Intact Sarcoplasmic Reticulum Research Articles

Can a chloride channel blocker mitigate muscle fatigue?

Can a chloride channel blocker mitigate muscle fatigue?

Excitation-contraction coupling in zebrafish ventricular myocardium is regulated by trans-sarcolemmal Ca2+ influx and sarcoplasmic reticulum Ca2+ release.

Zebrafish (Danio rerio) have become a popular model in cardiovascular research mainly due to identification of a large number of mutants with structural defects. In recent years, cardiomyopathies and other diseases influencing contractility of the heart have been studied in zebrafish mutants. However, little is known about the regulation of contractility of the zebrafish heart on a tissue level. The aim of the present study was to elucidate the role of trans-sarcolemmal Ca2+-flux and sarcoplasmic reticulum Ca2+-release in zebrafish myocardium. Using isometric force measurements of fresh heart slices, we characterised the effects of changes of the extracellular Ca2+-concentration, trans-sarcolemmal Ca2+-flux via L-type Ca2+-channels and Na+-Ca2+-exchanger, and Ca2+-release from the sarcoplasmic reticulum as well as beating frequency and β-adrenergic stimulation on contractility of adult zebrafish myocardium. We found an overall negative force-frequency relationship (FFR). Inhibition of L-type Ca2+-channels by verapamil (1 μM) decreased force of contraction to 22±7% compared to baseline (n=4, p<0.05). Ni2+ was the only substance to prolong relaxation (5 mM, time after peak to 50% relaxation: 73±3 ms vs. 101±8 ms, n=5, p<0.05). Surprisingly though, inhibition of the sarcoplasmic Ca2+-release decreased force development to 54±3% in ventricular (n=13, p<0.05) and to 52±8% in atrial myocardium (n=5, p<0.05) suggesting a substantial role of SR Ca2+-release in force generation. In line with this finding, we observed significant post pause potentiation after pauses of 5 s (169±7% force compared to baseline, n=8, p<0.05) and 10 s (198±9% force compared to baseline, n=5, p<0.05) and mildly positive lusitropy after β-adrenergic stimulation. In conclusion, force development in adult zebrafish ventricular myocardium requires not only trans-sarcolemmal Ca2+-flux, but also intact sarcoplasmic reticulum Ca2+-cycling. In contrast to mammals, FFR is strongly negative in the zebrafish heart. These aspects need to be considered when using zebrafish to model human diseases of myocardial contractility.

Spontaneous Ca2+ Waves in Rabbit Corpus Cavernosum: Modulation by Nitric Oxide and cGMP

Detumescent tone and subsequent relaxation by nitric oxide (NO) are essential processes that determine the erectile state of the penis. Despite this, the mechanisms involved are incompletely understood. It is often assumed that the tone is associated with a sustained high cytosolic Ca(2+) level in the corpus cavernosum smooth muscle cells, however, an alternative possibility is that oscillatory Ca(2+) signals regulate tone, and erection occurs as a result of inhibition of Ca(2+) oscillations by NO. The aim of this study is to determine if smooth muscle cells displayed spontaneous Ca(2+) oscillations and, if so, whether these were regulated by NO. Male New Zealand white rabbits were euthanized and smooth muscle cells were isolated by enzymatic dispersal for confocal imaging of intracellular Ca(2+) (using fluo-4AM) and patch clamp recording of spontaneous membrane currents. Thin tissue slices were also loaded with fluo-4AM for live imaging of Ca(2+). Cytosolic Ca(2+) was measured in isolated smooth muscle cells and tissue slices. Results. Isolated rabbit corpus cavernosum smooth muscle cells developed spontaneous Ca(2+) waves that spread at a mean velocity of 65 microm/s. Dual voltage clamp/confocal recordings revealed that each of the Ca(2+) waves was associated with an inward current typical of the Ca(2+)-activated Cl(-) currents developed by these cells. The waves depended on an intact sarcoplasmic reticulum Ca(2+) store, as they were blocked by cyclopiazonic acid (Calbiochem, San Diego, CA, USA) and agents that interfere with ryanodine receptors and IP(3)-mediated Ca(2+) release. The waves were also inhibited by an NO donor (diethylamine NO; Tocris Bioscience, Bristol, Avon, UK), 3-(5-hydroxymethyl-2-furyl)-1-benzyl indazole (YC-1) (Alexis Biochemicals, Bingham, Notts, UK), 8-bromo-cyclic guanosine mono-phosphate (Tocris), and sildenafil (Viagra, Pfizer, Sandwich, Kent, UK). Regular Ca(2+) oscillations were also observed in whole tissue slices where they were clearly seen to precede contraction. This activity was also markedly inhibited by sildenafil, suggesting that it was under NO regulation. These results provide a new basis for understanding detumescent tone in the corpus cavernosum and its inhibition by NO.

