Effects of modulators of sarcoplasmic Ca2+ release on the development of skeletal muscle fatigue.

The reduced release of Ca2+ from sarcoplasmic reticulum (SR) is considered a major determinant of muscle fatigue. In the present study, we investigated whether the presence of dantrolene, an established inhibitor of SR Ca2+ release, or caffeine, a drug facilitating SR Ca2+ release, modifies muscle fatigue development. Accordingly, the effects of Ca2+ release modulators were analyzed in vitro in mouse fast-twitch [extensor digitorum longus (EDL)] and slow-twitch (soleus) muscles, fatigued by repeated short tetani (40 Hz for 300 ms, 0.5 s(-1) in soleus and 60 Hz for 300 ms, 0.3 s(-1) in EDL, for 6 min). Caffeine produced a substantial increase of tetanic tension of both EDL and soleus muscles, whereas dantrolene decreased tetanic tension only in EDL muscle. In both EDL and soleus muscles, 5 microM dantrolene did not affect fatigue development, whereas 20 microM dantrolene produced a positive staircase during the first 3 min of stimulation in EDL muscle and a slowing of fatigue development in soleus muscle. The development of the positive staircase was abolished by the addition of 15 microM ML-7, a selective inhibitor of myosin light chain kinase. On the other hand, caffeine caused a larger and faster loss of tension in both EDL and soleus muscles. The results seem to indicate that the changes in fatigue profile induced by caffeine or dantrolene are mainly due to the changes in the initial tetanic tension caused by the drugs, with the resulting changes in the level of contraction-dependent factors of fatigue, rather than to changes in the SR Ca2+ release during fatigue development.

Expression of sarco(endo)plasmic reticulum Ca(2+)-ATPase slow (SERCA2) isoform in regenerating rat soleus skeletal muscle depends on nerve impulses.

We have examined the influence of innervation on the expression of different isoforms of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) in regenerating rat slow twitch muscle. The process of degeneration/regeneration was induced by injection of bupivacaine into rat soleus muscle under four different conditions: (1) in the presence of intact motor nerves, (2) after surgical denervation, (3) with nerve impulse conduction blocked by the Na(+)-channel blocker tetrodotoxin (TTX), and (4) with the axoplasmic flow blocked by vinblastine. Expression of SERCA isoforms was visualized by immunohistochemical and Western blot analysis. In regenerating innervated muscle, SERCA1, the isoform normally expressed in fast twitch fibres, was present after 5 days and was then progressively replaced by SERCA2, the isoform typical of slow twitch fibres. The maximum Ca(2+) uptake, measured in single skinned fibres regenerating for 10-21 days, was similar to that of slow adult fibres and significantly lower than that of fast adult fibres. Denervation or TTX treatment prevented the expression of the SERCA2 isoform. Conversely, vinblastine did not affect the expression of SERCA isoforms. These data indicate that nerve impulses play a determinant role in the expression of the SERCA2 isoform.

Apoptosis in the skeletal muscle of rats with heart failure is associated with increased serum levels of TNF-alpha and sphingosine.

Skeletal muscle in congestive heart failure (CHF) is responsible for increased fatigability, decreased endurance and exercise capacity. A specific myopathy with increased expression of fast myosin heavy chains (MHCs), myocyte atrophy, secondary to myocyte apoptosis, that is triggered by high levels of circulating tumor necrosis factor (TNF-alpha) has been described. However, a direct effect of TNF-alpha on skeletal muscle has not been described yet. In this paper we put forward the hypothesis that TNF-alpha plays an indirect effect on skeletal myocytes. In an animal model of CHF, the monocrotaline-treated rat, we have observed a significant (P<0.001) increase of circulating TNF-alpha that is paralleled by increased serum levels of the endogenous second messenger, sphingosine (SPH), (from 0.71+/-0.15 to 1.32+/-0.39 nmoles/ml, P<0.01). In the tibialis anterior (TA) muscle we found a marked increase of myocyte apoptosis (from 1.4+/-2.4 to 40.1+/-39.5 nuclei/mm(3), P<0.04). We correlated plasma levels of TNF-alpha with those of SPH and in turn with the magnitude of apoptosis. Linear regression showed a significant correlation between TNF-alpha, SPH, and apoptosis (r(2)=0.74, P=0.004 and r(2)=0.87, P=0.001 respectively). Analysis of covariance showed that TNF-alpha and SPH were independently correlated with the number of apoptotic nuclei (P=0.0001). In parallel in vitro experiments, where increasing concentrations of SPH were applied to skeletal muscle cells in culture, we observed a dose-dependent increase in apoptosis. These results suggest that TNF-alpha-induced SPH production may be responsible for skeletal muscle apoptosis. The link between TNF-alpha and skeletal muscle apoptosis could be represented by the second messenger SPH, which can directly induce apoptosis in these cells.

