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(Hypertension. 1996;27:1312-1317.)
© 1996 American Heart Association, Inc.

Articles

Platelet Ca²⁺ Is Not Increased in Stroke-Prone Spontaneously Hypertensive Rats

Comparative Study With Spontaneously Hypertensive Rats

Norihisa Ono; Tetsuya Oshima; Mari Ishida; Takafumi Ishida; Hideo Matsuura; Masayuki Kambe; Goro Kajiyama

From the First Department of Internal Medicine (N.O., M.I., T.I., H.M., G.K.) and Department of Clinical Laboratory Medicine (T.O., M.K.), Hiroshima (Japan) University School of Medicine.

Correspondence to Norihisa Ono, MD, First Department of Internal Medicine, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-ku, Hiroshima, 734, Japan.

	Abstract

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Abstract We have reported that cytosolic Ca²⁺ concentration ([Ca²⁺]_i) is increased in platelets from spontaneously hypertensive rats (SHR) in both basal and thrombin-stimulated conditions. To determine whether the correlation between blood pressure and cellular Ca²⁺ metabolism exists in stroke-prone SHR (SHRSP), we investigated Ca²⁺ handling using fura 2 and aggregation response in platelets of 12- to 13-week-old male SHRSP, SHR, and Wistar-Kyoto rats (WKY). Systolic pressure was highest in SHRSP and lowest in WKY (213±8, 172±7, and 135±5 mm Hg, respectively). Basal [Ca²⁺]_i was significantly higher in SHR than WKY (45.9±4.5 versus 41.2±4.8 nmol/L, P<.05), and that in SHRSP (40.2±2.8 nmol/L) was similar to that in WKY. Thrombin (0.1 IU/mL)–stimulated [Ca²⁺]_i rise was greater in SHR and smaller in SHRSP than in WKY in the presence of extracellular Ca²⁺ (530±50 and 408±52 versus 475±50 nmol/L, respectively; P<.05). The recovery rate from the peak [Ca²⁺]_i response to thrombin was greatest in SHRSP and least in WKY. Ionomycin (5 µmol/L)–stimulated [Ca²⁺]_i rise was similar in WKY, SHR, and SHRSP (731±97, 743±88, and 683±70 nmol/L, respectively). Thrombin-induced maximum platelet aggregation response was higher in SHR and lower in SHRSP than WKY (82±4% and 61±15% versus 73±6%, respectively; P<.05). In contrast to SHR, basal [Ca²⁺]_i in SHRSP was similar to that in WKY, and thrombin-stimulated [Ca²⁺]_i was attenuated. These results suggest that platelet Ca²⁺ handling differs between SHR substrains and that an increased [Ca²⁺]_i is not obligatory in genetically hypertensive rats.

Key Words: calcium • fura-2 • platelet aggregation • rats, inbred SHR • rats, inbred, SHRSP

	Introduction

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Abnormal calcium handling has been extensively reported in various cells of patients with essential hypertension^{1 2 3} and of genetically hypertensive rats^{3 4 5 6 7 8 9 10 11 12 13} and has been proposed to be involved in the pathogenesis of primary hypertension.¹⁴ Platelets are widely used for studies of cellular calcium metabolism because they are easy to handle and their contractile function is similar to that of vascular smooth muscle cells.^{15 16} Thus, there have been many reports of increased [Ca²⁺]_i in platelets of patients with essential hypertension^{1 3 17} and SHR^{3 7 8 9 10 18 19} compared with normotensive controls and of a positive correlation between basal [Ca²⁺]_i and BP.^{1 3 19 20}

SHRSP, a substrain of SHR, develop more severe hypertension spontaneously than SHR and die of massive cerebral hemorrhage or infarction.²¹ In contrast to SHR, little information exists about platelet Ca²⁺ handling in SHRSP. If the concept of a positive correlation between BP and platelet Ca²⁺ is correct, abnormal Ca²⁺ handling should be augmented in SHRSP. The purpose of the present study was to determine whether SHRSP handle platelet Ca²⁺ in a fashion similar to that of SHR. We determined platelet Ca²⁺ handling and aggregation response in SHRSP, SHR, and WKY in relation to BP.

