Hyperaldosteronemia in rabbits inhibits the cardiac sarcolemmal Na(+)-K(+) pump

Aldosterone upregulates the Na(+)-K(+) pump in kidney and colon, classical target organs for the hormone. An effect on pump function in the heart is not firmly established. Because the myocardium contains mineralocorticoid receptors, we examined whether aldosterone has an effect on Na(+)-K(+) pump function in cardiac myocytes. Myocytes were isolated from rabbits given aldosterone via osmotic minipumps and from controls. Electrogenic Na(+)-K(+) pump current, arising from the 3:2 Na(+):K(+) exchange ratio, was measured in single myocytes using the whole-cell patch clamp technique. Treatment with aldosterone induced a decrease in pump current measured when myocytes were dialyzed with patch pipette solution containing Na(+) in a concentration of 10 mmol/L, whereas there was no effect measured when the solution contained 80 mmol/L Na(+). Aldosterone had no effect on myocardial Na(+)-K(+) pump concentration evaluated by vanadate-facilitated [(3)H]ouabain binding or by K(+)-dependent paranitrophenylphosphatase activity in crude homogenates. Aldosterone induced an increase in intracellular Na(+) activity. The aldosterone-induced decrease in pump current and increased intracellular Na(+) were prevented by cotreatment with the mineralocorticoid receptor antagonist spironolactone. Our results indicate that hyperaldosteronemia decreases the apparent Na(+) affinity of the Na(+)-K(+) pump, whereas it has no effect on maximal pump capacity.     

Impact of HMG CoA reductase inhibition on small GTPases in the heart

OBJECTIVE: Members of the Rho GTPase family, Rac1 and RhoA have been suggested to be mediators of cardiac hypertrophy in mice. Rho proteins are posttranslationally isoprenylated. In addition to cholesterol-lowering, statins inhibit the isoprenylation of small G proteins. Therefore, it was tested if these drugs inhibit Rac1 and RhoA activity in cardiomyocytes and, thereby, prevent angiotensin II-mediated expression of atrial natriuretic factor (ANF) and myosin light chain (MLC)-2 in the heart. METHODS AND RESULTS: Western and Northern analysis of rat neonatal cardiomyocytes and H9C2 cells showed inhibition of basal and angiotensin-stimulated Rac1 expression, membrane-translocation and activity after statin treatment. Similarly, basal and stimulated RhoA membrane expression was inhibited. Statins concentration- and time-dependently downregulated basal as well as angiotensin-induced expression of ANF by 86+/-2.3% and 89+/-1.7%, as well as MLC-2 by 75+/-4.1% and 84+/-6%, respectively. Direct inhibition of Rac GTPase by overexpression of the dominant negative mutant RacN17 or by Clostridium sordellii lethal toxin in rat H9C2 cells inhibited ANF expression by 70+/-4.9% and 78+/-10%, respectively. Inhibition of RhoA by Clostridium botulinum C3 transferase or the dominant negative mutant RhoN19 reduced ANF mRNA by 19+/-11% and 23+/-8%, respectively. To test these findings in vivo, spontaneously hypertensive rats were treated with atorvastatin, leading to a decrease in cardiac Rac1 and RhoA activity as determined by [35S]-GTP gamma S-binding assays by 61+/-16% and 72+/-24%, and downregulation of MLC-2 as well as ANF mRNA expression by 31+/-16% and 80+/-24%, respectively. CONCLUSIONS: (1) Statins downregulate the activity of small G proteins in cardiomyocytes in culture as well as in vivo. (2) Inhibition of Rac1 and RhoA by statins reduces myocardial expression of ANF and MLC-2. (3) Targeting myocardial Rho GTPases by statins may be a novel treatment strategy to prevent cardiac hypertrophy.     

