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 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.     

Activation of the Na(+)-H+ exchanger modulates angiotensin II-stimulated Na(+)-dependent Mg2+ transport in vascular smooth muscle cells in genetic hypertension.

This study investigated the role of the Na(+)-H+ exchanger (NHE) on angiotensin II (Ang II)-induced activation of Na(+)-dependent Mg2+ transport in vascular smooth muscle cells (VSMCs) from Wistar-Kyoto rats (WKY; n=20) and spontaneously hypertensive rats (SHR; n=20). Intracellular free concentrations of Mg2+ ([Mg2+]i) and Na+ ([Na+]i) and intracellular pH (pHi) were measured with the specific fluorescent probes mag-fura 2-AM, SBFI-AM, and BCECF-AM, respectively. Na+ dependency of Mg2+ transport was assessed in Na(+)-free buffer, and the role of the NHE was determined with the highly selective NHE blocker 5-(N-methyl-N-isobutyl) amiloride (MIA). Basal [Mg2+]i was lower in SHR than WKY (0.59+/-0.01 versus 0.71+/-0.01 mmol/L, P<0.05). Basal pHi and [Na+]i were not different between the 2 groups. Ang II dose dependently increased [Na+]i and pHi and decreased [Mg2+]i. Responses were significantly greater (P<0.05) in SHR versus WKY ([Na+]i E(max)=37.5+/-1.1 versus 33.7+/-1.9 mmol/L; pHi E(max)=7.35+/-0.04 versus 7.20+/-0.01; [Mg2+]i E(min)=0. 28+/-0.09 versus 0.53+/-0.02 mmol/L, SHR versus WKY). In Na(+)-free buffer, Ang II-elicited [Mg2+]i responses were inhibited. MIA (1 micromol/L) inhibited Ang II-stimulated responses in WKY and normalized responses in SHR ([Mg2+]i E(min)=0.49+/-0.02). Ang II-stimulated activation of NHE was significantly increased (P<0.05) in SHR (0.07+/-0.002 DeltapH(i)/s) compared with WKY (0.05+/-0.004 DeltapH(i)/s). These data demonstrate that in VSMCs [Mg2+]i regulation is Na+ dependent, that activation of NHE modulates Na(+)-Mg2+ transport, and that increased activity of NHE may play a role in altered Na(+)-dependent regulation of [Mg2+]i in SHR.     

Effect of inhibition of Na, K-ATPase on cytosolic free sodium and calcium in platelets of spontaneously hypertensive rats.

Cytosolic free sodium concentrations ([Na+]i) in intact platelets of 18 spontaneously hypertensive rats (SHR) and of 18 age-matched normotensive Wistar-Kyoto rats (WKY) were measured using the sodium-sensitive fluorescent dye sodium-binding-benzofuran-isophthalate. In resting platelets [Na+]i tended to be higher in SHR compared to WKY (20.5 +/- 3.5 mmol/L v 15.1 +/- 1.9 mmol/L, mean +/- SEM), but the differences were not statistically significant. Stimulation of the Na-H-exchange by 1.0 U/mL thrombin increased [Na+]i in SHR by 22.9 +/- 4.3 mmol/L and in WKY by 35.0 +/- 5.6 mmol/L in a similar way. After inhibition of Na, K-ATPase by 1 mmol/L ouabain there was a significant rise of [Na+]i both in platelets of SHR to 38.0 +/- 5.1 mmol/L (P < .01 compared to resting platelets) and in platelets of WKY to 26.5 +/- 4.3 mmol/L (P < .01). However, no significant difference could be observed between these two groups. Using the calcium-sensitive dye fura-2, resting cytosolic free calcium concentrations ([Ca2+]i) were found to be significantly higher in platelets of SHR compared to WKY (171.9 +/- 21.5 nmol/L v 93.14 +/- 19.7 nmol/L, P < .05). After the addition of ouabain [Ca2+]i was significantly higher in SHR compared to WKY (245.5 +/- 32.6 nmol/L v 159.6 +/- 22.5 nmol/L, P < .05). The results do not support the hypothesis that altered sodium-calcium exchange causes elevated cytosolic free calcium 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)     