	Methods

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Animals
WKY and SHR were obtained from Charles River Japan Inc (Atsugi, Japan). SHRSP were provided by Takeda Chemical Industries, Ltd (Osaka, Japan) and had been maintained by brother-sister breeding in our laboratory. Rats had free access to standard rat chow (MF, Oriental Yeast Co, Ltd) and tap water. The rat chow contained (in grams per 100 g) calcium 1.15, potassium 0.89, sodium 0.26, protein 24.6, and phosphorous 0.88, with 360 kcal/100 g. Male WKY, SHR, and SHRSP at 12 to 13 weeks of age were used. Systolic BP was determined by the tail-cuff method.

Materials
Fura 2–acetoxymethyl ester (AM) was obtained from Molecular Probes. Thrombin and ionomycin were purchased from Sigma Chemical Co.

Platelet Aggregation
The degree of platelet aggregation in gel-filtered platelets was measured by a turbidimetric method with a six-channel NBS Hematracer 601 (Niko, Bioscience). Blood was drawn from the right atrium into a syringe containing 3.8% trisodium citrate anticoagulant, with rats under thiamyral sodium anesthesia (30 mg/kg IP). Platelet-rich plasma was prepared by centrifugation at 350g for 7 minutes at room temperature. Platelet-rich plasma was applied to a Sepharose 2B-CL column (Pharmacia LKB Biotechnology AB) that had been equilibrated with an elution medium containing (mmol/L) NaCl 145, KCl 5, MgSO₄ 1, HEPES 10, and glucose 5 (pH 7.4). A suspension (290 µL, 2x10⁸ cells per milliliter) of gel-filtered platelets was stimulated with 10 µL thrombin (final concentration, 0.1 IU/mL) in the presence of 1.0 mmol/L CaCl₂ at 37°C with constant stirring, and maximum aggregation was measured.

[Ca²⁺]_i Measurements
[Ca²⁺]_i measurements with fura 2 were made as previously described.^{7 8 9} The platelet suspension (5x10⁸ cells per milliliter) was prepared in the same way as for platelet aggregation measurements. The washed platelets were incubated with 2 mmol/L fura 2-AM for 30 minutes at room temperature. After gel filtration for removal of any extraneous fura 2-AM, the platelets were suspended in the buffer at a concentration of 5x10⁷ cells per milliliter, and CaCl₂ was added to the cell suspension at a final concentration of 1 mmol/L. For fluorescence measurements, 2.5-mL aliquots of cell suspension were stirred continuously by a magnetic stir bar in a fluorescence-free acrylic cuvette at 37°C, and fluorescence was recorded with a dual excitation wavelength fluorometer (DM3000 system, SPEX Industries Inc) with excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm. After fluorescence was recorded in the basal state, the [Ca²⁺]_i responses to thrombin were evaluated both in the presence of 1 mmol/L Ca²⁺ and in Ca²⁺-free (<10 nmol/L) buffer prepared by the addition of 5 mmol/L EGTA (Dojindo Laboratories). Furthermore, detailed analysis of the kinetics of the return of calcium (uptake and extrusion) in thrombin-stimulated platelets in the absence of extracellular calcium was performed as previously reported.⁹ We calculated the calcium transient at 10-second intervals for 60 seconds after the peak response to thrombin. With the use of the seven data points taken in the 60-second period for each rat, the calcium transient equation, [Ca²⁺]_i=Ae^-kt, was derived by regression analysis (StatView II software) with a personal computer, where A represents compartment size, k is the rate constant, and t equals time. We also evaluated the [Ca²⁺]_i response to ionomycin (5 µmol/L, the concentration at which a maximal response was elicited) in Ca²⁺-free buffer as an index of the intracellular Ca²⁺ discharge capacity. [Ca²⁺]_i was calculated with a general formula.²² Corrections were applied for extracellular fura 2 leakage with the use of EGTA and for autofluorescence by subtraction of the fluorescence of the unloaded platelets and the test reagents.⁷ Basal [Ca²⁺]_i was calculated as the mean of triplicate measurements. The thrombin or ionomycin-stimulated [Ca²⁺]_i rise was a single measurement.