1H-NMR Studies on Nucleotide Binding to the Sarcoplasmic Reticulum Ca2+ ATPase

The glycosidic bond torsion angles and the conformations of the ribose of Mg2+ATP, Mg2+ADP and Mg2+AdoPP[NH]P (magnesium adenosine 5'-[beta, gamma-imido]triphosphate) bound to Ca2+ATPase, both native and modified with fluorescein isothiocyanate (FITC), in intact sarcoplasmic reticulum have been determined by the measurement of proton-proton transferred nuclear Overhauser enhancements by 1H-NMR spectroscopy. This method shows clearly the existence of a low-affinity ATP binding site after modification of the high-affinity site with FITC. For all three nucleotides bound to both the high-affinity (catalytic) site and the low-affinity site, we find that the conformation about the glycosidic bond is anti, the conformation of the ribose 3'-endo of the N type and the conformation about the ribose C4'-C5' bond either gauche-trans or trans-gauche. The values for the glycosidic bond torsion angles chi (O4'-C1'-N9-C4) for Mg2+ATP, Mg2+ADP and Mg2+AdoPP[NH]P bound to the low-affinity site of FITC-modified Ca2+ATPase are approximately equal to 270 degrees, approximately equal to 260 degrees and approximately equal to 240 degrees respectively. In the case of the nucleotides bound to the high-affinity (catalytic) site of native Ca2+ATPase, chi lies in the range 240-280 degrees.

Cross-bridge blocker BTS permits direct measurement of SR Ca2+ pump ATP utilization in toadfish swimbladder muscle fibers.

Because the major processes involved in muscle contraction require rapid utilization of ATP, measurement of ATP utilization can provide important insights into the mechanisms of contraction. It is necessary, however, to differentiate between the contribution made by cross-bridges and that of the sarcoplasmic reticulum (SR) Ca2+ pumps. Specific and potent SR Ca2+ pump blockers have been used in skinned fibers to permit direct measurement of cross-bridge ATP utilization. Up to now, there was no analogous cross-bridge blocker. Recently, N-benzyl-p-toluene sulfonamide (BTS) was found to suppress force generation at micromolar concentrations. We tested whether BTS could be used to block cross-bridge ATP utilization, thereby permitting direct measurement of SR Ca2+ pump ATP utilization in saponin-skinned fibers. At 25 microM, BTS virtually eliminates force and cross-bridge ATP utilization (both <4% of control value). By taking advantage of the toadfish swimbladder muscle's unique right shift in its force-Ca2+ concentration ([Ca2+]) relationship, we measured SR Ca2+ pump ATP utilization in the presence and absence of BTS. At 25 microM, BTS had no effect on SR pump ATP utilization. Hence, we used BTS to make some of the first direct measurements of ATP utilization of intact SR over a physiological range of [Ca2+]at 15 degrees C. Curve fits to SR Ca2+ pump ATP utilization vs. pCa indicate that they have much lower Hill coefficients (1.49) than that describing cross-bridge force generation vs. pCa (approximately 5). Furthermore, we found that BTS also effectively eliminates force generation in bundles of intact swimbladder muscle, suggesting that it will be an important tool for studying integrated SR function during normal motor behavior.

