Histochemical study of calcium on T-tubule membranes and in the sarcoplasmic reticulum, in frog twitch muscle fibres at rest and during activity

The trigger calcium hypothesis of signal transmission between T-tubules and terminal cisternae (TC) of the sarcoplasmic reticulum (SR) in twitch muscle fibres implies the presence of calcium along T-tubule membranes at rest and its release upon excitation. To test this hypothesis, calcium was immobilised using a fixing and precipitating solution of glutaraldehyde in phosphate buffer at pH 8.0 and the calcium was substituted for by lead. Simultaneous tension recordings revealed the occurrence of contractions or a burst of twitches upon perfusion with the fixative. Procaine or tetrodotoxin (TTX) was used to inhibit this activity. In fibres without fixative-induced activity, precipitates were observed along T-tubules and in adjoining parts of TC. In activated fibres, tubular and TC precipitates were absent. These results are consistent with the trigger calcium hypothesis. In fibres activated by depolarisation, calcium returned to TC after passing successively through different parts of the SR.     

Effects of resting membrane potential and intactness of the T-tubules on caffeine contractures in rat skeletal muscle

We have studied the effects of changes in the resting membrane potential (Vm) and T-tubules on caffeine contracture (25 mM) elicited in rat soleus muscle in vitro at 34 degrees C. In high [K]o (30-140 mM, [K]o X [Cl]o constant) caffeine contractures were reduced by about 40-50% and had a faster time course than in normal Krebs ([K]o = 5 mM). Detubulation of the muscles by an osmotic treatment produces a reduction of about 30% in the caffeine contracture tension. Our results with high K solutions suggest a reduced sensitivity of the myofibrils to calcium released by caffeine. The effects of detubulation on caffeine contracture suggest that caffeine may have a direct effect on sarcolemma in addition to its well known action on the sarcoplasmic reticulum (SR). However, a depletion of the calcium content in the SR of depolarized muscle fibres as well as an anatomical damage produced by the osmotic treatment can not be ruled out as an explanation for the reduced caffeine contracture.     

Fiber-type morphology and function of the triads in frog (Rana esculenta) skeletal muscle)]

1. Owing to differing structural characteristics of the contractile substance, the muscle fibres have been divided into the three types A, B and C in former papers. This distinction seems to be corroborated by our investigations into the different structure regarding the traids. As for the A-fibres, they are structured in terms of the T-system being connected in its entire length with the SR-cisternae and circling the myofibrils at the level of the Z-layer. In the B-fibres, this permanent couping of the two membrane systems is partially interrupted so that the T-tubules are arranged round the myofibrils in such a way that they are isolated or only coupled on one side with the SR-cisternae. Apart from the triads we also find diads. There is a totally different arrangement of the membrane systems in the C-fibres. In this instance the T-tubules are not only arranged transversally but also vertically along the contractile elements. They are surrounded by an "SR-labyrinth" which forms individual minor cisternae which are lateraly coupled with the T-tubules. So the axis of these triads is turned by 90 degrees as compared to the A and B fibres. As a result of this different arrangement, these triads always appear in cross-sections, not however, in longitudinal sections as is the case with the A and B fibres. The tirads have a varying shape in the cross-sections depending on the level of the section and due to the fact that the cisternae are not always coupled congruently with the T-tubules. 2. In our discussion we have tried to related these differing shapes and arrangements of the triads in the fibre types A, B and C to known physiological findings. Therefore we deduced that the excitation transmittance and calcium release can be correlated with the anomal rectification of the triads, which has been localized in the region where the T-tubules and SR-cisternae are coupled. However, we can only reckon with a solution once the morphology and function of the "feet" and the eletronmicroscopically "blank" spaces which fill the gap-junction between the T-tubules and the SR-cisternae have been explained. Whatever function the free surface of the T-tubules has remains open. It is directly adjoining the sarcoplasm and we are tryping to relate it to the delayed rectification which appears on the fibre membrane. 3. Moreover from the arrangement of the SR-cisternae i- the individual fibre types we can deduce th intrafibrillar directions of expansion of the calcium after its release and thus the process of the excitation in the A, B and C fibres. It appears that calcium is being directed homogeneously from the SR-cisternae of the A-fibres to the actinfilaments. here the morphological appearance of the twitch fibre presents itself to us. In principle this pattern of expansion of calcium in the B-fibres remains consistent. Owing to the interruption between the T-system and the SR-cisternae we may assume that the process of contraction is delayed in contrast to the A-fibres...     