Loss of dystrophin and some dystrophin-associated proteins with concomitant signs of apoptosis in rat leg muscle overworked in extension.

This study investigated the basis for the high severity of damage to skeletal muscle due to eccentric exercise, i.e., to muscles generating force while lengthened. Fast and slow rat leg muscles maintained in an extended position were examined after 2-24 h of continuous stimulation. The treatment caused the injury to some regions of both muscles. Within the better preserved parts of the muscles, i.e., those without signs of necrotic processes, dystrophin, spectrin, and some of the dystrophin-associated proteins (beta-dystroglycan, alpha-sarcoglycan, and gamma-sarcoglycan) disappeared from sarcolemma of many fibers. The reduction or loss of dystrophin from the sarcolemma was more evident than that of other proteins examined, with sarcoglycans apparently being the most preserved. Several muscle fibers devoid of dystrophin contained apoptotic nuclei. Simultaneously, Bax, Bcl-2 and caspase-3 proteins appeared in many fibers. Our results indicate that a normal muscle overworking in an extended position undergoes the loss of several membrane skeletal proteins because of the excessive stress to the membrane cytoskeleton, which can lead to fiber death by either apoptosis or necrosis. This experimental model may represent a good model for mimicking the pathogenetic events in several muscular dystrophies.

Expression and characterization of Edg-1 receptors in rat cardiomyocytes: calcium deregulation in response to sphingosine 1-phosphate.

Recent evidence indicates that sphingolipids are produced by the heart during hypoxic stress and by blood platelets during thrombus formation. It is therefore possible that sphingolipids may influence heart cell function by interacting with G-protein-coupled receptors of the Edg family. In the present study, it was found that sphingosine 1-phosphate (Sph1P), the prototypical ligand for Edg receptors, produced calcium overload in rat cardiomyocytes. The cDNA for Edg-1 was cloned from rat cardiomyocytes and, when transfected in an antisense orientation, effectively blocked Edg-1 protein expression and reduced the Sph1P-mediated calcium deregulation. Taken together, these results demonstrate that cardiomyocytes express an extracellular lipid-sensitive receptorsystem that can respond to sphingolipid mediators. Because the major source of Sph1P is from blood platelets, we speculate that Edg-mediated Sph1P negative inotropic and cardiotoxic effects may play important roles in acute myocardial ischemia where Sph1P levels are probably elevated in response to thrombus.

Effects of fatigue on sarcoplasmic reticulum and myofibrillar properties of rat single muscle fibers.

Force decline during fatigue in skeletal muscle is attributed mainly to progressive alterations of the intracellular milieu. Metabolite changes and the decline in free myoplasmic calcium influence the activation and contractile processes. This study was aimed at evaluating whether fatigue also causes persistent modifications of key myofibrillar and sarcoplasmic reticulum (SR) proteins that contribute to tension reduction. The presence of such modifications was investigated in chemically skinned fibers, a procedure that replaces the fatigued cytoplasm from the muscle fiber with a normal medium. Myofibrillar Ca(2+) sensitivity was reduced in slow-twitch muscle (for example, the pCa value corresponding to 50% of maximum tension was 6.23 +/- 0.03 vs. 5.99 + 0.05, P < 0.01, in rested and fatigued fibers) and not modified in fast-twitch muscle. Phosphorylation of the regulatory myosin light chain isoform increased in fast-twitch muscle. The rate of SR Ca(2+) uptake was increased in slow-twitch muscle fibers (14.2 +/- 1.0 vs. 19.6 +/- 2. 5 nmol. min(-1). mg fiber protein(-1), P < 0.05) and not altered in fast-twitch fibers. No persistent modifications of SR Ca(2+) release properties were found. These results indicate that persistent modifications of myofibrillar and SR properties contribute to fatigue-induced muscle force decline only in slow fibers. These alterations may be either enhanced or counteracted, in vivo, by the metabolic changes that normally occur during fatigue development.