Low density lipoprotein density and composition in hypercholesterolaemic men treated with HMG CoA reductase inhibitors and gemfibrozil

The effects of HMG CoA reductase inhibitor (lovastatin or simvastatin) and gemfibrozil treatment on low density lipoprotein (LDL) density distribution and composition were studied in male patients with heterozygous familial hypercholesterolaemia (FH) (n = 17) or non-familial hypercholesterolaemia (non-FH) (n = 14). In FH patients the HMG CoA reductase inhibitors reduced 'light' LDL particles (density 1.022-1.033 g ml-1) significantly more than 'heavy' LDL (density 1.033-1.059 g ml-1), while a more uniform reduction of 'light' and 'heavy' LDL occurred in non-FH patients. HMG CoA reductase inhibitor treatment increased the apolipoprotein B (apoB) content and decreased the cholesterol/apoB ratio of LDL in FH patients. Gemfibrozil significantly reduced 'heavy' LDL but not the 'light' LDL fraction in both FH and non-FH patients, and the mean cholesteryl ester content of LDL increased, while the Tg content decreased, in both FH and non-FH patients. The results indicate that HMG CoA reductase inhibitor and gemfibrozil treatment have distinctly different effects on the physico-chemical properties of LDL, reflecting their different modes of action on lipoprotein metabolism.     

Singlet oxygen-induced inhibition of cardiac sarcolemmal Na+K(+)-ATPase.

We investigated the susceptibility of sarcolemmal Na+K(+)-ATPase to singlet oxygen. The role of this enzyme is regulation of Na+ concentration and thereby membrane potential. Inhibition of Na+ pump would lead to intracellular Ca2+ overload therefore further aggravating the injury caused by free radicals. Incubation of isolated sarcolemmal vesicles with irradiated rose bengal (150 nM) resulted in 86 +/- 1% inhibition of Na+K(+)-ATPase activity and histidine (25-100 mM) protected the enzyme in a dose-dependent fashion whereas SOD, catalase or mannitol (.OH radical scavenger) did not have any effect. Also, the inhibition of Na+K(+)-ATPase activity was dependent on rose bengal concentration, intensity of irradiation, duration of light exposure, showing that inhibition was directly related to amount of singlet oxygen generated. These results show that singlet oxygen may have significant disruptive effects on sarcolemmal function and may represent an important mechanism by which the oxidative injury to the myocardium induces arrhythmogenesis.     

Development of tachyphylaxis among patients taking HMG CoA reductase inhibitors

The purpose of this study was to determine if long-term use of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (atorvastatin, fluvastatin, lovastatin, pravastatin, or simvastatin) resulted in tachyphylaxis (a decreasing response to a physiologically active agent). To determine this, the charts of 254 patients treated with statins from the years 1996 to 1998 were retrospectively reviewed. During treatment, the low-density lipoprotein (LDL) cholesterol levels of patients were followed for a minimum of 300 days. To characterize LDL cholesterol changes during statin therapy, linear and nonlinear kinetic models were generated. Tachyphylaxis, defined as a positive slope of LDL cholesterol over time, after maximum LDL cholesterol reduction, was identified in patients treated with atorvastatin at exposure doses of 10 or 20 mg/day. All other statins, at all doses reviewed, showed no [corrected] evidence of tachyphylaxis. LDL cholesterol tachyphylaxis appeared to be a unique response to prolonged use of long half-life atorvastatin therapy at exposure dosages.     

HMG CoA reductase inhibitor (statin) and aortic valve calcium

There is no known pharmacological therapy for calcific aortic valvular sclerosis or stenosis. Because leaflet calcification occurs in areas of lipoprotein deposition, we hypothesised that 3-hydroxy-3-methylglutaryl-coenzyme A (HMG COA) reductase inhibitors (statins) might slow aortic valve calcium (AVC) accumulation. We retrospectively identified 65 patients who had undergone two electron-beam computed tomography scans at a mean (SD) interval of 2.5 (1.6) years. 28 (43%) patients were receiving statins. Patients who were treated with statins had a 62-63% lower median rate of AVC accumulation (p=0.006) and 44-49% fewer statin patients had definite AVC progression (p=0.043). These findings suggest that statins may decrease AVC accumulation.     

Treatment of hypercholesterolemia with the HMG CoA reductase inhibitor,simvastatin