Triad formation: organization and function of the sarcoplasmic reticulum calcium release channel and triadin in normal and dysgenic muscle in vitro

Excitation-contraction (E-C) coupling is thought to involve close interactions between the calcium release channel (ryanodine receptor; RyR) of the sarcoplasmic reticulum (SR) and the dihydropyridine receptor (DHPR) alpha 1 subunit in the T-tubule membrane. Triadin, a 95- kD protein isolated from heavy SR, binds both the RyR and DHPR and may thus participate in E-C coupling or in interactions responsible for the formation of SR/T-tubule junctions. Immunofluorescence labeling of normal mouse myotubes shows that the RyR and triadin co-aggregate with the DHPR in punctate clusters upon formation of functional junctions. Dysgenic myotubes with a deficiency in the alpha 1 subunit of the DHPR show reduced expression and clustering of RyR and triadin; however, both proteins are still capable of forming clusters and attaining mature cross-striated distributions. Thus, the molecular organization of the RyR and triadin in the terminal cisternae of SR as well as its association with the T-tubules are independent of interactions with the DHPR alpha 1 subunit. Analysis of calcium transients in dysgenic myotubes with fluorescent calcium indicators reveals spontaneous and caffeine-induced calcium release from intracellular stores similar to those of normal muscle; however, depolarization-induced calcium release is absent. Thus, characteristic calcium release properties of the RyR do not require interactions with the DHPR; neither do they require the normal organization of the RyR in the terminal SR cisternae. In hybrids of dysgenic myotubes fused with normal cells, both action potential- induced calcium transients and the normal clustered organization of the RyR are restored in regions expressing the DHPR alpha 1 subunit.     

On the specificity of the histochemical technique for sarcoplasmic reticular adenosine triphosphatase: a light and electron microscopic study.

The specificity of the histochemical localization of the calcium activated adenosine triphosphatase (ATPase) activity of the sarcoplasmic reticulum (SR) at pH 7.4 was studied using a calcium-citro-phosphate technique. The latter involves the splitting of ATP by ATPase producing phosphate ions which then react with calcium and citrate to form an insoluble reaction product. This reaction product was detected by both light and electron microscopy. Light microscopic examination showed a darkly stained continuous reticular pattern of reaction product which surrounded individual myofibrils. This reticular pattern of reaction product was distinctly dissimilar to that found when the histochemical reactions for mitochondrial or myofibrillar ATPase were performed. Ultrastructural investigations demonstrated the presence of discrete foci of electron dense reaction product in close association with the membranes of the SR in striated muscle fibres. Only occasional flecks were seen in the vicinity of mitochondria or myofilaments. The possibility is considered that the reticular pattern of staining achieved by the calcium-citro-phosphate technique may reflect the distribution of the "extra ATPase" of the SR, an enzyme implicated in the process of calcium uptake and muscle relaxation.     

The effects of sphingosine on sarcoplasmic reticulum membrane calcium release.

