Rudolf Locher

Is there a Role for the ob Gene Product Leptin in Essential Hypertension

In this study we wanted to evaluate the relationship between the ob gene product leptin and blood pressure, as well as plasma renin activity and plasma aldosterone levels. We studied 139 subjects with a mean+/-SD age of 50 +/-14 years and a body mass index of 26.5+/-5.3 kg/m2; 110 subjects had essential hypertension and 29 were healthy nonhypertensive controls. Blood pressure was measured in resting conditions in the morning and blood was drawn for the determination of the plasma renin activity, aldosterone, and leptin levels. The mean blood pressure of the population was 155/97 mm Hg. The relationship between these parameters was studied by univariate regression analysis according to gender and, whenever indicated, adjusted for age and body mass. The mean+/-SEM plasma leptin level in the whole population was 9.5+/-0.6 ng/mL (range, 1.1-43.3). Subjects with stage I hypertension had significantly higher plasma leptin levels than normotensive subjects. Systolic blood pressure correlated with the plasma leptin levels and the leptin levels adjusted for body weight in women (r = 0.422, P < .01) and nonhypertensive men (r = 0.644, P = .03) only. Plasma renin activity (r = 0.329, P = .03) and aldosterone levels (r = 0.342, P = .026) correlated with the leptin concentration. A significant relationship between the peripheral expression of the ob gene product leptin and systolic blood pressure was found in women and nonhypertensive men. In view of the multiple functions of leptin a causal relationship is postulated and potential mechanisms may involve modulatory effects of leptin on neuropeptide Y, angiotensinogen gene expression, the modulation of the autonomous nervous system, or effects on the pituitary adrenal axis. Direct relationships between both plasma renin activity and aldosterone levels and leptin support the potential importance of the relationship between leptin and blood pressure. Our observation may be of future importance for the understanding of the link between the increase in blood pressure and increasing body weight.

Novel cellular activities for low density lipoprotein in vascular smooth muscle cells.

Hyperlipidemia and hypertension play important roles in the pathogenesis of atherosclerosis. To investigate the underlying intraceiiular mechanisms, we studied the effect of various concentrations of low density lipoprotein from normolipidemic subjects on concentrations of free intracellular calcium, intraceiiular pH, DNA synthesis, and vascular tone in vascular smooth muscle cells and rings from rat aortas. Low density lipoprotein in the range of 1–15 μg/ml induced a dose-dependent increase of concentration of free intraceiiular calcium and a biphasic change of the intraceiiular pH. Similar concentrations of low density lipoprotein led to an enhanced DNA synthesis. Furthermore, cumulative addition of 1–15 μg/ml low density lipoprotein produced a dose-dependent increase in contractile tension of thoracic aortic rings from rats. The maximal low density lipoprotein-induced contractile response was approximately 70% of that induced by 40 mM KCI. These findings indicate that low concentrations of low density lipoprotein occurring, for example, in the extravascular fluid might contribute to the pathogenesis of cardiovascular diseases by enhancing cell proliferation and vasoconstriction by changing intraceiiular calcium and intracellular pH.

The platelet-derived growth factor isomers, PDGF-AA, PDGF-AB and PDGF-BB, induce contraction of vascular smooth muscle cells by different intracellular mechanisms

The effect of human recombinant platelet‐derived growth factor (PDGF) isoforms, (r)PDGF‐AA, PDGF‐AB and PDGF‐BB, on contractility of rat aortic rings as well as on intercellular free Ca2+ ([Ca2+]i), intracellular pHi (pHi) and thromboxane A2 (TXA2) formation in cultured vascular smooth muscle cells (VSMC) was examined. PDGF‐BB behaved similar to PDGF‐AB and both have features characteristic of conventional vasocontrictor‐agonists that directly increase [Ca2+]i, activate the Na+/H+ exchanger, stimulate the TXA2 formation, and induced contraction in VSMC whereas PDGF‐AA induced contraction without increasing of [Ca2+]i, pHi, and TXA2 formation.

Oxidation of low density lipoprotein enhances its potential to increase intracellular free calcium concentration in vascular smooth muscle cells.