Summary We report the results of a two center study on the use of the HMG Co A reductase inhibitor, simvastatin, in 44 patients suffering from familial hypercholesterolemia or from primary hypercholesterolemia of unknown etiology. The study included two separate phases: Phase I was part of a multicenter, 4-week, placebo-controlled trial; phase II was a 6-month, open extension trial, the object of which was to reduce low density lipoprotein (LDL) cholesterol levels to below the 50th percentile by increasing the dose of simvastatin, by the use of additional lipid-lowering medication, or both. Our phase I results were commensurate with those reported for the entire international cohort of 272 patients, indicating a clear dose-response relationship, with approximately 75% of the maximum reduction in LDL-C levels being achieved with 20 mg/day and over 90% of the maximum being achieved with 40 mg of simvastatin per day. In the open extension trial, the results from the 2 centers were essentially similar. Total cholesterol fell by 29% on the 20 mg/day dose and by 34% on the full dose of 40 mg/day. LDL-C levels were reduced by 40% on the 40 mg/day schedule, and triglycerides also fell to between 20% and 40% below baseline values. HDL-C concentration rose by 14% and 17.6%. The effects of simvastatin were uniform, both within and between the two cohorts. The addition of cholestyramine caused a further substantial reduction in LDL-cholesterol to below 55% of the initial value in four patients, whereas bezafibrate further enhanced the fall in triglycerides and the increase in high-density lipoprotein cholesterol, but had only a slight effect on LDL-C levels. Adverse reactions included asymptomatic increases in plasma creatine kinase activity, generally associated with previous physical exertion, and transient rises in transaminase levels. In one patient with a history of alcoholic excess, transaminase levels were persistently greater than normal. These results indicate that the HMG Co A reductase inhibitor, simvastatin, is a powerful therapeutic agent for the lowering of plasma cholesterol levels in patients with genetic hypercholesterolemia.     

Na(+)-K+ pump and Na(+)-K+ co-transport in cultured vascular smooth muscle cells from spontaneously hypertensive and normotensive rats: baseline activity and regulation.

OBJECTIVE: This paper examines the hypothesis that aberrations in vascular smooth muscle univalent ion transport systems play an important role in the pathogenesis of hypertension. DESIGN: Baseline Na(+)-K+ pump and Na(+)-K(+)-2Cl- co-transport activities and the regulation of these ion transport systems by angiotensin II and second messenger molecules have been studied in cultured aortic smooth muscle cells (VSMC) from normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). METHODS: Ion transport was studied using isotopic univalent cations (86Rb and 22Na). RESULTS: Baseline Na(+)-K+ pump activity was comparable between SHR- and WKY-derived VSMC. Baseline Na(+)-K(+)-2Cl- and K(+)-Cl- co-transport activity as well as K+ leakage were significantly greater in SHR VSMC. Baseline Na(+)-K(+)-2Cl- co-transport was sensitive to inhibition by forskolin and ethyleneglycol-bis-(beta-amino ethylester)-N,N,N',N'-tetraacetic acid, whereas cyclic guanosine monophosphate and phorbol 12-myristate, 13-acetate had no effect. Angiotensin II-stimulated Na(+)-K(+)-2Cl- co-transport activity did not differ between WKY and SHR VSMC. Angiotensin II increased Na(+)-K(+)-pump activity to a significantly greater extent in SHR VSMC. The stimulatory effect of angiotensin II upon Na(+)-K+ pump activity was reduced under Na(+)-free buffer conditions and in the presence of the Na(+)-H+ exchange inhibitor, ethylisopropyl amiloride. Na(+)-K+ pump activity was also stimulated by the protein kinase C activator, phorbol 12-myristate, 13-acetate, and this was completely inhibited under Na(+)-free buffer conditions. CONCLUSIONS: SHR VSMC exhibit anomalous Na(+)-K(+)-pump and Na(+)-K(+)-2Cl- co-transport activities. The influence of these univalent ion transport systems upon cellular Na+ and Ca2+ homeostasis invoke their participation in the pathogenesis of hypertension.     

Na+-K+ pump inhibition caused by chronic amiodarone in guinea pig myocardium

Summary Although it is known that amiodarone inhibits myocardial Na⁺-K⁺ pump activity, the potency and the time course of this inhibition are unknown. The aim of this study was to investigate these aspects with reference to digoxin, using guinea pigs treated with either intraperitoneal amiodarone (20mg/kg per week, up to 12 weeks,n = 26) or the same amount of vehicle as a control (n = 24). ECG recording and microelectrode experiments were conducted every 2 weeks. QT interval corrected by heart rate and action potential duration were prolonged as a function of the time of exposure to amiodarone. Hyperpolarization observed immediately after the overdrive (1.0Hz) termination or K⁺-replenishment following K⁺-depletion in the presence of 0.1mM Ba²⁺ was compared in the amiodarone-treated and untreated groups, as an index of the Na⁺-K⁺ pump activity. The resting membrane potential recovery from overdrive-induced depolarization was slower and the amplitude of K⁺-induced hyperpolarization was smaller in the amiodarone-treated group than in the untreated group. These changes were evident as the chronic amiodarone treatment progressed, although the changes in these parameters were greater in the case of acute application of 50µM digoxin. In conclusion, this study indicates that treatment with amiodarone for longer than several weeks moderately inhibits the myocardial Na⁺-K⁺ pump.This work was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (11877125).     

