APPS November 2002 Meeting Abstract 1330


Anastasia S. Mihailidou, Mahidi Mardini, Tafline Fraser, John W. Funder, Department of Cardiology, Royal North Shore Hospital, NSW and Baker Medical Institute, Vic.

Previously we have shown aldosterone causes an increase in Na+/K+/2Cl- cotransport activity in cardiac myocytes within 15 minutes of exposure. Aldosterone-induced increased cotransporter activity results in a rise in intracellular Na+ concentration and increased Na+-K+ pump activity. Since aldosterone has been shown to activate protein kinase C (PKC), we examined the potential role of PKC mediating rapid aldosterone-induced cotransport activation and PKC isoform specificity in this process.

Single rabbit ventricular myocytes were isolated, voltage-clamped and internally perfused using the whole-cell patch clamp technique. Pipette solutions were Na+-free and we used activity of the sarcolemmal Na+-K+ pump to indicate the presence of intracellular Na+. Electrogenic Na+-K+ pump current (Ip) arising from 3Na:2K exchange ratio, was used to measure pump activity. Ip (normalised for membrane capacitance) was identified as an ouabain-induced shift in holding current.

Mean Ip (±SE) of myocytes not exposed to aldosterone was very low (0.02 ± 0.01 pA/pF, n=6). In contrast, mean Ip of myocytes exposed to 10 nM aldosterone (0.22 ± 0.01 pA/pF, n=7) was significantly higher (p<0.001). Isoform-specific modulation of PKC was achieved by including in pipette solutions 100 nM peptide blockers to anchoring proteins for activated PKC. We selected α-, ε- and δ-isoform peptide blockers since the α-isoform is translocated by aldosterone and the δ-isoform regulates cotransporter activity in non-cardiac cells, while ε-isoform is the most abundantly expressed isoform in the heart. PKCε peptide inhibitor abolished aldosterone-induced increased pump activity (0.06 ± 0.02 pA/PF, n=7), whereas PKCα and PKCδ peptide inhibitors did not change the effect of aldosterone (0.18 ± 0.02 pA/pF, n=5 and 0.19 ± 0.01 pA/pF, n=9). Activation of PKCε had an effect on pump activity similar to that of aldosterone (0.31 ± 0.02 pA/pF, n=6).

These studies show PKCε mediates aldosterone-induced activation of Na+/K+/2Cl- cotransport activity in the heart with short latency.

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