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(Hypertension. 1995;25:377-383.)
© 1995 American Heart Association, Inc.

Articles

Increased Calcium Stores in Platelets From African Americans

Jwa Hwa Cho; Frederic Nash; Zoltan Fekete; Masayuki Kimura; John P. Reeves; Abraham Aviv

From the Hypertension Research Center, Departments of Internal Medicine and Physiology, University of Medicine and Dentistry of New Jersey, Newark.

Correspondence to Abraham Aviv, MD, Hypertension Research Center, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, 185 S Orange Ave, MSB F-464, Newark, NJ 07103-2714.

	Abstract

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Abstract Differences in cation transport have been observed between African Americans and whites. These differences may underlie the increased predisposition of African Americans to essential hypertension. To further explore these racial differences, we used platelets as a cellular model for calcium regulation. We measured ⁴⁵Ca fluxes in platelets from 21 African American and 25 white men. Additionally, using fura 2, we measured cytosolic free calcium levels in resting platelets and platelets treated with ouabain and thrombin. Platelet ⁴⁵Ca uptake was described by two exchangeable pools: a small, rapidly exchangeable pool and a larger, slowly exchangeable pool. Both pools were larger in platelets from African Americans than from whites (263 versus 185 pmol per 1x10⁸ platelets for the rapidly exchangeable pool, P<.05; 744 versus 532 pmol per 1x10⁸ platelets for the slowly exchangeable pool, P<.01). ⁴⁵Ca washout was described by a rapidly exchangeable pool and a static pool. The former was also higher in platelets from African Americans than from whites (246 versus 202 pmol per 1x10⁸ platelets, P<.01). The cytosolic free calcium concentrations in resting platelets were lower in African Americans than in whites. After treatment with ouabain and thrombin, the sustained posttransient levels of cytosolic free calcium increased to a greater extent in platelets from African Americans (46.7 nmol/L) than from whites (34.5 nmol/L, P=.033). Platelets from African Americans demonstrate higher intracellular calcium stores than platelets from whites. This racial difference could explain the sensitivity of African Americans to vasoactive agents acting through calcium mobilization from intracellular stores and cytosolic calcium.

Key Words: calcium pump • calcium channels • whites • hypertension, essential • ouabain • parathyroid hormone • thrombin

	Introduction

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Ion regulation at the cellular and systemic levels differs between African Americans and whites.¹ The mechanisms responsible for these racial differences are poorly understood but could relate to both genetic and environmental factors. These mechanisms may underlie the increased predisposition of African Americans to essential hypertension and its cardiovascular complications.^{2 3}

Using platelets as a cellular model, we recently showed that cellular calcium regulation differs in African Americans and whites.⁴ Moreover, the results suggested that calcium stores in the dense tubules are higher in platelets from African Americans. In our previous work, we used thapsigargin, an inhibitor of the calcium-ATPase in the endo(sarco)plasmic reticulum (SERCA-ATPase), to unravel differences in platelet calcium regulation between African Americans and whites. Inhibition of the SERCA-ATPase results in the gradual depletion of calcium from the endo(sarco)plasmic reticulum (or dense tubules in platelets) and the consequent opening of plasma membrane calcium channels.^{5 6 7} The rate of calcium entry to the cytosol from the external medium and from intracellular organelles and the rate of calcium extrusion can then be monitored. Under these circumstances, we demonstrated an increased calcium turnover rate (namely, enhanced calcium entry into and extrusion from the cytosol) in platelets from African Americans. The results also suggested that the amount of calcium in the dense tubules was greater in African Americans than in whites. However, these racial differences in platelet calcium homeostasis were demonstrated under specific, non–steady-state conditions. It is conceivable that such racial differences could arise from mechanisms that do not operate under physiological, steady-state conditions. For instance, it is possible that platelets from African Americans are more sensitive to thapsigargin than platelets from whites. Another possibility is that in response to thapsigargin the relay mechanisms between the dense tubules and the plasma membrane to open the calcium channels operate differently in African Americans than in whites. It was therefore imperative to examine calcium turnover rate and intracellular calcium stores in platelets from African Americans under steady-state conditions and without the inhibition of SERCA-ATPase. This has been the central aim of the present work. Additionally, using fura 2, we examined the effects of ouabain and thrombin on the platelet cytosolic calcium profile. The results strongly support the concept of increased calcium stores in platelets (and presumably other cells) from African Americans compared with those from whites. These racial differences in cellular calcium regulation could play a key role in predisposing African Americans to essential hypertension.

