HMG CoA reductase inhibition reduces sarcolemmal Na(+)-K(+) pump density.

OBJECTIVES: HMG CoA reductase inhibitors reduce cellular availability of mevalonate, a precursor in cholesterol synthesis. Since the cholesterol content of cell membranes is an important determinant of Na(+)-K(+) pump function we speculated that treatment with HMG CoA reductase inhibitors affects Na(+)-K(+) pump activity. METHODS: We treated rabbits and rats for 2 weeks with the HMG CoA reductase inhibitor lovastatin and measured Na(+)-K(+) pump current (I(p)) in isolated rabbit cardiac myocytes using the whole cell patch-clamp technique, K-dependent p-nitrophenyl phosphatase (p-NPPase) activity in crude myocardial and skeletal muscle homogenates, and vanadate-facilitated 3H-ouabain binding in intact skeletal muscle samples from rats. RESULTS: Treatment with lovastatin caused statistically significant reductions in I(p), myocardial and skeletal muscle K-dependent p-NPPase activity and 3H-ouabain binding in the myocardium and skeletal muscle. The lovastatin-induced decrease in I(p) was eliminated by parenteral co-administration of mevalonate. However, this was not related to cardiac cholesterol content. CONCLUSIONS: Treatment with lovastatin reduces Na(+)-K(+) pump activity and abundance in rabbit and rat sarcolemma.

Na+ influx and Na(+)-K+ pump activation during short-term exposure of cardiac myocytes to aldosterone.

To examine the effect of aldosterone on sarcolemmal Na+ transport, we measured ouabain-sensitive electrogenic Na(+)-K+ pump current (Ip) in voltage-clamped ventricular myocytes and intracellular Na+ activity (alpha iNa) in right ventricular papillary muscles. Aldosterone (10 nM) induced an increase in both Ip and the rate of rise of alpha iNa during Na(+)-K+ pump blockade with the fast-acting cardiac steroid dihydroouabain. The aldosterone-induced increase in Ip and rate of rise of alpha iNa was eliminated by bumetanide, suggesting that aldosterone activates Na+ influx through the Na(+)-K(+)-2Cl- cotransporter. To obtain independent support for this, the Na+, K+, and Cl- concentrations in the superfusate and solution of pipettes used to voltage clamp myocytes were set at levels designed to abolish the inward electrochemical driving force for the Na(+)-K(+)-2Cl- cotransporter. This eliminated the aldosterone-induced increase in Ip. We conclude that in vitro exposure of cardiac myocytes to aldosterone activates the Na(+)-K(+)-2Cl- cotransporter to enhance Na+ influx and stimulate the Na(+)-K+ pump.

Voltage-dependent stimulation of the Na(+)-K(+) pump by insulin in rabbit cardiac myocytes.

Insulin enhances Na(+)-K(+) pump activity in various noncardiac tissues. We examined whether insulin exposure in vitro regulates Na(+)-K(+) pump function in rabbit ventricular myocytes. Pump current (I(p)) was measured using the whole-cell patch-clamp technique at test potentials (V(m)s) from -100 to +60 mV. When the Na(+) concentration in the patch pipette ([Na](pip)) was 10 mM, insulin caused a V(m)-dependent increase in I(p). The increase was approximately 70% when V(m) was at near physiological diastolic potentials. This effect persisted after elimination of extracellular voltage-dependent steps and when K(+) and K(+)-congeners were excluded from the patch pipettes. When [Na](pip) was 80 mM, causing near-maximal pump stimulation, insulin had no effect, suggesting that it did not cause an increase in membrane pump density. Effects of tyrphostin A25, wortmannin, okadaic acid, or bisindolylmaleimide I in pipette solutions suggested that the insulin-induced increase in I(p) involved activation of tyrosine kinase, phosphatidylinositol 3-kinase, and protein phosphatase 1, whereas protein phosphatase 2A and protein kinase C were not involved.

