MATERIALS AND METHOD

Subjects.

We studied 30 patients with essential hypertension (15 men, 15 women, mean age 51 ± 11 yr) and 30 sex- and age-matched normotensive controls (15 men, 15 women, mean age 50 ± 13 yr). Normotensive controls were recruited from healthy subjects who underwent annual physical examinations. Hypertension was defined as systolic blood pressure ≥160 mmHg and/or diastolic blood pressure ≥95 mmHg on each of three consecutive clinical visits. We measured blood pressure with a mercury sphygmomanometer in sitting subjects at least five times during each clinical visit and used the average value of these measurements. The blood pressure in normotensives was consistently <140/90 mmHg. None of the hypertensives or normotensives had received any medication for at least 4 wk before the study. Subjects with secondary forms of hypertension were excluded by careful clinical examination. Hypertensive patients and normotensive controls were maintained on a regular diet with an intake of 170 mmol/day NaCl to allow stabilization of the systemic Na⁺ balance, and they ingested constant amounts of K⁺ (2,000 mg/day), Ca²⁺ (500 mg/day), and calories (40 kcal/kg) for 7 days before the study. Venous blood was collected from fasting and resting subjects, slowly and steadily via a 19-gauge needle into a syringe containing 3.8% trisodium citrate (1:9 by vol, total 30 ml), using a two-syringe method (21) to separate platelets for measurement of [Mg²⁺]_iand [Ca²⁺]_i. Blood samples were centrifuged at room temperature for 5 min at 800g, and [Mg²⁺]_iand [Ca²⁺]_iwere measured by use of the resultant platelet-rich plasma. To minimize any time-dependent effects on platelet responsiveness and leakage of dyes, measurements of platelet [Mg²⁺]_iand [Ca²⁺]_iwere performed separately by two independent investigators (HH measured [Ca²⁺]_i;MY measured [Mg²⁺]_i) in the same blood samples within 2 h after blood collection. Serum concentrations in electrolytes and lipids and mean platelet volume were measured by automated methods, and plasma renin activity and plasma aldosterone concentration were assayed by RIA in another blood sample.

Measurement of platelet [Ca²⁺]_i.

Platelet [Ca²⁺]_iwas measured as described previously (13, 21, 22). In short, platelet-rich plasma prepared as described above was layered onto a Sepharose 2B-CL column (Pharmacia LKB Biotechnology, Uppsala, Sweden) that had been equilibrated with medium containing (in mM) 145 NaCl, 10 HEPES, 5 KCl, 5 glucose, and 1 MgSO₄ (pH 7.4) at room temperature. Washed platelets were eluted from this column with buffer and incubated at 37°C with 1 μM fura 2-AM (Molecular Probes, Eugene, OR) and 0.02% Pluronic F-127 (Molecular Probes) for 30 min at a platelet concentration of 10⁸cells/ml. After platelets had again been washed by gel filtration to remove any extracellular fura 2-AM, the platelet count was adjusted to 10⁷ cells/ml, and CaCl₂ was added to the cell suspension at a final concentration of 1 mM. Incubation at 37°C for 7 min was performed to complete the hydrolysis of fura 2-AM, and platelet suspensions were then placed in cuvettes with stirrers at 37°C. Fluorescence was measured with a dual-excitation wavelength fluorometer (RF-5000, Shimadzu, Kyoto, Japan), using excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm. After fluorescence in the basal state had been recorded, the [Ca²⁺]_iresponses to thrombin (0.03, 0.1, 0.3, and 1.0 U/ml; Sigma Chemical, St. Louis, MO) were measured in the presence of 1 mM extracellular Ca²⁺ and in Ca²⁺-free buffer prepared by the addition of 10 mM EGTA (Dojindo Laboratories, Kumamoto, Japan; Fig.1). The discharge capacity of Ca²⁺ from intracellular storage sites was estimated by the [Ca²⁺]_iresponse to 5 μM ionomycin (Sigma) in the Ca²⁺-free medium (13, 22). [Ca²⁺]_iwas calculated using the following equation from Grynkiewicz et al. (7)

