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

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°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 ~ 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°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.