In this study, we report that sphingosine is a potent inhibitor of sarcoplasmic reticulum (SR) calcium release. Evidence is presented demonstrating a direct effect of sphingosine on the SR ryanodine receptor. Calcium release from "skinned" rabbit skeletal muscle fibers and isolated junctional SR derived from the terminal cisternae (TC) was measured in response to caffeine, doxorubicin, 5'-adenylyl-beta,gamma-imidodiphosphate or calcium. Sphingosine inhibited caffeine-induced release in a dose-dependent manner with an IC50 of 0.1 microM for the single muscle fibers and 0.5 microM for the isolated TC vesicles. Near complete blockage of TC calcium release rate was observed with 3 microM sphingosine. Neither sphingomyelin nor sphingosylphosphorylcholine had any effect at the 3 microM level, suggesting that the sphingosine effect was specific. Doxorubicin-induced calcium release and spontaneous calcium release were also blocked by sphingosine. Sphingosine was also capable of stimulating calcium transport in the isolated TC vesicles without an effect on Ca-ATPase activity. Ruthenium red was not capable of substantial additional stimulation of calcium transport nor inhibition of calcium release beyond the action of sphingosine. Sphingosine's blockage of calcium release was not reversed by the protein kinase inhibitor, 1-(5-isoquinolinesulfonyl)-2- methylpiperazine dihydrochloride, suggesting that the action of sphingosine on calcium release was not dependent on ryanodine receptor phosphorylation. Sphingosine significantly increased (8-fold) the Kd for specific [3H]ryanodine binding to TC membranes and decreased the Bmax with a dose dependence similar to the inhibition of calcium release, but sphingosine did not affect the pCa tension relationship of skinned skeletal muscle fibers. These data are consistent with a direct effect of submicromolar sphingosine on the ryanodine receptor. Substantially higher concentrations of sphingosine (30-50 microM) or sphingosylphosphorylcholine (10-20 microM) were capable of inducing calcium release by themselves. Preliminary data indicate that the transverse tubule and not the SR contain substantial sphingomyelinase activity consistent with a transverse tubule source of sphingosine production. Considering that sphingosine is found in micromolar concentrations in some cells, our data indicate that sphingosine generated by the transverse tubule membranes may be a physiologically relevant mechanism for modulating SR calcium release.     

Molecular organization of transverse tubule/sarcoplasmic reticulum junctions during development of excitation-contraction coupling in skeletal muscle.

The relationship between the molecular composition and organization of the triad junction and the development of excitation-contraction (E-C) coupling was investigated in cultured skeletal muscle. Action potential-induced calcium transients develop concomitantly with the first expression of the dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR), which are colocalized in clusters from the time of their earliest appearance. These DHPR/RyR clusters correspond to junctional domains of the transverse tubules (T-tubules) and sarcoplasmic reticulum (SR), respectively. Thus, at first contact T-tubules and SR form molecularly and structurally specialized membrane domains that support E-C coupling. The earliest T-tubule/SR junctions show structural characteristics of mature triads but are diverse in conformation and typically are formed before the extensive development of myofibrils. Whereas the initial formation of T-tubule/SR junctions is independent of association with myofibrils, the reorganization into proper triads occurs as junctions become associated with the border between the A band and the I band of the sarcomere. This final step in triad formation manifests itself in an increased density and uniformity of junctions in the cytoplasm, which in turn results in increased calcium release and reuptake rates.     

Calcium release and ionic changes in the sarcoplasmic reticulum of tetanized muscle: an electron-probe study

Approximately 60-70% of the total fiber calcium was localized in the terminal cisternae (TC) in resting frog muscle as determined by electron-probe analysis of ultrathin cryosections. During a 1.2 s tetanus, 59% (69 mmol/kg dry TC) of the calcium content of the TC was released, enough to raise total cytoplasmic calcium concentration by approximately 1 mM. This is equivalent to the concentration of binding sites on the calcium-binding proteins (troponin and parvalbumin) in frog muscle. Calcium release was associated with a significant uptake of magnesium and potassium into the TC, but the amount of calcium released exceeded the total measured cation accumulation by 62 mEq/kg dry weight. It is suggested that most of the charge deficit is apparent, and charge compensation is achieved by movement of protons into the sarcoplasmic reticulum (SR) and/or by the movement of organic co- or counterions not measured by energy dispersive electron-probe analysis. There was no significant change in the sodium or chlorine content of the TC during tetanus. The unchanged distribution of a permeant anion, chloride, argues against the existence of a large and sustained transSR potential during tetanus, if the chloride permeability of the in situ SR is as high as suggested by measurements on fractionated SR. The calcium content of the longitudinal SR (LSR) during tetanus did not show the LSR to be a major site of calcium storage and delayed return to the TC. The potassium concentration in the LSR was not significantly different from the adjacent cytoplasmic concentration. Analysis of small areas of I-band and large areas, including several sarcomeres, suggested that chloride is anisotropically distributed, with some of it probably bound to myosin. In contrast, the distribution of potassium in the fiber cytoplasm followed the water distribution. The mitochondrial concentration of calcium was low and did not change significantly during a tetanus. The TC of both tetanized and resting freeze-substituted muscles contained electron-lucent circular areas. The appearance of the TC showed no evidence of major volume changes during tetanus, in agreement with the estimates of unchanged (approximately 72%) water content of the TC obtained with electron-probe analysis.     

