New methods for the isolation of skeletal muscle sarcolemma and sarcoplasmic reticulum allowing a comparison between the mammalian and amphibian β₂‐adrenergic receptors and calcium pumps

Abstract

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.

Feeding wet distillers grains plus solubles contributes to sarcoplasmic reticulum membrane instability

“…The SR membrane was extracted from muscle tissue according to the method described by Hemmings (2001) with modifications. Ten g of powdered samples were suspended in 35 mL of ice-cold homogenisation buffer (10 mM NaHCO 3 , 2 mM sodium azide, 10 mM Tris-Cl, and 1 mM dithiothreitol) at pH 7.5 and homogenised using a Polytron homogeniser (Kinematica) at setting 6 for 15 s. Homogenate was transferred into a 50-mL plastic centrifuge tube and centrifuged at 2000g for 10 min at 4 C. The supernatant was collected and centrifuged at 10 000g (Sorvall RC5B Superspeed Centrifuge; Thermo Scientific, Rockford, IL, USA) for 30 min at 4 C. Four and a half percent KCl (w/v) was added to the supernatant and stirred for 30 min on ice.…”

Characterization of sarcolemma and sarcoplasmic reticulum isolated from skeletal muscle of the freeze tolerant wood frog, Rana sylvatica: the β2‐adrenergic receptor and calcium transport systems in control, frozen and thawed states

“…The isolation procedure followed was that described for the rapid isolation of sarcolemma from small amounts of frog tissue, detailed in the preceding paper 19 starting with 0.5± 2.0-g portions of trimmed skeletal muscle for each fraction. Sarcolemma fractions were used immediately for b 2 -adrenergic receptor binding and calcium transport assays.…”

CD38-cADPR-SERCA Signaling Axis Determines Skeletal Muscle Contractile Force in Response to β-Adrenergic Stimulation

“…Skeletal muscle SR was prepared from WT or CD38 KO mice gastrocnemius muscle using a modification of the described previously [24]. 0.5∼1 g portion of trimmed gastrocnemius muscle was transferred to 10 ml round bottom tube to which was added 5 volumes of SR homogenization buffer (10 mM NaHCO 3 , 2 mM sodium azide, 10 mM Tris-HCl, pH 7.5).…”