Thermal instability of rat muscle sarcoplasmic reticulum Ca2+-ATPase function

To examine the thermal instability and the role of sulfhydryl (SH) oxidation on sarcoplasmic reticulum (SR) Ca2+-ATPase function, crude homogenates were prepared from the white portion of the gastrocnemius (WG) adult rat muscles (n = 9) and incubated in vitro for less than or equal to60 min either at a normal resting body temperature (37degreesC) or at a temperature indicative of exercise-induced hyperthermia (41degreesC) with DTT and without DTT (CON). In general, treatment with DTT resulted in higher Ca2+-ATPase and Ca2+ uptake values (nmol.mg protein(-1).min(-1), P < 0.05), an effect that was not specific to time of incubation. Incubations at 41 degrees C resulted in lower (P < 0.05) Ca2+ uptake rates (156 +/- 18 and 35.9 +/- 3.3) compared with 37degreesC (570 +/- 54 and 364 +/- 26) at 30 and 60 min, respectively. At 37degreesC, ryanodine (300 muM), which was used to block Ca2+ release from the calcium release channel, prevented the time-dependent decrease in Ca2+ uptake. A general inactivation (P < 0.05) of maximal Ca2+-ATPase activity (V-max) in CON was observed with incubation time (0 > 30 > 60 min), with the effect being more pronounced (P < 0.05) at 41 degrees C compared with 37 degrees C. The Hill slope, a measure of co-operativity, and the pCa(50), the cytosolic Ca2+ concentration required for half-maximal activation of Ca2+ ATPase activity, decreased (P < 0.05) at 41degreesC only. Treatment with DTT attenuated the alterations in enzyme kinetics. The increase in V-max with the Ca2+ ionophore A-23187 was less pronounced at 41degreesC compared with 37degreesC. It is concluded that exposure of homogenates to a temperature typically experienced in exercise results in a reduction in the coupling ratio, which is mediated primarily by lower Ca2+ uptake and occurs as a result of increases in membrane permeability to Ca2+. Moreover, the decreases in Ca2+-AT-Pase kinetics in WG with sustained heat stress result from SH oxidation.