	Methods

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General Procedures
We studied 46 men (21 African Americans and 25 whites). All whites were of European extraction. Subjects were without any apparent disease (including sickle-cell trait in African Americans) and received no medications (including antiplatelet agents) for at least 2 weeks before the study. African Americans reporting that one of their parents or grandparents was white were excluded. All subjects originated from the staff and student body of the University of Medicine and Dentistry of New Jersey. Each subject was examined by the same investigator (F.N.) between 8 and 9:30 AM in the outpatient clinic of the University of Medicine and Dentistry of New Jersey, New Jersey Medical School. After measurements of height and weight were taken, the subject was comfortably seated for at least 10 minutes. During this period he was questioned in detail concerning family history of essential hypertension and non–insulin-dependent diabetes mellitus. Then, informed consent was obtained, and three consecutive blood pressure measurements were taken at 1-minute intervals from the dominant arm with a mercury sphygmomanometer. Seventy milliliters of fasting blood was then drawn from the antecubital vein. The first 50 mL was taken into acid citrate dextrose buffer (20/1, vol/vol) consisting of (mmol/L) sodium citrate 14, citric acid 11.8, and dextrose 18 (final pH 6.5). The remaining blood was taken for measurements of serum lipids, including lipoprotein(a), serum insulin, intact parathyroid hormone, and plasma glucose.

Blood samples were numerically labeled (by F.N.) and immediately transferred to the facilities of the Hypertension Research Center of the University of Medicine and Dentistry of New Jersey where platelet calcium parameters were measured by an investigator (J.H.C.) who did not know the origin of the samples. The remaining samples were stored at -20°C between 3 and 6 months for further measurements (by J.H.C. and Z.F.). The numerical code of the samples was broken only at the completion of the study after platelet calcium parameters for all subjects had been analyzed.

Platelet Preparation
Platelets were isolated at room temperature by differential centrifugation using a slightly modified procedure that has been previously described.⁸ Briefly, platelet-rich plasma was obtained by centrifugation at 200g for 10 minutes. Plasma was then centrifuged at 1000g for 10 minutes, and the platelet pellet was washed three times by centrifugation at 1000g for 10 minutes in buffer consisting of (mmol/L) NaCl 140, KCl 5, glucose 10, HEPES 10, acetylsalicylic acid 0.1, and EGTA 0.2 (pH 7.4, adjusted by NaOH). Bovine serum albumin (0.1%, wt/vol) was added to the third washing. Platelets were resuspended in HEPES buffer consisting of (mmol/L) NaCl 140, KCl 5, MgCl₂ 1, glucose 10, HEPES 10, and EGTA 0.1 (pH 7.4 at 37°C).

⁴⁵Ca Uptake and Washout Experiments
Platelets were incubated for 30 minutes at 37°C in HEPES buffer, except for the omission of EGTA and addition of 1 mmol/L CaCl₂ (pH 7.4), and their number was adjusted to 6x10⁸/mL. Multiple 20-µL aliquots of the platelet suspension were dispensed into 7.5-mL glass tubes. Then, the uptake experiments were initiated by the addition of 20 µL of buffer containing ⁴⁵Ca (specific activity, 12.7 to 18.7 mCi/mg; NEZ-013, NEN-DuPont Biotechnology; final activity, 50 to 100 µCi/mL). Platelet suspensions were incubated at 37°C for up to 240 minutes. To stop the ⁴⁵Ca uptake, 5 mL ice-cold HEPES buffer (plus 0.3 mmol/L EGTA) was added to each tube, and the extracellular radioactivity was removed by rapid filtration through HAWP filters (pore size, 0.45 µm; Millipore) using two additional 5-mL aliquots of ice-cold washing buffer. The instantaneous ⁴⁵Ca uptake (⁴⁵Ca added and extracellular radioactivity immediately removed by filtration) was subtracted from the uptake values.