CD43 polarization in unprimed T cells can be dissociated from raft coalescence by inhibition of HMG CoA reductase

Movement of T-lymphocyte cell surface CD43 is associated with both antigen activation of T-cell clones and chemokine induction of T-lymphocyte motility. Here, we demonstrate that CD43 movement away from the site of T-cell receptor ligation occurs in unprimed CD4(+) T cells as well as T-cell clones. The T-cell receptor (TCR)-dependent movement of CD43 in unprimed T cells is associated with a polarized morphology and CD43 accumulation at the uropods of the cells, unlike that reported for primed T cells. The polarization of CD43 has a requirement for Src kinases and occurs in conjunction with lipid raft coalescence. Thymocytes and T-cell hybridomas, cells that have altered responses to TCR activation and lack lipid raft coalescence, do not polarize CD43 as readily as unprimed T cells. The movement of CD43 depends on the cholesterol biosynthetic pathway enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase. Blockade of this enzyme can specifically prevent CD43 redistribution without affecting cell shape polarization. The likely mechanism of this alteration in CD43 redistribution is through decreased protein prenylation because the cholesterol-dependent lipid rafts still coalesce on activation. These findings suggest that the polarization of cell shape, lipid raft coalescence, and CD43 redistribution on T-cell activation have signaling pathway distinctions. Dissecting out the relationships between various stages of molecular redistribution and lymphocyte activation may facilitate fine-tuning of immunologic responses.     

Recovery of erythrocyte Na(+)-K(+)-Cl- cotransport activity by enalapril

We studied total exchangeable sodium, ion transport activity at maximal rate, and erythrocyte Na+ content in response to angiotensin converting enzyme inhibition in mild-to-moderate essential hypertensive patients with normal renal function. Twenty-five patients (mean age 56 years, range 40-62 years) who had abnormal red blood cell Na(+)-K(+)-Cl- cotransport or red blood cell Li(+)-Na+ countertransport were treated with either enalapril (20 mg daily) or hydrochlorothiazide (50 mg daily) during a 30-day period. During the period of enalapril treatment, Na(+)-K+ pump and Na(+)-K(+)-Cl- cotransport increased significantly from 4,282 +/- 255 to 5,236 +/- 325 mumol/l red blood cell/hr (p less than 0.01) and 166 +/- 21 to 220 +/- 24 mumol/l red blood cell/hr (p less than 0.05), respectively. Mean intracellular Na+ content in erythrocytes decreased from 11.4 +/- 0.40 to 10.0 +/- 0.33 mmol/l (p less than 0.01) and exchangeable Na+ from 39.8 +/- 0.6 mmol/kg to 35.6 +/- 0.6 mmol/kg (p less than 0.001). Sodium reduction correlated with the recovery of Na(+)-K(+)-Cl- cotransport activity (r = -0.65, p less than 0.01). During treatment, systolic and diastolic blood pressures were reduced significantly (p less than 0.01). In 12 patients treated with hydrochlorothiazide, Na(+)-K(+)-Cl- cotransport, Na(+)-K+ pump, Na(+)-Li+ countertransport, and Na+ permeability did not change significantly while Na+ content decreased from 11.7 +/- 0.3 to 10.3 +/- 0.2 mmol/l (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

A general assessment of the safety of HMG CoA reductase inhibitors (statins)

Few principles in medicine today are as well accepted as the benefits of using statins to reduce cardiovascular risk factors and to prevent cardiovascular disease. The recent withdrawal of a statin (cerivastatin) has lead to a re-examination of all statins, and an attempt to better assess the risk-benefit ratio of this class of drugs. Although the benefits of statin treatment are well known from a myriad of clinical trials, the safety of these compounds has not been an issue since lovastatin was first introduced in 1987. This report provides a brief review of statin safety, and reiterates the excellent profile of this class of drugs.     

