.21 +/- 0.36 in SHR and 8.75 +/- 0.26 in WKY). The dissociation between aortic smooth muscle relaxation and calcium influx blockade after nitrendipine was more pronounced in the SHR strain. This suggests that mechanisms other than or in addition to calcium influx blockade play a role in the relaxation of potassium-contracted vascular smooth muscle with dihydropyridine compounds, but not with other calcium antagonists.     

Ca2+ buffering function of the sarcoplasmic reticulum is increased in the carotid artery from spontaneously hypertensive rats.

To clarify whether the Ca2+ uptake function of the sarcoplasmic reticulum (SR) during arterial contraction is altered in hypertension, the effects of cyclopiazonic acid (CPA) and thapsigargin, which inhibit SR Ca2+-ATPase, on the contractile responses to Bay k 8644, an agonist of L-type Ca2+ channels, were compared in endothelium-denuded strips of carotid arteries from 13-week-old spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). The addition of Bay k 8644 (1-300 nM) to the strips caused a concentration-dependent contraction that was larger in SHR than in WKY. The contractile responses to Bay k 8644 were augmented by CPA (10 microM) or thapsigargin (100 nM) in both strains. This augmentation was greater in SHR. Each of CPA and thapsigargin induced a relatively transient contraction, and both of these contractions were larger in SHR than in WKY. The basal 45Ca influx in this artery was larger in SHR than in WKY. The addition of caffeine (1-20 mM) caused a transient contraction that was larger in SHR than in WKY. Our results indicate that 1) the large Ca2+ influx during rest in the SHR carotid artery is strongly buffered by Ca2+ uptake into the superficial SR; and 2) the Ca2+ uptake function of the SR during the contraction with Bay k 8644 was increased in SHR compared with WKY. We conclude that the SHR carotid artery has an increased total capacity of SR for Ca2+ storage as an attempt to compensate for the large Ca2+ influx.     

Increased Ca2+ buffering function of sarcoplasmic reticulum in small mesenteric arteries from spontaneously hypertensive rats.

We compared the Ca2+ buffering function of the superficial sarcoplasmic reticulum (SR) during rest and during contraction in endothelium-denuded strips of small mesenteric arteries from 13-week-old spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). The addition of caffeine (1-20 mM) caused a transient contraction in both strains, and the contraction was significantly larger in SHR. When the SR Ca2+ buffering function was eliminated by cyclopiazonic acid (CPA; 10 microM) or thapsigargin (100 nM), both of which inhibit SR Ca2+-ATPase, or by ryanodine (10 microM), which depletes the SR Ca2+, there was a larger contraction in SHR than in WKY, suggesting that the Ca2+ buffering function of the SR during rest is more important in SHR than in WKY. Judging from the augmenting effects of these three agents on the contractile responses to Bay k 8644 (1-300 nM), an agonist of L-type Ca2+ channels, or norepinephrine (10(-9)-10(-4) M), an alpha-adrenoceptor agonist, the effects were significantly greater in SHR than in WKY. We conclude that 1) the Ca2+ influx during rest and during stimulation with Bay k 8644 or norepinephrine is strongly buffered by Ca2+ uptake into the superficial SR in the small mesenteric arteries from SHR and WKY; and 2) these Ca2+ buffering functions are increased in SHR because of the larger capacity of SR for Ca2+ storage.     

Reduced calcium sensitivity of dihydropyridine binding to calcium channels in spontaneously hypertensive rats.

To explore the role of calcium channels in hypertension, dihydropyridine ([3H]PN200-110) binding to heart, brain, and skeletal muscle microsomes of 4-, 8- and 15-week-old spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats was measured. At a constant Ca2+ ion concentration (pCa 3.0), maximal binding (Bmax) of dihydropyridine binding to heart and brain microsomes was significantly enhanced in 8- and 15-week-old SHR compared with WKY rats (p less than 0.01), whereas this phenomenon was not observed in 4-week-old SHR and WKY rats. Bmax and dissociation constant (Kd) values for skeletal muscle microsomes from SHR showed no difference compared with WKY rats irrespective of age. Dihydropyridine binding to heart microsomes, brain microsomes, and solubilized skeletal muscle microsomes exhibited strong calcium dependence. The Ca2(+)-dependent dihydropyridine binding curves for heart showed a Hill slope, and pK 0.5 values for 15-week-old SHR and WKY rats were 0.70 +/- 0.12 and 4.66 +/- 0.12 versus 0.72 +/- 0.12 and 5.66 +/- 0.08 (n = 4, mean +/- SD), respectively, indicating that 15-week-old SHR require 10-fold higher calcium concentration than WKY rats to promote dihydropyridine binding. The pK 0.5 values of calcium for brain and solubilized skeletal muscle calcium channels in 15-week-old SHR were also significantly lower than in WKY rats. This difference first became apparent in SHR and WKY rats as early as 4 and 8 weeks after birth. These results suggest that enhancement of calcium channel density might occur in the heart and brain of SHR in response to elevated blood pressure and that reduced calcium sensitivity of dihydropyridine binding to calcium channels might be a primary characteristic of this rat strain.     