Protein kinase Cepsilon contributes to regulation of the sarcolemmal Na(+)-K(+) pump.

A reduction in angiotensin II (ANG II) in vivo by treatment of rabbits with the angiotensin-converting enzyme inhibitor, captopril, increases Na(+)-K(+) pump current (I(p)) of cardiac myocytes. This increase is abolished by exposure of myocytes to ANG II in vitro. Because ANG II induces translocation of the epsilon-isoform of protein kinase C (PKCepsilon), we examined whether this isozyme regulates the pump. We treated rabbits with captopril, isolated myocytes, and measured I(p) of myocytes voltage clamped with wide-tipped patch pipettes. I(p) of myocytes from captopril-treated rabbits was larger than I(p) of myocytes from controls. ANG II superfusion of myocytes from captopril-treated rabbits decreased I(p) to levels similar to controls. Inclusion of PKCepsilon-specific blocking peptide in pipette solutions used to perfuse the intracellular compartment abolished the effect of ANG II. Inclusion of psiepsilonRACK, a PKCepsilon-specific activating peptide, in pipette solutions had an effect on I(p) that was similar to that of ANG II. There was no additive effect of ANG II and psiepsilonRACK. We conclude that PKCepsilon regulates the sarcolemmal Na(+)-K(+) pump.

Angiotensin regulates the selectivity of the Na+-K+ pump for intracellular Na+.

Treatment of rabbits with angiotensin-converting enzyme (ACE) inhibitors increases the apparent affinity of the Na+-K+ pump for Na+. To explore the mechanism, we voltage clamped myocytes from control rabbits and rabbits treated with captopril with patch pipettes containing 10 mM Na+. When pipette solutions were K+ free, pump current (Ip) for myocytes from captopril-treated rabbits was nearly identical to that for myocytes from controls. However, treatment caused a significant increase in Ip measured with pipettes containing K+. A similar difference was observed when myocytes from rabbits treated with the ANG II receptor antagonist losartan and myocytes from controls were compared. Treatment-induced differences in Ip were eliminated by in vitro exposure to ANG II or phorbol 12-myristate 13-acetate or inclusion of the protein kinase C fragment composed of amino acids 530-558 in pipette solutions. Treatment with captopril had no effect on the voltage dependence of Ip. We conclude that ANG II regulates the pump's selectivity for intracellular Na+ at sites near the cytoplasmic surface. Protein kinase C is implicated in the messenger cascade.

Amiodarone inhibits the Na(+)-K+ pump in rabbit cardiac myocytes after acute and chronic treatment.

Amiodarone has been shown to affect cell membrane physicochemical properties, and it may produce a state of cellular hypothyroidism. Because the sarcolemmal Na(+)-K+ pump is sensitive to changes in cell membrane properties and thyroid status, we examined whether amiodarone affected Na(+)-K+ pump function. We measured Na(+)-K+ pump current (Ip) using the whole-cell patch-clamp technique in single ventricular myocytes isolated from rabbits. Chronic treatment with oral amiodarone for 4 weeks reduced i.p. when myocytes were dialyzed with patch-pipettes containing either 10 mM Na+ or 80 mM Na+. In myocytes from untreated rabbits, acute exposure to amiodarone in vitro reduced i.p. when patch pipettes contained 10 mM Na+ but had no effect on i.p. at 80 mM Na+. Amiodarone had no effect on the voltage dependence of the pump or the affinity of the pump for extracellular K+ either after chronic treatment or during acute exposure. We conclude that chronic amiodarone treatment reduces overall Na(+)-K+ pump capacity in cardiac ventricular myocytes. In contrast, acute exposure of myocytes to amiodarone reduces the apparent Na+ affinity of the Na(+)-K+ pump. An amiodarone-induced inhibition of the hyperpolarizing Na(+)-K+ pump current may contribute to the action potential prolongation observed during treatment with this drug.