Statistical Analysis
Values are expressed as mean±SD unless otherwise indicated. Data were analyzed by a standard personal computer with one-factor ANOVA followed by Fisher's protected least significant difference test as a multiple comparison procedure. A value of P<.05 was considered statistically significant.

	Results

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No abnormal signs or symptoms suggestive of stroke were observed in any of the rats used. Furthermore, serum creatinine levels measured by the alkaline picrate method did not differ significantly among the three strains, demonstrating that there was no significant organ damage in the rats used (Table 1). Systolic BP, heart rate, and body weight for the three strains are shown in Table 1. Systolic BP was significantly higher in SHR than WKY and higher in SHRSP than SHR and WKY. The differences of systolic BP in each strain were about 40 mm Hg. Body weight was highest in WKY and lowest in SHRSP. SHRSP had a significantly higher heart rate than SHR. Thrombin (0.1 IU/mL)–induced maximum platelet aggregation responses are shown in Fig 1. Maximum aggregation response was significantly lower in SHRSP (61±15%) and higher in SHR (82±4%) than in WKY (73±6%, P<.05).

View this table: [in this window] [in a new window]   Table 1. Systolic Blood Pressure, Heart Rate, Body Weight, and Serum Creatinine in Three Rat Strains

View larger version (50K): [in this window] [in a new window]   Figure 1. Thrombin (0.1 IU/mL)–induced maximum aggregation in platelets from WKY (n=15), SHR (n=15), and SHRSP (n=15). Values are expressed as mean±SD. *P<.05.

Intracellular fura 2 concentration, R_max and R_min, the ratio of fluorescence at excitation wavelength 340 and 380 nm under Ca²⁺-saturated and Ca²⁺-free conditions, respectivly, did not differ among the three strains, indicating that platelets were loaded with the dye to a similar extent (Table 2). Platelet size (mean diameter) measured by a Coulter counter (Coulter Channelyzer 256) was similar among the three strains (2.02±0.05, 2.07±0.08, and 2.06±0.07 µm, WKY, SHR, and SHRSP, respectively). The extracellular fura 2 leakage in SHR was significantly greater than in the other strains (Table 2), indicating the necessity of correcting for fura 2 leakage to prevent misleading estimates of [Ca²⁺]_i.

View this table: [in this window] [in a new window]   Table 2. Platelet Intracellular Fura 2 Metabolism in Three Rat Strains