In the preliminary study, to determine whether citrate alone is sufficient to prevent cell activation during the preparation of platelets, we studied the effects of other anticoagulant agents on Ca²⁺ handling by gel-filtered platelets of essential hypertensives and normotensives. Platelet-rich plasma was divided into two batches. Apyrase (20 μg/ml), hirudin (0.05 U/ml), and PGI₂ (1 μM) were added to one batch, and no agent was added to the other batch. These conditions were maintained during fura 2 loading. After the cells were gel filtered, [Ca²⁺]_iwas determined in the two batches. There was no effect of the anticoagulant cocktail on basal [Ca²⁺]_ior thrombin (0.1 U/ml)-stimulated [Ca²⁺]_iin seven subjects with essential hypertension (percentage of control: 101 ± 3 and 98 ± 5%, respectively) and eight normotensive controls (100 ± 4 and 99 ± 4%, respectively). We thus concluded that the use of citrate alone may be sufficient to inhibit [Ca²⁺]_ielevation induced by cell activation, when gel filtration is used to separate platelets.

Measurement of platelet [Mg²⁺]_i.

The washed platelets were incubated at 37°C with 2 μM mag-fura 2-AM (Molecular Probes) and 0.02% Pluronic F-127 for 30 min at a platelet concentration of ∼5 × 10⁷ cells/ml. Platelets were then washed again to remove extracellular mag-fura 2-AM, and CaCl₂ was added at a final concentration of 1 mM after resuspension of platelets in HEPES buffer at a platelet concentration of 10⁷cells/ml. Platelet suspension (3 ml) was incubated at 37°C for 7 min to complete the hydrolysis of mag-fura 2-AM, and samples were then placed in cuvettes and stirred magnetically at 37°C. Fluorescence was measured with a dual-excitation wavelength fluorometer (DM3000CM, SPEX, Edison, NJ), as described above. [Mg²⁺]_iwas calculated using the equation from Raju et al. (24)

Statistics.

Data are presented as means ± SD. Statistical comparisons were made using the Mann-Whitney U test. Curves were compared by using ANOVA for repeated measures. Statistical significance was defined as P < 0.05.

RESULTS

Basic information on the study subjects is shown in Table1. There were no significant differences in serum total cholesterol, triglyceride, high-density lipoprotein-cholesterol, plasma renin activity, plasma aldosterone concentration, or mean platelet volume between the hypertensive and normotensive control groups. No differences were detected in the concentrations of platelet intracellular mag-fura 2 or fura 2 (hypertensives vs. normotensives: 402 ± 42 vs. 390 ± 45, 493 ± 81 vs. 512 ± 61 μM, respectively) or in extracellular leakage of mag-fura 2 or fura 2 (31.0 ± 4.4 vs. 30.0 ± 3.7, 8.6 ± 1.7 vs. 9.3 ± 1.7%), R_max of mag-fura 2 or fura 2 (31.9 ± 3.8 vs. 32.4 ± 3.5, 39.3 ± 10.0 vs. 35.7 ± 9.7), or R_min of mag-fura 2 or fura 2 (0.72 ± 0.02 vs. 0.73 ± 0.02, 0.83 ± 0.05 vs. 0.83 ± 0.05), indicating that platelets were loaded with the dyes to a similar extent in the two groups.

Platelet basal [Ca²⁺]_iwas significantly higher in the hypertensive group than in the normotensive group (22.3 ± 5.3 vs. 17.8 ± 5.3 nM; Fig.2), although there was a considerable overlap in distribution between groups. Thrombin (0.03, 0.1, 0.3, and 1.0 U/ml)-evoked [Ca²⁺]_iresponses were significantly enhanced in the hypertensive group in the presence or absence of extracellular Ca²⁺ (Fig.3, A andB). Differences in [Ca²⁺]_iincrease between the presence and absence of extracellular Ca²⁺, representing thrombin-evoked external Ca²⁺ influx, were also enhanced in the hypertensive group (Fig.3C); i.e., external Ca²⁺ influx and discharge of Ca²⁺ from intracellular stores were both enhanced in thrombin-stimulated platelets from the hypertensive group. The discharge capacity of Ca²⁺ from intracellular storage sites, which was assessed by the [Ca²⁺]_i response to the addition of 5 μM ionomycin in a Ca²⁺- free medium, was greater in the hypertensive than the normotensive group (743.0 ± 250.4 vs. 624.2 ± 144.2 nM). However, basal [Mg²⁺]_iwas significantly higher in hypertensives than in normotensives (436.6 ± 109.9 vs. 353.0 ± 85.3 μM, Fig.4), whereas serum total Mg²⁺ was similar in the two groups (Table 1).