Increased membrane permeability of skin fibroblasts from the spontaneously hypertensive rat

Recently, we have demonstrated several abnormalities in Na+ and K+ homeostasis in cultured vascular smooth muscle cells derived from spontaneously hypertensive rats (SHR). To study whether similar defects can be identified in other cells of this rat strain, 86Rb and 22Na flux experiments as well as measurements of intracellular Na+ and K+ levels were performed in cultured skin fibroblasts of SHR and normotensive Wistar-Kyoto rats (WKY). The efflux rate constant (ke) for Rb+ (K+ analogue) was higher (p less than 0.001) in fibroblasts of SHR than in those of WKY (2.11 +/- 0.03 and 1.66 +/- 0.02 X 10-2/min; mean +/- SEM). The ouabain-insensitive influx rate constant (ki) for Rb+ was also higher (p less than 0.001) in fibroblasts of SHR than in those of WKY (13.26 +/- 0.41 and 10.71 +/- 0.27 X 10-2/min. On the other hand, the activity of the Na+-K+ pump of cells of SHR (44.81 +/- 0.81 X 10-2/min) was not different from that of cells of WKY (44.72 +/- 0.47 X 10-2/min). This parameter was obtained by calculating the ouabain-sensitive Rb+ influx rate constant. There was also no difference in the Na+ uptake (in the presence of ouabain) between cells of the two rat strains. Although there was no statistically significant difference in the measured intracellular total K+ levels between the two groups, on the basis of equilibrium distribution of 86Rb+, we calculated a significantly lower (p less than 0.001) level of exchangeable intracellular K+ in fibroblasts of SHR (98.2 +/- 1.2 mEq/L) as compared with cells of WKY (115.3 +/- 1.5 mEq/L). These findings indicate increased membrane permeability to K+ in fibroblasts of SHR and that this defect is likely to be innate to their membrane structure.     

Mitogen-activated protein/extracellular signal-regulated kinase inhibition attenuates angiotensin II-mediated signaling and contraction in spontaneously hypertensive rat vascular smooth muscle cells

This study investigates the role of extracellular signal-regulated kinases (ERKs) in angiotensin II (Ang II)-generated intracellular second messengers (cytosolic free Ca2+ concentration, ie, [Ca2+]i, and pHi) and in contraction in isolated vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) and control Wistar Kyoto rats (WKY) using the selective mitogen-activated protein (MAP)/ERK inhibitor, PD98059. VSMCs from mesenteric arteries were cultured on Matrigel basement membrane matrix. These cells, which exhibit a contractile phenotype, were used to measure [Ca2+]i, pHi, and contractile responses to Ang II (10(-12) to 10(-6) mol/L) in the absence and presence of PD98059 (10(-5) mol/L). [Ca2+]i and pHi were measured by fura-2 and BCECF methodology, respectively, and contraction was determined by photomicroscopy. Ang II-stimulated ERK activity was measured by Western blot analysis using a phospho-specific ERK-1/ERK-2 antibody and by an MAPK enzyme assay. Ang II increased [Ca2+]i and pHi and contracted cells in a dose-dependent manner. Maximum Ang II-elicited contraction was greater (P<0.05) in SHR (41.9+/-5.1% reduction in cell length relative to basal length) than in WKY (28.1+/-3.0% reduction in cell length relative to basal length). Basal [Ca2+]i, but not basal pHi, was higher in SHR compared with WKY. [Ca2+]i and pHi effects of Ang II were enhanced (P<0.05) in SHR compared with WKY (maximum Ang II-induced response [Emax] of [Ca2+]i, 576+/-24 versus 413+/-43 nmol/L; Emax of pHi, 7.33+/-0.01 versus 7.27+/-0.03, SHR versus WKY). PD98059 decreased the magnitude of contraction and attenuated the augmented Ang II-elicited contractile responses in SHR (Emax,19. 3+/-3% reduction in cell length relative to basal length). Ang II-stimulated [Ca2+]i (Emax, 294+/-55 nmol/L) and pHi (Emax, 7. 27+/-0.04) effects were significantly reduced by PD98059 in SHR. Ang II-induced ERK activity was significantly greater (P<0.05) in SHR than in WKY. In conclusion, Ang II-stimulated signal transduction and associated VSMC contraction are enhanced in SHR. MAP/ERK inhibition abrogated sustained contraction and normalized Ang II effects in SHR. These data suggest that ERK-dependent signaling pathways influence contraction and that they play a role in vascular hyperresponsiveness in SHR.     