⁴⁵Ca washout experiments were initiated after 120 minutes of ⁴⁵Ca uptake. For this purpose, the 40 µL of ⁴⁵Ca uptake medium plus platelets was diluted into 5 mL assay buffer (37°C) and the extracellular radioactivity periodically removed by filtration with three additional 5 mL of ice-cold washing buffer. The washout experiments lasted for 120 minutes. Measurements for the uptake and washout experiments were obtained in quadruplicate.

Because of concern regarding platelet stability, ⁴⁵Ca transport experiments were carried out for a maximum of 4 hours. The capacity of platelets to maintain stable total calcium within 4 hours was previously shown⁹ ; however, only a small portion of total platelet calcium is exchangeable during this period. Thus, the size of the calcium pools should be considered in relative terms.

Monitoring Cytosolic Free Calcium
Platelets were loaded for 30 minutes at 37°C with 5 µmol/L fura 2 acetoxymethyl ester (Molecular Probes) in calcium-containing HEPES buffer. The final centrifugation (to remove the extracellular dye) can activate some of the platelets and raise the resting cytosolic free calcium. It was therefore possible that different responses of the platelets to the final centrifugation could account for racial differences in the resting cytosolic free calcium. We used three approaches to ascertain that this is not the case. (1) We measured cytosolic calcium after removal of the extracellular dye by centrifugation. This was accomplished by a 7-second spin at maximal 3000g, rapid resuspension of the platelets in 100 µL HEPES buffer, and the immediate injection of the platelets into cuvettes with 3 mL of the same buffer for fluorescence monitoring. (2) We measured cytosolic calcium by injecting 100 µL platelets loaded with dye (without removal of the extra-cellular dye) into 3 mL HEPES buffer plus 0.5 mmol/L MnCl₂. This maneuver resulted in an initial sharp drop followed by a gradual decline in the fluorescence signal. The instantaneous drop results from the quenching of the extracellular fura 2 by manganese. The subsequent gradual decline in the signal reflects manganese influx. The junction between the instantaneous and gradual declines in the signal represents the resting cytosolic calcium. (3) We combined the first two methods.

Cytosolic free calcium was also measured after 60 minutes of preincubation with 0.1 mmol/L ouabain before and after treatment with 0.1 NIH U/mL of human thrombin (No. T9135, Sigma Chemical Co). In these experiments, cytosolic calcium levels were monitored for 15 seconds in HEPES buffer with 1 mmol/L calcium or calcium-free HEPES buffer (CaCl₂ removed and 0.3 mmol/L EGTA added). Thrombin was then added, and the thrombin-evoked cytosolic calcium transient and posttransient cytosolic calcium were monitored for a total of 300 seconds. The following variables were recorded (Fig 1): cytosolic calcium before treatment with thrombin, the peak thrombin-evoked cytosolic calcium transient, the posttransient cytosolic calcium at 5 minutes of monitoring in calcium-free buffer, and the posttransient cytosolic calcium at 5 minutes of monitoring in calcium-containing buffer after correction with manganese for dye leakage.

View larger version (15K): [in this window] [in a new window]   Figure 1. Line graph shows parameters used for the evaluation of the thrombin-evoked cytosolic calcium response. Cytosolic calcium response is shown in HEPES buffer containing 1 mmol/L calcium (solid line) and calcium-free buffer (dotted line). The letter a indicates resting cytosolic free calcium concentration before the addition of thrombin; b and b', peak thrombin-evoked (transient) cytosolic calcium response in calcium-containing and calcium-free buffer, respectively; c and c', posttransient cytosolic calcium levels at 5 minutes of monitoring in calcium-containing and calcium-free buffer, respectively. For experiments in 1 mmol/L calcium, 0.5 mmol/L manganese was added (d) to correct for dye leakage during the duration of the experiment. Values of c'-a and d-a were used to evaluate the posttransient cytosolic calcium response compared with the resting cytosolic calcium.

