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Background: Previous studies have shown that farnesol, a 15-carbon nonsterol derivative of mevalonic acid, inhibits vasoconstriction. Because of its lipophilic properties, we hypothesized that farnesol increased membrane dynamics, thus reducing uptake of Ca2+ and contraction., Objective: To characterize the effect of farnesol on cell membrane fluidity., Design: The study was conducted using A7r5 cells, a rat aortic vascular smooth muscle cell line. Inhibition of Ca2+ uptake by farnesol was first established in these cells. Then, the effect of farnesol on membrane dynamics was determined. Finally, to ascertain that activation of Ca2+ extrusion and reuptake processes by farnesol did not occur, Ca2+-ATPase activity was examined., Methods: Membrane fluidity in cell homogenates was estimated using two fluorescent dyes (1,6-diphenyl-1,3,5-hexatriene) and (1-[-(trimethylamino)-phenyl]-6-phenyl-1,3,5-hexatriene). Ca2+ uptake was determined by monitoring the changes in cytosolic Ca2+ concentration ([Ca2+]i) in fura-2-loaded cells after addition of KCI. Ca2+-ATPase activity was measured in 100000 x g cell fractions., Results: Farnesol reduced KCI-induced (Ca2+]i transients significantly (P < 0.001), but did not modify membrane dynamic properties [0.214+/-0.007 versus 0.218+/-0.007 (n = 10) and 0.142+/-0.002 versus 0.146+/-0.003 (n = 5) for 1 -[-(trimethylamino)-phenyl]-6-phenyl-1,3,5-hexatriene and 1,6-diphenyl-1,3,5-hexatriene anisotropies, respectively; NS]. Administration of up to 30 micromol/l farnesol did not affect Ca2+-ATPase activity., Conclusion: Farnesol inhibits KCI-dependent rise of [Ca2+]i in A7r5 cells. This effect of farnesol is not related to a global change in plasma membrane lipid organization or to activation of Ca2+ pumps. Other mechanisms such as direct inhibition of voltage-dependent Ca2+ channels could therefore explain the biologic action of farnesol in the vascular tissue.

Objective: To study the relationship between high blood pressure and hyperlipidaemia and the cytosolic calcium concentration in unstimulated platelets, focusing on the effects of an alteration in membrane dynamics., Materials and Methods: Basal cytosolic calcium concentrations were determined in the presence and the absence of a significant calcium influx in platelets of 47 untreated hypertensive patients and 26 normotensive subjects. Membrane microviscosity was investigated by fluorescence depolarization of diphenylhexatriene and trimethylaminodiphenylhexatriene. To study the influence of plasma factors, unstimulated platelets were loaded in the presence of plasma with Quin-2, which forms a relatively strong intracellular calcium buffer. The cytosolic calcium concentration was then determined at two extracellular calcium concentrations (1 mmol/l and in the absence of a Ca2+ influx)., Results: Irrespective of the external calcium concentration, the cytosolic calcium concentration increased significantly with diastolic blood pressure (P = 0.026 in the presence and P = 0.003 in the absence of Ca2+ influx) and with plasma triacylglycerols (P = 0.03 and 0.001, respectively). Multiple regression analysis indicated that the cytosolic Ca+ concentration was independently related to these two factors [Ca2+ = 35 + (18.6 +/- 4.6). In triacylglycerols (mmol/l) + (0.45 +/- 0.15) mmHg diastolic blood pressure; P < 0.001]. The relationship between the cytosolic calcium concentration and diphenylhexatriene or trimethylaminodiphenylhexatriene anisotropies was not independent of blood pressure and plasma triacylglycerol levels., Conclusions: The present results confirm the link between blood pressure and the platelet cytosolic calcium concentration and indicate that plasma triacylglycerols directly or indirectly modulate the ex vivo efficacy of platelet calcium storage and/or extrusion mechanisms. They could facilitate cell stimulation.

