EXPERIMENTAL
AND
Modifications
PATHOLOGY
induced
C. ROMANINI, Department
MOLECULAR
54, 122-128 (191)
by Gestational Hypertension Calcium Transport
on Platelet
A. L. TRANQUILLI, N. CESTER, H. VALENSISE, R. A. RABINI,~ AND L. MAZZANTI~
A. M. CUGINI,
of Obstetrics
Received
and Gynecology University August
and ‘Department of Biochemistry, of Ancona, Ancona, Italy
6, 1990, and in revised
form
October
School
of Medicine,
25, 1990
Several studies have recently demonstrated that the platelets of subjects affected by essential hypertension have, in their basal state, an elevated cellular caicium content. Such data appear particularly interesting with regard to gestational hypertension (GH). Supposing that the intracellular calcium may be involved in the regulation of blood pressure we have studied the cytosolic calcium concentration, Na+/K+-ATPase activity, Ca’+-ATPase activity, fluidity, and the cholesteroVphospholipid (C/P) molar ratio of the plasma membranes in platelets from 20 normotensive pregnant women and 20 women affected by mild gestational hypertension without pharmacological treatment, near term. We observed an increased Ca’+-ATPase activity and a decreased Na+/K’-ATPase activity in GH compared to the controls, accompanied by an increased Ca ‘+ intraplatelet concentration in the same patients. The fluidity and the C/P molar ratio were also increased. Our study gives indirect support to the hypothesis, supposing a reduced Na+/K’-ATPase activity which might cause increased intracellular Na+ content and decreased Ca” efflux through the Na+/Ca’+ exchange. However, out data can not rule out the other hypotheses explaining the increased cellular Ca” content. The present data indicate that GH is accompanied by a membrane structural abnormality that alters its physical state and modifies the membrane-related cellular functions. 0 1991 Academic Press. Inc.
INTRODUCTION The importance of calcium ions in the pathogenesis of arterial hypertension has long been studied (2, 17). An elevation of cytosolic calcium concentration is correlated with an increase in the active tension of smooth muscle cells in vessel walls, thus increasing arterial resistance. The intracellular concentration of calcium is also important in the regulation of the functional state of other cells involved in blood pressure homeostasis (aldosterone-secreting adrenal cortical cells and renin-secreting juxtaglomerular cells). For these reasons intracellular calcium may have a primary role in the regulation of blood pressure. The measurement of intracellular calcium has in the past been carried out using ion-specific microelectrodes introduced into cells of adequate dimensions, but substantial progress in this study has only come with the utilization of techniques based on fluorescent indicators (20). It has recently been demonstrated that platelets of subjects affected by essential hypertension have, in their basal state, an elevated cellular calcium content (5, 9). Antihypertensive therapy normalizes the intracellular calcium concentration along with a corresponding reduction in arterial blood pressure and peripheral vascular resistance (9). Such data appear particularly interesting with regard to gestational hypertension (GH) because platelets constitute an easily accessible and homogeneous cell population and possess many characteristics similar to those of smooth muscle cells. In fact platelets respond to agonists with an eleva122 0014-4800/91 $3.00 Copyright All rights
0 1991 by Academic Press. Inc. of reproduction m any form reserved.
CALCIUM
AND
GESTATIONAL
HYPERTENSION
123
tion of intracellular calcium stimulating the phosphorylation of the myosin light chain with subsequent shape change and aggregation (3). Previous studies on erythrocytes from subjects affected by GH have shown alterations of membrane ionic transport, with a reduced Na+/K+-ATPase activity and increased Ca’+-ATPase activity of plasma membranes and an elevated intracellular Na+ concentration that may cause, through Na+/Ca2+ exchange, a secondary elevation of intracellular Ca2’ (26, 27). The aim of the work presented here was to study intraplatelet calcium concentration, membrane fluidity, and membrane-related transport enzymatic activities (Na+/K+-ATPase and Ca 2+-ATPase) in mild gestational hypertension in light of their possible relationship with the pathogenesis of the hypertensive disorders of pregnancy. MATERIALS
AND METHODS
We have studied the cytosolic calcium concentration, Na+/K+-ATPase activ2+-ATPase activity, fluidity, and the cholesterol/phospholipid (C/P) molar ity, Ca ratio of the plasma membrane in platelets from 20 normotensive pregnant women and 20 women affected by mild gestational hypertension without pharmacological treatment, near term. Patients were considered mild gestational hypertensive, according to Davey and MacGillivray (6), when diastolic blood pressure was found to be >90 mm Hg, but < 110 mm Hg, in two consecutive occasions four or more hours apart, after the 20th week gestation, in previously normotensive, nonproteinuric, women. Cytosolic Free Calcium
Concentrations
Blood was drawn in plastic tubes containing citrate-citric acid-dextrose (CCD: citrate 0.1 M, citric acid 7 mM, dextrose 140 mM, pH 6.5) in a ratio of 9: 1. Ionized calcium in blood platelets was measured according to the method of Rao (21). Platelet rich plasma (PRP) was obtained after centrifugation of whole blood for 20 min at 140g and room temperature. The platelet count was adjusted to 300 x log/liter by adding platelet poor plasma (PPP). Platelets were washed twice in antiaggregating buffer (Tris-HCl 10 mM, NaCl 150 m&f, EDTA 1 mM, glucose 5 mM, pH 7.3). For the loading of the calciumsensitive probe platelets were subsequently incubated at 37°C for 45 min with fura-2-acetoxymethylester (Fura- AM) 1 pM in a solution containing 145 mM NaCl, 5 miV KCl, 1 mM MgSO,, 10 mM Hepes, 10 n&f glucose, pH 7.4. The cells were then washed again in the same solution to remove the excess dye. The determination of intracellular Ca2+ levels was performed in a Perkin-Elmer MPF-66 fluorescence spectrophotometer at 37°C according to the method of Grynkiewicz et al. (14). The fluorescence intensity was read at a constant emission wavelength (490 nm) with changes in the excitation wavelength (340 and 380 nm). The calibration was carried out as described by Grynkiewicz et al. (14) using the following equation: R-R& sl-2 [Ca2+li = Kd X R max -RXSb2’ where Kd is the dissociation constant of the Ca 2f-Fura 2 interaction in the cytosolic environment; R is the ratio of the fluorescence intensities at the excitation
124
ROMANINI
ET
AL.