Cytosolic calcium monitoring was performed with a Fluorolog II spectrofluorometer (model CM-3, SPEX Industries) at excitation wavelengths of 340 and 380 nm and an emission wavelength of 505 nm. The ratio of the maximal and minimal fluorescence was determined by the addition of 50 µmol/L digitonin followed by 15 mmol/L EGTA (pH 8.5). Autofluorescence was determined at the end of each experiment by the addition of 0.5 mmol/L MnCl₂ and 50 µmol/L digitonin.

Other Measurements
Intact parathyroid hormone (PTH-1-84) was measured using an immunoradiometric assay (Nichols Institute Diagnostics). Total cholesterol, high-density lipoprotein cholesterol, and triglycerides were measured with a Kodac Ectachem DT 60 analyzer and lipoprotein(a) with an Apo-Tek Lp(a) ELISA test system (Organon Teknika/Biotechnology Research Institute). Immunoreactive insulin was measured by a radioimmunoassay (Coat-A-Count, Diagnostic Products Corp).

Data Analysis
⁴⁵Ca uptake and washout data were best fitted to biexponential and monoexponential functions, respectively (Fig 2). Differences in the ⁴⁵Ca uptake and washout parameters between platelets from African Americans and whites were evaluated by the Johnson and Milliken method of modified least squares to fit parameters of nonlinear models.¹⁰ Other statistical methods included Pearson's correlation analysis, unpaired Student's t test, and Wilcoxon's rank sum test [in the case of lipoprotein(a), which demonstrated a non-Gaussian distribution in whites].

View larger version (18K): [in this window] [in a new window]   Figure 2. Line graphs show 45Ca uptake and washout. Open and closed symbols denote African Americans and whites, respectively. Data were fitted (lines) to the following models: 45Ca uptake=au1(1-exp 1/ku1t)+au2(1-exp 1/ku2t), where au1 and au2 are the small, rapidly exchangeable and the larger, slowly exchangeable 45Ca pools, respectively, and ku1 and ku2 are their respective uptake rate constants; 45Ca washout=aw exp 1/kwt+C, where aw and kw are the rapidly exchangeable 45Ca pool and its washout rate constant, respectively, and C is the static 45Ca pool. For the 45Ca uptake data (top), calcium accumulation in platelets was calculated based on the extracellular calcium concentration and medium activity of the 45Ca. Vertical bars denote SEM. Table 3 presents the parameters derived from the data with these models.

Because of technical difficulties, a complete set of cytosolic calcium profiles and ⁴⁵Ca transport measurements could not be obtained in platelets from all subjects.

	Results

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General Characteristics
Characteristics of the sample population are presented in Tables 1 and 2. There were no significant differences between African Americans and whites in age, body mass index, and blood pressure (Table 1). There were also no differences between the groups in glucose, insulin, and intact parathyroid hormone levels and the lipid profiles, except for lipoprotein(a), which was significantly higher in African Americans (Table 2). Higher levels of serum lipoprotein(a) in African Americans were observed in previous studies.^{4 11 12} Body mass index positively correlated with systolic pressure (r=.525, P=.001), diastolic pressure (r=.373, P=.0106), mean arterial pressure (r=.474, P=.0009), serum triglycerides (r=.525, P=.002), total cholesterol (r=.424, P=.0037), and low-density lipoprotein cholesterol (r=.366, P=.0134). Age weakly correlated with systolic pressure (r=.294, P=.047), but there was no significant correlation between age and body mass index in this sample population.

View this table: [in this window] [in a new window]   Table 1. Subject Characteristics

View this table: [in this window] [in a new window]   Table 2. Blood Chemistries

⁴⁵Ca Transport
⁴⁵Ca uptake was described by two exchangeable calcium pools (Fig 2, Table 3). The more rapidly exchangeable pool (a_u1) was substantially smaller than the slowly exchangeable pool (a_u2). Platelets from African Americans showed a greater accumulation of ⁴⁵Ca than platelets from whites. These racial differences related primarily to the greater pool sizes, which were higher by 42.4% for a_u1 and 39.8% for a_u2 in platelets from African Americans (Table 3).

