Membrane fluidity and hypertension:

Abnormalities in physical properties of the cell membranes may underlie the defects that are strongly linked to hypertension, stroke, and other cardiovascular diseases.^1,2 The abnormalities appear to be involved not only in vascular smooth muscle cells but also in circulating blood cells such as erythrocytes, lymphocytes and platelets. Membrane fluidity (the reciprocal value of membrane microviscosity) is a physicochemical feature of biomembranes that is an important factor in modulating cell rheologic behavior. Altered membrane fluidity and microviscosity might also affect membrane permeability, transport systems, receptor functions or enzyme activities.¹ There are suggestions that membrane fluidity can be modified by various factors: for example the state of membrane components, the cytoskeletal proteins, the intracellular calcium (Ca) and sodium (Na) contents, or the diverse number of neurohumoral molecules.¹ Electron paramagnetic resonance (EPR) using fatty acid spin-label agents, as well as fluorescence polarization anisotropy of diphenylhexatriene (DPH) derivatives, have been developed to evaluate membrane fluidity and perturbations of the membrane functions by external agents. In this article, we focus on the alterations in membrane fluidity determined by the EPR method in both human and experimental hypertension, and further elucidate the possible relationships between the membrane properties and some vasoactive substances that might contribute to the pathophysiology of hypertension.

Membrane fluidity in humans with essential hypertension and genetic hypertensive rats

Our early studies by using the EPR and fatty acid spin-labeling method have shown that membrane fluidity was significantly decreased (membrane microviscosity was increased) in the erythrocytes of patients with essential hypertension (EH) compared with those of normotensive subjects.^3,4,5,6,7 The membrane fluidity of erythrocytes and vascular smooth muscle cells was also lower in spontaneously hypertensive rats (SHR) than in normotensive Wistar-Kyoto (WKY) rats at both young and adult ages.⁸ The findings suggest that the cell membranes might be stiffer and less fluid in genetic hypertension. This change was not observed in secondary hypertension,⁵ indicating that the abnormality may not be a consequence of a state of high blood pressure. Erythrocyte membrane functions are critically important for rapid and homogenous perfusion of oxygen in the microcirculation. If the deformability of erythrocytes may be highly dependent on the membrane fluidity,⁹ the reduction in membrane fluidity could cause a disturbance in blood rheologic behavior and in the microcirculation, which might contribute to the pathophysiology of genetic hypertension.

Role of calcium, sodium, and humoral factors in regulation of membrane fluidity in hypertension

It is well recognized that intracellular Ca may regulate the size, shape, and other physical properties of the cells. Kuettner et al¹⁰ observed that the exposure of erythrocytes to the Ca ionophore A23187 caused marked changes in membrane viscoelastic properties, including development of rigidity and increased resistance to aspiration into micropipettes. These investigators proposed that these changes in erythrocyte behavior could be due to the Ca induced precipitation or cross-linkage of the proteins located inside the membranes. In addition, it has been shown that cellular ionic disturbances of Ca metabolism may have a crucial role in the pathophysiology of hypertension.¹¹ On the basis of these previous observations, the Ca induced changes in membrane fluidity of erythrocytes were examined in hypertension. Treatment of erythrocytes with the Ca ionophore and Ca decreased the membrane fluidity to a greater extent in EH and SHR than in the normotensive control subjects.⁴ Furthermore, the lower the plasma renin activity, the greater the Ca induced changes in membrane fluidity in EH.⁵ Although the precise mechanisms responsible for the alterations in membrane fluidity are still uncertain, it is possible that the Ca influx across the cell membranes or interactions between Ca and erythrocyte membranes might be pronounced in hypertension, particularly in individuals with low renin activity. If the same phenomenon occurs in the cells of other tissues such as vascular smooth muscle, it would cause vascular resistance to increase, given that Ca has a major role in vasoconstriction and membrane fluidity may be inversely correlated with membrane stiffness.

Recent studies have shown that hyperinsulinemia may be associated with hypertension. It was demonstrated that fasting and postprandial insulin levels were higher in hypertensive subjects than in normotensive subjects. With regard to the relationship between insulin and membrane functions, it was shown that insulin influenced several transmembrane ionic transport systems, including the Ca adenosine triphosphatase (ATPase), Na, K-ATPase, and Na-Ca exchange systems. In addition, insulin itself has primary direct cellular ionic actions to increase intracellular Ca, not only in vascular smooth muscle cells but also in platelets and erythrocytes. We previously demonstrated that the higher plasma insulin level was associated with the lower membrane fluidity of erythrocytes, which may support the idea that insulin could be a determinant of membrane fluidity of erythrocytes.¹² In an in vitro study, it was shown that insulin decreased membrane fluidity of erythrocytes in humans. In addition, the effects of insulin were significantly potentiated in the presence of extracellular Ca and, in contrast, were antagonized by the Ca channel blocker.¹³ These results are consistent with the hypothesis that insulin may actively participate in the regulation of membrane fluidity of erythrocytes in hypertensive subjects by modulating intracelluar Ca kinetics.

There is much interest in the hypothesis that endogenous Na, K-ATPase inhibitor (digitalis-like substance [DLS]) may serve as a specific regulator of the Na pump of the membranes and may be implicated in the pathogenesis of hypertension. It was demonstrated that this compound increased the intracellular Ca content, probably through the Na-Ca exchange mechanisms or through a direct effect upon Ca influx into the cells. We previously showed that the plasma content of DLS was significantly higher in hypertensive patients than in normotensive subjects.⁷ Moreover, the higher the plasma DLS values, the lower the erythrocyte membrane fluidity. Although the precise mechanisms underlying the effects of DLS are still uncertain, the results suggest the possible role of the Na pump as well as the intracellular Na content in the regulation of membrane fluidity of erythrocytes in hypertension.