Tonic component of myocardial contraction

Calcium transients and contractions of cardiac myocytes consist of phasic component, relaxing spontaneously independently of membrane voltage and of the tonic component (TC) relaxing only upon repolarization. Experimental data reviewed in this article suggest that most Ca(2+) activating TC is released from sarcoplasmic reticulum (SR) via the ryanodine receptors (RyRs). Most likely these RyRs are activated by sustained Ca(2+) influx. However, its route may differ depending on species and state of the cells. It seems that in rat RyRs responsible for TC are activated by the sustained Ca(2+) current. In guinea-pig the blockers of Ca(2+) current or reverse mode Na(+)/Ca(2+) exchange do not inhibit TC, so these routes seem unlikely. In myocytes of the failing human hearts TC is activated mostly via the reverse mode Na(+)/Ca(2+) exchange and contribution of SR is negligible. The mechanism of TC in the normal human cardiomyocytes has not been investigated. Thus, despite investigation of TC for half a century many problems concerning the mechanism of its activation and maintenance as well as its physiological meaning remain unsolved.     

Isolation of transverse tubules by fractionation of sarcoplasmic reticulum preparations in ion-free sucrose density gradients

A new method for the preparation of transverse tubules (T-tubules) from rabbit skeletal muscles is reported. When crude sarcoplasmic reticulum (SR) preparations were centrifuged on sucrose density gradients containing buffering ions (buffered gradients) 70-80% of the material sedimented as a single heavy band in the region of 43% sucrose. When this fraction (or crude SR) was recentrifuged on sucrose gradients prepared free of buffer or other ions (ion-free gradients) the heavy band dissociated into three fractions of different densities. The lightest fraction sedimented at 28% sucrose and was identified as T-tubules on the basis of its nitrendipine and ouabain binding properties. The enzymatic properties, cholesterol contents, and protein compositions of the fractions obtained when SR is centrifuged on buffered and ion-free sucrose density gradients were measured. The T-tubules were enriched in cholesterol and in marker enzymes for surface membranes while the other fractions were shown to be terminal cisternae and longitudinal cisternae on the basis of their (Ca2+,Mg2+)-ATPase activities and characteristic protein profiles.     

Theory of excitation-contraction coupling in cardiac muscle.

The consequences of cardiac excitation-contraction coupling by calcium-induced calcium release were studied theoretically, using a series of idealized models solved by analytic and numerical methods. "Common-pool" models, those in which the trigger calcium and released calcium pass through a common cytosolic pool, gave nearly all-or-none regenerative calcium releases (in disagreement with experiment), unless their loop gain was made sufficiently low that it provided little amplification of the calcium entering through the sarcolemma. In the linear (small trigger) limit, it was proven rigorously that no common-pool model can give graded high amplification unless it is operated on the verge of spontaneous oscillation. To circumvent this problem, we considered two types of "local-control" models. In the first type, the local calcium from a sarcolemmal L-type calcium channel directly stimulates a single, immediately opposed SR calcium release channel. This permits high amplification without regeneration, but requires high conductance of the SR channel. This problem is avoided in the second type of local control model, in which one L-type channel triggers a regenerative cluster of several SR channels. Statistical recruitment of clusters results in graded response with high amplification. In either type of local-control model, the voltage dependence of SR calcium release is not exactly the same as that of the macroscopic sarcolemmal calcium current, even though calcium is the only trigger for SR release. This results from the existence of correlations between the stochastic openings of individual sarcolemmal and SR channels. Propagation of regenerative calcium-release waves (under conditions of calcium overload) was analyzed using analytically soluble models in which SR calcium release was treated phenomenalogically. The range of wave velocities observed experimentally is easily explained; however, the observed degree of refractoriness to wave propagation requires either a strong dependence of SR calcium release on the rate of rise of cytosolic calcium or localization of SR release sites to one point in the sarcomere. We conclude that the macroscopic behavior of calcium-induced calcium release depends critically on the spatial relationships among sarcolemmal and SR calcium channels, as well as on their kinetics.     