Hyperreactivity of platelets from spontaneously hypertensive rats. Role of external magnesium

Thrombin-induced serotonin secretion from platelets from age-matched spontaneously hypertensive rats (SHR) and control Wistar-Kyoto rats (WKY) was compared in the presence of different Ca2+ and Mg2+ concentrations. Platelets from SHR were more reactive than those of WKY, and the difference was more marked in 11-week-old than in younger rats. The responses to three concentrations of extracellular Ca2+ and one extracellular Mg2+ concentration of 10(-3) M were compared. A high external Ca2+ concentration (2 X 10(-3) M) increased secretion in platelets of both strains without suppressing the difference between them. Platelets from SHR were more sensitive than those from WKY to a low external Ca2+ concentration (2 X 10(-6) M). Platelet secretion which is independent of external Ca2+ concentration was higher in platelets from SHR than in those from WKY. External Mg2+ exerted an inhibitory effect on serotonin secretion in both types of platelets, but platelets from SHR were less sensitive to Mg2+ than were those from WKY. This inhibitory effect appeared to be complex. It could be observed in the absence of external Ca2+, and in this case, the difference in reactivity between platelets SHR and WKY depended on the external Mg2+ concentration (up to 2 X 10(-3) M). Furthermore, a Mg2+ -induced antagonism of the stimulatory effect of external Ca2+ concentration appeared at higher concentrations of extracellular Mg2+ and was more potent in platelets from WKY than in those from SHR.(ABSTRACT TRUNCATED AT 250 WORDS)     

Active calcium and sodium transport by cardiac plasma membranes in the genetically hypertensive rat

Active Na+ and Ca2+ transports by sarcolemmal vesicles from young spontaneously hypertensive rats (SHR) and their normotensive controls (WKY) were compared. The effects of the calmodulin and the calcium antagonist nifedipine on Ca2+ binding ATP-dependent accumulation of Ca2+ were studied at free Ca2+ concentrations of 2.10(-8)M and 4.10(-7)M. 2.10(-7)M calmodulin stimulated Ca2+ binding to SHR membranes up to a level equivalent to that in WKY, whereas it enhanced active Ca2+ transport more in WKY than in SHR, thus suppressing the difference between the two substrains. At a 2.10(-8)M free Ca2+ concentration low concentrations of nifedipine (10(-7) to 10(-6)M) induced an increases in ATP-dependent Ca2+ transport by SHR vesicles. Inhibition of NA+, K+-adenosine triphosphatase activity by ouabain was also studied. Na+, K+ATPase activity in SHR membranes was double that in membranes from WKY (22.1 +/- 2.8 v.s. 11.3 +/- 1.1. mumole Pi/h/mg protein). These differences, observed on 3 week-old rats, before a significant rise blood pressure, may reflect genetic characteristics of these hypertensive-prone rats.     

Alterations of calcium channels in vascular smooth muscle cells from spontaneously hypertensive rats.