wavelengths of 340 and 380 nm; Rtii, and R,, are ratios of the fluorescence intensities without Ca*+ and with saturating levels of Ca*+ , respectively; Sf2 and Sb2 are fluorescence intensities at 380 nm without Ca*+ and with saturating levels of Ca*+, respectively. Rmin and St2 were measured after lysis and addition of CaCl, 10 m&f. Ca* f -ATPase
Assay
In order to obtain the platelet plasma membranes blood was drawn into CCD anticoagulant and PRP was separated by centrifugation at 14Og for 20 min at room temperature. Platelets were then washed in a buffer containing 8 miU Na,HPO,, 2 mM NaH,P04, 10 mM EDTA, 5 mM KCl, 135 rniV NaCl, pH 7.0, and pelleted by centrifugation at 3000g for 30 min. The cells were then lysed by ultrasonication and the platelet membrane fraction corresponding to the plasma membrane was isolated as described by Enouf et al. (8). The activity of the Ca*+-ATPase was then determined according to the method of Davis ef al. (7) by measuring inorganic phosphate (Pi) hydrolyzed from 1 n&f Na,ATP at 37°C in the presence and absence of 0.15 mJ4 Ca*‘. The reaction medium contained 0.1 mM EGTA, 75 mM NaCl, 25 mM KCI, 1 mM MgCl,, and 25 mM Tris-HCl, pH 7.4. The ATPase activity assayed in the absence of Ca*+ was subtracted from the total ATPase activity to calculate the activity of Ca*+-ATPase. The results are expressed as micromoles Pi/(mg membrane proteins X 90 min). Inorganic phosphate was measured according to Fiske and Subbarow (11). Protein concentration was determined by the Lowry method (IS), using albumin as standard. Na+lK+-ATPase
Assay
The Na+, K+-activated Mg*+-dependent ATPase activity was determined on platelet membranes by the Kitao method (16). The ATPase activity was assayed by incubating membranes at 37°C in 1 ml of medium containing MgCI, (5 n&f), NaCl(140 mM), KC1 (14 nuV) in 40 mM Tris-HCl, pH 7.7. The ATPase reaction was started by the addition of 3 n-&Y Na,ATP and stopped 20 min later by the addition of 1 ml of 15% trichloracetic acid. Inorganic phosphate (Pi) hydrolyzed from reaction was measured as previously described (1 I). Enzyme activity was expressed as the difference in organic phosphate released in the presence and absence of 10 mM ouabain. The ATPase activity assayed in the presence of ouabain was subtracted from the total Mgzf-dependent ATPase activity to calculate the activity of the ouabain-sensitive Nat/K+-ATPase. The results are expressed as micromoles Pil(mg membrane proteins x 60 min). Protein concentration was determined as previously described (18). Membrane Fluidity
The fluidity of the platelet membranes was determined by means of the measurement of the fluorescence polarization (P) of the lipophilic probe 1,6diphenyl-1,3,5-hexatriene (DPH), according to the method of Schachter et al. (23).
