Spontaneously Hypertensive Rats Platelets Research Articles

Upregulation of junctional adhesion molecule-A is a putative prognostic marker of hypertension

AimsEstablishing biochemical markers of pre-hypertension and early hypertension could help earlier diagnostics and therapeutic intervention. We assess dynamics of junctional adhesion molecule-A (JAM-A) expression in rat models of hypertension and test whether JAM-A expression could be driven by angiotensin (ANG) II and whether JAM-A contributes to the progression of hypertension. We also compare JAM-A expression in normo- and hypertensive humans.Methods and resultsIn pre-hypertensive and spontaneously hypertensive rats (SHRs), JAM-A protein was overexpressed in the brainstem microvasculature, lung, liver, kidney, spleen, and heart. JAM-A upregulation at early and late stages was even greater in the stroke-prone SHR. However, JAM-A was not upregulated in leucocytes and platelets of SHRs. In Goldblatt 2K-1C hypertensive rats, JAM-A expression was augmented before any increase in blood pressure, and similarly JAM-A upregulation preceded hypertension caused by peripheral and central ANG II infusions. In SHRs, ANG II type 1 (AT1) receptor antagonism reduced JAM-A expression, but the vasodilator hydralazine did not. Body-wide downregulation of JAM-A with Vivo-morpholinos in juvenile SHRs delayed the progression of hypertension. In the human saphenous vein, JAM-A mRNA was elevated in hypertensive patients with untreated hypertension compared with normotensive patients but reduced in patients treated with renin–angiotensin system antagonists.ConclusionBody-wide upregulation of JAM-A in genetic and induced models of hypertension in the rat precedes the stable elevation of arterial pressure. JAM-A upregulation may be triggered by AT1 receptor-mediated signalling. An association of JAM-A with hypertension and sensitivity to blockers of ANG II signalling were also evident in humans. We suggest a prognostic and possibly a pathogenic role of JAM-A in arterial hypertension.

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.

Abnormal Ca2+ handling and increased Mg2+ permeability in platelets of hypertensive rats.

In order to test the hypothesis that intracellular Na+ accumulation and cellular Mg2+ deficiency may be involved in the abnormalities in Ca2+ handling and reactivity in spontaneously hypertensive rats (SHR) platelets, the metabolism of Na+, Ca2+ and Mg2+ was determined in fluorescent dye loaded platelets from 15 SHR and 15 Wistar-Kyoto rats (WKY) at 12 weeks of age. Mg2+ leak was estimated as the Mg2+ influx induced by an increase in extracellular [Mg2+] (from 1 to 5 mmol/l) and Mg2+/Na+ exchange activity was estimated as the Mg2+ influx induced by a decrease in extracellular [Na+] (from 140 to 50 mmol/l). Cellular metabolism of the fluorescent dye was similar in the two groups. Mean platelet [Ca2+]i was significantly increased under basal and thrombin (0.1 U/ml)-stimulated conditions in SHR compared to WKY, both in the presence and absence of extracellular Ca2+. Mean Ca2+ discharge capacity was similar between the two groups. There was no difference in mean [Na+]i between the two groups. Basal [Mg2+]i was also increased in SHR platelets. Mg2+ leak was higher in SHR than in WKY, while Mg2+/Na+ exchange activity was similar in the two groups. There was no difference in serum Mg2+ concentration between SHR and WKY. These data suggest that abnormal Ca2+ handling is accompanied by elevation in [Mg2+]i via increased permeability of platelet cell membranes to Mg2+ in SHR without any alteration in [Na+]i, and do not support the Mg2+ deficiency hypothesis in genetically hypertensive rats.

Platelet sarco/endoplasmic reticulum Ca2+ATPase isoform 3b and Rap 1b: interrelation and regulation in physiopathology.