We examined the possible alterations in calcium handling through the calcium channels of spontaneously hypertensive rats (SHR) using 45Ca2+ uptake measurements in cultured aortic cells. Primary cultures of vascular smooth muscle cells (VSMC) were obtained by enzymatic dissociation of the thoracic aortas from 8-week-old SHR and age-matched Wistar-Kyoto rats (WKY). The functions of voltage sensitive calcium channels (VSCC) and receptor operated calcium channels (ROCC) were estimated from the activated 45Ca2+ uptake in VSMC with high K+ depolarization and arginine vasopressin (AVP), respectively. Compared to basal conditions, depolarization with 55 mM KCl increased 45Ca2+ uptake at 20 min by 94 +/- 17 (SE) % in SHR and 38 +/- 6% in WKY. The activated 45Ca2+ uptake was significantly greater in SHR than in WKY (p < 0.01). There was no significant difference in 45Ca2+ uptake at 20 min in the presence of 5 x 10(-8)M AVP between SHR and WKY. These results suggest that calcium uptake, at least through VSCC, is increased in VSMC of SHR. This enhanced activity may be implicated in the hypertensive mechanisms in this model of hypertension.     

Abnormal platelet and lymphocyte calcium handling in prehypertensive rats

We have reported that the basal and stimulated cytosolic free calcium concentrations [( Ca2+]i) are elevated in platelets isolated from 12-14-week-old spontaneously hypertensive rats (SHR) as compared with normotensive Wistar-Kyoto (WKY) rats. To determine whether altered cell calcium metabolism precedes the development of overt hypertension, we measured [Ca2+]i under resting and stimulated conditions in blood platelets and thymic lymphocytes isolated from 4-week-old prehypertensive SHR and WKY rats. Blood pressure was similar in both groups (SHR 95 +/- 8 versus WKY rats 92 +/- 7 mm Hg). Basal [Ca2+]i in platelets was higher in SHR than WKY rats (63.4 +/- 3.9 versus 54.8 +/- 3.1 nM, p less than 0.003). Also the [Ca2+]i response to thrombin was greater in SHR than WKY rats in both the presence and absence of extracellular calcium. For lymphocytes, although no difference was detected in basal [Ca2+]i, the concanavalin A-induced peak [Ca2+]i was higher for SHR than WKY rats in both calcium-containing and calcium-free media. These results suggest that agonist-stimulated calcium influx and calcium discharge from intracellular stores are enhanced in both platelets and lymphocytes of 4-week-old SHR. We conclude that abnormalities in calcium metabolism in two different cell types precede the development of overt hypertension in the SHR.     

Effect of atrial natriuretic factor on calcium fluxes in adrenal glomerulosa cells

We studied the effect of atrial natriuretic factor (ANF) on calcium influx and efflux in rat adrenal glomerulosa cells stimulated by angiotensin II (Ang II) or potassium ion, and observed how ANF inhibits the initial and sustained phases of the aldosterone response to Ang II or K+ using a superfusion system of dispersed adrenal glomerulosa cells. K+ (8 mM) significantly increased Ca2+ influx rate compared with basal rate (0.91 +/- 0.10 vs 0.42 +/- 0.04 nmol/min/10(6) cells; p less than 0.01). ANF (10(-8) M) did not inhibit the K+-induced increase in Ca2+ influx rate (0.99 +/- 0.18 nmol/min/10(6) cells). Ang II (10(-9) and 10(-8) M) stimulated Ca2+ influx rate (10(-9) M Ang II, 0.62 +/- 0.02; 10(-8) M Ang II, 0.71 +/- 0.09 vs basal, 0.44 +/- 0.03 nmol/min/10(6) cells; p less than 0.05), while ANF (10(-8) M) did not change the Ca2+ influx rate increased by Ang II (ANF + 10(-9) M Ang II, 0.62 +/- 0.06; ANF + 10(-8) M Ang II, 0.69 +/- 0.14 nmol/min/10(6) cells). In the Ca2+ efflux study ANF (10(-8) M) was perfused through the cells 10 minutes before the start of perfusion with Ang II (10(-9) M) or K+ (12 mM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of antihypertensive agents on proliferation of vascular smooth muscle cells from spontaneously hypertensive rats.