The fluorescence polarization measurements were made in a Perkin-Elmer spectrofluorimeter MPF 44A equipped with two quartz prism polarizers, with light excitation at 365 nm. The P level is a quantitative index of the freedom of rotation
CALCIUM
AND GESTATIONAL
125
HYPERTENSION
of the fluorescent probe; a decrease in the P value indicates a higher mobility of the DPH in the deeper part of the membrane hydrophobic bilayer (i.e., an increased membrane fluidity). Membrane
Cholesterol
and Phospholipid
Content
Lipids were extracted from platelet membranes according to the method of Folch (12), by using 10 ml chloroform/methanol (2/l, vol/vol) per milliliter of membrane suspension. Cholesterol concentrations were then determined by the method of Zak (29) and total phospholipids according to Bartlett (1). The data were analyzed by Student’s t test. RESULTS Platelets obtained from women affected by mild GH showed higher intracellular Ca” concentration in comparison to control subjects (Table I). The study of membrane-bound active transport systems demonstrated higher Ca2+-ATPase activity but lower Na+/K+ ATPase activity in women affected by GH in comparison to the controls (Table I). Values of fluorescence polarization determined on platelet membranes by DPH were decreased during GH, thus indicating higher fluidity of the platelet membrane (Table II). A significant increase in the cholesterol/phospholipid molar ratio and in the cholesterol concentration was also observed in the platelet membranes from GH women compared with normotensive ones, while no difference was observed in the membrane phospholipid concentration (Table II). DISCUSSION Abnormalities in the sodium transport across the cellular membrane have been widely described in gestational hypertension, where increased intracellular concentration of Naf and decreased Na+/K+-ATPase activity have been reported (26, 27). On the basis of similar studies performed in essential hypertension (3, 5, 9, 17, 22) it has been hypothesized that also in GH elevated intracellular Na+ content may cause, via an activation of Na+/Ca2+ exchange, an increase in the intracellular Ca2+ level. The present study demonstrates that platelet Ca” is significantly higher in untreated GH patients in comparison to control pregnant women and that this increase is not due to a reduced function of the active mechanism for Ca2’ efflux from the cell, namely the Ca 2+ ATPase. In fact, Ca2’ ATPase shows increased TABLE I Platelet Cytosolic Calcium Concentration ([Ca*+]J, Ca*+-ATPase Activity and Na+lK+ ATPase Activity of the Platelet Membranes in Normal Pregnant Women and in Women Affected by Mild Gestational Hypertension (GH)
[Ca”], (nmol/liter) Ca*+-ATPase (pmol P,lmg protein x 90 min) Na+/K+ ATPase (pm01 Pi/mg protein X h) Nore. Means f SD are shown. * P < 0.01.
Controls (?I = 20)
GH women
94.7 2 27.0 0.226 + 0.054 1.22 2 0.35
161.1 k 32.3* 0.491 k 0.059* 0.72 ” 0.35*
(n = 20)
126
ROMANINI
ET AL.
TABLE II Fluidity, Expressed as Fluorescence Polarization (P), Cholesterol and Phospholipid Concentration, and CholesteroVPhospholipid (C/P) Molar Ratio of the Platelet Membranes Obtained from Normal Pregnant Women and from Women Affected by Mild Gestational Hypertension (GH) Controls (n = 20) Fluidity (P) C/P (mol/mol) Cholesterol concentration (nmol/mg protein) Phospholipid concentration (nmoVmg protein)
0.298 0.60 102.8 171.4
” 2 f +
0.004 0.06 4.6 3.2
GH women (n = 20) 0.279 0.74 128.1 173.1
k 2 2 Ii
O.OlB* 0.07* 5.3* 3.7
Note. Means 2 SD are shown. * P < 0.01.
activity in the platelet membranes from GH women, being probably stimulated by the high intracellular calcium. Several alternative mechanisms may account for the observed CaZf increase in GH platelets during the resting state: increased release from intracellular pools, increased influx through membrane Ca*+ channels or decreased efflux via the Na+/Ca’+ exchange. Our study gives indirect support to the third hypothesis, demonstrating a reduced Na+/K+ ATPase activity which might cause increased intracellular Na+ content and decreased Ca2+ efflux through the Na+/Ca*+ exchange (2). However, our data cannot rule out the other hypotheses explaining the increased cellular Ca2+ content. The possibility of a relationship between changes in Na+/H+ exchange and the increased internal concentration of Ca*+ should also be taken into consideration, even if the role of cytosolic alkalinization in the induction of Ca2+ influx is still controversial in platelets (25). The present data also indicate that a membrane structural abnormality accompanies and/or precedes GH. In fact, a significant increase in membrane fluidity and in the cholesterol/phospholipid molar ratio has been observed in platelets from GH women, in agreement with previous studies performed on erythrocyte membranes (4). The increase in cholesterol content is in contrast with the increased membrane fluidity, as a higher C/P molar ratio should cause a reduction in platelet membrane fluidity, according to data in the literature (24). In order to explain the increased fluidity in GH platelet membranes, it might be hypothesized that-although the amount of membrane phospholipids is unchanged in this pathological condition-qualitative changes in the phospholipid composition of the membranes may offset the rigidifying effect of the increased cholesterol content. In fact, an increased membrane concentration of c&unsaturated fatty acids has been demonstrated to cause a decrease in DPH polarization, i.e., enhanced fluidity, of intact platelets and of platelet membranes (15, 19). Both the alteration in membrane fluidity and the elevated cholesterol content might directly influence the activity of sodium transport, as described by other authors (IO, 28). In conclusion our study demonstrates increased Ca2+ concentration in the platelets from women affected by GH. Platelets might reflect a general alteration of calcium transport, explaining the elevated peripheral vascular resistance and the increased response of vascular smooth muscle to vasoconstrictor stimuli present in GH (13).
CALCIUM
AND
GESTATIONAL
HYPERTENSION
127
ACKNOWLEDGMENTS The authors thank Dr. A. Malgaroli of the Department of Pharmacology, University of Milano, and Dr. G. Benedetti and R. Staffolani for help during the work.