View this table: [in this window] [in a new window]   Table 3. Kinetics of 45Ca Uptake and Washout

⁴⁵Ca washout was also described by two calcium pools: a pool that rapidly exchanged ⁴⁵Ca with the external medium (a_w) and a static pool (C) with no apparent ⁴⁵Ca exchange with the external medium within the monitoring period (Fig 2, Table 3). As ⁴⁵Ca uptake for 120 minutes was higher in platelets from African Americans, the pool size at the initiation of the ⁴⁵Ca washout was greater for platelets of African Americans than for those of whites. When ⁴⁵Ca washout data were normalized and expressed as a percentage of the initial activity (100% at the initiation of the ⁴⁵Ca washout; Fig 2, Table 3), ⁴⁵Ca washout was essentially identical in platelets from African Americans versus whites. However, in absolute values, the size of the rapidly exchangeable ⁴⁵Ca washout pool was greater in platelets from African Americans than in those from whites (246.5±7.5 versus 201.7±7.2 pmol per 1x10⁸ cells, P<.01). This difference reflects the higher levels of ⁴⁵Ca in platelets of African Americans at the initiation of washout. No racial differences were observed in absolute terms in the size of the apparently static pool of the ⁴⁵Ca washout (173.5±8.3 and 152.3±7.9 pmol per 1x10⁸ cells for African Americans and whites, respectively).

Cytosolic Free Calcium Monitoring With Fura 2
The low levels of resting platelet cytosolic free calcium recorded in this work (Table 4) and our previous work^{4 5} are in agreement with recent findings by other researchers.^{13 14 15} A lower resting free calcium concentration in African Americans than in whites was observed with the use of the three approaches described under "Methods," namely, platelets undergoing final centrifugation to remove the extracellular dye, platelets without the final centrifugation but subjected to manganese in the monitoring solution, and platelets subjected to both the final centrifugation and manganese in the monitoring solution (Table 4). These results concur with our previous observations,⁴ although the absolute values of resting cytosolic calcium in the present study were slightly higher than those reported previously.⁴

View this table: [in this window] [in a new window]   Table 4. Resting Cytosolic Free Calcium

There were no racial differences in the peak thrombin-evoked cytosolic calcium transient between African Americans and whites (289.3±7.5 versus 277.2±8.0 nmol/L in 1 mmol/L calcium and 185.4±7.2 versus 196.3±5.9 nmol/L in calcium-free buffer). Moreover, no racial differences were observed in the cytosolic calcium levels after 1 hour of treatment with ouabain, before exposure to thrombin (42.9±1.6 versus 42.1±1.7 nmol/L for whites and African Americans, respectively). The peak thrombin-evoked cytosolic calcium response of ouabain-treated platelets was also not different between the two groups (307.0±9.0 versus 296.3±7.7 nmol/L with calcium and 287.5±8.4 versus 283.6±8.6 nmol/L in calcium-free buffer, whites versus African Americans). However, the difference between the posttransient cytosolic calcium at 300 seconds (after correction for dye leak by manganese in 1 mmol/L CaCl₂) and the cytosolic calcium levels before the addition of thrombin was significantly higher for ouabain-treated platelets from African Americans than from whites (for calcium-containing buffer, 46.7±4.2 versus 34.5±3.7 nmol/L, P=.033; for calcium-free buffer, 3.0±1.3 versus -1.0±1.4 nmol/L, P=.046).

Correlations Between Platelet Calcium Parameters With Blood Chemistries
A negative correlation was observed between serum intact parathyroid hormone and the rate constant (k_w) for ⁴⁵Ca washout (Fig 3). No significant correlations were noted between platelet calcium parameters and other blood chemistries, blood pressure parameters, and family history of essential hypertension and non–insulin-dependent diabetes mellitus.

View larger version (16K): [in this window] [in a new window]   Figure 3. Scatterplot shows the relation between kw (45Ca washout rate constant) and circulating intact parathyroid hormone (PTH). Open and closed symbols denote African Americans and whites, respectively.