We have previously shown that Ca-antagonists and alpha-blockers substantially inhibit the cellular proliferation of cultured rat vascular smooth muscle cells (VSMC). This study explored whether these inhibitory effects on cellular proliferation differ between cultured VSMC from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). SHR VSMC proliferated much faster than WKY VSMC in 10% FCS. Cellular proliferation, determined by both cell number count and 3H-thymidine incorporation, was significantly blunted in the presence of either nifedipine (Nif) or bunazosin (Bun). The magnitude of these inhibitory effects was more pronounced for SHR cells than WKY cells (% reduction of 3H-thymidine uptake with Nif: 62.1 +/- 7.8% for SHR vs 75.3 +/- 10.2% for WKY, n = 6, p less than 0.05, and with Bun: 70.2 +/- 7.8% for SHR vs 82.1 +/- 9.9% for WKY, n = 6, p less than 0.05). In contrast, the intracellular water volume was unaffected by these antihypertensive agents based on equilibrium distribution of 3-O-methyl-D-glucose14C. It is concluded that SHR VSMC grow much faster than WKY VSMC and that this abnormality is innate to the SHR cells. It is also concluded that both Ca-antagonists and alpha-blockers exerted a substantial inhibitory effect on cellular proliferation of the cultured VSMC of either SHR or WKY. Furthermore, the greater inhibition of proliferation in the SHR VSMC suggests that Ca mediated- and/or alpha-receptor mediated processes of cellular proliferation of SHR could differ from that of WKY and that these abnormalities may contribute to the hyperproliferative changes of VSMC in this model.     

Calcium buffering of resting, voltage-dependent Ca2+ influx by sarcoplasmic reticulum in femoral arteries from spontaneously hypertensive rats at prehypertensive stage.

We examined the Ca2+-buffering function of the sarcoplasmic reticulum (SR) in the resting state of arteries from spontaneously hypertensive rats (SHR) at a prehypertensive stage. Differences in the effects of cyclopiazonic acid (CPA) and thapsigargin, agents that inhibit SR Ca2+-ATPase, and of ryanodine, which depletes SR Ca2+, on tension and cellular Ca2+ level were assessed in endothelium-denuded strips of femoral arteries from 4-week-old SHR and normotensive Wistar-Kyoto rats (WKY). Addition of CPA, thapsigargin or ryanodine to the resting state of the strips caused an elevation of cytosolic Ca2+ level and a contraction in both WKY and SHR. These responses were larger in SHR than in WKY. The contractions were inhibited strongly by 100 nM nifedipine or 3 microM verapamil and were abolished by Ca2+-free solution. Nifedipine, verapamil or Ca2+-free solution itself caused a relaxation from the resting state of SHR strips, but not from that of WKY strips. The resting Ca2+ influx in arteries measured by a 5-min incubation with 45Ca was significantly larger in SHR than in WKY. This influx was decreased by 10 microM CPA or 10 microM ryanodine in both WKY and SHR. These results suggest that in the resting state of the femoral artery from 4-week-old SHR, the greater part of the increased Ca2+ influx via L-type Ca2+ channels is buffered by Ca2+ uptake into the SR, while some Ca2+ reaches the myofilaments, resulting in the maintenance of resting tone.     

Cytosolic Ca2+ attenuates ANF-induced cyclic GMP response in vascular smooth muscle cells

Despite a high density of atrial natriuretic factor (ANF) receptors, cultured vascular smooth muscle cells of the spontaneously hypertensive rat (SHR) manifest a blunted cyclic GMP (cGMP) response to ANF. We explored the role of cytosolic free Ca2+ ([Ca2+]i) in the ANF-induced cGMP response of cultured aortic vascular smooth muscle cells from SHR and two normotensive rat strains: Wistar-Kyoto (WKY) and American Wistar. Exposure to 500 nmol/l A23187 in Ca2+-containing but not in Ca2+-deficient medium resulted in a decline in the ANF-induced cGMP response at maximal ANF concentration (500 nmol/l; SHR from 1004 +/- 98 to 423 +/- 67, P less than 0.001; WKY from 1791 +/- 209 to 625 +/- 90, P less than 0.001; American Wistar from 1496 +/- 125 to 559 +/- 96 fmol/10(6) cells/4 min, P less than 0.001). The same phenomenon was observed by depolarization with 50 mmol/l KCl in Ca2+-containing medium. There were no significant differences among the rat strains in basal levels of [Ca2+]i. If Ca2+ plays a role in the blunted cGMP response to ANF in vascular smooth muscle cells of the SHR, this effect may be exerted by a distinct pool of the ion in the submembrane domain which is associated with the particulate guanylate cyclase system.     

Effect of amlodipine and cilazapril treatment on platelet Ca2+ handling in spontaneously hypertensive rats.