Fig 2 shows the resting level of [Ca²⁺]_i in platelets from the three strains. Basal [Ca²⁺]_i was significantly higher in SHR than WKY (45.9±4.5 versus 41.2±4.8 nmol/L, P<.05), as we previously reported,^{7 8 9 10} and basal [Ca²⁺]_i in SHRSP (40.2±2.8 nmol/L) was at the same level as in WKY. Thrombin (0.1 IU/mL)–stimulated [Ca²⁺]_i rises in the absence and presence of extracellular Ca²⁺ are shown in Figs 3 and 4, respectively. The [Ca²⁺]_i response to thrombin in the absence of extracellular Ca²⁺, which indicated a release from internal Ca²⁺ stores, was significantly greater in SHR than WKY and SHRSP (282±43 versus 245±22 and 223±30 nmol/L, respectively; P<.05). No significant difference existed between WKY and SHRSP. An elevation in [Ca²⁺]_i evoked by thrombin in the presence of extracellular Ca²⁺ was significantly higher in SHR and lower in SHRSP than in WKY (530±50 and 408±52 versus 475±50 nmol/L, respectively; P<.05). We assessed thrombin-induced intraplatelet Ca²⁺ influx by subtraction of the thrombin-stimulated [Ca²⁺]_i rise in the absence of extracellular Ca²⁺ from that in the presence of extracellular Ca²⁺ (Fig 5). Intraplatelet Ca²⁺ influx was smaller in SHRSP than WKY and SHR (185±39 versus 229±48 and 248±73 nmol/L, respectively; P<.05). The rate of decay of the Ca²⁺ transient in Ca²⁺-free medium was greater in SHR than WKY and greater in SHRSP than SHR (Fig 6, Table 3). We also evaluated the internal Ca²⁺ discharge capacity, assessed by the intracellular Ca²⁺ response to the addition of 5 µmol/L ionomycin, a calcium ionophore, in a Ca²⁺-free medium. No significant differences were found in the ionomycin-evoked [Ca²⁺]_i rise among WKY, SHR, and SHRSP (731±97, 743±88, and 683±70 nmol/L, respectively; Fig 7).

View larger version (13K): [in this window] [in a new window]   Figure 2. Resting [Ca2+]i in platelets from WKY (n=15), SHR (n=15), and SHRSP (n=15). Resting [Ca2+]i was significantly higher in SHR than WKY and SHRSP. No significant difference was detected between WKY and SHRSP. *P<.05.

View larger version (15K): [in this window] [in a new window]   Figure 3. Rise in platelet [Ca2+]i in response to 0.1 IU/mL thrombin in the absence of extracellular Ca2+ in WKY (n=15), SHR (n=15), and SHRSP (n=15). The [Ca2+]i rise in SHR was significantly greater than in WKY and SHRSP. No difference was found between SHRSP and WKY. *P<.05.

View larger version (16K): [in this window] [in a new window]   Figure 4. Rise in platelet [Ca2+]i in response to 0.1 IU/mL thrombin in the presence of extracellular Ca2+ in WKY (n=15), SHR (n=15), and SHRSP (n=15). The [Ca2+]i rise in SHR was significantly greater than in WKY and SHRSP. The [Ca2+]i rise in SHRSP was significantly smaller than in WKY. *P<.05.

View larger version (16K): [in this window] [in a new window]   Figure 5. Thrombin (0.1 IU/mL)–stimulated Ca2+ influx in platelets from WKY (n=15), SHR (n=15), and SHRSP (n=15). Each data point was calculated by subtraction of the thrombin (0.1 IU/mL)–stimulated [Ca2+]i increase in the absence of extracellular Ca2+ from the [Ca2+]i increase in the presence of extracellular Ca2+. Intraplatelet Ca2+ influx evoked by thrombin in SHRSP was significantly smaller than WKY and SHR. *P<.05.

View larger version (16K): [in this window] [in a new window]   Figure 6. Rate of recovery from peak intracellular Ca2+ response in Ca2+-free medium induced by 0.1 IU/mL thrombin in platelets from WKY (n=15), SHR (n=15), and SHRSP (n=15). Values are expressed as mean±SEM.

View this table: [in this window] [in a new window]   Table 3. Curve Fit Parameters in Three Rat Strains

View larger version (16K): [in this window] [in a new window]   Figure 7. Rise in platelet [Ca2+]i in response to 5 µmol/L ionomycin in the absence of extracellular Ca2+ in WKY (n=15), SHR (n=15), and SHRSP (n=15). Ionomycin-evoked [Ca2+]i rise did not differ significantly among the three strains.

