New methods for the isolation of skeletal muscle sarcolemma and sarcoplasmic reticulum allowing a comparison between the mammalian and amphibian beta(2)-adrenergic receptors and calcium pumps.

New methods were established for the rapid and simultaneous isolation of multiple sarcolemmal and sarcoplasmic reticular fractions from very small amounts (0.25-2.0 g) of skeletal muscle. Thebeta(2)-adrenergic receptor and calcium transport systems were used as indices of purity and functional integrity as well as being the focal points of the study. These methods were found to be suitable for the special needs of small tissue samples, allowed rapid preparation and were appropriate for skeletal muscle from various species, frogs to mammals. The sarcolemmalbeta(2)-adrenergic receptor was expressed in frogs and mammals at similar levels of expression (336-454 fmol. x mg(-1)). The calcium pump was also present in sarcolemmal and sarcoplasmic reticular fractions in all species but notable species differences were found. In sarcolemmal fractions, while calcium binding was uniformly low (<1 nmol. x mg(-1)), oxalate stimulation was variable: low in frogs ( approximately 1.05-fold) high in mammals (120-450-fold). In sarcoplasmic reticular fractions, calcium binding was low in frogs (4-9 nmol. x mg(-1)) and much higher in mammals (322-383 nmol. x mg(-1)); oxalate stimulated calcium transport to a much greater extent in frogs (<70-fold) than in mammals (1.6-2-fold). It is concluded that thebeta(2)-adrenergic receptor appears to be strongly conserved in skeletal muscle while the use of calcium pumps evolves from reliance in Amphibia on the sarcoplasmic reticular calcium pump to the use in Mammalia of calcium pumps from both the sarcoplasmic reticulum and the plasma membrane.

Characterization of sarcolemma and sarcoplasmic reticulum isolated from skeletal muscle of the freeze tolerant wood frog, Rana sylvatica: the beta(2)-adrenergic receptor and calcium transport systems in control, frozen and thawed states.

In freeze tolerant wood frog Rana sylvatica, the freeze-induced liberation of glucose plays a critical role in survival in response to sub-zero temperature exposure. We have shown that the glycaemic response is linked to selective changes in the expression of hepatic adrenergic receptors through which catecholamines act to produce their hepatic glycogenolytic effects. The purpose of the present study was to determine if skeletal muscle, another catecholamine-sensitive tissue with glycogenolytic potential, displayed similar or different changes. In order to achieve these objectives, skeletal muscle derived from Rana sylvatica was studied in control, frozen and thawed states. In isolated sarcolemmal fractions, freezing effected an 88% decrease in beta(2)-adrenergic receptor expression but was without effect on the calcium pump; while thawing resulted in a recovery of the beta(2)-adrenergic receptor to 60% of control levels and a 2.4-fold increase in calcium transport. In isolated sarcoplasmic reticular fractions, freezing effected a 52% decrease in calcium binding and a 92% decrease in oxalate-stimulated calcium uptake; while thawing elicited partial normalization to control levels to 70% with respect to calcium binding and to 47% with respect to calcium uptake. Freezing and thawing were associated with increases and decreases, receptively, in blood glucose levels but were without effect on skeletal muscle glycogen content. Thus these muscle changes in Rana sylvatica in freezing and thawing are not linked to glycogen breakdown, are different from those previously seen in liver, and may provide a role in recovery of muscle function during thawing by protecting glycogen stores for contraction and maximizing extracellular calcium for excitation-contraction coupling in the frozen state. The involvement of thyroid hormone in triggering these muscle changes is discussed.

cADP-ribose activates reconstituted ryanodine receptors from coronary arterial smooth muscle.

The present study was designed to test the hypothesis that cADP-ribose (cADPR) increases Ca(2+) release through activation of ryanodine receptors (RYR) on the sarcoplasmic reticulum (SR) in coronary arterial smooth muscle cells (CASMCs). We reconstituted RYR from the SR of CASMCs into planar lipid bilayers and examined the effect of cADPR on the activity of these Ca(2+) release channels. In a symmetrical cesium methanesulfonate configuration, a 245 pS Cs(+) current was recorded. This current was characterized by the formation of a subconductance and increase in the open probability (NP(o)) of the channels in the presence of ryanodine (0.01-1 microM) and imperatoxin A (100 nM). A high concentration of ryanodine (50 microM) and ruthenium red (40-80 microM) substantially inhibited the activity of RYR/Ca(2+) release channels. Caffeine (0.5-5 mM) markedly increased the NP(o) of these Ca(2+) release channels of the SR, but D-myo-inositol 1,4,5-trisphospate and heparin were without effect. Cyclic ADPR significantly increased the NP(o) of these Ca(2+) release channels of SR in a concentration-dependent manner. Addition of cADPR (0.01 microM) into the cis bath solution produced a 2.9-fold increase in the NP(o) of these RYR/Ca(2+) release channels. An eightfold increase in the NP(o) of the RYR/Ca(2+) release channels (0.0056 +/- 0.001 vs. 0.048 +/- 0.017) was observed at a concentration of cADPR of 1 microM. The effect of cADPR was completely abolished by ryanodine (50 microM). In the presence of cADPR, Ca(2+)-induced activation of these channels was markedly enhanced. These results provide evidence that cADPR activates RYR/Ca(2+) release channels on the SR of CASMCs. It is concluded that cADPR stimulates Ca(2+) release through the activation of RYRs on the SR of these smooth mucle cells.