Platelet Ca2+ signalling involves intracellular Ca2+ pools, whose content is controlled by sarco/endoplasmic reticulum Ca2+ATPases (SERCAs). Among these, a key role is played by the inositol trisphosphate-sensitive Ca2+ pool, associated with the SERCA 3b isoform. We have investigated the control of this Ca2+ pool through the cAMP-dependent phosphorylation of the GTP-binding protein, Rap (Ras-proximate) 1b. We first looked for this Ca2+ pool target of regulation by studying the expression of the different SERCA and Rap 1 proteins in human platelets and various cell lines, by Western blotting and reverse transcription-PCR. Since co-expression of Rap 1b and SERCA 3b was obtained, we looked for their protein-protein interaction as a function of the cAMP-dependent phosphorylation of Rap 1b. Co-immunoprecipitations of SERCA 3b and Rap 1b proteins were found in the absence of phosphorylation, induced by the catalytic subunit of the cAMP-dependent protein kinase (csPKA). In contrast, upon pre-treatment of platelet membranes with csPKA, the SERCA 3b dissociated from the Rap 1b protein, in agreement with a role of its phosphorylated state in their interaction. Finally, we looked for adaptation of this complex in a platelet pathological model of hypertension. We investigated the expression of both proteins, as well as the cAMP-dependent phosphorylation of Rap 1b and SERCA 3b activity in platelets from control normotensive Wistar-Kyoto rats and from spontaneously hypertensive rats (SHRs). A decrease in SERCA 3b activity was associated with a decrease in Rap 1b endogenous phosphorylation in SHR platelets, consistent with a functional role in the regulation of the SERCA 3b-associated Ca2+ pool.

Dietary Calcium Attenuates Platelet Aggregation and Intracellular Ca 2 Mobilization in Spontaneously Hypertensive Rats

Spontaneously hypertensive rats (SHR) are known to be blood pressure sensitive to dietary calcium. The effects of dietary calcium on platelet aggregation and intracellular Ca 2+ mobilization were assessed by turbidimetric methods and fura-2 methods, respectively, in washed platelets of SHR. Ca 2+ ATPase activity was examined in aortic membrane fractions. Six weeks of dietary calcium supplementation attenuated the increase of systolic blood pressure (SBP 199 ± 16 v 170 ± 9 mm Hg, P < .001) and thrombin-induced platelet aggregation (84.5 ± 3.7 v 73.7 ± 7.4%, P < .004) at 9 weeks of age. The ionomycin-induced intracellular calcium ([Ca 2+] i) peak in the absence of external Ca 2+, which reflects [Ca 2+] i storage size, and thrombin-evoked [Ca 2+] i release from [Ca 2+] i storage were decreased by 2.0% Ca diet (472 ± 55 v 370 ± 23 nmol/L, P < .001, 339 ± 29 v 278 ± 33 nmol/L, P < .002). In addition, SBP was positively correlated with platelet aggregation (r = 0.703, P = .0088), thrombin-evoked [Ca 2+] i (r = 0.739, P = .0044), and ionomycin-induced [Ca 2+] i (r = 0.591, P = .0415), respectively. However, there was no significant effect of dietary calcium on Ca 2+-ATPase activity in aortic membranes. These results suggest that dietary calcium supplementation had a beneficial effect on platelets of SHR by attenuating [Ca 2+] i mobilization from [Ca 2+] i storage. The hypotensive effect of dietary calcium might be associated with attenuated [Ca 2+] i mobilization in SHR.

The rat platelet 97-kDa Ca2+ATPase isoform is the sarcoendoplasmic reticulum Ca2+ATPase 3 protein.