Cytosolic free calcium of aorta in hypertensive rats. Chronic inhibition of angiotensin converting enzyme

Cytosolic free calcium concentration ([Ca2+]i) and muscle tension were simultaneously measured in aortic tissue isolated from spontaneously hypertensive rats (SHR), normotensive Wistar-Kyoto (WKY) rats, and SHR chronically treated with a novel angiotensin converting enzyme inhibitor, CS-622. In the presence of 2.5 mM Ca2+ in the bathing solution, aortic [Ca2+]i measured with fura-2 was higher in SHR than in WKY rats, and it was almost the same in CS-622-treated SHR and untreated WKY rats. Increase of external Ca2+ concentration from zero to 2.5 mM elicited a contraction in SHR aortas but not in aortas from both CS-622-treated SHR and untreated WKY rats. When the aortas were contracted by 60 mM K+, however, [Ca2+]i as well as developed tension was similar in the three groups. CGP-28392 (10(-6) M), a Ca2+ channel activator, induced a rhythmic activity superimposed on a gradual increase of [Ca2+]i and tension in SHR aortas but not in the aortas of CS-622-treated SHR or untreated WKY rats. Nicardipine (10(-7) M) decreased the resting [Ca2+]i and the resting tone in SHR aortas, but not in WKY rat aortas. These results suggest that SHR aortas have a higher myogenic tone due to increased [Ca2+]i than WKY rat aortas and that the increased [Ca2+]i is attributed to alterations of dihydropyridine-sensitive Ca2+ channels in SHR aortas. Further, the decrease of the vascular tone induced by long-term administration of the angiotensin converting enzyme inhibitor may be due to a reduction of increased [Ca2+]i in SHR.     

Increased function of the voltage-dependent calcium channels, without increase of Ca2+ release from the sarcoplasmic reticulum in the arterioles of spontaneous hypertensive rats

It has been reported that the increased function of the voltage-dependent calcium channels (VDCC) in the artery is involved in the increase of peripheral resistance in hypertension, and that the sarcoplasmic reticulum (SR) in the artery plays an important role in preventing the development of hypertension via a buffering effect. However, no reports have described the role of VDCC and SR in resistance arterioles in the development or maintenance of hypertension. We investigated the function of VDCC and of SR in the cremaster arterioles of spontaneous hypertensive rats (SHR) and age-matched Wistar Kyoto rats (WKY). The changes in diameter and the intracellular calcium ion concentration ([Ca2+]i) in the microdissected arterioles, using fluorescent dyes, were measured with videomicroscopy. The KCl concentration-response curves were analyzed in 4- to 5- and 7- to 8-week-old SHR and WKY. The changes in the vascular diameter and [Ca2+]i in response to ryanodine, an alpha-1 adrenoceptor, and angiotensin-II stimulation were compared between the 7- to 8-week-old SHR and WKY. We found an increase in the Ca2+ influx by VDCC in the early hypertensive stage, but not in prehypertensive SHR. However, after the onset of hypertension, there were no significant differences from WKY in the SR function mediated by Ca2+-induced Ca2+ release or inositol 1,4,5-trisphosphate-induced Ca2+ release. In conclusion, an increased influx of Ca2+ in the cell membrane, without a buffering effect of SR, was associated with progression of hypertension in the cremaster arterioles of SHR.     

Thapsigargin-insensitive calcium pools in vascular smooth muscle cells.