荔枝雌蕊发育过程中钙分布变化与细胞程序性死亡

应用焦锑酸钾沉淀法研究了荔枝雌花和雄花雌蕊发育过程中钙的分布变化。在大孢子母细胞阶段,雌花近珠孔内珠被细胞和花柱细胞的钙沉淀颗粒主要分布在细胞壁和细胞间隙,少部分在液泡;雌花花柱维管细胞中含有很多的钙沉淀颗粒;在雄花的近珠孔内珠被细胞钙沉淀颗粒大多在液泡中;雄花花柱细胞和维管细胞中钙沉淀颗粒很少。大孢子母细胞减数分裂后,雌花雌蕊继续发育,雄花雌蕊败育。雌花维管中的钙沉淀颗粒数量减少,可能被转运到将要发生花粉萌发和受精的部位。雌花近珠孔内珠被细胞壁的钙沉淀颗粒分布增加,花柱细胞从上（近柱头）到下（近子房）钙沉淀颗粒量递增。雄花近珠孔内珠被细胞发生程序性死亡：液泡中的钙进入细胞核启动细胞程序性死亡,核周隙与质膜腔形成连续的通道,钙在核与细胞质之间的流动不受限制;在特定的时间段,钙沉淀颗粒出现在线粒体、过氧化物体和线型内质网的外膜上。钙在细胞中重新分布可能触发和调节细胞程序性死亡的进程。缺乏钙沉淀颗粒的雄花花柱细胞迅速解体。     

Effects of ruthenium red on excitation and contraction in muscle fibres with Ca2+ electrogenesis.

The effect of ruthenium red (RR) on the electrical and contractile responses, membrane Ca currents, staining patterns of the external and internal membrane system were tested in intact and mechanically skinned muscle fibres of the crayfish Astacus fluviatilis. The following results were obtained: 1. Depression of the contractile responses following membrane depolarization (twitch, tetanus, potassium contractures). 2. Caffeine contractures were unaffected in intact (100 mumol/l - 1 mmol/l RR) and blocked in skinned fibres (30 mumol/l RR). 3. Mechanical threshold and mechanical latency were increased and/or prolonged. 4. The rate of depolarization of the action potentials (AP) was decreased and decremental spread of AP was recorded. 5. Both fast and slowly inactivating Ca ionic currents were decreased and the time constants of activation (tau(m] and inactivation (tau(h] were prolonged after RR (100 mumol/l) pretreatment. 6. The penetration of RR into the T-system was inversely related to its binding to the sarcolemma. The depression of depolarization-induced contractions was most pronounced in fibres with unstained sarcolemma and stained T-tubules. In intact fibres, neither terminal cisternae nor other elements of SR were stained. On the contrary, all internal membrane structures were stained in skinned fibres. There was a gradient of staining intensity from surface toward the interior.     

3H]PN200-110 and [3H]ryanodine binding and reconstitution of ion channel activity with skeletal muscle membranes

Skeletal muscle membranes derived either from the tubular (T) network or from the sarcoplasmic reticulum (SR) were characterized with respect to the binding of the dihydropyridine, [3H]PN200-110, and the alkaloid, [3H]ryanodine; polypeptide composition; and ion channel activity. Conditions for optimizing the binding of these radioligands are discussed. A bilayer pulsing technique is described and is used to examine the channels present in these membranes. Fusion of T-tubule membranes into bilayers revealed the presence of chloride channels and dihydropyridine-sensitive calcium channels with three distinct conductances. The dihydropyridine-sensitive channels were further characterized with respect to their voltage dependence. Pulsing experiments indicated that two different populations of dihydropyridine-sensitive channels existed. Fusion of heavy SR vesicles revealed three different ion channels; the putative calcium release channel, a potassium channel, and a chloride channel. Thus, this fractionation procedure provides T-tubules and SR membranes which, with radioligand binding and single channel recording techniques, provide a useful tool to study the characteristics of skeletal muscle ion channels and their possible role in excitation-contraction coupling.     