We recently showed that human and rat platelets express two types of SERCAs (Sarco Endoplasmic Reticulum Ca2+ATPases): a 100-kDa SERCA2b isoform and a 97-kDa SERCA isoform. Here, we explored the possibility that the rat 97-kDa isoform is identical to the SERCA3 protein. For this purpose, we first attempted to detect SERCA3 mRNA in rat platelet total RNA by reverse transcription-polymerase chain reaction using SERCA3-specific primers, and demonstrated the presence of this mRNA species by sequencing the amplification product. We then searched for a relationship between the expression of the SERCA3 mRNA and of the 97-kDa protein using either rat aortic smooth muscle cells, previously found not to express the 97-kDa SERCA isoform (negative model), or platelets of spontaneously hypertensive rats (SHR), which overexpress this isoform (overexpression model) but express the 100-kDa SERCA2b isoform normally. No expression of SERCA3 mRNA was detectable by analysis of smooth muscle cell RNA, but comparison by reverse transcription-polymerase chain reaction of the SERCA2b and SERCA3 mRNAs from the platelets of normotensive (Wistar-Kyoto, WKY) rats and SHR clearly demonstrated a 238 +/- 43% increase in the expression of the SERCA3 mRNA in SHR platelets only. Last, by comparative Western blotting of WKY rat and SHR platelet membranes using a recently developed polyclonal anti-SERCA3 antibody, we established that the 97-kDa SERCA and the SERCA3 protein are identical, as immunostaining of the 97-kDa protein revealed a 230 +/- 25% increase in the expression of this protein in SHR versus WKY rat platelets. It is concluded that the 97-kDa platelet SERCA isoform, which is up-regulated in SHR, is the SERCA3 protein. As far as we know, this constitutes the first demonstration of the actual presence of this Ca2+ATPase isoform in normal cells, in addition to the artificial transfection systems.

Platelets from spontaneously hypertensive rats exhibit decreased expression of inhibitory guanine nucleotide regulatory protein. Relation with adenylyl cyclase activity.

We have recently demonstrated an enhanced expression of inhibitory guanine nucleotide regulatory protein (Gi) in the heart and aorta from spontaneously hypertensive rats (SHR) as compared with control Wistar-Kyoto (WKY) rats; this enhanced Gi expression was associated with an increased inhibition of adenylyl cyclase by inhibitory hormones and decreased stimulation of adenylyl cyclase by stimulatory hormones. In the present studies, we have determined the levels of stimulatory and inhibitory guanine nucleotide regulatory proteins (Gs and Gi, respectively) in platelets from SHR by cholera toxin- and pertussis toxin-catalyzed ADP-ribosylations, respectively, as well as by immunoblotting techniques using specific antibodies for Gs and Gi. Cholera toxin catalyzed the ADP-ribosylation of a single protein of M(r) 45,000 in rat platelets from SHR and WKY rats, and the labeling of this band was not altered in SHR as compared with WKY rats. Pertussis toxin, on the other hand, catalyzed the ADP-ribosylation of a single protein band of M(r) 41,000 in platelets from SHR and WKY rats, and unlike the response in heart and aorta, the labeling of this band was significantly decreased in SHR as compared with WKY rats. Furthermore, immunoblotting experiments using AS/7 antibody, which is specific for Gi alpha-1 and Gi alpha-2, showed a decrease in Gi alpha-2 in platelets from SHR as compared with WKY rats. In addition, the inhibitory effects of angiotensin II and atrial natriuretic factor on adenylyl cyclase and cAMP levels were completely abolished in SHR platelets, whereas the stimulatory effects of GTP, N-ethylcarboxamide adenosine, prostaglandin E1, and forskolin on adenylyl cyclase and cAMP levels were enhanced.(ABSTRACT TRUNCATED AT 250 WORDS)

Spontaneously hypertensive rats and platelet Ca2+-ATPases: specific up-regulation of the 97 kDa isoform