Since sarcoplasmic Ca2+-ATPase may play an important role for the regulation of cytosolic free calcium concentration ([Ca2+]i) and may be altered in primary hypertension, the effects of thapsigargin and bradykinin on intracellular calcium pools in cultured vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats of the Münster strain (SHR) and normotensive Wistar-Kyoto (WKY) rats were investigated. VSMC were cultured on glass cover slips and [Ca2+]i was measured using the fluorescent dye fura2. To exclude transplasmamembrane calcium influx all experiments were performed in a calcium free medium. Thapsigargin, a selective inhibitor of the sarcoplasmic Ca2+-ATPase, and bradykinin, that is known to induce inositol trisphosphate release, dose dependently caused an increase of [Ca2+]i by emptying intracellular Ca2+ stores. The peak increase of [Ca2+]i after addition of saturation doses of thapsigargin (1 micromol/L) was not significantly different in the two strains (SHR: 69 +/- 11 nmol/L, n=24; WKY: 58 +/- 12 nmol/L, n=20; mean +/- SEM). When 10 micromol/L bradykinin was added after depletion of the thapsigargin-sensitive pools, still a release of [Ca2+]i could be observed. The bradykinin-induced [Ca2+]i increase was similar in the absence and presence of thapsigargin in VSMC from SHR (62 +/- 12 nmol/L, n=20; vs 52 +/- 18 nmol/L, n=22). In contrast, in the VSMC from WKY a significant reduction of the bradykinin induced [Ca2+]i-increase could be observed after the depletion of the thapsigargin sensitive calcium pools (70 +/- 8 nmol/L, n=21, vs. 33 +/- 7, n=20; p<0.002). It is concluded that bradykinin releases calcium from a pool that is not refilled by the common, thapsigargin-sensitive Ca2+-ATPase. In contrast to VSMC from normotensive WKY, in VSMC from spontaneously hypertensive rats thapsigargin and bradykinin sensitive pools may be regulated separately.     

Cytosolic calcium in platelets of spontaneously hypertensive rats

To test the hypothesis that an abnormality of the intracellular concentration of ionized calcium, [Ca2+]i, is associated with high blood pressure, we measured [Ca2+]i in the platelets of spontaneously hypertensive (SHR) and Wistar-Kyoto control (WKY) rats using the Quin 2 technique after separation of the platelets in calcium-free medium, during calcium repletion, and upon exposure to agonists which increase platelet [Ca2+]i (thrombin, adenosine diphosphate, serotonin and ionomycin). Despite clear-cut changes in [Ca2+]i during these manipulations, there were no differences between the SHR and WKY rats in baseline levels of [Ca2+]i or in the kinetics of changes in [Ca2+]i. These results do not support the hypothesis that high levels of [Ca2+]i at rest or abnormal kinetics of changes in [Ca2+]i play a pathophysiological role in the hypertension of SHR.     

Thrombin-Stimulated Calcium Uptake is Altered in Platelets from Spontaneously Hypertensive Rats. Effect of External Magnesium

Thrombin-induced calcium uptake and serotonin release were measured on platelets from spontaneously hypertensive rats (SHR) and normotensive control animals (WKY). Both ⁴⁵Ca uptake and serotonin release induced by thrombin are enhanced in platelets from SHR in the presence of various Ca2+ concentrations.

In the presence of [Ca2+] 10-⁶M a correlation exists between thrombin-induced calcium uptake and serotonin release; platelets from SHR and WKY do not differ in this respect, which demonstrates that the anomaly observed in platelets from SHR concerns an early reaction in the stimulation-response coupling.

The inhibitory effect of magnesium on thrombin-induced serotonin release, less important in platelets from SHR than in control ones, seems to pass through two phenomena: an inhibition of calcium influx occurring at high concentrations and less effective on SHR platelets and a stimulation of calcium influx, probably corresponding to intracellular calcium sequestering, observed in the presence of low magnesium concentrations and enhanced in platelets from SHR. This study therefore evidence a relationship between the increases observed in both serotonin secretion and calcium influx on thrombin-stimulated SHR platelets and suggests cellular mechanisms involved in the inhibitory action of magnesium impaired in these SHR platelets.     

