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To examine the effect of short term intense activity on sarcoplasmic reticulum (SR) Ca2+ sequestering function, the gastrocnemius (G) muscles of 11 anaesthetized male rats (weight, 411 +/- 8 g, X +/- SE) were activated using supramaximal, intermittent stimulation (one train of 0.2 msec impulses per sec of 100 msec at 100 Hz). Homogenates were obtained from stimulated white (WG-S) and red (RG-S) tissues, assayed for Ca2+ uptake and maximal Ca2+ ATPase activity and compared to contralateral controls (WG-C, RG-C). Calcium uptake (nmoles/mg protein/min) determined using Indo-1 and at [Ca2+]i concentrations between 300-400 nM was unaffected (p > 0.05) by activity in both WG (6.14 + 0.43 vs 5.37 + 0.43) and RG (3.21 + 0.18 vs 3.07 + 0.20). Similarly, no effect (p > 0.05) of contractile activity was found for maximal Ca2+ ATPase activity (mumole/mg protein/min) determined spectrophotometrically in RG (0.276 + 0.03 vs 0.278 + 0.02). In WG, Ca2+ ATPase activity was 15% higher in WG-S compared to WG-C (0.412 + 0.03 vs 0.385 + 0.04). Repetitive stimulation resulted in a reduction in tetanic tension of 74% (p < 0.05) by 2 min in the G muscle. By the end of the stimulation period, ATP concentration was reduced (p < 0.05) by 57% in the WG and by 47% in the RG.(ABSTRACT TRUNCATED AT 250 WORDS)

A multiple measurement system for assessing sarcoplasmic reticulum (SR) Ca(++)-ATPase activity and Ca(++)-uptake was used to examine the effects of SR fractionation and quick freezing on rat white (WG) and red (RG) gastrocnemius muscle. In vitro measurements were performed on whole muscle homogenates (HOM) and crude microsomal fractions (CM) enriched in SR vesicles before and after quick freezing in liquid nitrogen. Isolation of the CM fraction resulted in protein yields of 0.96 +/- 0.1 and 0.99 +/- 0.1 mg/g in WG and RG, respectively. The percent Ca(++)-ATPase recovery for CM compared to HOM was 14.5% (WG) and 10.1% (RG). SR Ca(++)-activated Ca(++)-ATPase activity was not affected by quick freezing of HOM or CM, but basal ATPase was reduced (P < 0.05) in frozen HOM (5.12 +/- 0.18-3.98 +/- 0.20 mole/g tissue/min in WG and from 5.39 +/- 0.20-4.48 +/- 0.24 mumole/g tissue/min in RG). Ca(++)-uptake was measured at a range of physiological free [Ca++] using the Ca++ fluorescent dye Indo-1. Maximum Ca(++)-uptake rates when corrected for initial [Ca++]f were not altered in HOM or CM by quick freezing but uptake between 300 and 400nM free Ca++ was reduced (P < 0.05) in quick frozen HOM (1.30 +/- 0.1-0.66 +/- 0.1 mumole/g tissue/min in WG and 1.04 +/- 0.2-0.60 +/- 0.1 mumole/g tissue/min in RG). Linear correlations between Ca(++)-uptake and Ca(++)-ATPase activity measured in the presence of the Ca++ ionophore A23187 were r = +0.25, (P < 0.05) and r = +0.74 (P < 0.05) in HOM and CM preparations, respectively, and were not altered by freezing. The linear relationships between HOM and CM maximum Ca(++)-uptake (r = +0.44, P < 0.05) and between HOM and CM Ca(++)-ATPase activity (r = +0.34, P < 0.05) were also not altered by tissue freezing. These data suggest that alterations in maximal SR Ca(++)-uptake function and maximal Ca(++)-ATPase activity may be measured in both HOM and CM fractions following freezing and short term storage.