RyR1/SERCA1 cross-talk regulation of calcium transport in heavy sarcoplasmic reticulum vesicles.

We investigated the functional interdependence of sarco-endoplasmic reticulum Ca2+ ATPase isoform 1 and ryanodine receptor isoform 1 in heavy sarcoplasmic reticulum membranes by synchronous fluorescence determination of extravesicular Ca2+ transients and catalytic activity. Under conditions of dynamic Ca2+ exchange ATPase catalytic activity was well coordinated to ryanodine receptor activation/inactivation states. Ryanodine-induced activation of Ca2+ release channel leaks also produced marked ATPase activation in the absence of measurable increases in bulk free extravesicular Ca2+. This suggested that Ca2+ pumps are highly sensitive to Ca2+ release channel leak status and potently buffer Ca2+ ions exiting cytoplasmic openings of ryanodine receptors. Conversely, ryanodine receptor activation was dependent on Ca2+-ATPase pump activity. Ryanodine receptor activation by cytosolic Ca2+ was (i) inversely proportional to luminal Ca2+ load and (ii) dependent upon the rate of presentation of cytosolic Ca2+. Progressive Ca2+ filling coincided with progressive loss of Ca2+ sequestration rates and at a threshold loading, ryanodine-induced Ca2+ release produced small transient reversals of catalytic activity. These data indicate that attainment of threshold luminal Ca2+ loads coordinates sensitization of Ca2+ release channels with autogenic inhibition of Ca2+ pumping. This suggests that Ca2+-dependent control of Ca2+ release in intact heavy sarcoplasmic reticulum membranes involves a Ca2+-mediated "cross-talk" between sarco-endoplasmic reticulum Ca2+ ATPase isoform 1 and ryanodine receptor isoform 1.

A new approach for measuring real-time calcium pumping and SR function in muscle fibers.

Different muscles are designed to perform a wide variety of motor activities that extend over a large range of frequencies. To increase our understanding of the molecular basis for these different designs, we are attempting to model the activation-relaxation cycle of different muscles by obtaining kinetic information about each of the constituent processes (1, 2). An important component in muscle relaxation is Ca*+ sequestration by sarcoplasmic reticulum (SR)-Ca*’ pumps, but the pump turnover rate in different fiber types is not known precisely. Previous measurements of SR function have generally involved one of two approaches. One approach involves homogenizing muscle, making SR preparations and measuring the rate of Ca*+ accumulation in the presence of oxalate (e.g., 3). It is unclear, however, whether homogenization alters the pumping rate compared to that of intact SR in muscle fibers. Further, the presence of oxalate probably increases the rate of accumulation above that in normal fibers by reducing the free Ca*+ concentration inside the SR. An alternate approach involves physiological measurements of intracellular free [Ca*+] using calcium sensitive dyes (e.g., 1, 4). However, this approach provides direct information only about the small amount of Ca*+ that is actually free in the myoplasm. The total movements of Ca*+ can only be estimated by modelling the large quantities of other Ca*+ buffers in the cell (e.g., parvalbumin and troponin; 4), but the amounts and the kinetics of these calcium buffers are not always known in detail. We have developed a technique and an instrument that permit direct measurements of Ca*+ sequestration by intact SR of muscle fibers as a function of [Ca2+rree]. Single muscle fibers are exposed to 50 wg/ml saponin (20 min), which permeablizes the cell membrane, but leaves the SR membrane intact (5). The “skinned” muscle fibers are then bathed in solutions that contain various concentrations of Ca2+rree; and calcium sequestration (i.e., Ca *+ leaving the bath) is monitored by using a Ca*+ sensitive dye, FURA-2. This approach solves two important problems. First, during the “skinning” procedure, most of the intracellular Ca*+-buffer (the soluble parvalbumin) leaks out of the cell membrane and is washed away. Second, the amounts of remaining intracellular buffers (e.g., troponin), become very small compared to the total buffer in the bath, which is well defined in terms of amounts and kinetics, and can be accurately calibrated. Hence Ca*+ sequestration by the SR can be accurately measured as a removal of Ca*+ from the bathing solution.

