Fiber splitting in tonic and phasic skeletal muscles following denervation, chloroquine and triton WR-1339 treatments.

Fiber splitting was studied in the tonic, anterior (ALD) and phasic, posterior (PLD) latissimus dorsi muscles using three experimental models, denervation, chloroquine administration and Triton WR-1339 administration. Fiber splitting was observed in the PLD in all three models, but in the ALD only after chloroquine administration. A basement membrane was always present in the split fibers, and acid phosphatase was localized between the split fibers in the PLD. However, no evidence of lysosomal involvement in the fiber splitting process was observed.

Further studies on a unique T-tubular acid phosphatase in avian skeletal muscle.

With the unique observation, using conventional cytochemistry, of acid phosphatase reaction production in the T-tubules of the posterior latissimus dorsi muscle of the chicken, the possibility of endocytosis of lysosomal enzymes by muscle cells came into question. After testing the substrate specificity of this T-tubular phosphatase, it was clear that the enzyme hydrolysed glucose 6-phosphate and beta-glycerophosphate at pH 5.0 but not cytidine-5'-monophosphate which was hydrolysed by dense bodies and autophagic vacuoles. The cytochemical evidence points to a unique phosphatase present on muscle cell membranes which apparently does not belong to the vacuolar apparatus of skeletal muscle and is not 5'-nucleotidase.

Isopycnic-zonal centrifugation of plasma membrane, sarcoplasmic reticular fragments, lysosomes, and cytoplasmic proteins from phasic skeletal muscle.

Homogenates of the posterior latissimus dorsi muscle, a phasic muscle, were fractionated by a one-step zonal centrifugation technique into four major organelle populations and cytoplasmic constituents. These were: (1) Plasma membrane fragments with a modal equilibrium density of 1.10 and containing 5'-nucleotidase, alkaline phosphodiesterase, p-nitrophenylphosphatase and acid phosphatase (beta-glycerophosphate was used as the substrate). (2) Sarcoplasmic reticular fragments which could be further subdivided into calcium transport vesicles, with a model equilibrium density of 1.16, that exhibited calcium uptake; K+-ATPase; leucyl-bet-naphthylamidase; acid phosphodiesterase; acid phosphatase (using cytidine monophosphate as the substrate); and sarcoplasmic reticular lysosomes, with a model equilibrium density of 1.18, possessing dipeptidyl-aminopeptidase II, cathepsin D, alpha-glucosidase, N-acetyl-beta-glucosaminidase, and NADH oxidase activity. (3) Mitochondria with a modal equilibrium density of 1.21. (4) Catalase-containing vesicles with a modal equilibrium density of 1.22; and cytoplasmic constituents (modal density of 1.25) with phosphorylase, pyruvate kinase, myosin-ATPase, aldolase, and protein and RNA content. The purity of these organelles was equal to or better than previous efforts, with a 30-fold purification achieved for 5'-nucleotidase and alkaline phosphodiesterase. These results lend support to the hypothesis that the sarcoplasmic reticulum of phasic muscle, in addition to its specialized role in excitation-contraction coupling, represents a multifunctional membrane system, and that, similar to the smooth endoplasmic reticulum of other cells, it includes some membrane-bound lysosomal enzymes and NADH oxidase.

Degeneration and regeneration in denervated tonic and phasic skeletal muscle: morphology and acid phosphatase cytochemistry.

Following denervation, ultrastructural alterations were observed in the tonic, anterior (ALD) and phasic posterior (PLD) latissimus dorsi muscles of the chicken. In the ALD muscle these changes were characteristic of both degeneration and regeneration, while in the PLD muscle, the changes were characteristic only of degeneration. Acid phosphatase positive structures, which included dense bodies in the ALD and PLD as well as T-tubules in the PLD, were observed intact with no evidence of release of enzyme into the sarcoplasm. No evidence of an increase in the number of autophagic vacuoles was found. The morphological evidence presented in this communication does not support the hypothesis that lysosomes are involved in denervation atrophy through autophagy of muscle cell constituents.

Skeletal muscle myopathy caused by triton WR-1339.