HMG CoA reductase inhibitors lower LDL cholesterol without reducing Lp(a) levels

Lp(a) is a plasma lipoprotein particle consisting of a plasminogenlike protein [apo(a)] disulfide bonded to the apo B moiety of low-density lipoprotein (LDL). Increased plasma levels of Lp(a), either independently or interactively with LDL levels, have been shown to be a risk factor for atherosclerosis. Recently, a new class of lipid-lowering drugs, HMG CoA reductase inhibitors, have been introduced. These drugs act by decreasing liver cholesterol synthesis resulting in up-regulation of LDL receptors, increased clearance of LDL from plasma, and diminution of plasma LDL levels. In this study, we examined the effect of HMG CoA reductase inhibitors on Lp(a) levels in three groups of subjects, five volunteers and two groups of five and 14 patients. In all 24 subjects, mean decreases were observed in total cholesterol (43 +/- 5%), total triglyceride (35 +/- 8%), very low-density lipoprotein (45 +/- 9%), and LDL cholesterol (43 +/- 5%). The mean change in high-density lipoprotein cholesterol was an increase of 7 +/- 8%. Despite the very significant decrease in LDL cholesterol levels (p less than 0.001), Lp(a) levels increased by 33 +/- 12% (p less than 0.005). This was not associated with a measurable change in the chemical composition or size of the Lp(a) particle. This emphatically suggests that Lp(a) particles, despite consisting principally of LDL, are cleared from plasma differently than LDL. The surprising finding of an increase in Lp(a) levels suggests this class of drugs may have a direct effect on Lp(a) synthesis or clearance independent of its effect on LDL receptors.     

HMG CoA reductase inhibitors are related to improved systemic endothelial function in coronary artery disease

The hypocholesterolemic and anti-atherosclerotic effect of TS-962, a specific ACAT inhibitor, was investigated in a hamster model fed a high fat diet containing 10% coconut oil and 0.05% cholesterol. Lipid accumulated atherosclerotic lesions were detected by using oil red O staining in the lesion-prone aortic arch. A high dose, estimated to be 15 mg/kg, of TS-962 decreased serum cholesterol to normal levels with reduction of liver cholesterol contents below normal levels, and as a consequence, entirely inhibited the lipid accumulation in the aortic arch. Furthermore, a low dose, estimated to be 1.5 mg/kg, of TS-962 remarkably inhibited aortic lipid accumulation by 73% compared with the control group, without changing either serum cholesterol level or liver cholesterol content. These findings suggest that TS-962 is effective in the primary prevention of atherosclerosis by directly suppressing the formation of foam cells in arteries.     

Treating hypercholesterolaemia with HMG CoA reductase inhibitors: a direct comparison of simvastatin and pravastatin

Abstract Background: Simvastatin and pravastatin are both competitive inhibitors of the rate limiting enzyme for cholesterol biosynthesis (HMG CoA) reductase, but data from individual clinical trials suggest significant differences in potency for cholesterol reduction between the two drugs. Aim: To assess any differences in efficacy and safety between simvastatin and pravastatin in a direct, comparative study. Methods: A double-blind, double-dummy, randomised study design was used, involving 48 patients with primary hypercholesterolaemia. Following a 6 week placebo baseline period, patients were randomly allocated to treatment with either simvastatin or pravastatin, commencing at a dose of 10 mg daily. The dose levels were titrated up to the recommended maximum effective dose of 40 mg daily at 6 weekly intervals if LDL cholesterol levels remained 3.4 mmol/L. After 18 weeks of therapy, all patients were transferred to simvastatin therapy for a further 6 weeks, continuing at their week 18 dose level. Patients complied with a standard lipid lowering diet (containing < 30% of energy as total fat) throughout the study period. Results: Over the 18 week direct comparison of the two drugs, there was a significant difference (p< 0.001) in response between simvastatin and pravastatin for reduction in levels of total cholesterol (32%vs21% respectively), LDL cholesterol (38%vs27%) and apolipoprotein B levels (34%vs23%). No significant difference in drug effect was seen for the small reduction in levels of apolipoprotein AI (5%vs6% respectively), nor for the increased levels of apolipoprotein All (14%vsll%) and HDL cholesterol (1 l%vs7%). Lp(a) levels remained unchanged. When pravastatin was replaced with simvastatin for the final 6 weeks of the study in the 23 patients initially randomised to pravastatin, there were further reductions (p < 0.01) in total and LDL cholesterol, and apolipoprotein B. These results establish the advantage of simvastatin over pravastatin in terms of efficacy, for the treatment of primary hypercholesterolaemia.