REFERENCES 1. BARTLETT, G. R. (1959). Phosphorus assay in column chromatography. .I. Biol. Chem. 234,466468. 2. BLAUNSTEIN, M. P. (1977). Sodium ions, calcium ions, blood pressure regulation and hypertension: A reassessment and a hypothesis. Amer. J. Physiol. 232, 165-173. 3. BUHLER, F. R., and RESINK, T. 3. (1988). Platelet abnormalities and the pathophysiology of essential hypertension. Experientia 44, 94-%. 4. CESTER, N., BENEDETTI, G., CUGINI, A. M., GALEAZZI, M., TOCCHINI, M., PELLEGRINI, S., and MAZZANTI, L. (1989). XV World Congress of Anatomic and Clinical Pathology Abstract Book, Nuova Gratica Fiorentina, Firenze. 5. COOPER, R. S., SHAMSI, N., and KATZ, S. (1987). Intracellular calcium and sodium in hypertensive patients. Hypertension 9, 224-229. 6. DAVEY, D. A., and MACGILLIVRAY, I. (1986). The classification and definition of hypertensive disorders of pregnancy. Clin. Exper. Hypertens. Hypertens. Pregnancy BS, 87-102. 7. DAVIS, F. B., DAVIS, P. J., NAT, G., BLAS, S. D., MACGILLIVRAY, M., GUTMAN, S., and FELDMAN, M. J. (1985). The effect of in vivo glucose administration on human erythrocyte Ca2 + -ATPase activity and on enzyme responsiveness in vitro to thyroid hormone and calmodulin. Diabetes 34, 639-646, 8. ENOUF, J., BREDOUX, R., BOURDEAU, N., SARKADI, B., and LEVY-T• LEDANO, S. (1989). Further characterization of the plasma membrane- and intracellular membrane-associated platelet Ca2 + transport systems. Biochem. J. 263, 547-552. 9. ERNE, P., BOLLI, P., BURGISSER, F., and BUHLER, F. R. (1984). Correlation of platelet calcium with blood pressure: Effect of antihypertensive therapy. N. Engl. J. Med. 310, 10841088. 10. FARIAS, R. N. (1980). Membrane cooperative enzymes as a tool for the investigation of membrane structure and related phenomena. Adv. Lipid Res. 17, 251-282. 11. FISKE, C., and SUBBAROW, Y. (1925). The calorimetric determination of phosphorus. J. Biof. Chem. 66,375-W. 12. FOLCH, J., LESS, M., and SLOANE-STANLEY, G. H. (1957). A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 466-468. 13. GANT, N. F., and WORLEY, R. K. (1980). “Hypertension in Pregnancy.” Appleton-Century-crofts, New York. 14. GRYNKIEWICZ, G., POENIE, M., and TSIEN, R. Y. (1985). A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 6, 3440-3450. 15. KITAGAWA, S., ENW, J., and KAMETANI, F. (1985). Effects of long-chain cis-unsaturated fatty acids and their alcohol analogs on aggregation of bovine platelets and their relation with membrane fluidity change. Biochim. Biophys. Acta 818, 391-397. 16. KITAO, T., and HATTORI, K. (1983). Inhibition of erythrocyte ATPase activity by aclacynomycin and reverse affects of ascorbate on ATPase activity. Experientia 39, 1362-1364. 17. LECHI, A., LECHI, C., BONADONNA, G., and DE TOGNI, P. (1987). Increased basal and thrombininduced free calcium in platelets of essential hypertensive patients. Hypertension 9, 230-236. 18. LOWRY, 0. H., ROSENBURG, M. Y., FARR, A. L., and RANDALL, R. T. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 192, 265-275. 19. MACINTYRE, D. E., HOOVER, R. L., SMITH, M., STEER, M., LYNCH, C., KARNOVSKY, M. J., and SALZMAN, E. W. (1984). Inhibition of platelet function by cis-unsaturated fatty acids. Blood 63, 848-857.
MALGAROLI, A., MILANI, D., MELDOLESI, J., and POZZAN, T. (1987). Fura- measurements of cytosolic free Ca + + in monolayers and suspensions of various types of animal cells. J. Cell Biol. 105, 2145-2155. 21. RAo, G. H. R. (1988). Measurement of ionized calcium in normal human blood platelets. Anal. Biochem. 169,400-404. 22. RINALDI, G., BOHR, D. (1988). Plasma membrane and its abnormality in hypertension. Amer. J. Med. Sci. 295, 389-395.
20.
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23. SCHACHTER, D., and SHINITZKY, M. (1977). Fluorescence polarization studies of rat intestinal microvillus membrane. J. Clin. Invest. 59, 536-548. 24. SHATTIL, S. J., and COOPER, R. A. (1976). Membrane microviscosity and human platelet function. Biochemistry 15, 4832-4837. 25. SIFFERT, W., and ACKERMAN, J. W. N. (1989). Na+/H + exchange and Ca2+ influx. FEBS Left. 259, 14. 26. TESTA, I., RABINI, R. A., TRANQUILLI, A. L., CESTER, N., MAZZANTI, L., BERTOLI, E., ROMANINI, C., and DANIELI, G. (1988). Abnormal membrane cation transport in pregnancy-induced hypertension. Stand. J. Clin. Lab. Invest. 48, 7-13. 27. TIUNQUILLI, A. L., MAZZANTI, L., BERTOLI, E., and ROMANINI, C. (1988). Sodium/Potassium adenosine triphosphatase on erythrocyte ghosts from pregnant women and its relationship to pregnancy-induced hypertension. Obstet. Gynecol. 71, 627-631. 28. YEAGLE, P. L. (1983). Cholesterol modulation of (Na+,K +)-ATP hydrolyzing activity in the human erythrocyte. Biochim. Biophys. Acta 727, 39-44. 29. ZAK, B. (1957). Simple rapid microtechnique for serum total cholesterol Amer. J. Clin. Pathol. 27, 583-588.