	Discussion

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The results of the present and previous work⁴ are consistent with studies by other researchers showing lower cytosolic calcium in resting platelets of blacks compared with whites.^{16 17} More important, this work complements the previous investigation⁴ that used the fluorescent dye fura 2 to demonstrate a higher calcium turnover rate in thapsigargin-treated platelets from African Americans compared with those from whites. The results of that study also suggested that intracellular calcium stores are higher in platelets from African Americans. However, these results were obtained under special circumstances, namely, non–steady-state conditions with respect to cytosolic calcium and a dense tubule calcium-ATPase that was inhibited by thapsigargin.⁴ Findings in platelets also concur with the study showing that on stimulation with serum, cultured skin fibroblasts from African Americans manifested a higher calcium turnover rate than fibroblasts from whites, a process that was primarily due to greater calcium mobilization from intracellular organelles in the former group.¹⁸ Thus, racial differences in calcium turnover rate are not limited to a single cell type.

The nature of the pools containing more calcium in platelets from African Americans than from whites cannot be readily ascertained from the mathematical analyses of the ⁴⁵Ca uptake or washout data. It is likely that a_u1 is associated with the cytosol and intracellular organelles that exchange calcium rapidly with the cytosol. In the nonstimulated state, a_u2 may be associated with cellular structures such as the dense tubules and secretory granules, which could slowly exchange calcium with the cytosol. However, these are only rough extrapolations from the mathematical models to distinct biological structures. As pointed out previously,⁹ the distribution of calcium within the cytosol and intracellular organelles such as the dense tubules is unlikely to be homogeneous. Thus, the cellular pools expressed in the ⁴⁵Ca uptake and washout are likely to reflect the input of groups of pools. This concept is illustrated by the observation that cytosolic free calcium concentrations are slightly lower yet a_u1 is larger in platelets of African Americans than in those of whites. It is possible that despite lower cytosolic calcium concentrations, the pool size of cytosolic calcium is larger in African Americans. Alternatively, and perhaps more likely, a_u1 may not entirely represent the cytosol but only a portion of this biological compartment. It is of note, nonetheless, that despite a number of experimental differences between this study and that of Brass,⁹ the size of the rapidly exchangeable ⁴⁵Ca pool in both studies is approximately one third that of the slowly exchangeable pool. A major portion of a_u1 probably overlaps with a_w of the washout experiments. Likewise, a_u2 may in part overlap with C of the washout experiments.

As the capacity to sequester calcium in intracellular organelles is higher in platelets from African Americans than in those from whites, we expected that inhibition of calcium efflux by the sodium-calcium exchange, which is present in human platelets,^{19 20} would promote a greater accumulation of calcium in platelets from African Americans. We explored this concept by monitoring cytosolic free calcium (using fura 2) after treatment with ouabain. Ouabain inhibits the sodium pump and reduces the transmembrane sodium gradient. This results in the inhibition of the forward mode of the sodium-calcium exchanger, which depends on the transmembrane sodium gradient.

Treatment with ouabain did raise the cytosolic calcium. However, cytosolic free calcium concentrations before treatment with thrombin and the peak thrombin-evoked cytosolic calcium transients were not different in ouabain-treated platelets from African Americans compared with those from whites. Several reasons may account for these unexpected findings. (1) Platelets from African Americans appear to possess not only increased sequestering capacity but also a greater capacity to extrude calcium, thereby masking an influence of increased releasable calcium stores on cytosolic free calcium.⁴ Although the steady-state efflux experiments in Fig 2 did not reveal any difference in the rate constants of calcium efflux between African Americans and whites, the magnitude of steady-state calcium efflux was greater in the African Americans by virtue of their larger exchangeable calcium pools. Thus, the efflux results are consistent with this possibility. (2) Thrombin stimulation mobilizes only a fraction of stored calcium in both ouabain-treated and ouabain-untreated cells, and this partial mobilization, usually reflected in the peak of the thrombin-evoked calcium transients, could be insufficient to reveal racial differences in the size of calcium stores. (3) An increased sequestering capacity of the dense tubules in African Americans could attenuate the effects of calcium release on cytosolic free calcium concentrations because of increased reuptake of calcium into the dense tubules. (4) The increased intracellular calcium in platelets from African Americans may reside in thrombin-insensitive or nonreleasable compartments. In any event, ouabain-treated platelets from African Americans demonstrated a lag in cytosolic calcium recovery toward resting calcium after the transient phase of calcium release. This might reflect the influence of increased calcium influx, the effects of a greater calcium load on the extrusion process, or a greater dependence of calcium efflux on sodium-calcium exchange in African Americans compared with whites. It is clear that further studies will be required to address this issue.