Determinants of relaxation rate in skinned frog skeletal muscle fibers.

The influences of sarcomere uniformity and Ca2+ concentration on the kinetics of relaxation were examined in skinned frog skeletal muscle fibers induced to relax by rapid sequestration of Ca2+ by the photolysis of the Ca2+ chelator, diazo-2, at 10 degreesC. Compared with an intact fiber, diazo-2-induced relaxation exhibited a faster and shorter initial slow phase and a fast phase with a longer tail. Stabilization of the sarcomeres by repeated releases and restretches during force development increased the duration of the slow phase and slowed its kinetics. When force of contraction was decreased by lowering the Ca2+ concentration, the overall kinetics of relaxation was accelerated, with the slow phase being the most sensitive to Ca2+ concentration. Twitchlike contractions were induced by photorelease of Ca2+ from a caged Ca2+ (DM-Nitrophen), with subsequent Ca2+ sequestration by intact sarcoplasmic reticulum or Ca2+ rebinding to caged Ca2+. These twitchlike responses exhibited relaxation kinetics that were about twofold slower than those observed in intact fibers. Results suggest that the slow phase of relaxation is influenced by the degree of sarcomere homogeneity and rate of Ca2+ dissociation from thin filaments. The fast phase of relaxation is in part determined by the level of Ca2+ activation.

The effects of essential oil of Mentha x villosa on skeletal muscle of the toad

The effects of the essential oil of Mentha x villosa (EOMV) was investigated in saponin-permeabilized fibres and in intact sartorius muscle of the toad. In intact muscle EOMV at concentrations from 1 to 100 μg/mL blocked the contraction induced by high (80 mM) potassium and induced potentiation of caffeine-induced muscle contraction. From 1 to 6 mg/mL EOMV elicited contraction in both depolarized (by 110 mM K+) and non-depolarized (4 mM K+) muscle fibres. This contraction was blocked by procaine. At 3 mg/mL EOMV depolarized the normal resting potential from −80.77 to −71.21 mV, but did not alter the transmembrane potential in the presence of 60 mM K+. EOMV (1 mg/mL) induced contraction of saponin-permeabilized muscle fibres with an intact sarcoplasmic reticulum (SR). These data suggest that EOMV: (i) induces muscle contraction by releasing Ca2+ from SR; (ii) impairs the excitation–contraction coupling at a step before Ca2+ release from the SR. © 1997 John Wiley & Sons, Ltd.

The effects of essential oil ofMentha x villosa on skeletal muscle of the toad

The effects of the essential oil of Mentha x villosa (EOMV) was investigated in saponin-permeabilized fibres and in intact sartorius muscle of the toad. In intact muscle EOMV at concentrations from 1 to 100 μg/mL blocked the contraction induced by high (80 mM) potassium and induced potentiation of caffeine-induced muscle contraction. From 1 to 6 mg/mL EOMV elicited contraction in both depolarized (by 110 mM K+) and non-depolarized (4 mM K+) muscle fibres. This contraction was blocked by procaine. At 3 mg/mL EOMV depolarized the normal resting potential from −80.77 to −71.21 mV, but did not alter the transmembrane potential in the presence of 60 mM K+. EOMV (1 mg/mL) induced contraction of saponin-permeabilized muscle fibres with an intact sarcoplasmic reticulum (SR). These data suggest that EOMV: (i) induces muscle contraction by releasing Ca2+ from SR; (ii) impairs the excitation–contraction coupling at a step before Ca2+ release from the SR. © 1997 John Wiley & Sons, Ltd.