AND
Modifications
PATHOLOGY
induced
C. ROMANINI, Department
MOLECULAR
54, 122-128 (191)
by Gestational Hypertension Calcium Transport
on Platelet
A. L. TRANQUILLI, N. CESTER, H. VALENSISE, R. A. RABINI,~ AND L. MAZZANTI~
A. M. CUGINI,
of Obstetrics
Received
and Gynecology University August
and ‘Department of Biochemistry, of Ancona, Ancona, Italy
6, 1990, and in revised
form
October
School
of Medicine,
25, 1990
Several studies have recently demonstrated that the platelets of subjects affected by essential hypertension have, in their basal state, an elevated cellular caicium content. Such data appear particularly interesting with regard to gestational hypertension (GH). Supposing that the intracellular calcium may be involved in the regulation of blood pressure we have studied the cytosolic calcium concentration, Na+/K+-ATPase activity, Ca’+-ATPase activity, fluidity, and the cholesteroVphospholipid (C/P) molar ratio of the plasma membranes in platelets from 20 normotensive pregnant women and 20 women affected by mild gestational hypertension without pharmacological treatment, near term. We observed an increased Ca’+-ATPase activity and a decreased Na+/K’-ATPase activity in GH compared to the controls, accompanied by an increased Ca ‘+ intraplatelet concentration in the same patients. The fluidity and the C/P molar ratio were also increased. Our study gives indirect support to the hypothesis, supposing a reduced Na+/K’-ATPase activity which might cause increased intracellular Na+ content and decreased Ca” efflux through the Na+/Ca’+ exchange. However, out data can not rule out the other hypotheses explaining the increased cellular Ca” content. The present data indicate that GH is accompanied by a membrane structural abnormality that alters its physical state and modifies the membrane-related cellular functions. 0 1991 Academic Press. Inc.
INTRODUCTION The importance of calcium ions in the pathogenesis of arterial hypertension has long been studied (2, 17). An elevation of cytosolic calcium concentration is correlated with an increase in the active tension of smooth muscle cells in vessel walls, thus increasing arterial resistance. The intracellular concentration of calcium is also important in the regulation of the functional state of other cells involved in blood pressure homeostasis (aldosterone-secreting adrenal cortical cells and renin-secreting juxtaglomerular cells). For these reasons intracellular calcium may have a primary role in the regulation of blood pressure. The measurement of intracellular calcium has in the past been carried out using ion-specific microelectrodes introduced into cells of adequate dimensions, but substantial progress in this study has only come with the utilization of techniques based on fluorescent indicators (20). It has recently been demonstrated that platelets of subjects affected by essential hypertension have, in their basal state, an elevated cellular calcium content (5, 9). Antihypertensive therapy normalizes the intracellular calcium concentration along with a corresponding reduction in arterial blood pressure and peripheral vascular resistance (9). Such data appear particularly interesting with regard to gestational hypertension (GH) because platelets constitute an easily accessible and homogeneous cell population and possess many characteristics similar to those of smooth muscle cells. In fact platelets respond to agonists with an eleva122 0014-4800/91 $3.00 Copyright All rights
0 1991 by Academic Press. Inc. of reproduction m any form reserved.
CALCIUM
AND
GESTATIONAL
HYPERTENSION
123
tion of intracellular calcium stimulating the phosphorylation of the myosin light chain with subsequent shape change and aggregation (3). Previous studies on erythrocytes from subjects affected by GH have shown alterations of membrane ionic transport, with a reduced Na+/K+-ATPase activity and increased Ca’+-ATPase activity of plasma membranes and an elevated intracellular Na+ concentration that may cause, through Na+/Ca2+ exchange, a secondary elevation of intracellular Ca2’ (26, 27). The aim of the work presented here was to study intraplatelet calcium concentration, membrane fluidity, and membrane-related transport enzymatic activities (Na+/K+-ATPase and Ca 2+-ATPase) in mild gestational hypertension in light of their possible relationship with the pathogenesis of the hypertensive disorders of pregnancy. MATERIALS
AND METHODS
We have studied the cytosolic calcium concentration, Na+/K+-ATPase activ2+-ATPase activity, fluidity, and the cholesterol/phospholipid (C/P) molar ity, Ca ratio of the plasma membrane in platelets from 20 normotensive pregnant women and 20 women affected by mild gestational hypertension without pharmacological treatment, near term. Patients were considered mild gestational hypertensive, according to Davey and MacGillivray (6), when diastolic blood pressure was found to be >90 mm Hg, but < 110 mm Hg, in two consecutive occasions four or more hours apart, after the 20th week gestation, in previously normotensive, nonproteinuric, women. Cytosolic Free Calcium
Concentrations
Blood was drawn in plastic tubes containing citrate-citric acid-dextrose (CCD: citrate 0.1 M, citric acid 7 mM, dextrose 140 mM, pH 6.5) in a ratio of 9: 1. Ionized calcium in blood platelets was measured according to the method of Rao (21). Platelet rich plasma (PRP) was obtained after centrifugation of whole blood for 20 min at 140g and room temperature. The platelet count was adjusted to 300 x log/liter by adding platelet poor plasma (PPP). Platelets were washed twice in antiaggregating buffer (Tris-HCl 10 mM, NaCl 150 m&f, EDTA 1 mM, glucose 5 mM, pH 7.3). For the loading of the calciumsensitive probe platelets were subsequently incubated at 37°C for 45 min with fura-2-acetoxymethylester (Fura- AM) 1 pM in a solution containing 145 mM NaCl, 5 miV KCl, 1 mM MgSO,, 10 mM Hepes, 10 n&f glucose, pH 7.4. The cells were then washed again in the same solution to remove the excess dye. The determination of intracellular Ca2+ levels was performed in a Perkin-Elmer MPF-66 fluorescence spectrophotometer at 37°C according to the method of Grynkiewicz et al. (14). The fluorescence intensity was read at a constant emission wavelength (490 nm) with changes in the excitation wavelength (340 and 380 nm). The calibration was carried out as described by Grynkiewicz et al. (14) using the following equation: R-R& sl-2 [Ca2+li = Kd X R max -RXSb2’ where Kd is the dissociation constant of the Ca 2f-Fura 2 interaction in the cytosolic environment; R is the ratio of the fluorescence intensities at the excitation
124
ROMANINI
ET
AL.