Fiber Ultrastructure and Contraction Kinetics in Insect Fast Muscles

SYNOPSIS. Isometric contraction kinetics were measured and fiberstructure was quantified in tymbal muscles from different cicadaspecies. Twitch duration is directly correlated with the sizeof the myofibrils and with the ratio of the fraction of fibervolume which is myofibril to that which is sarcoplasmic reticulum(SR) and T-tubules (fast muscles have small myofibrils and arelatively large volume of SR and T-tubules). Twitch durationis not significantly correlated with fiber size, with sarcomerelength, nor with the fractional volume of the fibers which ismitochondria, indicating that these structural features arenot strongly involved in the determination of isometric contractionkinetics. In the tettigoniid Neoconocephalus robustus, twitchesfrom forewing muscles of male animals become progressively shorterover the first five days following the adult molt. This changein contraction kinetics is associated with an increase in therelative volume of SR and T-tubules. Denervation blocks theacquisition of rapid kinetics, indicating that neural inputis necessary for this transformation.     

Sequential docking, molecular differentiation, and positioning of T-Tubule/SR junctions in developing mouse skeletal muscle.

Skeletal muscle Ca(2+) release units (CRUs) are junctions of the surface membrane/T-tubule system and the sarcoplasmic reticulum (SR) that function in excitation-contraction coupling. They contain high concentrations of dihydropyridine receptors (DHPRs) in the T-tubules and of ryanodine receptors (RyR) in the SR and they are positioned at specific locations in the sarcomere. In order to characterize the sequence of developmental steps leading to the specific molecular and structural organization of CRUs, we applied a range of imaging techniques that allowed us to follow the differentiation of the membrane compartments and the expression of junctional proteins in developing mouse diaphragm muscle. We find that docking of the two membrane systems precedes the incorporation of the RyRs into the junctions, and that T-tubule/SR junctions are formed and positioned at the I-A interface at a stage when the orientation of T-tubule is predominantly longitudinal. Thus, the sequence of developmental events is first the docking of T-tubules and SR, secondly the incorporation of RyR in the junctions, thirdly the positioning of the junctions in the sarcomere, and only much later the transverse orientation of the T-tubules. These sequential stages suggests an order of inductive processes for the molecular differentiation and structural organization of the CRUs in skeletal muscle development.     

An examination of the ability of inositol 1,4,5-trisphosphate to induce calcium release and tension development in skinned skeletal muscle fibres of frog and crustacea

We have examined the ability of inositol 1,4,5-trisphosphate (InsP3) to cause contractions of mechanically skinned muscle fibres of frog and barnacle. InsP3 (10-500 microM) did not cause any tension development in 25 frog skinned fibres and 26 barnacle myofibrillar bundles, although contractions could be readily evoked by caffeine and by replacement of an impermeant anion by Cl-, treatments known to release calcium from the sarcoplasmic reticulum (SR). Four barnacle bundles did give responses to InsP3. InsP3 did not modify responses to caffeine or calcium-induced calcium release. Free Mg2+ was lowered to 40 microM and 15 mM D-2,3-diphosphoglycerate was added in order to inhibit the possible breakdown of InsP3 by inositol trisphosphatase. Neither measure revealed a response to InsP3. Arsenazo III absorbance measurements failed to detect any binding of Mg2+ (0-0.5 mM) by 0.35 mM InsP3 in our solutions. Inhibitors of SR calcium uptake (cadium, quercetin, furosemide), omission of EGTA from the solution and varying the temperature from 4 degrees to 22 degrees C also failed to reveal a response of frog skinned fibres to InsP3. The nucleotide GTP, which has been reported to enhance InsP3-induced calcium release from rat liver microsomes, had no effect at 50 microM on the response of frog fibres to InsP3. It is concluded that under conditions in which other calcium release mechanisms operate well, InsP3 is relatively ineffective at releasing calcium from the SR in amounts sufficient to induce contraction. Although we have been unable to find evidence to support the proposed role of InsP3 as an essential link in excitation-contraction coupling of skeletal muscle, we cannot entirely reject its role if essential cofactors are lost in the skinned preparations.     