There have been suggestions that oxidation of low density lipoproteins (LDL) might increase their atherogenic potential. Because changes in intracellular free calcium concentration [Ca2+]i have been linked to atherogenesis, we compared the influence of oxidized LDL (Ox-LDL) and native LDL (N-LDL) on [Ca2+]i in vascular smooth muscle cells cultured from rat aortas. For determination of [Ca2+]i, fura-2 fluorescence was used. LDL was isolated by ultracentrifugation from the sera of human donors (n = 17). In N-LDL, oxidation was prevented by addition of antioxidants, whereas Ox-LDL was obtained by auto-oxidation. The extent of oxidation was assessed by measurement of thiobarbituric acid-reactive substances. Addition of Ox-LDL (20 micrograms protein/ml) to the vascular smooth muscle cells induced a mean increase of 129 +/- 13% in [Ca2+]i compared with 81 +/- 7% with N-LDL (p less than 0.01). Dose-response curves from 1 to 20 micrograms/ml (six experiments) confirmed this difference within the entire dose range. These results indicate that a more pronounced increase in [Ca2+]i induced by Ox-LDL might be one of the cellular mechanisms responsible for the higher atherogenic potential of Ox-LDL compared with N-LDL, as [Ca2+]i is an important second-messenger system involved in many atherogenic processes such as hypertrophy, cell migration, and cell damage.

The cholesterol content of the human erythrocyte influences calcium influx through the channel.

In order to study the influence of the cholesterol content on the calcium entry channel, the human red blood cell was used as a model system. The cholesterol to lecithin ratio (C/L ratio) of the membrane was modified experimentally by incubating the cells (15h, 25 degrees) with liposomes of defined C/L ratios. Subsequently, net 45Calcium-influx into the cell was measured by inhibiting the Ca-ejecting ATPase with vanadate. Additionally, the use of nitrendipine, a potent calcium channel inhibitor, during incubation allowed the determination of Ca-influx through the calcium channel. A positive correlation between the 45Ca++-influx and the molar C/L ratio of the membrane was found over a wide C/L range. A molar C/L ratio of 1.4 in the membrane increased calcium influx by 150 % compared to controls (molar C/L ratio = 0.8, calcium influx rate = 100 %), while a molar C/L ratio at less than 0.75 decreased calcium influx by 50 %. We conclude, that the cholesterol content of the membrane greatly influences the calcium channel and thus plays a pivotal role for the availability of calcium as a second messenger. These findings may provide a link between high plasma cholesterol and the development of atherosclerosis as well as enhanced platelet aggregability.

Green tea polyphenols inhibit human vascular smooth muscle cell proliferation stimulated by native low-density lipoprotein.

This study investigated whether human vascular smooth muscle cell proliferation induced by native low-density lipoprotein (LDL) is affected by green tea catechins. Furthermore, the effects of native LDL on extracellular signal-regulated kinase (ERK) 1/2 activity were determined. Cell proliferation stimulated by native LDL was concentration-dependently inhibited by epigallocatechin, epigallocatechin-3-gallate, green tea polyphenon, and the nonspecific antioxidant N-acetylcysteine (P<0.05). Combined treatment of green tea polyphenon and N-acetylcysteine markedly potentiated the effect of each drug on vascular smooth muscle cell proliferation. ERK1/2 activity was only partly inhibited by green tea catechins alone or in combination with N-acetylcysteine (P<0.05). These data suggest that green tea constituents inhibit proliferation of human vascular smooth muscle cells exposed to high levels of native LDL. Green tea constituents and antioxidants may exert vascular protection by inhibiting human vascular smooth muscle cell growth associated with hypercholesterolemia.