wavelengths of 340 and 380 nm; Rtii, and R,, are ratios of the fluorescence intensities without Ca*+ and with saturating levels of Ca*+ , respectively; Sf2 and Sb2 are fluorescence intensities at 380 nm without Ca*+ and with saturating levels of Ca*+, respectively. Rmin and St2 were measured after lysis and addition of CaCl, 10 m&f. Ca* f -ATPase
Assay
In order to obtain the platelet plasma membranes blood was drawn into CCD anticoagulant and PRP was separated by centrifugation at 14Og for 20 min at room temperature. Platelets were then washed in a buffer containing 8 miU Na,HPO,, 2 mM NaH,P04, 10 mM EDTA, 5 mM KCl, 135 rniV NaCl, pH 7.0, and pelleted by centrifugation at 3000g for 30 min. The cells were then lysed by ultrasonication and the platelet membrane fraction corresponding to the plasma membrane was isolated as described by Enouf et al. (8). The activity of the Ca*+-ATPase was then determined according to the method of Davis ef al. (7) by measuring inorganic phosphate (Pi) hydrolyzed from 1 n&f Na,ATP at 37°C in the presence and absence of 0.15 mJ4 Ca*‘. The reaction medium contained 0.1 mM EGTA, 75 mM NaCl, 25 mM KCI, 1 mM MgCl,, and 25 mM Tris-HCl, pH 7.4. The ATPase activity assayed in the absence of Ca*+ was subtracted from the total ATPase activity to calculate the activity of Ca*+-ATPase. The results are expressed as micromoles Pi/(mg membrane proteins X 90 min). Inorganic phosphate was measured according to Fiske and Subbarow (11). Protein concentration was determined by the Lowry method (IS), using albumin as standard. Na+lK+-ATPase
Assay
The Na+, K+-activated Mg*+-dependent ATPase activity was determined on platelet membranes by the Kitao method (16). The ATPase activity was assayed by incubating membranes at 37°C in 1 ml of medium containing MgCI, (5 n&f), NaCl(140 mM), KC1 (14 nuV) in 40 mM Tris-HCl, pH 7.7. The ATPase reaction was started by the addition of 3 n-&Y Na,ATP and stopped 20 min later by the addition of 1 ml of 15% trichloracetic acid. Inorganic phosphate (Pi) hydrolyzed from reaction was measured as previously described (1 I). Enzyme activity was expressed as the difference in organic phosphate released in the presence and absence of 10 mM ouabain. The ATPase activity assayed in the presence of ouabain was subtracted from the total Mgzf-dependent ATPase activity to calculate the activity of the ouabain-sensitive Nat/K+-ATPase. The results are expressed as micromoles Pil(mg membrane proteins x 60 min). Protein concentration was determined as previously described (18). Membrane Fluidity
The fluidity of the platelet membranes was determined by means of the measurement of the fluorescence polarization (P) of the lipophilic probe 1,6diphenyl-1,3,5-hexatriene (DPH), according to the method of Schachter et al. (23).