A number of publications have suggested that circulating parathyroid hormone levels positively correlate with blood pressure and are elevated in some essential hypertensive individuals.^{21 22 23} As previous reports have also documented elevated parathyroid hormone levels in African Americans,^{16 24} we explored the relation between platelet calcium regulation and parathyroid hormone. We could identify neither racial differences in intact parathyroid hormone levels nor a positive correlation between intact parathyroid hormone levels and blood pressure. The reasons for the inconsistency among the studies are unclear. In this context, the study¹⁶ documenting racial differences in intact parathyroid hormone levels and the present work showed substantial scatter of the data for both African Americans and whites.

Nonetheless, the negative correlation in the present study between intact parathyroid hormone levels and the rate constant of ⁴⁵Ca washout from the rapidly exchangeable calcium pool suggests that mechanisms engaged in parathyroid hormone regulation and the effect of the hormone on target cells are somehow involved in platelet calcium homeostasis. It is doubtful that a direct interaction between parathyroid hormone and circulating platelets accounts for the findings, as in vitro no effect of human intact parathyroid hormone was demonstrated on platelet ⁴⁵Ca washout parameters at concentrations (10 and 100 pg/mL) within the range documented in the present work for endogenous levels of the hormone (not shown).

Essential hypertension may be marked by a state of insulin resistance and elevated fasting serum insulin (reviewed in Reference 25²⁵ ). Additionally, platelet function and cytosolic calcium homeostasis could be influenced by circulating lipoproteins.^{26 27} However, no correlations were observed in the present study between insulin, lipoproteins, and any of the platelet cytosolic calcium parameters.

Based on this study and our previous work,⁴ we propose the following to explain differences in platelet calcium regulation between African Americans and whites. Larger intracellular pools of exchangeable calcium are present in platelets of African Americans than in those from whites. As the resting cytosolic free calcium concentration under steady state is not higher and is in fact lower in platelets from African Americans, it is logical to conclude that the sequestering mechanisms of calcium into intracellular pools are more active in platelets from African Americans than in those from whites. The augmented capacity to sequester calcium probably involves the dense tubule calcium-ATPase.

Earlier studies demonstrated a positive relation between resting platelet calcium and blood pressure and substantially higher levels of resting platelet calcium in some patients with essential hypertension than in normotensive subjects (eg, see References 28²⁸ and 29²⁹ ). More recent reports documented small differences in resting platelet calcium between normotensive and hypertensive subjects or no correlation between this parameter and the systemic blood pressure (eg, References 30³⁰ and 31³¹ ). We also failed to show any relation between resting platelet calcium and the systemic blood pressure.³² Our findings therefore do not support the concept that a higher resting platelet calcium level reflects the presence of or the predisposition to essential hypertension. In this regard, the resting calcium levels in vitro may bear little relevance to the cytosolic calcium profiles in vivo, a state in which calcium continuously oscillates.

If the phenomenon of increased exchangeable calcium stores is not limited to platelets but includes cells of the vascular bed, it may result in increased sensitivity to vasoactive agents that raise the cytosolic calcium, including a number of hormones and autacoids and particularly endogenous ouabain³³ or other ouabainlike factors that inhibit the sodium pump. Moreover, increased intracellular calcium stores in African Americans could explain the greater effectiveness of calcium antagonists than ß-adrenergic blockers or angiotensin-converting enzyme inhibitors in the treatment of essential hypertension in this racial group.^{34 35 36}

	Acknowledgments

 
This work was supported by grants from the National Heart, Lung, and Blood Institute (HL-47906 and HL-49932). Z. Fekete is a Postdoctoral Research Fellow of the American Heart Association, New Jersey Affiliate; F. Nash is a National Institutes of Health Minority Postdoctoral Research Fellow. We thank Dr Joan Skurnick for the statistical analysis, Girgis Awad for subject recruitment, and Patricia Peluso for her secretarial skills.

Received April 29, 1994; first decision July 19, 1994; accepted October 2, 1994.

	References

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