Ecto-ATPase activity of alpha-sarcoglycan (adhalin).

alpha-Sarcoglycan is a component of the sarcoglycan complex of dystrophin-associated proteins. Mutations of any of the sarcoglycan genes cause specific forms of muscular dystrophies, collectively termed sarcoglycanopathies. Importantly, a deficiency of any specific sarcoglycan affects the expression of the others. Thus, it appears that the lack of sarcoglycans deprives the muscle cell of an essential, yet unknown function. In the present study, we provide evidence for an ecto-ATPase activity of alpha-sarcoglycan. alpha-Sarcoglycan binds ATP in a Mg2+-dependent and Ca2+-independent manner. The binding is inhibited by 3'-O-(4-benzoyl)benzoyl ATP and ADP. Sequence analysis reveals the existence of a consensus site for nucleotide binding in the extracellular domain of the protein. An antibody against this sequence inhibits the binding of ATP. A dystrophin.dystrophin-associated protein preparation demonstrates a Mg-ATPase activity that is inhibited by the antibody but not by inhibitors of endo-ATPases. In addition, we demonstrate the presence in the sarcolemmal membrane of a P2X-type purinergic receptor. These data suggest that alpha-sarcoglycan may modulate the activity of P2X receptors by buffering the extracellular ATP concentration. The absence of alpha-sarcoglycan in sarcoglycanopathies leaves elevated the concentration of extracellular ATP and the persistent activation of P2X receptors, leading to intracellular Ca2+ overload and muscle fiber death.

Functional roles of dystrophin and of associated proteins. New insights for the sarcoglycans.

The discovery of the dystrophin gene, whose mutations lead to Duchenne's and Becker's muscular dystrophy (DMD and BMD), represents the first important landmark by which, in the last ten years, molecular biology and genetic studies have revealed many of the molecular defects of the major muscular dystrophies. Very rapidly, several studies revealed the presence at skeletal and cardiac muscle sarcolemma of a group of proteins associated to dystrophin. This includes a set of five transmembrane glycoproteins, the sarcoglycans, whose physiological role, however, is still poorly understood. Dystrophin and the associated proteins are believed to play an important role in membrane stability and maintenance during muscle contraction and relaxation. However, the absence of sarcoglycans from sarcolemma does not appear to affect membrane integrity suggesting that these components of the dystrophin complex are recipients of other important functions. This review deals with recent advances in the knowledge of sarcoglycan function and organization that may give important insights into the pathogenetic mechanisms of muscular dystrophies.

The role of sphingolipids in the control of skeletal muscle function: a review.

In this review, potential roles for the endogenous sphingolipid, sphingosine, and its derivatives are described for muscle cells. Sphingosine modulates the function of important calcium channels in muscle, including the ryanodine receptor (RyR) calcium release channel of the sarcoplasmic reticulum (SR). Sphingosine blocks calcium release through the SR ryanodine receptor and reduces the activity of single skeletal muscle RyR channels reconstituted into planar lipid bilayers. Sphingosine-blocked calcium release is coincident with the inhibitory effects of sphingosine on [3H]ryanodine binding to the RyR. The sphingomyelin signal transduction pathway has also been identified in both skeletal and cardiac muscle. A neutral form of sphingomyelinase (nSMase) enzyme has been localized to the junctional transverse tubule membrane. The high turnover of the SMase is responsible for the production of ceramide and sphingosine. HPLC analyses indicate that significant resting levels of sphingosine are present in muscle tissue. A model of excitation-contraction coupling is presented suggesting a potential role for this endogenous sphingolipid in normal muscle function. Putative roles for sphingolipid mediators in skeletal muscle dysfunction are also discussed. We hypothesize that sphingosine plays important roles in malignant hyperthermia and during the development of muscle fatigue.