The use of platelets instead of smooth muscle cells (SMC) to study the abnormal Ca2+ handling found in hypertension was investigated using spontaneously hypertensive rats (SHR). We studied the regulation of platelet Ca(2+)-ATPases, as we have recently demonstrated that human platelets, like SMC, contain the Ca(2+)-ATPase isoform termed SERCA2-b (sarco-endoplasmic reticulum Ca(2+)-ATPase). In mixed membranes isolated from platelets of normotensive Wistar-Kyoto (WKY) rats and SHR, total Ca(2+)-ATPase activity was found to be 43% higher in SHR than in WKY rats. By the use of autophosphorylation of rat platelet Ca(2+)-ATPases with [gamma-32P]ATP, followed by SDS/PAGE and Western blotting, we found that rat platelets express two distinct Ca(2+)-ATPases: a 100 kDa isoform, recognized by a SERCA2-b-specific anti-peptide antibody, and a 97 kDa isoform, specifically recognized by a polyclonal anti-SERCA antibody. Comparative analysis of platelet membrane Ca(2+)-ATPases from WKY rats and SHR demonstrated that the expression of the SERCA2-b isoform did not change significantly (128 +/- 22%), whereas that of the 97 kDa isoform reached 300 +/- 35% in SHR when compared with WKY rats. We concluded that the upregulation of total platelet Ca(2+)-ATPases in SHR is not due to the 100 kDa SERCA2-b isoform found in SMC, but is specific to the 97 kDa Ca(2+)-ATPase isoform which is not present in SMC. Therefore platelets should be used with extreme caution as a surrogate model of vascular smooth muscle Ca2+ homeostasis.

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.

Vascular eicosanoids and platelet-aortic wall interactions in spontaneously hypertensive rats

We studied the aggregation of collagen and ADP-stimulated platelet-rich plasma (PRP) and the formation of thromboxane B 2 (TxB 2) by collagen-stimulated PRP in spontaneously hypertensive rats (SHR) and in Wistar-Kyoto control rats (WKY). In addition, we evaluated the inhibition of the aggregation of PRP following homologous or heterologous perfusions through isolated aortas, the release of 6-keto-prostaglandin (PG)F 1α from these arteries perfused with PRP, and the sensitivity of PRP to the antiaggregatory activity of the stable PGI 2 analogue, iloprost, in both SHR and WKY. The lower activities (aggregation induced by ADP and collagen, collagen-stimulated TxB 2 production) of SHR platelets, were not accompanied by morphological differences from WKY platelets. These changes were associated with a greater release of arterial 6-keto-PGF 1α, with greater platelet antiaggregatory activity of the arterial wall and with higher sensitivity of platelets to iloprost. The lower reactivity of platelets to aggregating agents, and the greater sensitivity to prostacyclin, associated with a greater production of arterial prostacyclin were the major changes observed in SHR animals These alterations in the SHR vs. normotensive WKY may lead to an enhanced risk of hemorrhngc in the hypertensive state.

Increased Platelet Membrane Sodium-Proton Exchange Rate in Spontaneously Hypertensive Rats

It has been proposed that increased Na-H exchange activity is involved in the pathophysiology of genetic hypertension. We studied platelets of spontaneously hypertensive rats (SHR), normotensive Wistar-Kyoto (WKY), and domestic Wistar rats (DWR), since platelets have similarities to smooth muscle cells and have been shown to have potentially related ion metabolism abnormalities, such as increased intracellular calcium activity. We determined the exchange rate by the intracellular acidification-dependent, extracellular sodium-dependent volume increase. The rate of increase during the first 3 min is linear and is shown to be higher in SHR (0.475 X min-1) than in WKY (0.410 X min-1) or DWR (0.389 X min-1). We conclude that the Na-H activity is increased in platelets of SHR. Similar findings in lymphocytes and neutrophils of SHR, and analogous findings in platelets of humans with essential hypertension, suggest that this abnormality is expressed by several cell types, some of which may be involved in the pathophysiology of genetic hypertension in both rats and humans.

Receptor dependent and -independent protein phosphorylation in platelets of spontaneously hypertensive rats

