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

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.

Sodium Influx Pathways during and after Anoxia in Rat Hippocampal Neurons

“…The tendency for bumetanide to augment the rise in [Na ϩ ] i after anoxia in 11-14, but not 6 -10, DIV hippocampal neurons (Table 2) may reflect the finding in cardiac myocytes that Na ϩ /K ϩ /2Cl Ϫ cotransport contributes to Na ϩ efflux at reperfusion (Anderson et al, 1996). Na ϩ /Ca 2ϩ exchange, operating in forward mode, may mediate Ca 2ϩ efflux after anoxia or ischemia (Breder et al, 2000) and thereby contribute to Na ϩ influx at this time.…”

Acute effects of 17β-estradiol on myocardial pH, Na+, and Ca2+ and ischemia-reperfusion injury

“…The methods used were modified from those previously reported (3,6,31). Briefly, 12-to 13-wk-old ovariectomized Sprague-Dawley rats (OVX) were purchased from Charles River 1 wk after ovariectomy.…”

“…Na i ϩ (in meq/kg dry wt) was calculated from the calibrated area of the "unshifted" intracellular peak after subtracting the shifted extracellular peak as previously described (3,5,31). This method measures Na i ϩ as an amount, and to minimize assumptions, we report it as such.…”

“…Anaerobic metabolism decreases cytosolic pH (pH i ), which stimulates pHregulatory Na ϩ /H ϩ exchange (NHE1) to increase Na ϩ uptake and thereby increase intracellular Na ϩ (Na i ϩ ). Increases in (3,7,31,37,46,49). In the heart, there are questions about whether the bulk of Na ϩ and Ca 2ϩ entry occurs during ischemia or reperfusion, but there is a growing consensus that much of the Ca 2ϩ entry is Na ϩ dependent (36).…”

“…Increases in Na i ϩ decrease the driving force for cytosolic Ca 2ϩ efflux via Na ϩ /Ca 2ϩ exchange (NCE) and thereby cause an increase in cytosolic [Ca 2ϩ ] ([Ca 2ϩ ] i ). Increases in [Ca 2ϩ ] i cause a cascade of events ending in injury, apoptosis, or necrosis (3,7,31,37,46,49). In the heart, there are questions about whether the bulk of Na ϩ and Ca 2ϩ entry occurs during ischemia or reperfusion, but there is a growing consensus that much of the Ca 2ϩ entry is Na ϩ dependent (36).…”