	Discussion

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In the present study, we confirmed our previous findings that basal and thrombin-stimulated [Ca²⁺]_i values were increased in platelets from SHR compared with those from WKY.^{7 8 9 10} BP was highest in SHRSP and lowest in WKY (SHRSP>SHR>WKY). If Ca²⁺ regulation is altered in a wide variety of tissues and cells and participates in the pathogenesis of hypertension, abnormal Ca²⁺ handling in SHR should be augmented in SHRSP. However, basal [Ca²⁺]_i did not differ between SHRSP and WKY. Furthermore, the thrombin-stimulated [Ca²⁺]_i rise in the presence of extracellular Ca²⁺ was significantly smaller in SHRSP than WKY. The positive correlation between BP and platelet [Ca²⁺]_i in SHR could not be extended to SHRSP. We cannot explain the differences in Ca²⁺ metabolism by the difference in cell size or intracellular fura 2 metabolism. However, previous studies indicated that platelet turnover was accelerated in SHRSP compared with SHR^{23 24} and WKY.²⁴ One explanation is increased platelet consumption caused by hypertensive vascular damage²⁴ ; another is that the primary defect of SHRSP platelets appears to be an impaired function of Ca²⁺ metabolism.²⁵ Decreased thrombin-stimulated responses in SHRSP platelets might be due to relatively immature and damaged platelets.

The thrombin-induced peak [Ca²⁺]_i and recovery rate of [Ca²⁺]_i were greater in SHR than WKY, as previously reported.⁹ An explanation for this observation is that the rate of decay of the thrombin-activated Ca²⁺ transient is greater in SHR because of the higher peak level of intracellular Ca²⁺ that was achieved. On the other hand, although thrombin-evoked peak [Ca²⁺]_i in the absence of extracellular Ca²⁺ did not differ significantly between WKY and SHRSP, the recovery rate of [Ca²⁺]_i was significantly greater in SHRSP than WKY. These findings suggest that the platelet Ca²⁺ extrusion process in SHRSP is enhanced with thrombin activation. Furthermore, thrombin-induced intraplatelet Ca²⁺ influx was attenuated in SHRSP in comparison with WKY. There is a possibility that an attenuated Ca²⁺ influx and enhanced Ca²⁺ extrusion might result in a reduced [Ca²⁺]_i response in SHRSP. Intracellular Ca²⁺ discharge capacity, assessed by the response of intracellular Ca²⁺ to a maximal dose of ionomycin in the absence of extracellular Ca²⁺, did not differ between WKY, SHR, and SHRSP. This finding supports our previous report that there was no relation between the size of the intracellular Ca²⁺ store assessed by the ionomycin and BP and that the size of the intracellular ionomycin-released Ca²⁺ fraction was similar in rat strains of Wistar origin but different between Wistar and other strains.¹⁰ The differences in thrombin-activated Ca²⁺ mobilization are thought to be associated with alterations in signal transduction rather than the different sizes of releasable intracellular Ca²⁺ stores.

As far as the [Ca²⁺]_i measurement is concerned, methodological issues are important in the assessment of Ca²⁺ handling in fura 2–loaded cells.^{7 8 9 26} First, variations in intracellular fura 2 concentration could affect the [Ca²⁺]_i response to agonists because of the Ca²⁺-buffering effects of the dye. In addition, the extent of dye ester hydrolysis also affects fluorescence dynamics. The presence of unhydrolyzed or incompletely hydrolyzed dye may lead to underestimation of [Ca²⁺]_i. However, the intracellular fura 2 concentration and ratio of R_max to R_min, an index of dye ester hydrolysis, did not differ significantly among the three strains. Second, the extracellular fura 2 leakage in the SHR was significantly greater than in SHRSP and WKY. Thus, the leakage of fura 2 from cells should be corrected for calculation of [Ca²⁺]_i. When no correction was made for dye leakage, [Ca²⁺]_i was overestimated in Ca²⁺-supplemented buffer and underestimated in Ca²⁺-free buffer, leading to increased variation in [Ca²⁺]_i. Because cellular dye metabolism was evaluated in the present study, we can safely compare the calculated [Ca²⁺]_i in fura 2–loaded platelets among different rat strains.