Low-density lipoprotein elevates intracellular calcium and pH in vascular smooth muscle cells and fibroblasts without mediation of LDL receptor

Low-density lipoprotein (7 micrograms/ml) induced in the absence or in the presence of 7, 35, 70 micrograms/ml monoclonal antibodies against the specific Low-density lipoprotein receptor an elevation of intracellular Ca2+ from 105 to approximately 210 nM in vascular smooth muscle cells from rat aorta. Moreover, in both human cultured fibroblasts from normocholesterolemic individuals and from patients with familial hypercholesterolemia homozygote class 1, Low-density lipoprotein (7 micrograms/ml) induced a rise of free intracellular calcium and a biphasic change of intracellular pH. Low-density lipoprotein (1,7,15,30 micrograms/ml) had no significant influence on the phosphatidylinositol-turnover in vascular smooth muscle cells and fibroblasts. Since homozygote class 1 fibroblasts lack specific Low-density lipoprotein receptors, and as antibodies against this receptor did not attenuate the Low-density lipoprotein-induced elevation of cytosolic calcium and pH, we conclude that these intracellular changes are independent from the classical Low-density lipoprotein receptor.

Stimulation of the phosphoinositide signalling system as a possible mechanism for glucocorticoid action in blood pressure control.

Cortisol stimulates the phosphoinositide signalling system in smooth muscle cells of the rat aorta. After stimulation of the cells with cortisol, epinephrine or both compounds, inositol-1,4,5-trisphosphate concentrations were analysed by standardized ionexchange chromatography procedure. A 15-min stimulation with physiological concentrations of cortisol (0.02–5.0 μg/ml) led to a dose-dependent increase of the inositol trisphosphate concentrations (up to 500%) and also to a translocation of the calcium-and lipid-dependent protein kinase C activity from the cytosolic to the membranous compartment. Incubation of smooth muscle cells with epinephrine (10-9 to -5 mol/l) did not lead to an increase in the inositol trisphosphate concentrations. However, after pre-incubation with an average dose of cortisol (0.2 μg/ml) the inositol trisphosphate response was potentiated by 10-7 mol/l epinephrine. Our results suggest that stimulation of the phosphoinositide system is a still unknown mechanism of glucocorticoid action in smooth muscle cells, which could influence intracellular free calcium and thus vascular reactivity and blood pressure.

Fish oil affects phosphoinositide turnover and thromboxane A metabolism in cultured vascular muscle cells.

Fish oil has been reported as having beneficial effects on cardiovascular diseases. Elevated serum lipoproteins, prostaglandins and intracellular free calcium concentrations [( Ca2+]i) of the vasculature and thus the phosphoinositide (PI) turnover may be involved in the pathogenesis of these disorders. Therefore, the effect of fish oil on the potency of both low-density lipoprotein (LDL) and angiotensin II (AII) to stimulate the PI turnover in cultured rat vascular smooth muscle cells (VSMC) has been studied. Furthermore, a possible link between PI turnover activity and thromboxane A2 (TXA2) metabolism in these cells has been investigated. In VSMC cultured for up to 7 weeks with either fish oil or n-3 eicosapentaenoic acid (EPA) a decrease to 5-48% of the LDL-induced inositol trisphosphate (IP3) formation (= 100%) was found. A similar range of decreased IP3 synthesis was observed, when AII was used instead of LDL. Both LDL- and AII-stimulated TXA2 synthesis was suppressed concomitantly within the range 34-60%. Blockade of VSMC TXA2 biosynthesis with either indomethacin or TXA2 synthetase blocker (SQ-80338) inhibited LDL-induced formation of IP3 in a dose-dependent manner. Similar results were obtained, when TXA2 receptor coupling antagonists (SQ-27427 or BM-13177) were used. However, blockers of TXA2 synthesis and of TXA2 receptor binding failed to affect AII-induced formation of IP3.

Lysolecithin actions on vascular smooth muscle cells.

Oxidation of low density lipoprotein increases its atherogenic potential. During oxidation there is an extensive conversion of lecithin to lysolecithin. In rat aortic smooth muscle cells, 2-25 micrograms/ml lysolecithin elevated cytosolic calcium concentration up to 560%. Lysolecithin (10-20 micrograms/ml) increased [3H]thymidine incorporation from 15 cpm/mg cell protein (controls) up to 189 cpm/mg cell protein. Lysolecithin (10 micrograms/ml) potentiated the PDGF-induced (50 ng/ml) [3H]thymidine incorporation up to 6.3 times. The results indicate that lysolecithin could induce mechanisms, by which oxidized low density lipoproteins could promote cell growth and thus contribute to atherosclerosis.