Glycosphingolipid expression in human skeletal and heart muscle assessed by immunostaining thin-layer chromatography

In this study the comparative TLC immunostaining investigation of neutral GSLs and gangliosides from human skeletal and heart muscle is described. A panel of specific polyclonal and monoclonal antibodies as well as the GM1-specific choleragenoid were used for the overlay assays, combined with preceding neuraminidase treatment of gangliosides on TLC plates. This approach proved homologies but also quantitative and qualitative differences in the expression of ganglio-, globo- and neolacto-series neutral GSLs and gangliosides in these two types of striated muscle tissue within the same species. The main neutral GSL in skeletal muscle was LacCer, followed by GbOse3Cer, GbOse4Cer, nLcOse4Cer and monohexosylceramide, whereas in heart muscle GbOse3Cer and GbOse4Cer were the predominant neutral GSLs beside small quantities of LacCer, nLcOse4Cer and monohexosylceramide. No ganglio-series neutral GSLs and no Forssman GSL were found in either muscle tissue. GM3(Neu5Ac) was the major ganglioside, comprising almost 70% in skeletal and about 50% in cardiac muscle total gangliosides. GM2 was found in skeletal muscle only, while GD3 and GM1b-type gangliosides (GM1b and GD1) were undetectable in both tissues. GM1a-core gangliosides (GM1, GD1a, GD1b and GT1b) showed somewhat quantitative differences in each muscle; lactosamine-containing IV3Neu5Ac-nLcOse4Cer was detected in both specimens. Neutral GSLs were identified in TLC runs corresponding to e.g. 0.1 g muscle wet weight (GbOse3Cer, GbOse4Cer), and gangliosides GM3 and GM2 were elucidated in runs which corresponded to 0.2 g muscle tissue. Only 0.02 g and 0.004 g wet weight aliquots were necessary for unequivocal identification of neolacto-type and GM1-core gangliosides, respectively. Muscle is known for the lowest GSL concentration from all vertebrate tissues studied so far. Using the overlay technique, reliable GSL composition could be revealed, even from small muscle probes on a sub-orcinol and sub-resorcinol detection level. Abbreviations: ATCC, American Type Culture Collection; GSL(s), glycosphingolipid(s); HPLC, high performance liquid chromatography; HPTLC, high performance thin layer chromatography; Neu5Ac, N-acetylneuraminic acid; Neu5Gc, N-glycolylneuraminic acid [78]; PBS, phosphate buffered saline. The designation of the following glycosphingolipids follows the IUPAC-IUB recommendations [79] and the ganglioside nomenclature system of Svennerholm [80]. Lactosylceramide or LacCer, Gal1-4Glc1-1Cer; gangliotriaosylceramide or GgOse3Cer, GalNAc1-4Gal1-4Glc1-1Cer; gangliotetraosylceramide or GgOse4Cer, Gal1-3GalNAc1-4Gal1-4Glc1-1Cer; globotriaosylceramide or GbOse3Cer, Gal1-4Gal1-4Glc1-1Cer; globoside or globotetraosylceramide or GbOse4Cer, GalNAc1-3Gal1-4Gal1-4Glc1-1Cer; Fo or Forssman GSL, GalNAc1-3GalNAc1-3Gal1-4Gal1-4Glc1-1Cer; paragloboside or lacto-N-neotetraosylceramide or nLcOse4Cer, Gal1-4GlcNAc1-3Gal1-4Glc1-1Cer; lacto-N-norhexaosylceramide or nLcOse6Cer, Gal1-4GlcNAc1-3Gal1-4GlcNAc1-3Gal1-4Glc1-1Cer; GM3, II3Neu5Ac-LacCer; GM2, II3Neu5Ac-GgOse3Cer; GM1 or GM1a, II3Neu5Ac-GgOse4Cer; GM1b, IV3Neu5Ac-GgOse4Cer; GD3, II3(Neu5Ac)2-LacCer; GD1a, IV3Neu5Ac,II3Neu5Ac-GgOse4Cer; GD1b, (II3Neu5Ac)2-GgOse4Cer; GD1, IV3Neu5Ac,III6Neu5Ac-GgOse4Cer; GT1b, IV3Neu5Ac,II3(Neu5Ac)2-GgOse4Cer; GQ1b, IV3(Neu5Ac)2, II3(Neu5Ac)2-GgOse4Cer.     

