The relation between cellular sodium, pH and volumes and the activity of Na/H antiport during hypothermic ischemia: multinuclear NMR studies of rat hearts

Abstract

The present study evaluates the activity of the Na/H antiport during cold ischemia and aims to determine its influence on cellular sodium. pH and volumes. Cellular parameters; volumes, sodium, pH and high energy phosphates, were measured by multinuclear NMR spectroscopy in rat hearts during 12 h of storage at 4 degrees C and reperfusion, along with functional parameters. Cell volumes were measured by 1H and 59Co NMR using the extracellular marker cobalticyanide, pH and energetics by 31P NMR and sodium compartmental distribution by 23Na NMR spectroscopy using the shift reagent Dy(TTHA)-3. Three storage solutions were applied: Krebs-Henseleit (containing 144 mM sodium, KH), a solution supplemented with 0.20 mM amiloride (KH-ami) and a solution containing 23 mM sodium and 242 mM mannitol (KH-man). Inhibition of the Na/H antiport with amiloride reduced the cellular sodium accumulation by 56%. The end-ischemic concentrations were 45 mM (KH-ami) and 77 mM (KH). Amiloride also reduced the extent of cell swelling by 53% from an end-ischemic volume of 3.56 ml/gdw (KH) to 2.97 ml/gdw (KH-ami), however cell swelling persisted in both groups at reperfusion (33% increase in cell water). The molar ratio of sodium and water cellular accumulation was constant: Na/H2O approximately 3.7 x 10(-3) throughout the whole storage period. Inhibition of the antiport was protective for the high energy phosphates during ischemia and reperfusion. In KH-ami the pH acidified after 6 h of storage to an end-ischemic value of 6.35 (pH = 6.50 in KH): this difference persisted after 60 min of reperfusion, pH = 6.98 in KH-ami and pH = 7.1 in KH. Storage in the low-sodium solution was disadvantageous for the high energy phosphates during ischemia and reperfusion with a recovery of pH to 6.92 when reperfused with KH. Hearts stored with amiloride or mannitol solution failed to resume contraction at reperfusion. It is concluded: (a) the antiport is active at 4 degrees C; (b) during ischemia it mediates sodium influx and contributes to cell swelling with minor effects on the cytosolic pH; (c) at reperfusion the antiport is active it participates in the extrusion of excess protons, but has a minor impact on sodium and water homeostasis; (d) inhibition of the antiport does not protect the cardiac muscle at low temperatures.