Calcium transport in erythrocytes of rats with spontaneous hypertension

In Quin-2-loaded erythrocytes of two genetically hypertensive rat strains (spontaneously hypertensive rats, SHR, and the Milan hypertensive strain, MHS) intracellular Ca2+ (Ca2+i) concentration and 45Ca influx rate were increased by 25-30 and 15-20% respectively, in comparison with normotensive controls (Wistar-Kyoto rats, WKY, and rats of the Milan normotensive strain, MNS). After 4 h incubation in the presence of 5 mmol/l sodium vanadate (Na3VO4) as an inhibitor of Ca-ATPase, 45Ca content of intact erythrocytes of SHR was twofold higher while erythrocyte count of stroke-prone SHR (SHRSP) was threefold higher than in WKY. This increase was observed in SHR during the pre-hypertensive stage. Under the same conditions, no difference was noted between MHS and MNS rats. The rate of 32P influx, as well as the concentration of exchangeable chloride, was studied. We failed to detect any significant differences in either parameter between hypertensive and normotensive rats, suggesting that altered cell membrane potential was not responsible for allied Ca fluxes. Erythrocyte shrinking, however, resulted in a two to threefold increase in the rate of 45Ca influx. Neither the rate of 45Ca influx nor Ca2+i were modified by the inhibitor of calmodulin-dependent reactions, R24571 (10 mumol/l). It is suggested that the higher rate of Ca2+ influx in Quin-2-loaded erythrocytes of SHR, as well as the increment in 45Ca content in intact erythrocytes treated with orthovanadate, is due to a change in membrane skeleton organization and cell shrinkage.     

Effects of intracellular acidosis on [Ca2+]i transients, transsarcolemmal Ca2+ fluxes, and contraction in ventricular myocytes

We examined the effects of intracellular acidosis produced by washout of NH4Cl on [Ca2+]i transients (indo-1 fluorescence), cell contraction (video motion detector), and 45Ca and 24Na fluxes in cultured chick embryo ventricular myocytes. Exposure of cells to 10 mM NH4Cl produced intracellular alkalosis (pH 7.6), and subsequent washout resulted in a transient acidosis (pH 6.5). Exposure to 10 mM NH4Cl slightly decreased [Ca2+]i transients but increased the amplitude of cell contraction. Subsequent washout of NH4Cl initially increased diastolic [Ca2+]i and decreased the peak positive and negative d[Ca2+]i/dt, while the amplitude of cell contraction was markedly decreased. Subsequently, peak systolic [Ca2+]i increased with partial recovery of contraction. A similar increase in [Ca2+]i and decrease in contraction after washout of NH4Cl was observed in single paced adult guinea pig ventricular cells. Acidosis decreased 45Ca uptake by sarcoplasmic reticulum vesicles isolated from chick embryo ventricle. However, the [Ca2+]i increase caused by intracellular acidosis was also observed in the presence of 10 mM caffeine, suggesting that altered sarcoplasmic reticulum handling of calcium is not the only mechanism involved. Intracellular acidosis only slightly increased total 24Na uptake under these conditions, an effect resulting from the combination of a stimulation of amiloride-sensitive sodium influx (Na(+)-H+ exchange) and inhibition of sodium influx via Na(+)-Ca2+ exchange, manifested by a significant decrease in 45Ca efflux. Further support for a lack of involvement of an increased [Na+]i in the observed increase in [Ca2+]i during acidosis was low-sodium, nominal 0-calcium extracellular solution, an experimental condition that minimizes the possible effects of Na(+)-H+ exchange and Na(+)-Ca2+ exchange. We conclude that the [Ca2+]i increase caused by intracellular acidosis in cultured ventricular cells is primarily due to changes in [Ca2+]i buffering and [Ca2+]i extrusion, rather than to an increase in transsarcolemmal calcium influx. Intracellular acidosis also markedly decreases the sensitivity of the contractile elements to [Ca2+]i in cultured chick embryonic and adult guinea pig ventricular myocytes.     

Intracellular calcium transients in myocardium from spontaneously hypertensive rats during the transition to heart failure

