UBC Theses and Dissertations
Studies on the regulation of calcium transport in cardiac and skeletal muscle sarcoplasmic reticulum Eibschutz, Barry
The sarcoplasmic reticulum (SR) membrane plays a critical role in excitation-contraction coupling in both skeletal and cardiac muscle. In an attempt to examine the function and regulation of SR, we examined the unique role that it plays in both normal and diseased tissue. In our elucidation of the role of the SR in normal dog heart muscle, we attempted to "purify" the crude microsomes enriched in SR according to the method of Jones et al (1979), in order to minimize contamination by sarcolemmal and mitochondrial membranes; both of these membranes have been shown to contain ATPase activity, and may contribute to spurious results. Both sarcolemmal and mitochondrial membrane contamination were decreased in the Ca-oxalate loaded, purified SR preparation. In addition, a 3-5-fold enhancement of both Ca⁺⁺-uptake and (Ca⁺⁺-Mg⁺⁺)-ATPase activity was observed in the purified preparation compared to crude. When analyzed on SDS-PAGE, the absence of a 95,000 MW protein in the purified preparation was evident. Regulation of the purified SR preparation by cAMP-dependent protein kinase (cAMP-PK) and calmodulin (CAM) appeared identical to the stimulation typically observed in crude preparations with the exception of lower stimulation of Ca⁺⁺-uptake at higher (1.0 μM) free Ca⁺⁺ concentrations. A comparison of the two preparations with respect to membrane phosphorylation revealed that incubation of either purified or crude SR with cAMP-PK or the catalytic (C) subunit of cAMP-PK resulted in similar time-course profiles. In the presence of CAM, the total level of phosphoprotein incorporation was decreased in the crude preparation compared to the purified, and both the intensity and time to complete phosphorylation of phospholamban were markedly diminished in purified SR. Nevertheless, both crude and purified SR were similar in that CAM-dependent phosphorylation was both slower and decreased compared to that observed with either cAMP-PK or C subunit incubation. An examination of the role of CAM in normal rabbit skeletal SR was undertaken as a result of the disputed claims of the mode of CAM binding to the skeletal SR membrane. Our studies revealed that, unlike the claims of Campbell and MacLennan (1982), a considerable amount of CAM remained bound to the SR membrane following extensive washing with EGTA. SDS-PAGE of all supernatants revealed a number of bands which migrated in the region of CAM (15.5-19.5 Kdaltons); following high-speed centrifugation of all the supernatants, the molecular weight bands assumed to be CAM did not appear in the EGTA-washed fSR or sSR supernatants. An RIA for CAM revealed that measureable levels of CAM were present in all supernatants. Higher levels of CAM were released into the supernatant from sSR than from fSR. In addition, CAM was liberated from supernatants boiled in the presence or absence of EDTA regardless of prior EGTA-washing, although the levels of CAM derived from vesicles that been pre-treated with EGTA were approximately 33% less than the levels obtained from vesicles which were not treated with EGTA. CAM RIA revealed that this EGTA-extractable CAM was not present in the supernatant of EGTA-washed SR. It was concluded that the stimulator present in boiled supernatants of fast and slow SR was, indeed, CAM, whereas the stimulator reported by Campbell and MacLennan (1982) was most likely CAM that originated from the boiling of contaminant SR membrane fragments present in EGTA-washed SR supernatants. The final aspect of SR regulation investigated was Ca⁺⁺-uptake in the skeletal muscle of streptozotocin-diabetic rats. It was found that, similar to the reports in rat cardiac SR, skeletal SR from diabetic rats had significantly depresssed levels of Ca⁺⁺-uptake, and elevated levels of free carnitine and long-chain acylcarnitines, compared to controls. It was concluded that the diabetic state may result in generalized pathophysiology, a result of which may be the non-specific decrease in Ca⁺⁺-uptake in SR.
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