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.

Mechanisms of Na+-K+ pump regulation in cardiac myocytes during hyposmolar swelling.

We have previously demonstrated that the sarcolemmal Na+-K+ pump current (Ip) in cardiac myocytes is stimulated by cell swelling induced by exposure to hyposmolar solutions. However, the underlying mechanism has not been examined. Because cell swelling activates stretch-sensitive ion channels and intracellular messenger pathways, we examined their role in mediating Ip stimulation during exposure of rabbit ventricular myocytes to a hyposmolar solution. Ip was measured by the whole cell patch-clamp technique. Swelling-induced pump stimulation altered the voltage dependence of Ip. Pump stimulation persisted in the absence of extracellular Na+ and under conditions designed to minimize changes in intracellular Ca2+, excluding an indirect influence on Ip mediated via fluxes through stretch-activated channels. Pump stimulation was protein kinase C independent. The tyrosine kinase inhibitor tyrphostin A25, the phosphatidylinositol 3-kinase inhibitor LY-294002, and the protein phosphatase-1 and -2A inhibitor okadaic acid abolished Ip stimulation. Our findings suggest that swelling-induced pump stimulation involves the activation of tyrosine kinase, phosphatidylinositol 3-kinase, and a serine/threonine protein phosphatase. Activation of this messenger cascade may cause activation by the dephosphorylation of pump units.

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.