After administration (1 g/kg every other day for a total of five injections) of Triton WR-1339, the tonic, anterior (ALD) and phasic, posterior (PLD) latissimus dorsi muscles of the chicken underwent distinct pathological modifications. Some of the morphological alterations in the muscles paralleled those seen after administration of chloroquine, increased autophagic vacuole formation in the ALD muscle and swelling of the sarcoplasmic reticulum in the PLD muscle, but other changes were unique to Triton WR-1339. These included loss of myofilaments and whole myofibrils, indentation of the sarcolemma as well as increased numbers of ribosomes in the ALD muscle and swelling of the T-tubular system in the PLD muscle. These results are compared with other lysosome mediated pathologies, as well as with other myopathies.

Increased autophagy in chloroquine-treated tonic and phasic muscles: an alternative view.

Ultrastructural and cytochemical techniques were used to investigate autophagy in the tonic anterior(ALD) and phasic posterior (PLD) latissimus dorsi muscles of the chicken following chloroquine administration. Autophagic vacuoles were seen in the ALD after 1 day of chloroquine administration while no change was seen in the PLD until 3 days. In both muscles, autophagic vacuoles and myeloid bodies were found at the level of the I band. Myeloid bodies usually were found in the longitudinal rows of mitochondria in the ALD muscle. Some, but not all, of the autophagic vacuoles and myeloid bodies were cytochemically acid phosphatase positive, while the portion of the sarcoplasmic reticulum of both muscles which is normally acid phosphatase positive, while the portion of the sarcoplasmic reticulum of both muscles which is normally, acid phosphatase positive was devoid of activity following chloroquine administration. These observations are discussed in regard to accepted mechanisms of autophagy and the possible inhibition of autophagy in skeletal muscle tissue by chloroquine.

A unique acid phosphatase location: the transverse tubule of avian fast muscle.

Acid phosphatase activity was localized cytochemically in the posterior latissimus dorsi muscle of the chicken. Reaction product was observed in three distinct structures: T-tubules, sarcoplasmic reticulum and dense bodies. Examination of cross- and longitudinal sections confirmed that the reaction product was membrane-limited. Acid phosphatase activity was observed in sarcoplasmic reticulum adjacent to the A-I junction and the A-band, in termyofibrillar dense bodies located along the length of the fibre and in the T-tubules but not in the surface caveolae or in the lateral sacs of the sarcoplasmic reticulum. The uniqueness of the T-tubular localization with respect to cytochemical localizations in other muscles is discussed.

Cytochemical observations of two distinct acid phosphatase-reactive structures in anterior latissimus dorsi muscle of the chicken.

Acid phosphatase activity has been localized cytochemically in the avian anterior latissimus dorsi muscle in two distinct structures, the sarcoplasmic reticulum and membrane-limited dense bodies. Cross and longitudinal sections confirmed that the reaction product was membrane-bound. Acid phosphatase activity was observed in the longitudinal tubules of the sarcoplasmic reticulum in the region of the I band and in dense bodies located along the length of the fibre just beneath the sarcolemma and between the myofibrils. This dual localization is discussed in relationship to previous cytochemical and zonal centrifugation evidence for more than one acid phosphatase-containing organelle in skeletal muscle.

Alterations in lysosomes, catalase-containing organelles, mitochondria and plasma membrane fragments from hypertensive rat aorta and caudal artery.

Vascular smooth muscle (VSM) cells from hypertensive and normotensive rat aortae and caudal arteries were isolated by enzymatic techniques, homogenized, and fractionated by differential pelleting. By these techniques, only mitochondria could be enriched more than fivefold in any one fraction. The other organelles were distributed heterogeneously in almost all fractions. Hypertensive smooth muscle enzyme distribution patterns were different from the normotensive, suggesting that changes in sedimentation characteristics had occurred. Activity of the enzyme 5'-nucleotidase increased in whole tissue homogenates and in the 'microsomal' fraction of aortic and caudal artery of hypertensive VSM. The lysosomal protease, cathepsin D, of hypertensive animals decreased in activity for both vascular smooth muscles while N-acetyl-beta-glucosaminidase and pNPPase (acid phosphatase) increased. The possibility of a functional deficiency in protein degradation causing lysosomal overloading is discussed.

Potentials in mammalian skeletal muscle from collagenase-treated tissue.

Transmembrane potentials were recorded from skeletal muscle fibres dissected with the aid of collagenase perfusion. Collagenase treatment had little or no effect on the action or resting potentials.