Calcium uptake and release modulated by counter‐ion conductances in the sarcoplasmic reticulum of skeletal muscle

The sarcoplasmic reticulum (SR) plays the central role in regulating the free myoplasmic Ca2+ level for the contractile activation of skeletal muscle. The initial stages of the voltage-controlled Ca2+ release mechanism are known in molecular detail. However, there is still very little known about the later stages of Ca2+ uptake and total Ca2+ turnover in the contraction-relaxation cycle under normal physiological conditions or under conditions influenced by fatigue or disease. Ca2+ uptake and release are both accompanied by "counter-ion' movements across the SR membrane which prevent or reduce the generation of SR membrane potentials and balance for electroneutrality in the SR lumen. The SR membrane is permeable for the cations K+, Na+, H+ and Mg2+ and the anion Cl-. Using electron-probe X-ray microanalysis. It has been shown that during tetanic stimulation the Ca2+ release was mainly balanced by uptake of K+ and Mg2+ leaving a charge deficit that was assumed to be neutralized via H+ ion or organic counter-ion movement. The low time resolution of electron-probe X-ray microanalysis leaves the possibility of other transient concentration changes in the SR, e.g. for Cl- ions. Possible physiological roles of the SR counter-ion conductances can be tested using skinned muscle fibre preparations with intact sarcoplasmic reticulum and removed or chemically permeabilized outer sarcolemma. In skinned fibres, the SR K+ conductance can be effectively reduced with SR K+ channel blockers such as 4-aminopyridine, tetraethylammonium and decamethonium. Interestingly, these blockers increase Ca2+ loading as well as Ca2+ release, whereas other less specific blockers, such as 1.10-bis-quanidino-n-decane, seem to reduce Ca2+ release, possibly also via blocking Ca2+ release channels. Thus, it seems very important also to test the effects of counter-currents carried by K+, Mg2+, H+ or Cl- ions on intact and voltage-clamped single-fibre preparations.

Modulation of the Ca 2+,Mg 2+-ATPase of sarcoplasmic reticulum by the hypothalamic hypophyseal inhibitory factor

We have studied the effect of the endogenous inhibitor of the Na + and Ca 2+ pumps, HHIF, on sarcoplasmic reticulum (SR) vesicles. The effect of HHIF on the SR Ca 2+,Mg 2+-ATPase activity shows a biphasic pattern. Low HHIF concentrations activate the Ca 2+,Mg 2+-ATPase by dissipation of Ca 2+ gradient across the SR membrane. Higher concentrations irreversibly inhibit this activity following a slow kinetic process both in intact SR membranes and in purified Ca 2+,Mg 2+-ATPase. Differential scanning calorimetry shows that the Ca 2+,Mg 2+-ATPase is denatured after incubation with HHIF concentrations which produced full inhibition of its activity. Micromolar Ca 2+ and millimolar Mg t+ADP protect against the irreversible inhibition of the Ca 2+,Mg 2+-ATPase by HHIF. The concentration of HHIF which produces 50% inhibition depends upon SR membrane concentration and upon the lipid:protein ratio in purified Ca 2+,Mg 2+-ATPase. From this we have obtained a partition coefficient for binding of HHIF to SR membranes of 0.6 (μg SR protein/ml) −1.