This study investigates the role of protein kinase C and of myosin light chain kinase in mediating platelet hyperresponsiveness in spontaneously hypertensive rats (SHR). For this purpose, 32P-labeled washed platelets of both SHR and normotensive controls Wistar-Kyoto (WKY) were challenged either with a receptor-mediated agonist (thrombin) or with direct activators of myosin light chain kinase and protein kinase C. Such enzymatic activities were assessed by measuring changes in 32P-labeling of their respective target proteins, namely myosin light chain (20 KDa) and the 47 KDa protein. In resting platelets, the patterns of protein phosphorylation were similar between SHR and WKY, suggesting that the two cell types were in a comparable quiescent status. By contrast, in both dose-response and time-course studies, thrombin promoted a significantly greater phosphorylation of the 20- and 47 KDa proteins in platelets of SHR compared with that for WKY. Sensitivity of myosin light chain kinase to the calcium ionophore A23187 and of protein kinase C to both phorbol ester and dioctanoylglycerol was apparently not different between the two cell types. The data indicate that the exaggerated thrombin-induced protein phosphorylation observed for platelets of SHR is not linked to alterations in protein kinase C and/or myosin light chain kinase per se. These results therefore suggest that platelet hyperresponsiveness in SHR is likely to be related, at least in part, to abnormalities in receptor-mediated transmembrane signalling.

Abnormal calcium handling by platelets of spontaneously hypertensive rats.

There is controversy as to whether platelet intracellular free calcium ([Ca2+]i) is increased in spontaneously hypertensive rats (SHR) as compared with Wistar-Kyoto (WKY) rats. Discrepant results may be due to methodological problems including platelet activation during the collection process and leakage of intracellular dye used for [Ca2+]i measurement. To provide further insight into this problem, [Ca2+]i was estimated in fura-2-loaded platelets isolated from eight SHR and seven WKY rats at 12-14 weeks of age by using a two-syringe blood collection method and a correction method for fura-2 leakage. Basal [Ca2+]i was higher in SHR than in WKY rats (61.6 +/- 5.6 vs. 54.0 +/- 3.9 nM, p less than 0.02). However, the difference disappeared when a correction for fura-2 leakage was not used (109.7 +/- 18.4 vs. 94.9 +/- 9.2 nM, p less than 0.1). Thus, differences in [Ca2+]i between SHR and WKY rats may be obscured if dye leakage from platelets is not taken into account. Thrombin (0.1 units/ml) induced a rise in [Ca2+]i that was greater in SHR than WKY rats, both in the presence (491.4 +/- 31.6 vs. 377.5 +/- 21.7 nM, p less than 0.002) and absence (264.9 +/- 33.6 vs. 228.2 +/- 30.1 nM, p less than 0.05) of calcium in the media. These results indicate that thrombin-stimulated calcium influx as well as discharge of calcium from intracellular stores is increased in SHR platelets. Thus, under both basal and stimulated conditions, platelet calcium handling is abnormal in the SHR.

Responses of Cytosolic Free Calcium to ADP in Platelets of Spontaneously Hypertensive Rats

Abnormalities of Ca2+ handling have been reported in patients with essential hypertension and in spontaneously hypertensive rats (SHR). In this study, responses of cytosolic Ca2+ to ADP in platelets of SHR were examined. Four- and seven-week-old male SHR and age- and sex-matched Wistar-Kyoto rats (WKY) were used. Basal levels of the intracellular Ca2+ concentration in platelets and responses to ADP were estimated using fluorescent indicator fura-2 in the medium containing 1 mmol/L CaCl2 and Ca2(+)-free buffer with 1 mmol/L EGTA. Basal levels of platelet cytosolic Ca2+ of SHR were significantly higher than those of WKY at 4 and 7 weeks of age in the presence of external Ca2+. However, no significant difference was observed in basal levels of platelet cytosolic Ca2+ in the Ca2(+)-free EGTA-containing buffer between SHR and WKY. The peak cytosolic Ca2+ concentration evoked by ADP was significantly diminished in SHR compared with WKY in the absence of external Ca2+, whereas the responses of platelet cytosolic Ca2+ to ADP were similar in SHR and WKY in the presence of external Ca2+. These results suggest that release from intracellular Ca2+ store is reduced in SHR and that the regulation of cytosolic Ca2+ in SHR is more dependent on extracellular Ca2+ compared with WKY.