Gender- and thyroid hormone-related transitions of essential myosin light chain isoform expression in rat soleus muscle during ageing.

In this brief review, the modulatory influence of essential myosin light chain (MLC) isoforms on muscle cell contractility is discussed. Specific interest is focused on the expression of the MLC1Sa and MLC1Sb isoforms in the slow-twitch soleus muscle in male and female rats, during ageing and after thyroid hormone treatment. According to two-dimensional gel electrophoresis analysis, the MLC1Sa/MLC1SB ratio increased during ageing in both males and females in parallel with the age-related decrease in shortening velocity reported in muscle fibres expressing the slow (type 1) myosin heavy chain (MHC) isoform. However, the MLC1Sa and MLC1Sb isoform expression responded to thyroid hormone treatment in a complex manner which did not parallel the age-related changes in shortening velocity reported in hyperthyroid animals. Thus, if MLC1Sa and MLC1Sb isoforms modulate shortening velocity in type 1 fibres, then other modulators of shortening velocity are not regulated by thyroid hormone in co-ordination with these essential MLCs.

Early effects of denervation on sarcoplasmic reticulum properties of slow-twitch rat muscle fibres.

The Ca2+ release activity of the sarcoplasmic reticulum (SR) in chemically skinned single slow-twitch fibres from control, 2-day and 7-day denervated rat soleus muscle was studied. Histochemical fibre type composition of the whole muscle, electrophysiological properties and the Ca2+ sensitivity of tension development by single muscle fibres were also studied. All the data were correlated with contractile properties of the in vitro muscle. In the 2-day denervated muscle the SR Ca2+ capacity and the rate of Ca2+ uptake decreased from the control values of 0.384 +/- 0.030 micromol (mg fibre protein)-1 and 19.8 +/- 1.9 nmol min-1 (mg fibre protein)-1, respectively, to 0.210 +/- 0.016 micromol (mg fibre protein)-1 and 13.5 +/- 0.9 nmol min-1 (mg fibre protein)-1; the calculated amount of Ca2+ released upon stimulation by caffeine decreased from the control value of 0.148 to 0.078 micromol (mg fibre protein)-1. In the 7-day denervated muscle, the SR Ca2+ capacity and the rate of Ca2+ uptake increased to 0.517 +/- 0.06 micromol (mg fibre protein)-1 and 21.6 +/- 2.3 nmol min-1 (mg fibre protein)-1, respectively; the calculated amount of Ca2+ released increased to 0.217 micromol (mg fibre protein)-1. Both contraction time and tension of the isometric twitch decreased in 2-day denervated and increased in 7-day denervated muscles. Electrophysiological and histochemical changes, as well as changes in the Ca2+ sensitivity of the muscle fibres did not show any apparent correlation with mechanical changes. It is therefore concluded that the SR plays a prominent role in the early changes of contraction time and tension following denervation.

Sphingosylphosphocholine modulates the ryanodine receptor/calcium-release channel of cardiac sarcoplasmic reticulum membranes.

Sphingosylphosphocholine (SPC) modulates Ca2+ release from isolated cardiac sarcoplasmic reticulum membranes; 50 microM SPC induces the release of 70 80% of the accumulated calcium. SPC release calcium from cardiac sarcoplasmic reticulum through the ryanodine receptor, since the release is inhibited by the ryanodine receptor channel antagonists ryanodine. Ruthenium Red and sphingosine. In intact cardiac myocytes, even in the absence of extracellular calcium. SPC causes a rise in diastolic Ca2+, which is greatly reduced when the sarcoplasmic reticulum is depleted of Ca2+ by prior thapsigargin treatment. SPC action on the ryanodine receptor is Ca(2+)-dependent. SPC shifts to the left the Ca(2+)-dependence of [3H]ryanodine binding, but only at high pCa values, suggesting that SPC might increase the sensitivity to calcium of the Ca(2+)-induced Ca(2+)-release mechanism. At high calcium concentrations (pCa 4.0 or lower), where [3H]ryanodine binding is maximally stimulated, no effect of SPC is observed. We conclude that SPC releases calcium from cardiac sarcoplasmic reticulum membranes by activating the ryanodine receptor and possibly another intracellular Ca(2+)-release channel, the sphingolipid Ca(2+)-release-mediating protein of endoplasmic reticulum (SCaMPER) [Mao, Kim, Almenoff, Rudner, Kearney and Kindman (1996) Proc.Natl.Acad.Sci. U.S.A 93, 1993-1996], which we have identified for the first time in cardiac tissue.