Ca-release by phosphoinositides from sarcoplasmic reticulum of frog skeletal muscle

To clarify the biological role of phosphoinositides including inositol trisphosphate (IP3) in the skeletal muscle, we examined the Ca-releasing action on the heavy fraction of sarcoplasmic reticulum (HFSR) from bullfrog skeletal muscle of IP3, phosphatidylinositol monophosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2), and glycerophosphoinositol 4,5-bisphosphate (GPIP2). Only PIP2 caused dose-dependent Ca release. IP3 (up to 55 microM), PIP (up to 37 microM), and GPIP2 (up to 33 microM) were ineffective. The PIP2-induced Ca release is due to the direct action of PIP2, but not its metabolite(s). The properties of the PIP2-induced Ca release are unique and cannot be accounted for by the Ca release mechanisms already reported, such as Ca2+-induced, ionic substitution-induced, or IP3-induced Ca release. The rate of the PIP2-induced Ca release, however, is so slow that it may have no physiological relevance unless stimulating factors or agents exist.     

Acoustic signals from frog skeletal muscle.

Acoustic, force, and compound muscle action-potential signals were recorded simultaneously during maximal isometric twitches of frog gastrocnemius muscles. The onset of sound production occurred after the onset of muscle depolarization but before the onset of external force production. Acoustic waveforms consisted of oscillations that initially increased in amplitude, followed by decaying oscillations. The peak-to-peak acoustic amplitude increased with increasing temperature with a Q10 of 2.6 +/- 0.2 over a range of 7.0-25.0 degrees C. The acoustic amplitude increased with increasing muscle length up to approximately 90% of the optimal length for force generation. As length was increased further, the acoustic amplitude decreased. Microphones positioned on opposite sides of the muscle recorded acoustic signals that were 180 degrees out of phase. These results provided evidence that sound production is produced by lateral oscillations of muscle. The oscillation frequency may provide a measure of mechanical properties of muscle.     

Isolation and characterization of basement membrane from frog skeletal muscle

Isolation of basement membrane from frog skeletal muscle has been described. The membrane preparation contained 35 micrograms hexoses, 1.72 micrograms sialic acid, 6.8 micrograms phospholipids, 0.21 micrograms cholesterol/mg protein. Na + K-ATPase and 5'-nucleotidase could not be detected in the membrane preparation. Glycine accounted for about 20% of the total amino acids. On SDS-PAGE, the membrane resolved into 20-22 polypeptide bands.     

Protein phosphatase type-1 from skeletal muscle of the freeze-tolerant wood frog

We evaluated the effects of freezing, dehydration and anoxia stresses on muscle PP-1 activity in the freeze-tolerant amphibian, Rana sylvatica. In addition, PP-1 catalytic subunit (PP-1c) was purified to homogeneity to assess the biochemical properties of the enzyme from a freeze-tolerant vertebrate. Freezing stimulated a rise in the amount of active PP-1 (70% above the control) at 20 min post-nucleation. With longer freezing (1–12 h), the amount of active enzyme returned to control levels, and the amount of total PP-1 fell, decreasing by up to 43%. This decline in total PP-1 kept the % active at a high value throughout the freeze. Anoxia exposure (12 h) reduced the active PP-1 by 60%, but had no effect on total PP-1 activity. Neither dehydration nor rehydration had any significant effect on the amounts of either total or active PP-1. PP-1 activity associated with the myofibril fraction increased, while activity associated with the glycogen pellet decreased in response to freezing and dehydration, but not anoxia. Purified frog PP-1c showed a variety of properties that are typical of the enzyme from other sources. In addition, the enzyme was strongly inhibited by AMP and weakly by ADP and ATP; the physiological relevance of inhibition by nucleotides remains to be determined. Overall, the results suggest an important role for PP-1 in signal transduction in the skeletal muscle of freeze-tolerant amphibians.     