DISCUSSION

We (19) and other investigators (5, 9) have reported an increase in the intracellular concentration of Na⁺and in basal [Ca²⁺]_iin blood cells, such as platelets and lymphocytes, of subjects with essential hypertension. In the present study, the basal [Ca²⁺]_iin platelets was elevated in hypertensive subjects, confirming most previous results (5, 8, 11, 16, 30). However, there was a considerable overlap in distribution between hypertensives and normotensives. This overlap may be due to the heterogeneity of essential hypertension, which should not be regarded as a single disease entity. Essential hypertensive patients have heterogeneity in several factors, such as renin status (19), blood pressure level (5), age (2), and salt intake (20), each of which may influence intracellular cation characteristics and are difficult to control precisely.

The mechanisms that contribute to evoked [Ca²⁺]_iunder stimulated conditions are different from those regulating basal [Ca²⁺]_i. Most previous reports have been limited to the measurement of basal [Ca²⁺]_i. Even in a few previous studies with stimulated platelets, the status of the [Ca²⁺]_iresponse was controversial: an enhanced [Ca²⁺]_iresponse to thrombin was reported by Lechi et al. (16) and to ANG II by Touyz and Schiffrin (30) in platelets from hypertensive patients, whereas Haller et al. (8) showed that the change in [Ca²⁺]_iin response to thrombin was similar in platelets from hypertensives and those from normotensives. In the present study, the evoked [Ca²⁺]_iresponses to thrombin were enhanced in hypertensives, both in the absence and presence of extracellular Ca²⁺. Platelets from hypertensive subjects exhibited not only an increase in basal [Ca²⁺]_ibut also an enhanced Ca²⁺discharge from intracellular stores, an increase in Ca²⁺ influx under thrombin-stimulated conditions, and an increase in intracellular Ca²⁺ discharge capacity.

We have repeatedly emphasized that methodological issues are important in the assessment of [Ca²⁺]_iand [Mg²⁺]_iin fluorescent dye-loaded platelets (7, 13, 14, 18, 21). Accordingly, the present study was carried out so as to minimize platelet activation during blood collection by using a 19-gauge needle and a two-syringe method. Attention must be paid to the possible activation of platelets and the coagulation system under the conditions of blood collection. Second, corrections were applied for extracellular leakage of dye, which leads to overestimations of [Ca²⁺]_iand [Mg²⁺]_iin the presence of extracellular Ca²⁺ and Mg²⁺ when a cell suspension system is used. Corrections for extracellular fura 2 should be made by using EGTA in agonist-stimulated conditions. Because Mn²⁺ enters platelets via the Ca²⁺ channel, MnCl₂ may be unsuitable for correction for dye leakage in estimating agonist responses. Third, in any comparison of Mg²⁺ or Ca²⁺ handling between hypertensives and normotensives, all aspects of intracellular dye metabolism, such as cytosolic fluorescent dye concentration and the degree of hydrolysis of fluorescence, should be similar in the two groups. The extent of dye ester hydrolysis affects fluorescence dynamics. However, many investigators have failed to clearly define the method of blood collection, correction for extracellular dye leakage, and the comparison of fluorescent dye metabolism. In the many reports concerning basal [Ca²⁺]_iin human platelets, the findings have been variable, ranging from 20 to 200 nM (5, 8, 11, 13, 16, 30). Our basal [Ca²⁺]_ilevel probably reflects improved methods with minimal activation of platelets and correction for dye leakage from platelets. Recent data from careful investigations of methods have shown basal [Ca²⁺]_iin human platelets as low as the level determined in our study (6, 11,17, 27).