Changes in renal, platelet and cardiac nitrobenzylthioinosine binding in spontaneously hypertensive rats

In an attempt to investigate the role of nucleoside transporter function in the hypertensive state, we have compared the binding of [ 3H]nitrobenzylthionosine ([ 3H]NBMPR), a nucleoside transporter probe, in membranes prepared from platelet, renal, pulmonary, cardiac and brain tissues of spontaneously hypertensive rats (SHR) to those of age-matched Wistar-Kyoto (WKY) controls. At 4 weeks of age, ([ 3H]NBMPR) binding sites (Bmax) increased in the kidney of SHR but decreased in platelets, whereas no changes were found in the heart, lung or brain. At 18 weeks of age, [ 3H]NBMPR binding sites (Bmax) remained increased in the kidney and decreased in platelets with no changes in the other tissues. The only change in apparent binding affinity (K D) was an increase in the heart of SHR at 4 weeks. Age-dependent decreases were also observed in the heart and platelets of both SHR and WKY at 18 weeks. The results indicate that the changes in binding characteristics may be due to a combination of the pharmacodynamic differences between the strains, age, as well as to the pathogenesis of hypertension. Consequently, it cannot be concluded that the altered binding characteristics are the result of the elevated blood pressure.

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(-6)M 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.

Enhanced turnover of phosphatidylcholine in platelets of hypertensive rats. Possible involvement of a phosphatidylcholine-specific phospholipase C

In an attempt to determine the mechanism involved in the hyperreactivity of platelets in primary hypertension, the dynamic behavior of phospholipids was investigated in quiescent platelets of spontaneously hypertensive rats (SHR) compared to normotensive controls. By using 32P i, [methyl- 3H]choline or [ 3Higlycerol as the radioactive precursors, the labeling of phosphatidylcholine (PC) was shown to be markedly enhanced (10-20-times) in SHR compared to controls. This difference between SHR and controls could not be ascribed to differences either in the actual amount of PC or m the uptake of various labels, suggesting that PC turnover was markedly enhanced in platelets of SHR. The [methyl-H]choline labeling of phosphocholine and of CDP-choline was twice as high m SHR as in controls; chase experiments showed that when the label disappeared from phosphocholine, it was rapidly converted to PC. The results indicated that in rat platelets, PC synthesis occurred mainly via the CDP-choline pathway, and suggested that CTP: phosphocholine cytidylyltransferase was the rate-limiting step; they also indicated that the activity of this enzyme and that of choline kinase might be enhanced m SHR platelets compared to Wistar-Kyoto rat (WKY) platelets, and may thus be responsible for the enhanced PC synthesis. From these results, the existence of a PC-specific phospholipase C activity involved in PC turnover in SHR platelets can be envisaged.

Thrombin-induced abnormal platelet activation in spontaneously hypertensive rats is linked with phosphoinositides turnover and phosphorylation of 47,000 and 20,000 dalton proteins

We have shown earlier that abnormal platelet aggregation in spontaneously hypertensive rats (SHR) is not caused by prostaglandins. In this study platelets from SHR and normotensive (Wistar Kyoto, WKY) rats were used to examine the role of phosphoinositides and phosphorylation of 47,000 and 20,000 Dalton proteins in abnormal platelet activation in hypertension. Thrombin (0.05 U/ml) induced a rapid decrease in (32P)-PO4 labelled phosphatidylinositol-4,5-bisphosphate (PIP2), phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol (PI) in washed rat platelets. However, significantly greater loss of PIP2 and PI was seen in SHR platelets than in WKY platelets. For example the level of PIP2 declined by 32% in SHR platelets and only by 13% in WKY platelets at five seconds of incubation with thrombin. The loss of PI was similar in SHR and WKY platelets for the first five seconds of incubation with thrombin. However, by 15 seconds SHR platelets showed a significantly greater loss (24%) in PI than in WKY platelets (8%). Thrombin induced a 14% and 18% decrease in PIP at three seconds in WKY and SHR platelets respectively. In SHR platelets PIP level returned to the baseline in five seconds and then rose to 20% above the baseline by 30 seconds. In contrast PIP level in WKY platelets slowly reached the basal value by 30 seconds. Thrombin also produced a two- to three-fold greater accumulation of (32P)-phosphatidic acid (PA) in SHR platelets than in WKY platelets. Thrombin (0.05 U/ml) induced rapid phosphorylation of 47,000 Dalton (P47) and 20,000 Dalton (P20) proteins in both WKY and SHR platelets. Thrombin induced a four-fold greater increase in phosphorylation of P47 in SHR platelets than in WKY platelets in the first five seconds. Thrombin produced significantly greater increase in phosphorylation of P20 in SHR platelets (34% and 41%) than in WKY platelets (18% and 28%) at 5 and 15 seconds. Phosphorylation of P20 was followed by dephosphorylation in both WKY and SHR platelets. Aspirin (500 uM) did not affect phosphorylation of either P47 or P20 in SHR or WKY platelets. In other experiments prostaglandin El (0.5 uM), which stimulates adenylate cyclase via a guanine nucleotide regulatory protein termed Gs, caused an eighteen-fold increase in cyclic AMP level in SHR platelets as compared to a six-fold increase in WKY platelets. These data lead us to suggest that increased turnover of phosphoinositides and increased phosphorylation of P47 and P20 are involved in abnormal platelet activation in SHR platelets.