Sodium-calcium exchange in transverse tubules isolated from frog skeletal muscle

Transverse tubule vesicles isolated from frog skeletal muscle display sodium-calcium exchange activity, which was characterized measuring 45Ca influx in vesicles incubated with sodium. The initial rates of exchange varied as a function of the membrane diffusion potentials imposed across the membrane vesicles, increasing with positive intravesicular potentials according to an electrogenic exchange with a stoichiometry greater than 2 sodium ions per calcium ion transported. The exchange activity was a saturable function of extravesicular free calcium, with an apparent K0.5 value of 3 microM and maximal rates of exchange ranging from 3 to 5 nmol/mg protein per 5 s. The exchange rate increased when intravesicular sodium concentration was increased; saturation was approached when vesicles were incubated with concentrations of 160 mM sodium. The isolated transverse tubule vesicles, which are sealed with the cytoplasmic side out, had a luminal content of 112 +/- 39 nmol calcium per mg protein. In the absence of sodium, the exchanger carried out electroneutral calcium-calcium exchange, which was stimulated by increasing potassium concentrations in the intravesicular side. Calcium-calcium exchange showed an extravesicular calcium dependence similar to the calcium dependence of the sodium-calcium exchange, with an apparent K0.5 of 6 microM. Sodium-calcium and calcium-calcium exchange were both inhibited by amiloride. The sodium-calcium exchange system operated both in the forward and in the reverse mode; sodium, as well as calcium, induced calcium efflux from 45Ca-loaded vesicles. This system may play an important role in decreasing the intracellular calcium concentration in skeletal muscle following electrical stimulation.     

Activation of phosphorylase in frog muscle as determined by contractile activity

The state of activation of phosphorylation in muscle has been reinvestigated by combining the extraction procedures of Danforth, Helmreich, and Cori with the low-temperature techniques of this laboratory. In resting frog muscle, the phosphorylase-alpha content is usually below detectability. Upon contractile activity in series of twitches, activation of phosphorylase beta to alpha took place, without activation of phosphorylase beta kinase as defined by the assay procedure. Two different experimental designs were used to examine the relation between phosphorylase activation and the myothermally determined energy turnover per twitch, and these showed, identically, that the enzyme activation is proportional to the energy per twitch.     

The amino-acid sequence of troponin C from frog skeletal muscle.

The primary structure of the troponin C from skeletal muscle of the frog Rana esculenta has been determined. The amino acid sequence was deduced from amino acid determinations of peptides obtained after cleavage with cyanogen bromide. Overlapping peptides were isolated from tryptic digests of performic-acid-oxidized troponin C and phthalylated performic-acid-oxidized troponin C. All overlaps have been determined except for the Arg-Ile sequence at position 103--104, which has been obtained by comparison with homologous troponins C. Frog troponin C consists of one polypeptide chain containing 152 amino acids. The calculated molecular weight is 18299. There is a single cysteine residue at position 101 and a single tyrosine residue at position 112. No histidine or tryptophan residues are present. The amino-terminal amino acid is N-acetylated. The homology of frog troponin C with other skeletal and cardiac troponin C is briefly discussed.     

A rapid method for preparation of sarcolemma from frog skeletal muscle

A rapid method for the preparation of sarcolema from frog skeletal muscle has been described. The purified cell segments were transparent and devoid of contractile material. The Na⁺, K⁺ -ATPase and 5'-nucleotidase activities in sarcolemma purified by this method were comparable to those reported for sarcolemmal preparations purified by density gradient centrifugation. The preparation also possessed acid phosphatase, alkaline phosphatase and K⁺ -activated, ouabain-sensitive p-nitrophenyl phosphatase activities. The cholesterol to phospholipid ratio of the sarcolemma was 0.33, indicating its high purity; further, the preparation was free from mitochondria and contractile proteins.     