Ultrastructural organisation and the sites of calcium localisation in the lamprey striated muscle

The present paper examines the ultrastructure of the sarcoplasmic recitulum (SR) and the T system in the striated muscle of the lamprey. The pyroantimonate method was used to visualise the sites of intracellular calcium localisation. Characteristic for the muscle studied are the presence of numerous intricately shaped invaginations on the surface membrane of muscle fibres and peripheral contacts between SR cisternae and the sarcolemma. In addition to calcium localised in the terminal cisternae of SR and N-bands of the I-disk, as typical of vertebrate muscles, a great amount of calcium is present in the subsarcolemmal region, corresponding to the area of invaginations, and in longitudinal elements of SR.     

G-protein distribution in canine cardiac sarcoplasmic reticulum and sarcolemma: comparison to rabbit skeletal muscle membranes and to brain and erythrocyte G-proteins

Herein we describe the distribution of G-proteins in canine cardiac sarcolemma (SL) and sarcoplasmic reticulum (SR) and in rabbit skeletal muscle SL, T-tubules, and junctional and longitudinal SR in comparison to G-proteins of human erythrocyte and bovine brain. G-proteins were unequivocally present in cardiac SL and SR and in skeletal T-tubules. Both cardiac fractions had two substrates specifically ADP-ribosylated by cholera toxin migrating on a sodium dodecyl sulfate-polyacrylamide gel at about 42 and 45 kDa. In skeletal muscle membranes, cholera toxi-labeled substrates migrated at about 42 and 62 kDa. Three substrates for pertussis toxin were resolved by sodium dodecyl sulfate/urea-polyacrylamide gel electrophoresis in cardiac SL at about 38, 40, and 43 kDa. Only the two higher molecular weight substrates were detected in cardiac SR and in any of several skeletal muscle membrane fractions. Comparison of G-proteins in muscle membrane fractions with G-proteins isolated from bovine brain and human erythrocyte as well as their reaction with antisera to either a common sequence of alpha subunits of G-proteins (G alpha common antibody) or to a unique sequence of the alpha subunit of Go (G alpha o antibody) indicated that the two lower molecular weight bands in cardiac SL are Go or Go-like, and therefore the upper band is probably Gi. These data demonstrate that pertussis toxin substrates are more heterogeneous than previously described and have implications for studies attempting to attribute physiological functions to G-protein isolates.     

Coordinated development of myofibrils, sarcoplasmic reticulum and transverse tubules in normal and dysgenic mouse skeletal muscle, in vivo and in vitro.

We studied the development of transverse (T)-tubules and sarcoplasmic reticulum (SR) in relationship to myofibrillogenesis in normal and dysgenic (mdg/mdg) mouse skeletal muscle by immunofluorescent labeling of specific membrane and myofibrillar proteins. At E16 the development of the myofibrils and membranes in dysgenic and normal diaphragm was indistinguishable, including well developed myofibrils, a delicate network of T-tubules, and a prominent SR which was not yet cross-striated. In diaphragms of E18 dysgenic mice, both the number and size of muscle fibers and myofibrillar organization were deficient in comparison to normal diaphragms, as previously reported. T-tubule labeling was abnormal, showing only scattered tubules and fragments. However, many muscle fibers displayed cross striation of sarcomeric proteins and SR comparable to normal muscle. In cultured myotubes, cross-striated organization of sarcomeric proteins proceeded essentially in two stages: first around the Z-line and later in the A-band. Sarcomeric organization of the SR coincided with the first stage, while the appearance of T-tubules in the mature transverse orientation occurred infrequently, only after A-band maturation. In culture, myofibrillar and membrane organization was equivalent in normal and dysgenic muscle at the earlier stage of development, but half as many dysgenic myotubes reached the later stage as compared to normal. We conclude that the mdg mutation has little effect on the initial stage of membrane and myofibril development and that the deficiencies often seen at later stages result indirectly from the previously described absence of dihydropyridine receptor function in the mutant.