We have previously reported differences in abnormal Ca²⁺ handling by fura 2-loaded platelets from several types of hypertensive rats (12, 21, 23). Basal [Ca²⁺]_iis increased in spontaneously hypertensive rats (21, 23) but decreased in Dahl salt-sensitive and DOCA-salt hypertensive rats (12) and similar in stroke-prone spontaneously hypertensive rats (17) compared with those from normotensive control rats. The evoked [Ca²⁺]_iresponses to thrombin in the absence of extracellular Ca²⁺ were enhanced in these three strains of hypertensive rats, whereas in the presence of extracellular Ca²⁺, the [Ca²⁺]_iincrease was enhanced in spontaneously hypertensive rats (21, 23), decreased in Dahl salt-sensitive rats (12), and similar in DOCA-salt rats compared with values in normotensive control rats. Thus differences in platelet intracellular Ca²⁺ handling exist between strains of hypertensive rats, and platelets from models of hypertension do not always show an elevation of [Ca²⁺]_i. However, platelets from essential hypertensive subjects may be comparable to those from spontaneously hypertensive rats with respect to basal [Ca²⁺]_iand [Ca²⁺]_iresponses to thrombin.

Mg²⁺ is an important constituent of cells and an essential cofactor in many cell functions, including the regulation of receptor systems, transmembrane flux of cation, and activation of cellular enzyme, since certain enzyme activity, including Ca²⁺-ATPase and Na⁺-K⁺-ATPase, depends entirely on Mg²⁺ (3) and Na⁺ transport and cellular Ca²⁺ handling, which may be affected by [Mg²⁺]_i(25). Rat studies have shown that a deficiency of dietary Mg²⁺ is associated with the development of hypertension (1). Epidemiological studies suggest an inverse relationship between the dietary intake of Mg²⁺ and blood pressure (15). One could therefore hypothesize that an Mg²⁺ deficiency is associated with a decrease in Ca²⁺-ATPase and Na⁺-K⁺-ATPase activity, an elevated cytosolic Ca²⁺ level, and hence an increase in vascular resistance in hypertensive patients. To test this hypothesis, [Mg²⁺]_iand Ca²⁺ metabolism were studied in platelets from hypertensive patients and normotensive controls matched for age and gender. Unexpectedly, [Mg²⁺]_iwas elevated significantly in platelets from patients with essential hypertension; furthermore, serum total magnesium was similar in the hypertensive and normotensive groups. Thus we could not support the hypothesis that hypertension results from a cellular deficiency of Mg²⁺.

Resnick et al. (26, 27) previously described a decrease in intraerythrocyte concentration of free Mg²⁺ in essential hypertension based on studies employing nuclear magnetic resonance spectroscopy. Results contrary to ours may be due to differences in the cells used for [Mg²⁺]_imeasurement. Furthermore, a few previous reports (11, 30) using mag-fura 2 have shown the decrease in [Mg²⁺]_iin platelets from subjects with essential hypertension. This discrepancy may result from differences in the methods used, such as isolation of platelets or correction for extracellular dye. In other reports (28, 29), intracellular Mg²⁺ measurement by atomic absorption spectroscopy represents intracellular total magnesium concentration, and this may not accurately reflect cellular Mg²⁺ activity, since protein-bound and anion complex magnesium are unavailable for biochemical processes, whereas free Mg²⁺ does have biological activity. Similarly, we could not find a significant difference between hypertensives and normotensives in serum total magnesium, which may not represent intracellular Mg²⁺metabolism.

In summary, abnormal Ca²⁺handling, including higher basal [Ca²⁺]_i, enhanced thrombin-evoked [Ca²⁺]_iresponses in the presence or absence of extracellular Ca²⁺, and a greater Ca²⁺ discharge capacity was observed in platelets from hypertensive patients. Platelet [Mg²⁺]_iwas higher in hypertensives than in normotensives. Our data appear to negate the hypothesis that abnormal Ca²⁺ handling in platelets from hypertensive subjects results from a cellular Mg²⁺ deficiency.

Perspectives

Mg²⁺ deficiency has been recognized to be associated with the pathogenesis of several cardiovascular diseases, such as arrhythmia and coronary heart disease. Hypertension is an established risk factor for these cardiovascular diseases. In the present study, we have clarified the increased [Mg²⁺]_iin essential hypertension. Thus further studies are necessary to clarify the relation between systemic Mg²⁺ balance and cellular Mg²⁺ metabolism. The Mg²⁺ balance study might solve this problem. Furthermore, the reason for increased [Mg²⁺]_iin essential hypertension is not clear, since the precise mechanisms that regulate [Mg²⁺]_iare not fully understood. Intracellular ATP concentration and Mg²⁺/Na⁺ exchanger are reported to regulate [Mg²⁺]_i. These factors should be studied in the cardiovascular diseases and their risk factors.