Ionic movements mediated by monensin in frog skeletal muscle.

Monensin-mediated ionic movements were studied in frog skeletal muscle. The ionophore, which forms electrically neutral complexes with monovalent cations, induced dose dependent fluxes of Na+, K+ and H+ in and out of the fibers. Monensin concentrations ([MON]) ranged from 2 to 40 microM. In the presence of normal Ringer's solution the following maximum ionic exchanges were generated by monensin (in pmol cm-2 s-1): (1) Nai+/Nao+ 112, (2) Nai+/Ho+ 30.7, (3) Ki+/Nao+ 14.2 (4) Hi+/Nao+ 49. The maximum net fluxes produced by these exchanges (i.e. for [MON] = infinity) are (in pmol cm-2 s-1): Na+ (inward) 32.5, K+ (outward) 14.2, H+ (outward) 18.3. The last one appears to be largely offset by a passive (monensin-independent) H+ influx down an inwardly directed electrochemical gradient promoted by pH reduction of the T-tubular lumen content as a consequence of the monensin-mediated net H+ efflux. Maximum unidirectional cationic fluxes mediated by monensin amounted to 206 pmol cm-2 s-1 and had the following composition: influx: 85% Na+ and 15% H+; efflux: 69% Na+, 7% K+, 24% H+.     

The "intermediate component" of the radiosodium efflux from frog skeletal muscle

The influence of diversity in the size of the cells of the frog's sartorius on the radiosodium efflux from the muscle was investigated. Morphometric analyses of light micrographs of complete cross sections of the muscle were done in the proximal and distal regions. The results were used to predict the shape of the radiosodium washout curve under the following assumptions: the cells differ in size and shape, but each has a single internal pool of exchangeable sodium; the sodium exchange properties of the limiting membranes are the same for all cells; and the diversity of the true areas of the limiting membranes is reflected by the diversity of the apparent areas measured at the light microscopic level. Radiosodium efflux measurements were performed on similar muscles. The model correctly predicted the occurrence of a continuous decline of the fractional loss of radiosodium, which was not due to diffusional delay and which would be interpreted as a second internal compartment in a compartmental analysis, and an effect of short versus long isotope loading intervals on the efflux. It was concluded that the existence of cell size diversity satisfactorily explains the flux data. No "special region" must be postulated.     

Discoupling of excitation-contraction processes by urea treatment of frog skeletal muscle

On exposure (E) of frog semitendinosus muscle to 400 mmol/l urea (U) in sodium chloride Ringer's solution, the tension development to isoK+ solutions decreased, while in choline chloride Ringer it increased. On quick removal (R) of urea, always a block of excitation-contraction (E-C) coupling occurred accompanied by transient or persistent swelling of fibres and a similar but definite decrease of their resting membrane potential (Fig. 2). Muscle contraction could be elicited by caffeine even after UER-treatment but then only the slow tension increase (second phase of normal caffeine contraction) occurred (Fig. 3a). The fast tension increase to caffeine (first phase) could be restored if after UER-treatment 5 mmol/l mannitol (Fig. 3b), a 20 min treatment with choline chloride (Fig. 4a) or sodium isethionate (Fig. 4b) Ringer's solution of double osmolarity were applied. Caffeine contraction could not be elicited when sodium chloride Ringer's solution of double osmolarity was used under similar conditions (Fig. 5). E-C block to isoK+ solution persisted in all these experiments. E-C coupling could partially be restored by short treatment of muscle with caffeine (Figs 6a, b).     

Diffusible magnesium in frog skeletal muscle cells

Total diffusible magnesium concentration in frog skeletal muscle is 5.2 mM as determined by electron probe microanalysis of 0.2 nl liquid samples. The calculated free Mg concentration, 0.2 mM, is at the lower end of the range of values reported by others as calculated by methods using nuclear magnetic resonance, Mg-selective microelectrodes, and metallochromic indicator dyes. Magnesium is but one of many elements of physiological importance in muscle that can be analyzed using this novel liquid-sampling and x-ray spectroscopic method.