Effects of age on sarcoplasmic reticulum properties and histochemical composition of fast- and slow-twitch rat muscles.

Calcium release activity of sarcoplasmic reticulum and enzyme-histochemical properties were investigated in extensor digitorum longus (e.d.l.) and soleus muscles in young (4 months and old (24 months) male rats. With age, the caffeine threshold concentration for calcium release from the sarcoplasmic reticulum of soleus skinned muscle fibres showed only minor modifications. On the other hand, in e.d.l. skinned muscle fibres, the caffeine threshold concentration decreased significantly (P < 0.05). The histochemical fibre type composition changed with age both in soleus and in e.d.l. muscles, showing a common transformation toward a more oxidative histochemical profile. In fact, in aged soleus, a significant (P < 0.05) increase was observed of type 1 fibres to represent almost the totality of the muscle fibres (more than 98%), while types 2C and 2A were reduced in proportion. In aged e.d.l. the percentage of type 1 (P < 0.05), 2A and 2X (a recently identified fourth component of the fast-twitch muscle types) fibres increased, with a reduction of type 2B (P < 0.01) fibres. The present results suggest that the changes in contractile properties of aged muscles may be related to the changes not only in fibre composition but also in the mechanism of calcium release from sarcoplasmic reticulum.

Involvement of ryanodine receptors in sphingosylphosphorylcholine-induced calcium release from brain microsomes.

Sphingosylphosphorylcholine (SPC) releases Ca2+ from brain microsomes. SPC-induced CA2+ release differs from IP3-induced Ca2+ release in that it is more extensive in the cerebrum than in the cerebellum. SPC has little effect on [3H] IP3 binding but enhances [3H] ryanodine binding, as expected for an activator of ryanodine receptors. SPC-induced Ca2+ release is inhibited by ryanodine receptor blockers but not by selective blockers of IP3 receptors. We conclude that SPC releases Ca2+ from brain microsomes by activating ryanodine receptors rather than IP3 receptors. Activation of an additional SPC-sensitive pathway for releasing Ca2+ is not precluded.

Control of cardiac Ca2+ levels. Inhibitory actions of sphingosine on Ca2+ transients and L-type Ca2+ channel conductance.

The naturally occurring second messenger sphingosine (SPH) was examined for its ability to influence cardiac myocyte Ca2+ regulation. SPH inhibited intracellular Ca2+ transients in adult and neonatal rat ventricular myocytes. The inhibition was steeply dose dependent, with complete blockage of the Ca2+ transients occurring in the 20- to 25-mumol/L range. Whole-cell patch clamping revealed substantial inhibition of the L-type Ca2+ channel current (ICa) by SPH. The ability of SPH to block both the Ca2+ transients and ICa was not dependent on protein kinases, since the general protein kinase inhibitor H7 failed to prevent the actions of SPH. The specificity of the effect of SPH was determined in experiments showing that SPH analogues did not produce comparable effects. Neither the naturally occurring ceramide, N-stearoyl SPH, nor the cell-permeant ceramide, N-acetyl SPH, had SPH-like actions on the Ca2+ transients or L-type channel conductances. Caffeine-induced Ca2+ transients were also inhibited by the actions of SPH on cardiac sarcoplamic reticulum Ca2+ release, and the threshold for caffeine-induced Ca2+ release was raised. We conclude that SPH inhibits excitation-contraction coupling in cardiac myocytes by reducing the amount of entering "trigger Ca2+" for Ca(2+)-induced Ca2+ release and by simultaneously raising the threshold of the ryanodine receptor for Ca(2+)-induced Ca2+ release. Consequently, we propose that sphingolipids produced by the sphingomyelin signal transduction pathway could be physiologically relevant regulators of cardiac [Ca2+]i and therefore cardiac contractility.