The fluorescence polarization measurements were made in a Perkin-Elmer spectrofluorimeter MPF 44A equipped with two quartz prism polarizers, with light excitation at 365 nm. The P level is a quantitative index of the freedom of rotation
CALCIUM
AND GESTATIONAL
125
HYPERTENSION
of the fluorescent probe; a decrease in the P value indicates a higher mobility of the DPH in the deeper part of the membrane hydrophobic bilayer (i.e., an increased membrane fluidity). Membrane
Cholesterol
and Phospholipid
Content
Lipids were extracted from platelet membranes according to the method of Folch (12), by using 10 ml chloroform/methanol (2/l, vol/vol) per milliliter of membrane suspension. Cholesterol concentrations were then determined by the method of Zak (29) and total phospholipids according to Bartlett (1). The data were analyzed by Student’s t test. RESULTS Platelets obtained from women affected by mild GH showed higher intracellular Ca” concentration in comparison to control subjects (Table I). The study of membrane-bound active transport systems demonstrated higher Ca2+-ATPase activity but lower Na+/K+ ATPase activity in women affected by GH in comparison to the controls (Table I). Values of fluorescence polarization determined on platelet membranes by DPH were decreased during GH, thus indicating higher fluidity of the platelet membrane (Table II). A significant increase in the cholesterol/phospholipid molar ratio and in the cholesterol concentration was also observed in the platelet membranes from GH women compared with normotensive ones, while no difference was observed in the membrane phospholipid concentration (Table II). DISCUSSION Abnormalities in the sodium transport across the cellular membrane have been widely described in gestational hypertension, where increased intracellular concentration of Naf and decreased Na+/K+-ATPase activity have been reported (26, 27). On the basis of similar studies performed in essential hypertension (3, 5, 9, 17, 22) it has been hypothesized that also in GH elevated intracellular Na+ content may cause, via an activation of Na+/Ca2+ exchange, an increase in the intracellular Ca2+ level. The present study demonstrates that platelet Ca” is significantly higher in untreated GH patients in comparison to control pregnant women and that this increase is not due to a reduced function of the active mechanism for Ca2’ efflux from the cell, namely the Ca 2+ ATPase. In fact, Ca2’ ATPase shows increased TABLE I Platelet Cytosolic Calcium Concentration ([Ca*+]J, Ca*+-ATPase Activity and Na+lK+ ATPase Activity of the Platelet Membranes in Normal Pregnant Women and in Women Affected by Mild Gestational Hypertension (GH)
[Ca”], (nmol/liter) Ca*+-ATPase (pmol P,lmg protein x 90 min) Na+/K+ ATPase (pm01 Pi/mg protein X h) Nore. Means f SD are shown. * P < 0.01.
Controls (?I = 20)
GH women
94.7 2 27.0 0.226 + 0.054 1.22 2 0.35
161.1 k 32.3* 0.491 k 0.059* 0.72 ” 0.35*
(n = 20)
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TABLE II Fluidity, Expressed as Fluorescence Polarization (P), Cholesterol and Phospholipid Concentration, and CholesteroVPhospholipid (C/P) Molar Ratio of the Platelet Membranes Obtained from Normal Pregnant Women and from Women Affected by Mild Gestational Hypertension (GH) Controls (n = 20) Fluidity (P) C/P (mol/mol) Cholesterol concentration (nmol/mg protein) Phospholipid concentration (nmoVmg protein)
0.298 0.60 102.8 171.4
” 2 f +
0.004 0.06 4.6 3.2
GH women (n = 20) 0.279 0.74 128.1 173.1
k 2 2 Ii
O.OlB* 0.07* 5.3* 3.7
Note. Means 2 SD are shown. * P < 0.01.
activity in the platelet membranes from GH women, being probably stimulated by the high intracellular calcium. Several alternative mechanisms may account for the observed CaZf increase in GH platelets during the resting state: increased release from intracellular pools, increased influx through membrane Ca*+ channels or decreased efflux via the Na+/Ca’+ exchange. Our study gives indirect support to the third hypothesis, demonstrating a reduced Na+/K+ ATPase activity which might cause increased intracellular Na+ content and decreased Ca2+ efflux through the Na+/Ca*+ exchange (2). However, our data cannot rule out the other hypotheses explaining the increased cellular Ca2+ content. The possibility of a relationship between changes in Na+/H+ exchange and the increased internal concentration of Ca*+ should also be taken into consideration, even if the role of cytosolic alkalinization in the induction of Ca2+ influx is still controversial in platelets (25). The present data also indicate that a membrane structural abnormality accompanies and/or precedes GH. In fact, a significant increase in membrane fluidity and in the cholesterol/phospholipid molar ratio has been observed in platelets from GH women, in agreement with previous studies performed on erythrocyte membranes (4). The increase in cholesterol content is in contrast with the increased membrane fluidity, as a higher C/P molar ratio should cause a reduction in platelet membrane fluidity, according to data in the literature (24). In order to explain the increased fluidity in GH platelet membranes, it might be hypothesized that-although the amount of membrane phospholipids is unchanged in this pathological condition-qualitative changes in the phospholipid composition of the membranes may offset the rigidifying effect of the increased cholesterol content. In fact, an increased membrane concentration of c&unsaturated fatty acids has been demonstrated to cause a decrease in DPH polarization, i.e., enhanced fluidity, of intact platelets and of platelet membranes (15, 19). Both the alteration in membrane fluidity and the elevated cholesterol content might directly influence the activity of sodium transport, as described by other authors (IO, 28). In conclusion our study demonstrates increased Ca2+ concentration in the platelets from women affected by GH. Platelets might reflect a general alteration of calcium transport, explaining the elevated peripheral vascular resistance and the increased response of vascular smooth muscle to vasoconstrictor stimuli present in GH (13).
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ACKNOWLEDGMENTS The authors thank Dr. A. Malgaroli of the Department of Pharmacology, University of Milano, and Dr. G. Benedetti and R. Staffolani for help during the work.