Caffeine and Ca 2+ stimulate mitochondrial oxidative phosphorylation in saponin-skinned human skeletal muscle fibers due to activation of actomyosin ATPase

The rate of mitochondrial oxidative phosphorylation of saponin-skinned human muscle fibers from m. vastus lateralis in the presence of glutamate, malate and ATP is reported to be sensitive to caffeine and to changes of free calcium ion concentration. An approximately twofold increase in respiration was observed by the addition of 15 mM caffeine, because of the efflux of calcium from sarcoplasmic reticulum. Direct addition of a Ca 2+/CaEGTA buffer, containing 1.5 μM free calcium ions had a similar effect. The ATP-splitting activity of skinned fibers was also stimulated by caffeine or calcium. These observations can be explained exclusively by the calcium-induced activation of actomyosin ATPase. (i) Thapsigargin, an inhibitor of the sarcoplasmic reticulum Ca 2+-ATPase, had no influence. (ii) In myosin-extracted ‘ghost’ fibers containing intact mitochondria and an intact sarcoplasmic reticulum caffeine had a negligible effect on oxidative phosphorylation. (iii) The caffeine-induced increase in rate of fiber respiration was concomitant with a decrease in mitochondrial membrane potential and a decrease in the redox state of the mitochondrial NAD system. (iv) The calcium ionophore A 23187 caused a stimulation of respiration and ATP-splitting activity, similar to caffeine. (v) The calcium dependencies of respiration and ATP splitting activity of saponin-skinned human muscle fibers were in experimental error identical. Therefore it is concluded that calcium efflux from sarcoplasmic reticulum affects oxidative phosphorylation in skeletal muscle mostly via the stimulation of actomyosin ATPase.

Biochemical identification of transmembrane segments of the Ca(2+)-ATPase of sarcoplasmic reticulum.

The transmembrane segments of sarcoplasmic reticulum Ca(2+)-ATPase were determined by trypsinization of cytoplasmic side-out intact sarcoplasmic reticulum vesicles. The membrane portion of tryptic digest comprising the transmembrane fragments, joined by the intravesicular segments, was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after labeling with fluorescein 5-maleimide in the presence of sodium dodecyl sulfate. In this way, seven fluorescent bands of tryptic fragments below 11 kDa were observed which were derived from 4 pairs of membrane spanning segments and one hydrophobic sequence at the C-terminal end. Two peptides of 10.8 and 10.6 kDa had the identical N-terminal sequence beginning at Glu826, representing the transmembrane segments M7 and M8 and their connecting loop. A band at 8.1 kDa contained one peptide beginning at Tyr36 (M1/loop/M2). A 7.7-kDa peptide starting at Leu253 (M3/loop/M4) and a 7.3-kDa peptide beginning at Ala752 (M5/loop/M6) were also observed. A band at 6.7 kDa contained two peptides, one beginning at Ser48 (M1/loop/M2) and another beginning at Tyr763 (M5/loop/M6). In addition, a 4-kDa peptide beginning at Met925 was observed. The size of this peptide did not allow for a complete pair of transmembrane segments, but this peptide could have been derived from trypsinolysis between the last pair of membrane spanning segments. These data therefore provide biochemical evidence for at least 8 transmembrane segments and perhaps two more at the C-terminal end of the enzyme.

Relation between reverse sodium-calcium exchange and sarcoplasmic reticulum calcium release in guinea pig ventricular cells.

Exchange-inhibitory peptide (XIP) can inhibit sodium-calcium exchange without inhibiting L-type calcium current (ICa). We therefore used this compound to test the hypothesis that reverse sodium-calcium exchange can trigger contraction in guinea pig ventricular myocytes. When cells were dialyzed with 20 mmol/L sodium, rapid blockade of ICa with nifedipine had little effect on cell shortening. However, if reverse exchange was inhibited by first dialyzing the cells with XIP, blockade of ICa largely inhibited cell shortening. In cells dialyzed with 10 mmol/L sodium, about 51% of the maximum cell shortening remained after ICa was blocked. When both ICa and reverse exchange were significantly inhibited with nifedipine and XIP, only 24% of the cell shortening remained; ie, 27% was XIP inhibitable. Cells dialyzed with solutions deficient in sodium exhibited contractions that were largely dependent on ICa (ie, not XIP inhibitable). If the sarcoplasmic reticulum (SR) was disabled with ryanodine and thapsigargin, reverse exchange could not cause contraction. We therefore conclude that with intact SR, reverse sodium-calcium exchange activates contraction by triggering calcium release from the SR in cells dialyzed with either 10 or 20 mmol/L sodium. A scrambled sequence of XIP, sXIP, caused no measurable effect on contraction.

