Effects of Na-K-2Cl cotransport inhibition on myocardial Na and Ca during ischemia and reperfusion

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Abstract
In the context of the "pump-leak" hypothesis (37), changes in myocardial intracellular Na (Nai) during ischemia and reperfusion have historically been interpreted to be the result of changes in Na efflux via the Na-K pump. We investigated the alternative hypothesis that changes in Nai during ischemia are the result of changes in the Na "leak" rather than changes in the pump. More specifically, we hypothesize that the increase in Nai during ischemia is in part the result of increased Na uptake mediated by Na/H exchange. Furthermore, we present data consistent with the interpretation that the Na-K-2Cl cotransporter is active (or, alternatively, displaced from equilibrium) during ischemia and may contribute an additional Na efflux pathway during reperfusion. Thus inhibition of Na efflux via Na-K-2Cl cotransport during ischemia and reperfusion could result in increased Nai and therefore decreased force driving Ca efflux via Na/Ca exchange and ultimately increased intracellular Ca concentration ([Ca]i). Nai (in meq/kg dry wt) and [Ca]i (in nM) were measured in isolated Langendorff-perfused rabbit hearts using nuclear magnetic resonance spectroscopy. Except, during the 65 min of ischemia, hearts were perfused with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid-buffered Krebs-Henseleit solution equilibrated with 100% O2 at 23 degrees C and pH 7.4 +/- 0.05. During ischemia, Nai rose from 16.6 +/- 0.3 to 62.9 +/- 5.1 (delta Nai approximately 46) meq/kg dry wt and decreased during subsequent reperfusion (mean +/- SE, n = 3 hearts). To measure Na uptake ("leak") in the absence of efflux via the Na-K pump, in all of the protocols described below, the perfusate was nominally K-free solution containing 1 mM ouabain for 10 min before ischemia and during the 30-min reperfusion. After K-free perfusion, Nai rose from 20.2 +/- 0.5 to 79.1 +/- 5.3 (delta Nai approximately 59) meq/kg dry wt (n = 3) during ischemia and decreased during K-free reperfusion. When amiloride (1 mM) was added to the K-free perfusate to inhibit Na/H exchange, Nai rose from 16.3 +/- 0.9 to 44.7 +/- 5.1 (delta Nai approximately 28) meq/kg dry wt (n = 3) during ischemia; i.e., amiloride decreased Na uptake. When bumetanide (20 microM) was added to the nominally K-free perfusate to inhibit Na-K-2Cl contransport, Nai rose from 22.5 +/- 3.9 to 83.8 +/- 13.9 (delta Nai approximately 61 meq/kg dry wt (n = 3) during ischemia and did not decrease during reperfusion; i.e., bumetanide inhibited Na recovery during reperfusion (P < 0.05 compared with bumetanide free). For the same protocol, the presence of bumetanide resulted in increased [Ca]i during ischemia and reperfusion (P < 0.05); these increases in [Ca]i are interpreted to be the result of increased Nai. Thus the results are consistent with the hypotheses.