Relationship Between Enhanced Phosphoinositide Turnover and Cellular Responses in Platelets from Spontaneously Hypertensive Rats

Thrombin-induced aggregation and serotonin release were markedly enhanced in platelets from spontaneously hypertensive rats (SHR) when compared to normotensive Wistar-Kyoto (WKY) controls. Since phosphoinositides are involved in calcium-mediated platelet responses, the metabolism of these lipids was investigated in SHR and WKY rats by using 32P-labeled quiescent platelets. In unstimulated cells, both the rate and extent of 32P incorporation into individual inositol-containing phospholipids and phosphatidic acid (PA) were identical in SHR and WKY rats. This suggests that the pool size and basal turnover of phosphoinositides did not differ between the two strains. In contrast, early thrombin-induced phosphoinositide metabolism, when monitored as changes in 32P-PA, was significantly higher in SHR than in WKY rats. Following thrombin stimulation, 32P-PA formation likely reflects the initial agonist-receptor interaction; therefore, our results suggest that phospholipase C activity is enhanced in SHR platelets. Thus, it can be postulated that the observed hypersensitivity of SHR phospholipase C may play a role in the overall alteration of cell calcium handling and hence in the SHR platelet response.

Hypersensitivity of phospholipase C in platelets of spontaneously hypertensive rats.

Thrombin-induced aggregation and serotonin release were markedly enhanced in platelets from spontaneously hypertensive rats (SHR) when compared with those from normotensive Wistar-Kyoto rats (WKY). Since phosphoinositides are involved in calcium-mediated platelet responses, the metabolism of these lipids was investigated in SHR and WKY by using 32P-labeled quiescent platelets. In unstimulated cells, both the rate and extent of 32P incorporation into individual inositol-containing phospholipids and phosphatidic acid were identical in SHR and WKY. This finding suggests that the pool size and basal turnover of phosphoinositides did not differ between the two strains. In contrast, early thrombin-induced phosphoinositide metabolism, when monitored as changes in [32P]phosphatidic acid, was significantly higher in SHR than in WKY. For example, a 20-second exposure to thrombin, 0.3 U/ml, induced the formation of 1.6 times more [32P]phosphatidic acid in SHR than in WKY. These results provide evidence for a leftward shift of the dose-response and time-course curves of thrombin-induced [32P]phosphatidic acid formation in SHR. Moreover, the extent of the difference between SHR and WKY was independent of the extracellular calcium concentration. Following thrombin stimulation, [32P]phosphatidic acid formation likely reflects the initial agonist-receptor interaction; therefore, these results suggest that phospholipase C activity is enhanced in platelets of SHR and that the hypersensitivity of phospholipase C in SHR may play a role in the overall alteration of cell calcium handling and, hence, in the platelet responses of SHR.