Effects of Mg2+ on Ca2+ handling by the sarcoplasmic reticulum in skinned skeletal and cardiac muscle fibres.

The influence of myoplasmic Mg2+ (0.05-10 mM) on Ca2+ accumulation (net Ca2+ flux) and Ca2+ uptake (pump-driven Ca2+ influx) by the intact sarcoplasmic reticulum (SR) was studied in skinned fibres from the toad iliofibularis muscle (twitch portion), rat extensor digitorum longus (EDL) muscle (fast twitch), rat soleus muscle (slow twitch) and rat cardiac trabeculae. Ca2+ accumulation was optimal between 1 and 3 mM Mg2+ in toad fibres and reached a plateau between 1 and 10 mM Mg2+ in the rat EDL fibres and between 3 and 10 mM Mg2+ in the rat cardiac fibres. In soleus fibres, optimal Ca2+ accumulation occurred at 10 mM Mg2+. The same trend was obtained with all preparations at 0.3 and 1 microM Ca2+. Experiments with 2,5-di-(tert-butyl)-1,4-benzohydroquinone, a specific inhibitor of the Ca2+ pump, revealed a marked Ca2+ efflux from the SR of toad iliofibularis fibres in the presence of 0.2 microM Ca2+ and 1 mM Mg2+. Further experiments indicated that the SR Ca2+ leak could be blocked by 10 microM ruthenium red without affecting the SR Ca2+ pump and this allowed separation between SR Ca2+ uptake and SR Ca2+ accumulation. At 0.3 microM Ca2+, Ca2+ uptake was optimal with 1 mM Mg2+ in the toad iliofibularis and rat EDL fibres and between 1 and 10 mM Mg2+ in the rat soleus and trabeculae preparations. At higher [Ca2+] (1 microM), Ca2+ uptake was optimal with 1 mM Mg2+ in the iliofibularis fibres and between 1 and 3 mM Mg2+ in the EDL fibres.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for the lumenal location of the 53 kDa glycoprotein of sarcoplasmic reticulum

The 53 kDa glycoprotein from sarcoplasmic reticulum was shown to be protected from proteolysis by trypsin, V8 proteinase and proteinase K in intact vesicles yet readily digested in the presence of the non-denaturing detergent C 12E 8. Competitive ELISAs with a library of seven monoclonal antibodies raised against the 53 kDa glycoprotein showed that the epitopes for these antibodies were only accessible in C 12E 8 solubilised and not intact sarcoplasmic reticulum. When the monoclonal antibodies against the 53 kDa glycoprotein were assessed for their effect on the uptake of Ca 2+ by sarcoplasmic reticulum no effect was detected; neither were these antibodies able to augment the inhibitory influences of anti-(Ca 2+-Mg 2+)-ATPase monoclonal antibodies on Ca 2+ uptake. These data indicate that the 53 kDa glycoprotein is located in the lumen of the sarcoplasmic reticulum.

Binding of myotoxin a to sarcoplasmic reticulum calcium-ATPase: a structural study