Maximum shortening velocity and coexistence of myosin heavy chain isoforms in single skinned fast fibres of rat skeletal muscle.

Myosin heavy chain composition of a large number (288) of single fibres from slow (soleus), and fast (superficial part of tibialis anterior, and plantaris) muscles of adult (3-5-month-old) Wistar rats was determined. A combination of SDS-PAGE and monoclonal antibodies against myosin heavy chains allowed to identify four myosin heavy chain isoforms (1, 2A, 2X, and 2B) and to detect myosin heavy chain coexistence. Four groups of fibres containing only one myosin heavy chain (1 myosin heavy chain, 2A myosin heavy chain, 2X myosin heavy chain, and 2B myosin heavy chain), and five groups containing more than one myosin heavy chain (1 and 2A myosin heavy chains, 2A and 2X myosin heavy chains, 2X and minor amounts of 2B (2X-2B fibres), 2B and minor amounts of 2X (2B-2X fibres), and 2A, 2X, and 2B myosin heavy chain were identified and their relative percentages were assessed. Coexistence of fast myosin heavy chain isoforms was found to be very frequent (50% of the fibres in plantaris, and 30% in tibialis anterior), whereas coexistence of slow and fast (2A) myosin heavy chain was very rare. Maximum shortening velocity (V0) was determined using the slack-test procedure in a subset of 109 fast fibres from the above population. The values of V0 formed a continuum extending from 2A to 2X to 2X-2B to 2B-2X to 2B fibres. 2A fibres had the lowest value of V0 and 2B fibres the highest. Only the differences between 2A and 2B and 2A and 2B-2X fibres were statistically significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Unloaded shortening velocity and myosin heavy chain and alkali light chain isoform composition in rat skeletal muscle fibres.

1. This study aims to assess the role of myosin heavy chain (MHC) and alkali myosin light chain (MLC) isoforms in determining maximum velocity of shortening in fast skeletal muscle fibres. 2. The maximum velocity of shortening as determined by the slack test (Vo) was tested for its relationship with MHC composition and with alkali MLC isoform ratio of fast fibres of known MHC composition. 3. MHC isoform composition was determined using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and monoclonal antibodies against MHCs, and combining the results obtained using the two methods. Three groups of fast fibres containing only one MHC isoform were identified: IIA, IIX and IIB fibres containing respectively IIA MHC, IIX MHC and IIB MHC. Fibres containing more than one MHC isoform were discarded. 4. The mean Vo value of IIA fibres was 2.33 +/- 0.29 muscle lengths per second (L s-1; mean +/- S.D.), this was significantly lower than that for IIX fibres (3.07 +/- 0.70 L s-1) which in turn had a mean Vo value significantly lower than that for IIB fibres (3.69 +/- 1.01 L s-1). 5. The relative proportion of alkali MLC isoforms (MLC3f, MLC1f) was determined by means of electrophoretic separation and densitometric quantification and was expressed as MLC3f/MLC2f with reference to the dithio-nitrobenzoic acid (DTNB) light chain (MLC2f). The mean value of the MLC3f/MLC2f ratio was significantly lower in IIA than in IIX and IIB fibres. 6. Vo was found to be proportional to the relative content of MLC3f in IIA, IIX and IIB fibres.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of cardiac sarcoplasmic reticulum ryanodine receptor by sphingosine.