To investigate the mechanism of impaired myocardial function after long-term pressure overload, we studied cardiac muscle mechanical contraction and intracellular calcium transients using the bioluminescent indicator aequorin. Left ventricular papillary muscle preparations were examined from three groups of rats: 1) aging spontaneously hypertensive rats (SHR) with clinical and pathological evidence suggesting heart failure (SHR-F group), 2) age-matched SHRs with no evidence of heart failure (SHR-NF group), and 3) age-matched normotensive Wistar-Kyoto rats (WKY group). Isometric force development was depressed in both SHR groups relative to the WKY group. Resting [Ca2+]i was lower in the SHR-F group, and the time to peak [Ca2+]i was prolonged in this group. The relative increases in peak [Ca2+]i with the inotropic interventions of increased [Ca2+]o and the addition of isoproterenol were similar among groups. Although inotropy increased in all groups with increased [Ca2+]o, after isoproterenol, inotropy increased only in the WKY group. Thus, in SHR myocardium, [Ca2+]i increased after isoproterenol, but inotropy failed to increase. Myosin isozymes were shifted toward the V3 isoform in both SHR groups; the V3 isoform was virtually 100% in papillary muscles from the SHR-F group. These changes may reflect events directly contributing to the development of heart failure or represent adaptive changes to chronic pressure overload and heart failure.     

Reoxygenation-induced Ca2+ rise is mediated via Ca2+ influx and Ca2+ release from the endoplasmic reticulum in cardiac endothelial cells

OBJECTIVE: Conditions of ischemia-reperfusion disturb the homoeostasis of cytosolic Ca2+ in cardiac microvascular endothelial cells (CMEC), leading to numerous malfunctions of the endothelium. Reperfusion specifically aggravates the Ca2+ overload developed during sustained ischemia. The aim of this study was to identify the origin of the reperfusion-induced part of the Ca2+ overload. Our hypotheses were that this is either due to a Na+-dependent process, e.g. involving the Na+/H+ exchanger (NHE) and/or the Na+/Ca2+ exchanger (NCX), or a process involving the endoplasmic reticulum (ER) and store-operated channels (SOC). METHODS AND RESULTS: Cultured CMEC from rats were exposed to conditions of simulated ischemia (hypoxia, pH 6.4) and reperfusion (reoxygenation, pH 7.4). Cytosolic Ca2+ ([Ca2+]i) and cytosolic Na+ ([Na+]i) concentrations and cytosolic pH (pHi) were measured with the use of fluorescent indicators. Removal of Ca2+ from the extracellular media during reoxygenation prevented the [Ca2+]i rise. Neither the activation of the NHE nor of the NCX in reoxygenated CMEC caused a change in this [Ca2+]i rise. Complete or partial removal of Na+ from the external media also had no effect on the [Ca2+]i rise. In contrast, specific inhibition of the inositol trisphosphate (InsP3) receptor by xestospongin C (3 micromol/l), of phospholipase (PLC) by U73122 (1 micromol/l), or of SOC by the inhibitors gadolinium (10 micromol/l) or 2-APB (50 micromol/l) lowered or abolished the reoxygenation-induced [Ca2+]i rise. CONCLUSION: In CMEC exposed to reperfusion conditions, the enhanced Ca2+ overload is due to Ca2+ influx. The influx is not mediated by a Na+-dependent mechanism, but rather is due to activation of the InsP3 receptor of the ER and activation of SOC.     

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.     

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.     

Restoring nitric oxide cytosolic calcium regulation by cyclic guanosine monophosphate protein kinase I alpha transfection in coronary endothelial cells of spontaneously hypertensive rats

In microcoronary endothelial cells (RCEs) from spontaneously hypertensive rats (SHR), the nitric oxide (NO)/cyclic guanosine monophosphate (GMP)-dependent proteinkinase I (cGKI) pathway cannot regulate the cytosolic calcium ([Ca2+]i) dynamic as in RCEs from Wistar Kyoto rats (WKY). We investigated the altered downstream NO target in SHR cells and, since cGKI expression was low, whether the re-expression of cGKIα in SHR RCEs could restore NO calcium responsiveness. We measured [Ca2+]i dynamic by fura-2 imaging analysis and the cGKI level by RT-PCR and Western blot in SHR and WKY RCEs. Plasmids encoding for enhanced green fluorescence protein or cGKIα-enhanced green fluorescence protein were transiently transfected in SHR RCEs, and [Ca2+]i was evaluated. Angiotensin-II (AT-II) increased [Ca2+]i in a concentration-dependent way in both strains. Whereas in WKY, endogenously produced NO and cyclic GMP analog decreased the AT-II-induced [Ca2+]i transient, they were ineffective in SHR RCEs. The cGKI level was low in SHR cells. However, after cGKIα re-expression, endogenous NO decreased the AT-II-induced [Ca2+]i transient, while endothelial NO synthase and cGKI inhibition prevented it. The low expression of cGKI in SHR accounts for the absent regulation of the agonist-induced [Ca2+]i transient by the NO/cyclic GMP pathway. Studies on cGKI in humans could contribute to a better understanding of cardiovascular pathologies.     