Abnormal Ca2+ handling and enhanced aggregation response have been reported in platelets from spontaneously hypertensive rats (SHR) and patients with essential hypertension, and thought to be involved in the progression of target organ damage of hypertension. It is important to examine whether antihypertensive therapy can improve the abnormal platelet response in hypertension. We investigated the effect of antihypertensive treatment such as amlodipine and cilazapril on Ca2+ handling and aggregation response in SHR platelets. Four-week-old male SHR were divided into three groups. Each group was treated with amiodipine (A: 10 mg/kg/day), cilazapril (C: 10 mg/kg/day) or vehicle (V) for 8 weeks by gavage. At 12-week-old, platelet [Ca2+]i was measured with fura-2 in each group of SHR and age-matched Wistar-Kyoto rats (WKY) as normal control. Systolic blood pressure in amlodipine and cilazapril treated groups were similar with WKY and significantly lower than vehicle treated group (A: 124 +/- 9, C: 126 +/- 9, WKY: 122 +/- 10 and V: 180 +/- 9 mmHg, respectively). The basal [Ca2+]i in the three groups of SHR were similar and higher than WKY (A: 47 +/- 1.7, C: 47 +/- 1.2, V: 48 +/- 3.9 and WKY: 40 +/- 4.0 nmol/l, respectively). There were no significant differences in thrombin (0.1 U/ml)-stimulated [Ca2+]i rise in the presence or absence of extracellular Ca2+ among the three groups of SHR and these were higher than WKY. Intracellular Ca2+ discharge capacity, assessed by the ionomycinstimulation was similar in the all groups. Thrombin-induced maximum platelet aggregation responses in the three groups of SHR were similar and higher than WKY. The antihypertensive treatment of Ca2+ antagonist or ACE inhibitor gave no change in intraplatelet Ca2+ metabolism in SHR. These results support the hypothesis that an abnormal Ca2+ handling in SHR platelet is genetically determined and not improved by hypotensive therapy.     

Role of extracellular matrix in angiotensin II signalling in aortic smooth muscle cells: relationship with arterial hypertension

Angiotensin II (Ang II) is involved in hypertension-related arterial wall hypertrophy [1]. Regulation of AT II transduction pathway in vascular smooth muscle cells (VSMC) may involve cytoskeleton and extracellular matrix (ECM) [2]. We assessed the role of components of ECM on Cai2+ increase induced by Ang II in Wistar-Kyoto (WKY) and Spontaneously Hypertensive Rats (SHR) aortic VSMC. The effect of Ang II (1 mumol) on Ca2+ mobilization was studied in cultured VSMC isolated from the aorta of 6-wk old WKY (MAP (m +/- SE) = 98 +/- 4 mmHg) and SHR (136 +/- 5 mmHg; p < 0.05), using fluorescent imaging microscopy (Fura-2 AM). Cai2+ release from internal stores and Ca2+ influx were assessed in the absence and upon reintroduction of external Ca2+ respectively. Cells were cultured on uncoated glass coverslips (control) or coated with either collagen I (10 micrograms/mL), collagen IV (7 micrograms/mL), vitronectin (0.1 microgram/mL), fibronectin (3 micrograms/mL) and extracellular matrix extract (matrigel, 1/10) and studied at confluence. Paxillin was located in cells by indirect immunofluorescence micrography. Results are expressed in % of Control. Statistical significance (p < 0.05) was assessed with Student's t-test for unpaired data. The effects on Ang II-induced Ca2+ mobilization of growing cells on ECM are in Table. Paxillin in Control cells appeared as dots at the cell boundaries. Density increased in cells grown on collagen I with a diffuse distribution in the WKY cells. On matrigel, paxillin was located in a belt-like fashion at the periphery of the cell. These effects were not linked to differences in cell cycle (flux cytometry).     