Excitation contraction (EC) coupling in muscle cells involves the movement of calcium through the calcium release channel of the sarcoplasmic reticulum (SR) membrane known as the ryanodine receptor. We have recently shown that the novel second messenger, sphingosine, can block calcium release from skinned skeletal muscle fibers and from isolated skeletal muscle SR membranes (Sabbadini et al., J Biol Chem 267: 15475-15484, 1992). In this report, we demonstrate that sphingosine also inhibits calcium release from isolated canine cardiac SR membranes containing the ryanodine receptor when release is induced by caffeine, doxorubicin or by calcium. Sphingosine also prevents the augmentation of [3H]-ryanodine binding normally produced by caffeine and doxorubicin and exerts noncompetitive inhibition with regard to both releasing agents. Sphingosine significantly reduces in a dose-dependent manner [3H]-ryanodine binding to the high affinity site of the receptor and increases by several-fold the Kd for binding, which is consistent with a blocking action of sphingosine on the ryanodine receptor calcium channel. Sphingosine inhibits the extent of calcium-induced calcium release (CICR) and significantly shifts the threshold for CICR so that a higher level of trigger calcium is required to initiate CICR. The sphingosine inhibition of CICR is consistent with the near abolition of calcium dependent [3H]-ryanodine binding. HPLC analysis of cardiac sphingosine content indicates that sphingosine is present in the cardiac cell at moderately high levels (29.4 nmol/g wet wt for the entire cell and approximately 0.4 microM for the cytosol) which are sufficient to produce significant inhibition by sphingosine on calcium release and ryanodine binding. The data suggest that sphingosine acts on the cardiac ryanodine receptor by opposing the physiological stimulus (e.g. trigger calcium entering via the dihydropyridine receptor). We propose that sphingosine is produced by the T-tubule membranes and that sphingosine is released into the protected intracellular environment of the T-tubule/SR junction to negatively modulate calcium release. Consequently, it is possible that sphingosine is a physiologically relevant regulator of calcium levels in the heart.

Localization of protein kinase C in skeletal muscle T-tubule membranes.

Membrane fractions enriched in transverse tubules, either predominantly free or junctional, sarcoplasmic reticulum subfractions and purified sarcolemmal preparations have been isolated from rabbit skeletal muscle and examined for their contents of protein kinase C. Using activity measurements and immunoblotting methods, we have been able to detect substantial amounts of endogenous protein kinase C in T-tubules membranes and to a lesser extent, in muscle sarcolemma. Protein kinase C was found to be highest in junctional T-tubules and to be virtually absent from sarcoplasmic reticulum-derived membrane fractions. Immunofluorescence staining of muscle fibers is consistent with a T-tubule localization of the kinase. The T-tubule-associated protein kinase C enzyme phosphorylates several potentially important membrane proteins.

Dystrophin is phosphorylated by endogenous protein kinases.

Dystrophin, the protein coded by the gene missing in Duchenne muscular dystrophy, is assumed to be a component of the membrane cytoskeleton of skeletal muscle. Like other cytoskeletal proteins in different cell types, dystrophin bound to sarcolemma membranes was found to be phosphorylated by endogenous protein kinases. The phosphorylation of dystrophin was activated by cyclic AMP, cyclic GMP, calcium and calmodulin, and was inhibited by cyclic AMP-dependent protein kinase peptide inhibitor, mastoparan and heparin. These results suggest that membrane-bound dystrophin is a substrate of endogenous cyclic AMP- and cyclic GMP-dependent protein kinases, calcium/calmodulin-dependent kinase and casein kinase II. The possibility that dystrophin could be phosphorylated by protein kinase C is suggested by the inhibition of phosphorylation by staurosporin. On the other hand dystrophin seems not to be a substrate for protein tyrosine kinases, as shown by the lack of reaction of phosphorylated dystrophin with a monoclonal antiphosphotyrosine antibody. Sequence analysis indicates that dystrophin contains seven potential phosphorylation sites for cyclic AMP- and cyclic GMP-dependent protein kinases (all localized in the central rod domain of the molecule) as well as several sites for protein kinase C and casein kinase II. Interestingly, potential sites of phosphorylation by protein kinase C and casein kinase II are located in the proximity of the actin-binding site. These results suggest, by analogy with what has been demonstrated in the case of other cytoskeletal proteins, that the phosphorylation of dystrophin by endogenous protein kinases may modulate both self assembly and interaction of dystrophin with other cytoskeletal proteins in vivo.