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.     

Identification of a functional Na+/Mg2+ exchanger in human trophoblast cells

BACKGROUND: Ionized magnesium levels are elevated in fetal blood compared with maternal blood, suggesting that the placenta may possess an active transport mechanism for magnesium. In the present study, we sought to determine the existence of an active transport mechanism for magnesium in the placenta using cultured trophoblast cells. METHODS: Using choriocarcinoma cells as a model system, we attempted to demonstrate the presence of a functional Na+/Mg2+ exchanger. Human choriocarcinoma JEG-3 cells cultured on glass coverslips were loaded with MAG-Fura 2-AM (5 micromol/L x 30 min) to spectrofluorometrically assess kinetics of intracellular magnesium ([Mg2+]i). Cells were superfused with various concentrations of Na+, Mg2+, Ca2+ and imipramine, a blocker of erythrocyte Na/Mg exchange. [Mg2+]i calibration was determined via Triton X-100 and EDTA. RESULTS: Sequential lowering of extracellular Na+ caused progressively larger, transient increases in [Mg2+]i. These transient changes in [Mg2+]i were completely dependent on [Mg]o but was independent of extracellular calcium ([Ca]o). Although acute imipramine did not alter basal [Mg2+]i, imipramine eliminated the return-to-basal phase of the [Mg2+]i transient induced by low sodium medium. Increasing extracellular magnesium ([Mg]o) caused stepwise increases in [Mg2+]i. CONCLUSIONS: The JEG-3 cells appear to possess a functional Na/Mg exchanger that functions to maintain low [Mg2+]i in cytotrophoblast cells. In addition, [Mg2+]i is acutely regulated by [Mg]o. Because placental trophoblasts are sites of maternal-fetal ion exchange, and [Mg]o is altered in preeclampsia, derangements in or modulation of this exchanger may contribute to complications of pregnancy such as pregnancy-induced hypertension, pre-eclampsia, and preterm labor.     

Control of the erythrocyte free Ca2+ concentration in essential hypertension.

Since Ca2+ ions seem to directly participate in the control of erythrocyte membrane structure and deformability and because cell Ca2+ metabolism has been repeatedly proposed to be modified in hypertension, the intracellular calcium ion concentration ([Ca2+]i) was investigated in red blood cells from hypertensive and normotensive subjects. [Ca2+]i was measured by using the fluorescent Ca2+ chelator fura-2. Red blood cell [Ca2+]i was increased in hypertensive compared with normotensive subjects in the whole population and further increased when hypertensive were compared with age-matched normotensive subjects. An inverse relation between age and [Ca2+]i was observed when calculated with blood pressure adjusted. In hypertensive patients, high [Ca2+]i values were associated with a reduced erythrocyte deformability. The initial rate of 45Ca2+ uptake did not differ between the two blood pressure groups. Similarly, when the extracellular Ca2+ concentration was elevated from 1 to 2 mmol/l, [Ca2+]i increased by 16 +/- 4% (p less than 0.03) in red blood cells from both groups, thus maintaining a significant difference between hypertensive and normotensive subjects. Under these conditions, the addition of 10(-7) mol/l nicardipine, a dihydropyridine Ca2+ antagonist, decreased [Ca2+]i by 15 +/- 4% (p less than 0.05) and 7 +/- 5% in erythrocytes from hypertensive and normotensive subjects, respectively, thereby reducing the difference in [Ca2+]i observed between these two groups. This nicardipine effect was positively correlated to the initial [Ca2+]i. In the presence of 5 mumol/l W7, a calmodulin antagonist, [Ca2+]i increased significantly only in erythrocytes from hypertensive patients (26 +/- 6%, p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)