The interaction of myotoxin alpha with intact sarcoplasmic reticulum (SR) components was investigated, and two SR proteins were identified that associated with myotoxin a. One of the proteins has an apparent molecular weight similar to the Ca(2+)-ATPase, the major SR protein responsible for calcium loading. Ca(2+)-ATPase was purified, and its interaction with myotoxin a was studied. Evidence for specific binding of myotoxin a to Ca(2+)-ATPase was established by isolating chemically cross-linked myotoxin a-Ca(2+)-ATPase complexes and further proving their association with anti-myotoxin a antibodies. The binding region of myotoxin a was further delineated by cleaving the protein with cyanogen bromide (CNBr) into two fragments, a larger N-terminal fragment of 28 residues and a smaller C-terminal fragment of 14 residues. Competition experiments with 125I-myotoxin a showed that the C-terminal fragment competed better against 125I-myotoxin a than the N-terminal fragment for SR protein binding. Two overlapping peptides covering the sequence of the N-terminal fragment were synthesized to clarify the interaction of the N-terminal fragment of myotoxin a with SR proteins. A 16-residue peptide corresponding to residues 1-16 competed strongly with 125I-myotoxin a, while a second peptide (residues 13-28) did not.

Vanadate inhibition of ATP and p-nitrophenyl phosphate hydrolysis in the fragmented sarcoplasmic reticulum.

The vanadate inhibition of the Ca(2+)-ATPase activity was analysed both in intact sarcoplasmic reticulum vesicles and in the presence of low concentrations of Tween 20, using ATP and p-nitrophenyl phosphate as substrates. The saturation of the internal low-affinity calcium-binding sites protects the enzyme against vanadate inhibition, because: (1) p-nitrophenyl phosphate hydrolysis is not inhibited by vanadate in intact vesicles, but inhibition developed after solubilization with detergents; (2) the vanadate inhibition of the p-nitrophenyl phosphate hydrolysis in solubilized preparations is prevented by free Ca2+ concentrations higher than 10(-3) M and vanadate competes with calcium (10(-5)-10(-3) M); and (3) the vanadate inhibition of ATP hydrolysis is decreased with an increase in vesicular Ca2+ concentration. The presence of magnesium ions is indispensable for the vanadate effect. The vanadate inhibition is non-competitive with respect to Mg-p-nitrophenyl phosphate and uncompetitive with respect to Mg-ATP. However, in the presence of dimethyl sulfoxide, which facilitates phosphorylation of the enzyme, the inhibition is converted to a competitive one with respect to a substrate. The results suggest, that in the process of enzyme operation vanadate interacts with the unliganded E form of Ca(2+)-ATPase, occupying probably an intermediate position between the E2 and E1 forms, with the formation of an E2 Van complex, that imposes the inhibition on the Ca(2+)-ATPase activity.

GTP gamma S causes contraction of skinned frog skeletal muscle via the DHP-sensitive Ca2+ channels of sealed T-tubules.

We have investigated the involvement of G-proteins in excitation-contraction coupling of fast-twitch skeletal muscle, using a fibre preparation designed to retain intact T-tubules and sarcoplasmic reticulum. The nonhydrolysable analogue of guanosine triphosphate, GTP gamma S (50-500 microM) caused a strong, transient isometric contraction in this preparation. Reduction of ethylene-bis(oxonitrilo)tetraacete (EGTA) in the sealed T-tubules from 5 mM to 0.1 mM lowered the threshold to GTP gamma S and removal of sodium reversibly raised it. The dihydropyridine (DHP) calcium channel antagonists nicardipine and nifedipine allowed a first contraction and then blocked subsequent GTP gamma S action. The phenylalkylamine methoxyverapamil (D-600) did likewise, reversibly, at 10 degrees C. The guanosine diphosphate analogue, GDP beta S, and procaine reversibly blocked the action of GTP gamma S; pertussis toxin also blocked it. Photolytic release of 40-100 microM GTP gamma S within 0.1 s from S-caged GTP gamma S caused contraction after a latent period of 0.3-20 s. We conclude that GTP gamma S can activate contraction in frog skeletal muscle via a route requiring both the integrity of the T-tubular DHP-sensitive calcium channel (DHPr) and the presence of sodium in the sealed T-tubules. We propose that in this preparation GTP gamma S activates a G-protein, which in turn activates the DHPr as a calcium channel and releases stored calcium from within the sealed T-tubule. Implications of these results for the excitation-contraction coupling mechanism in skeletal muscle are discussed.