Objective: To verify that platelet cytosolic pH is altered in essential hypertension and to investigate the mechanisms involved., Methods: Cytosolic pH was determined in unstimulated platelets by the fluorescent indicator 2,7-bis-carboxyethyl-5(6)-carboxyfluorescein (BCECF). Membrane microviscosity was evaluated by the fluorescence anisotropies of diphenylhexatriene (DPH) and its cationic derivative trimethylamino-diphenylhexatriene (TMA-DPH)., Results: The cytosolic alkalinization previously observed in platelets from untreated hypertensive patients was confirmed. The buffering capacity appeared unaltered and the cytosolic pH was not modified by 50 mumol/l N-5-ethylisopropylamiloride, a specific inhibitor of the Na(+)-H+ exchange. Exposure to external Na(+)-free media produced an intracellular acidification that was similar in hypertensive and normotensive donors and maintained the cytosolic pH difference between the two groups. In the two blood pressure groups platelet cytosolic pH varied inversely with the steady-state anisotropy of TMA-DPH but not with that of DPH. Experimentally induced acidification of the cytosol by Na+ removal with or without nigericin treatment was accompanied by rises in TMA-DPH anisotropy., Conclusions: This study of platelet intracellular pH in essential hypertension confirms cytosolic alkalinization and demonstrates its association with changes in the dynamic properties of the platelet plasma membrane.

Cytosolic free calcium concentration [Ca2+] was studied in platelets of hypertensive patients with the use of the fluorescent indicator Quin 2/AM. Cytosolic free Ca2+ was significantly higher in platelets of hypertensive patients than in those of normotensive subjects (241 +/- 9 versus 192 +/- 7 nmol/l, n = 58 and 57, respectively P less than 0.001). When all 115 subjects were included, there was a significant correlation between cytosolic free Ca2+ and systolic or diastolic blood pressure (r = 0.262, P less than 0.0025 and r = 0.251, P less than 0.0025, respectively). Intracellular Quin 2 concentration was measured to evaluate the formaldehyde production (a product of Quin 2/AM hydrolysis which has been described as reducing the adenosine triphosphate (ATP) production). The Quin 2 concentrations in platelets of the two groups of subjects were observed to be similar (0.41 +/- 0.03 versus 0.38 +/- 0.03 mmol/l, n = 8 and 7 for hypertensives and normotensives, respectively). The effects of prostaglandin E1 (PGE1), an adenylate cyclase stimulator, on cytosolic free Ca2+ were studied. The presence of 10(-7) mol/l PGE1 lowered the Ca2+ in platelets of hypertensive patients only, suppressing the difference between the two groups.

Cytosolic free [Ca2+] and serotonin (5-HT) content were measured in platelets from 22 untreated hypertensive patients and 16 normotensive subjects. In hypertensive patients, cytosolic free [Ca2+] was significantly higher (239 +/- 13 nmol/l versus 186 +/- 7 nmol/l, n = 22 and 16, P less than 0.01) and 5-HT content was significantly lower than those measured in cells from control subjects (3.22 +/- 0.26 versus 4.99 +/- 0.38 X 10(-7) mol/10(11) cells, n = 22 and 16, P less than 0.001). These two parameters were closely correlated (r = -0.565, n = 38, P less than 0.001). These two concomitant changes in platelet characteristics might result from a common cause, such as cell membrane alterations. As ritanserine, a specific 5-HT2 receptor antagonist, did not modify the cytosolic free [Ca2+], a higher stimulation of 5-HT2 receptors is not likely to be responsible for the enhanced free Ca2+ levels.

Various abnormalities in platelet metabolism, including increased sensitivity to several aggregating agents, have been described in essential hypertension. Platelet response is controlled by Ca2+ and cyclic AMP-dependent mechanisms (stimulatory and inhibitory, respectively) which oppose one another. In the present study, the cyclic AMP contents of unstimulated platelets were measured by radio-immunoassay and observed to be lower in hypertensive than in normotensive subjects, either in the basal state or after prostaglandin E1 (PGE1) stimulation. In the presence of 7-bromo-1,5,dihydro-3,6-dimethylimidazo [2,1-b] quinazolin-2(3H)-one (Ro 15-2041), a specific inhibitor of phosphodiesterase, the increases in cyclic AMP content were similar in platelets from both groups, indicating that this enzyme was not responsible for the alterations in cyclic AMP metabolism observed in hypertension. Low external Ca2+ reduced basal and PGE1-stimulated cyclic AMP content in both normotensive and hypertensive groups but cyclic AMP levels remained lower in hypertensive patients than in normotensive subjects, indicating that Ca2+ influx is not responsible for this altered metabolism of cyclic AMP in hypertension. These data suggest that the reduced platelet cAMP content may participate in the hyperreactivity to various aggregating agents previously reported to accompany essential hypertension.