Skeletal Muscle Transverse Tubule Research Articles

ATP-energized Ca2+ pump in isolated transverse tubules of skeletal muscle.

A modified protocol for isolation of transverse tubules incorporated an extra stage of purification. The existence of an ATP-energized Ca2+ pump in transverse tubules isolated from rabbit skeletal muscle has been demonstrated. Isolated transverse tubules had a Ca-ATPase activity of 0.78 mu mol/min . mg; this was 300% in excess of that activity attributable to sarcoplasmic reticulum contamination. The distribution of part of the CaATPase activity and ATP-energized Ca2+ uptake coincided with the distribution of transverse tubules in isopycnic sucrose gradients loaded with mechanically disrupted triad junctions. Transverse tubules accumulated over 70 nmol of Ca2+/mg of protein; this uptake was abolished by the Ca2+ ionophore A23187. Neither digitoxin nor monensin inhibited Ca2+ uptake, indicating that Ca2+ accumulation did not occur through a sodium/calcium exchange. Conditions for half-maximal Ca2+ uptake were 5 micro M free Ca2+ and 10 micro M ATP. The Ca2+ pump of isolated transverse tubules was distinguished from the Ca2+ pump of sarcoplasmic reticulum and sarcolemma in that the transverse tubule Ca2+ pump: 1) was not enhanced by oxalate; 2) was not energized by acetyl phosphate, p-nitrophenyl phosphate, or 3-O-methylfluorescein phosphate; and 3) did not hydrolyze p-nitrophenyl phosphate or 3-O-methyl-fluorescein phosphate. Using Ca2+-dependent 3-O-methylfluorescein phosphatase as a marker for sarcoplasmic reticulum, the contamination of the transverse tubule preparation was calculated to be 6%. This agreed with a contamination level of 5% estimated by freeze-fracture electron microscopy.

Beta-adrenergic receptor and adenylate cyclase in transverse tubules of skeletal muscle.

Experiments were carried out to clarify the sites of action of beta-adrenergic agonists in skeletal muscle microsomes. Microsomes were fractionated into longitudinal reticulum, terminal cisternae, and isolated transverse tubules. Transverse tubules were selectively labeled and tracked with [3H]ouabain. beta-adrenergic receptor was identified by [3H]dihydroalprenolol binding. Assays of beta-adrenergic receptor, adenylate cyclase, and protein kinase-stimulated phosphorylation showed: 1) beta-adrenergic receptor was detected in transverse tubules with a receptor density of 0.61 pmol/mg of protein. No significant binding was detected in longitudinal reticulum or in terminal cisternae. 2) Isoproterenol-stimulated adenylate cyclase was present in microsomes but was similarly confined to the transverse tubular fraction. The activity of F- stimulated cyclase in transverse tubules was 2.3 nmol/mg of protein/min. 3) No phosphorylation of microsomes by cyclic AMP and protein kinase could be detected. We conclude that the action of epinephrine on skeletal muscle is mediated through receptors and adenylate cyclase in the external membrane.

A lesion of the transverse tubules of skeletal muscle.

1. A method is described by which a selective lesion can be made in vitro in the transverse tubules of frog skeletal muscle.2. The method consists of exposing the muscle for 1 hr or more to a buffered salt solution made hypertonic by the inclusion of 400 mM glycerol and then returning the muscle to an isotonic salt solution. The lesion is induced during the washout of the glycerol.3. Electron micrographs reveal that the lesion consists of a rearrangement of the T-system membranes in such a way that the continuity of the tubules is lost. The membranes appear to coalesce into large vesicles scattered irregularly throughout the sarcoplasm.4. The glycerol treatment results in a depression of the resting potential of up to 30 mV. The treated fibres are depolarized by high concentrations of K as are normal muscle fibres.5. T-tubule lesioned fibres are unable to respond mechanically either to electrical stimulation or to elevated K but they do contract in the presence of caffeine and relax when the caffeine is removed.6. Problems concerning the variability of the procedure are presented and certain considerations concerning the mechanism of the effect are discussed.

THE LOCATION OF SODIUM IN THE TRANSVERSE TUBULES OF SKELETAL MUSCLE

THE LOCATION OF SODIUM IN THE TRANSVERSE TUBULES OF SKELETAL MUSCLE