REFERENCES 1. BARTLETT, G. R. (1959). Phosphorus assay in column chromatography. .I. Biol. Chem. 234,466468. 2. BLAUNSTEIN, M. P. (1977). Sodium ions, calcium ions, blood pressure regulation and hypertension: A reassessment and a hypothesis. Amer. J. Physiol. 232, 165-173. 3. BUHLER, F. R., and RESINK, T. 3. (1988). Platelet abnormalities and the pathophysiology of essential hypertension. Experientia 44, 94-%. 4. CESTER, N., BENEDETTI, G., CUGINI, A. M., GALEAZZI, M., TOCCHINI, M., PELLEGRINI, S., and MAZZANTI, L. (1989). XV World Congress of Anatomic and Clinical Pathology Abstract Book, Nuova Gratica Fiorentina, Firenze. 5. COOPER, R. S., SHAMSI, N., and KATZ, S. (1987). Intracellular calcium and sodium in hypertensive patients. Hypertension 9, 224-229. 6. DAVEY, D. A., and MACGILLIVRAY, I. (1986). The classification and definition of hypertensive disorders of pregnancy. Clin. Exper. Hypertens. Hypertens. Pregnancy BS, 87-102. 7. DAVIS, F. B., DAVIS, P. J., NAT, G., BLAS, S. D., MACGILLIVRAY, M., GUTMAN, S., and FELDMAN, M. J. (1985). The effect of in vivo glucose administration on human erythrocyte Ca2 + -ATPase activity and on enzyme responsiveness in vitro to thyroid hormone and calmodulin. Diabetes 34, 639-646, 8. ENOUF, J., BREDOUX, R., BOURDEAU, N., SARKADI, B., and LEVY-T• LEDANO, S. (1989). Further characterization of the plasma membrane- and intracellular membrane-associated platelet Ca2 + transport systems. Biochem. J. 263, 547-552. 9. ERNE, P., BOLLI, P., BURGISSER, F., and BUHLER, F. R. (1984). Correlation of platelet calcium with blood pressure: Effect of antihypertensive therapy. N. Engl. J. Med. 310, 10841088. 10. FARIAS, R. N. (1980). Membrane cooperative enzymes as a tool for the investigation of membrane structure and related phenomena. Adv. Lipid Res. 17, 251-282. 11. FISKE, C., and SUBBAROW, Y. (1925). The calorimetric determination of phosphorus. J. Biof. Chem. 66,375-W. 12. FOLCH, J., LESS, M., and SLOANE-STANLEY, G. H. (1957). A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 466-468. 13. GANT, N. F., and WORLEY, R. K. (1980). “Hypertension in Pregnancy.” Appleton-Century-crofts, New York. 14. GRYNKIEWICZ, G., POENIE, M., and TSIEN, R. Y. (1985). A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 6, 3440-3450. 15. KITAGAWA, S., ENW, J., and KAMETANI, F. (1985). Effects of long-chain cis-unsaturated fatty acids and their alcohol analogs on aggregation of bovine platelets and their relation with membrane fluidity change. Biochim. Biophys. Acta 818, 391-397. 16. KITAO, T., and HATTORI, K. (1983). Inhibition of erythrocyte ATPase activity by aclacynomycin and reverse affects of ascorbate on ATPase activity. Experientia 39, 1362-1364. 17. LECHI, A., LECHI, C., BONADONNA, G., and DE TOGNI, P. (1987). Increased basal and thrombininduced free calcium in platelets of essential hypertensive patients. Hypertension 9, 230-236. 18. LOWRY, 0. H., ROSENBURG, M. Y., FARR, A. L., and RANDALL, R. T. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 192, 265-275. 19. MACINTYRE, D. E., HOOVER, R. L., SMITH, M., STEER, M., LYNCH, C., KARNOVSKY, M. J., and SALZMAN, E. W. (1984). Inhibition of platelet function by cis-unsaturated fatty acids. Blood 63, 848-857.
MALGAROLI, A., MILANI, D., MELDOLESI, J., and POZZAN, T. (1987). Fura- measurements of cytosolic free Ca + + in monolayers and suspensions of various types of animal cells. J. Cell Biol. 105, 2145-2155. 21. RAo, G. H. R. (1988). Measurement of ionized calcium in normal human blood platelets. Anal. Biochem. 169,400-404. 22. RINALDI, G., BOHR, D. (1988). Plasma membrane and its abnormality in hypertension. Amer. J. Med. Sci. 295, 389-395.
20.
128
ROMANINI
ET AL.
23. SCHACHTER, D., and SHINITZKY, M. (1977). Fluorescence polarization studies of rat intestinal microvillus membrane. J. Clin. Invest. 59, 536-548. 24. SHATTIL, S. J., and COOPER, R. A. (1976). Membrane microviscosity and human platelet function. Biochemistry 15, 4832-4837. 25. SIFFERT, W., and ACKERMAN, J. W. N. (1989). Na+/H + exchange and Ca2+ influx. FEBS Left. 259, 14. 26. TESTA, I., RABINI, R. A., TRANQUILLI, A. L., CESTER, N., MAZZANTI, L., BERTOLI, E., ROMANINI, C., and DANIELI, G. (1988). Abnormal membrane cation transport in pregnancy-induced hypertension. Stand. J. Clin. Lab. Invest. 48, 7-13. 27. TIUNQUILLI, A. L., MAZZANTI, L., BERTOLI, E., and ROMANINI, C. (1988). Sodium/Potassium adenosine triphosphatase on erythrocyte ghosts from pregnant women and its relationship to pregnancy-induced hypertension. Obstet. Gynecol. 71, 627-631. 28. YEAGLE, P. L. (1983). Cholesterol modulation of (Na+,K +)-ATP hydrolyzing activity in the human erythrocyte. Biochim. Biophys. Acta 727, 39-44. 29. ZAK, B. (1957). Simple rapid microtechnique for serum total cholesterol Amer. J. Clin. Pathol. 27, 583-588.