Effects of nitroglycerin and nifedipine on coronary and systemic hemodynamics during transient coronary artery occlusion

Nitroglycerin (NTG) and nifedipine (NIF) have the potential to augment coronary blood flow in addition to reducing peripheral determinants of myocardial oxygen demand as a synergistic protective mechanism during ischemia. To examine these effects, systemic and coronary hemodynamic responses were measured continuously before and during brief periods of myocardial ischemia induced by left anterior descending coronary balloon occlusion in 26 patients undergoing angioplasty (PTCA). Data were compared for two matched occlusion periods, one control and one "drug" occlusion. In 17 patients (NTG group), 200 micrograms of intracoronary NTG was given immediately before coronary occlusion. In nine patients (NIF group), 10 mg of sublingual NIF was given 15 minutes before the "drug" occlusion. NTG significantly but transiently reduced mean arterial pressure (91 +/- 11 to 82 +/- 15 mm Hg, p less than 0.05) and augmented basal coronary blood flow (95 +/- 38 to 127 +/- 54 ml/min, p less than 0.05) but did not alter great vein blood flow (59 +/- 29 vs 61 +/- 29 ml/min) or coronary occlusion pressure (25 +/- 7 to 24 +/- 7 mm Hg) during ischemia. NIF significantly reduced systolic, diastolic, and mean arterial pressure (119 +/- 21 to 95 +/- 8 mm Hg, p less than 0.001) and heart rate-pressure product from control. NIF maintained basal great vein blood flow (125 +/- 41 to 106 +/- 57 ml/min) during reduced myocardial oxygen demand, but did not affect great vein blood flow (73 +/- 29 to 79 +/- 37 ml/min) or coronary occlusion pressures during ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Contractile response of aorta to alpha 1-adrenergic stimulation in Ca(2+)-free medium is reduced in spontaneous hypertension.

Vascular responses of aortic rings to alpha 1-adrenergic stimulation by phenylephrine (Phe) from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) were studied in Ca(2+)-containing medium and Ca(2+)-free medium plus 50 mumol/L EGTA. Although there was no difference in the sustained force development between SHR and WKY vessels in response to 100 mmol/L KCl or 10 mumol/L Phe in Ca(2+)-containing medium, the transient contractile response to 10 mumol/L Phe in Ca(2+)-free medium was substantially smaller in SHR compared to that in WKY. Subsequent addition of 2.5 mmol/L Ca2+ restored the sustained contractile response to a similar level in both SHR and WKY vessels. The transient contractile response to Phe in Ca(2+)-free medium containing EGTA, presumably due to the release of intracellular Ca2+, decreased progressively with preincubation time in Ca(2+)-free medium, indicating intracellular Ca2+ depletion. Such a temporal change of aortic response was more pronounced in SHR than in WKY. The subsequent response to Ca2+ repletion in the presence of Phe, on the other hand, increased progressively with Ca(2+)-depletion period and was higher in SHR than in WKY. The rate of relaxation after washout of Phe was slower in SHR aorta compared to WKY aorta. These results, together with our earlier findings, collectively suggest that the previous known deficiency in Ca2+ pumping mechanisms of vascular muscle microsomes leading to a reduced functional size of intracellular Ca2+ pool may account for the smaller contractile response of SHR aorta to alpha 1-adrenergic stimulation in Ca(2+)-free medium and the slower rate of relaxation.     

Effect of nitrate tolerance and dipyridamole on the response to SIN1 in the human isolated saphenous vein

The relaxant effects of nitroglycerin (NTG) and SIN1 on human vena saphena magna were studied in vitro. Nitrate tolerance was produced after incubation of the preparation with nitroglycerin (NTG 10 microM for 10 minutes). Vessels precontracted by serotonin (0.25 microM) and made tolerant to NTG exhibited a slight but significant shift (p less than 0.01) to the right of the dose-response curve to SIN1 (EC50 increased from 1.12 +/- 0.21 microM to 2.74 +/- 0.32 microM). The maximal relaxation was unaltered. On the contrary, there was a marked attenuation of the maximal relaxation to NTG in the nitrate-tolerant preparation (maximal relaxation decreased from 73 +/- 2% to 35 +/- 1%). Dipyridamole, a phosphodiesterase (PDe) inhibitor, significantly potentiated the responses to SIN1 on control rings (EC50 = 57.1 +/- 1.8 nM), and on NTG-tolerant rings it reversed the responsiveness to SIN1 (EC50 = 88.9 +/- 9.2 nM), which suggests that nitrate tolerance may be partially due to an increase in PDe activity. In conclusion we have demonstrated a slight cross-tolerance between SIN1 and NTG on human vena saphena magna. Nevertheless, after induction of in vitro NTG tolerance, the attenuation of responses to SIN1 is much less pronounced that the alteration of NTG relaxations.