Defense mechanisms against abnormal membrane fluidity in hypertension

Adenylate cyclase activity with a concomitant increase in the intracellular cyclic adenosine 3′,5′-monophosphate (cAMP) level is thought to be a common factor through which prostaglandins (PG) and other humoral substances exert their biologic effects. The presence of adenylate cyclase was demonstrated in human erythrocyte membranes, and it was observed that the enzymatic activity was stimulated by prostaglandin E₁ (PGE₁), and was inhibited by Ca. Our previous study showed that PGE₁ and the cAMP-analog dibutyryl cAMP increased membrane fluidity of erythrocytes in humans in a dose-dependent manner.¹⁴ Adrenomedullin is a peptide that has vasorelaxant and long lasting hypotensive effects, and has been shown to increase the intracellular cAMP level. In the EPR study, adrenomedullin significantly increased membrane fluidity of erythrocytes in hypertensive patients, and the effect was enhanced in by PGE₁ and dibutyryl cAMP, suggesting that adrenomedullin improved membrane microviscosity in hypertension, probably by a cAMP-dependent mechanism.¹⁴ This finding may be consistent with the idea that the peptide could, at least to some extent, be involved in the regulation of membrane functions in hypertension.

Recently there has been much evidence showing that nitric oxide (NO) may actively participate in the pathophysiology of hypertension. Jubelin and Gierman¹⁵ have shown that erythrocytes of rats and humans are positive for NO synthase, which indicates that erythrocytes possess all of the cellular machinery to synthesize their own NO. They proposed that erythrocytes would synthesize and use NO to modulate their own physiology. We demonstrated that the plasma level of the NO metabolites (nitrite and nitrate) was significantly lower in hypertensive postmenopausal women than in normotensive postmenopausal women.¹⁶ Although the sources of plasma NO are not fully understood, the decreased circulating plasma NO may result from a diffuse endothelial dysfunction throughout the body in hypertensive postmenopausal women. In addition, we showed that the lower membrane fluidity of erythrocytes was associated with the lower plasma NO level.¹⁶ In an in vitro study, it was also demonstrated that exogenously applied NO donor (S-nitroso-N-acetylpenicillamine) significantly increased membrane fluidity of erythrocytes in hypertensive subjects.¹⁷ These findings support the idea that NO may be a determinant of the membrane fluidity of erythrocytes in hypertension.

It has been shown that the effects of a variety of vasoactive substances such as estrogen, progesterone, and leptin might, at least in part, be mediated by the production of NO. We demonstrated that estrogen increased the membrane fluidity and improved the membrane microviscosity of erythrocyte membranes, partially mediated by an NO- and cGMP-dependent pathway in hypertensive postmenopausal women.¹⁸ Leptin, the product of the obesity gene, also ameliorated the rigidity of cell membranes to some extent via an NO-dependent mechanism.¹⁹ One hypothesis is that these cAMP- and NO-related compounds may have a crucial role in the regulation of rheologic behavior of erythrocytes and microcirculation and may contribute to the defense against further increase in membrane microviscosity in hypertension.

Intervention with nonpharmacologic and pharmacologic tools

As previously mentioned, sodium ions may have a crucial role in the regulation of membrane fluidity of erythrocytes. To investigate the effects of salt intake on membrane fluidity of erythrocytes in EH, hospitalized patients were first given a normal diet (5 to 8 g of salt/day) for 7 days, a low salt diet (2 to 3 g/day) for 7 days thereafter, and a high salt diet (15 to 20 g/day) for the following week. The membrane fluidity of erythrocytes was increased with a low salt intake. In contrast, a high salt intake significantly reduced the membrane fluidity to a greater degree.⁶ The finding indicated that the abnormality in membrane fluidity in hypertension might be attenuated by a low salt diet, and by contrast, accelerated by a high salt diet.

Many studies have suggested fairly well that aerobic physical exercise may be beneficial for the prevention and treatment of hypertension and other cardiovascular diseases, although the influence of exercise on the membrane functions is not fully understood. In an in vitro study, Ca channel blockers were believed possibly to restore the decrease in membrane fluidity of erythrocytes in EH.⁴ However, it is still uncertain whether pharmacologic intervention with various antihypertensive agents or estrogen (hormone replacement therapy) can improve the membrane fluidity in hypertensive subjects in vivo. Further studies should be performed to assess more thoroughly the relationship between antihypertensive therapies and membrane functions in hypertension.

In summary, the demonstration in the EPR study that the membrane fluidity may be decreased in hypertension indicates a disturbance in blood rheologic behavior and microcirculation, which might partially explain the vascular complications in hypertension. Although we have a very limited understanding of the mechanisms accounting for the abnormalities in membrane fluidity, our data suggest that the diverse number of neurohumoral molecules may have a crucial modulatory action on membrane fluidity that may also be of considerable biologic and clinical significance in determining rheologic properties of the cell membranes in hypertension. Moreover, a better knowledge of the cellular mechanisms underlying membrane abnormalities could provide useful information concerning the development of a more specific and more physiologic approach to hypertension research.