In this study, the resting [Ca²⁺]_i level of each strain (45.9±4.5 nmol/L in SHR and 41.2±4.8 nmol/L in WKY) was lower than levels we had previously reported (61.6±5.6 versus 54.0±3.9,⁷ 63.4±3.9 versus 54.8±3.1,⁸ 59.9±5.7 versus 52.4±3.9,⁹ and 70.9±4.1 versus 62.2±4.6 nmol/L¹⁰ in SHR and WKY, respectively). If fura 2-AM was incompletely hydrolyzed, [Ca²⁺]_i would be underestimated. However, this possibility can be rejected because the ratio of R_max to R_min did not differ between fura 2–loaded platelets and the fura 2 solution. The difference in [Ca²⁺]_i may have resulted from differences in the rat supplier, type of cuvette, or cell number of fura 2–loaded platelets. In this study, increased basal and thrombin-stimulated [Ca²⁺]_i values in SHR platelets were observed, as previously reported. Therefore, the differences in calculated [Ca²⁺]_i level between investigations may not be so important.

A positive correlation between BP and basal [Ca²⁺]_i in platelets has been repeatedly reported.^{1 3 19 20} However, we have demonstrated in our series of studies that platelet [Ca²⁺]_i does not simply correlate with BP. First, we demonstrated increased basal and thrombin-stimulated [Ca²⁺]_i in SHR compared with WKY at 12 to 14 weeks of age.^{7 9 10} However, this abnormality already existed in 4-week-old prehypertensive SHR, whose BP was the same as that of control WKY.⁸ These results indicate that the processes controlling [Ca²⁺]_i are already modified in blood platelets before the onset of overt hypertension. Second, in deoxycorticosterone acetate–salt hypertensive rats, an experimental model of acquired hypertension, basal [Ca²⁺]_i level was decreased and thrombin-stimulated [Ca²⁺]_i with extracellular Ca²⁺ was similar to that in control rats.²⁷ In another model of salt-induced hypertension, the Dahl salt-sensitive hypertensive rat, basal and thrombin-stimulated [Ca²⁺]_i values were decreased by a high NaCl diet.²⁸ In the present study, basal [Ca²⁺]_i levels did not differ between SHRSP and WKY. Thrombin-stimulated [Ca²⁺]_i in SHRSP was significantly decreased compared with that in WKY. These results indicate that elevated basal [Ca²⁺]_i and enhanced thrombin-stimulated [Ca²⁺]_i rise are limited to SHR among hypertensive rat strains and not obligatory to hypertension.

This study is the first to examine intracellular Ca²⁺ metabolism in SHRSP platelets with the use of the fura 2 method and to detect attenuated intracellular Ca²⁺ mobilization. It is widely accepted that platelet aggregation responses in SHRSP are decreased.^{24 29 30} The decreased platelet aggregation response in SHRSP can be explained by the attenuated Ca²⁺ metabolism because [Ca²⁺]_i is an important second messenger that regulates aggregation, shape change, and the secretional response.

In summary, in contrast to SHR, basal [Ca²⁺]_i in SHRSP was similar to that in WKY and the thrombin-stimulated [Ca²⁺]_i rise and aggregation response were attenuated. These results suggest that platelet Ca²⁺ handling differs between SHR substrains and that an increased [Ca²⁺]_i is not obligatory in genetically hypertensive rats.

	Selected Abbreviations and Acronyms

 

[Ca2+]i = cytosolic free Ca2+ concentration BP = blood pressure SHR = spontaneously hypertensive rat(s) SHRSP = stroke-prone spontaneously hypertensive rat(s) WKY = Wistar-Kyoto rat(s)

	Acknowledgments

 
This study was supported by a Grant-in-Aid for Scientific Research (No. 07407065). The authors wish to thank A. Nagaoka, Takeda Chemical Industries Ltd, Osaka, Japan, and M. Yoshimura for technical support. The authors also thank Yuko Omura for the secretarial assistance.

Received June 23, 1995; first decision September 21, 1995; accepted February 5, 1996.

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