Mechanical noise of the myocardium as an index of sarcoplasmic reticulum activity

The effects of caffeine (0.5-12 mM) and cooling on the mechanical noise (MN) and relaxation rate of contractile response (RR) were studied in experiments on rat papillary muscles. Mean frequency of MN and RR were found to have similar temperature dependences from 12 to 32 degrees C. MN amplitude was more sensitive to the action of sarcoplasmic reticulum activity blocker, caffeine, than RR one. MN as an index of sarcoplasmic reticulum activity made it possible to demonstrate a competition between caffeine and local anaesthetics described earlier for skeletal muscles.     

Chemical modification of calcium release from the sarcoplasmic reticulum of mechanically skinned skeletal bullfrog muscle fibers

Several types of reagents that react with amino acid side chains induced repetitive phasic contracture of skinned skeletal muscle from frogs. The presence of 10 mM procaine or 5 mM magnesium in the medium or disruption of the sarcoplasmic reticulum (SR) eliminated this contracture, indicating that the calcium-induced calcium-release mechanism of SR is involved in the contraction. Dithiothreitol inhibited the contracture induced by chloramine T, N-acetylimidazole, or p-chloromercuriphenylsulfonic acid (pCMPS) but not in the case of carbodiimide, phenylglyoxal, trinitrobenzenesulfonic acid, diethylpyrocarbonate (DEP), or N-chlorosuccinimide (NCS). Therefore, modification of groups other than the sulfhydryl ones seems to induce contractures under such conditions. The amplitude of the caffeine-induced contracture decreased after treatment with pCMPS, DEP, or NCS. NCS shifted the pCa-tension curve toward low pCa in the SR-disrupted fibers. This shift would explain the decrease in the caffeine contracture. It is tentatively concluded that pCMPS and DEP release a large amount of calcium from SR.     

Relationships between the sarcoplasmic reticulum and sarcolemmal calcium transport revealed by rapidly cooling rabbit ventricular muscle

Rabbit right ventricular papillary muscles were cooled from 30 to approximately 1 degree C immediately after discontinuing electrical stimulation (0.5 Hz). This produced a contracture that was 30-50% of the preceding twitch magnitude and required 20-30 s to develop. The contractures were identical in cooling solutions with normal (144 mM) or low (2.0 mM) Na. They were therefore not Na-withdrawal contractures. Contracture activation was considerably slower than muscle cooling (approximately 2.5 s to cool below 2 degrees C). Cooling contractures were suppressed by caffeine treatment (10.0 mM). Rapid cooling did not cause sufficient membrane depolarization (16.5 +/- 1.2 mV after 30 s of cooling) to produce either a voltage-dependent activation of contracture or a gated entry of Ca from the extracellular space. Contractures induced by treating resting muscles with 5 X 10(-5) M strophanthidin at 30 degrees C exhibited pronounced tension noise. The Fourier spectrum of this noise revealed a periodic component (2-3 Hz) that disappeared when the muscle was cooled. Cooling contractures decayed with rest (t1/2 = 71.0 +/- 9.3 s). This decay accelerated in the presence of 10.0 mM caffeine and was prevented and to some extent reversed when extracellular Na was reduced to 2.0 mM. 20 min of rest resulted in a net decline in intracellular Ca content of 1.29 +/- 0.38 mmol/kg dry wt. I infer that cooling contractures are principally activated by Ca from the sarcoplasmic reticulum (SR). The properties of these contractures suggest that they may provide a convenient relative index of the availability of SR Ca for contraction. The rest decay of cooling contractures (and hence the decay in the availability of activating Ca) is consistent with the measured loss in analytic Ca during rest. The results suggest that contraction in heart muscle can be regulated by an interaction between sarcolemmal and SR Ca transport.     

Effects of rapid application of caffeine on intracellular calcium concentration in ferret papillary muscles

In this paper we investigate the effects of caffeine (5-20 mM) on ferret papillary muscle. The intracellular Ca2+ concentration ( [Ca2+]i) was measured from the light emitted by the photoprotein aequorin, which had previously been microinjected into superficial cells. Isometric tension was measured simultaneously. The rapid application of caffeine produced a transient increase of [Ca2+]i, which decayed spontaneously within 2-3 s and was accompanied by a transient contracture. The removal of extracellular Na+ or an increase in the concentration of intracellular Na+ (produced by strophanthidin) increased the magnitude of the caffeine response. Cessation of stimulation for several minutes or stimulation at low rates decreased the magnitude of the stimulated twitch and Ca2+ transient. These maneuvers also decreased the size of the caffeine response. These results are consistent with the hypothesis that the caffeine-releasable pool of Ca2+ (sarcoplasmic reticulum) is modulated by maneuvers that affect contraction. Ryanodine (10 microM) decreased the magnitude of the caffeine response as well as that of the stimulated twitch. In contrast, the rapid removal of external Ca2+ abolished the systolic Ca2+ transient within 5 s, but had no effect on the caffeine response. From this we conclude that the abolition of twitch by Ca2+-free solutions is not due to depletion of the sarcoplasmic reticulum of Ca2+, but may be due to a requirement of Ca2+ entry into the cell to trigger Ca2+ release from the sarcoplasmic reticulum.     

The mechanism of ryanodine action in rabbit ventricular muscle evaluated with Ca-selective microelectrodes and rapid cooling contractures

Cellular Ca uptake and efflux in rabbit ventricular muscle was measured using double-barreled Ca microelectrodes in the extracellular space. When repetitive stimulation was stopped there was a slow loss of cellular Ca. Upon resumption of stimulation Ca was taken up by the cells. These Ca movements are thought to represent the loss of Ca from the sarcoplasmic reticulum and the cell during rest and the refilling of the sarcoplasmic reticulum during stimulation. Ryanodine (100 nM) greatly enhanced both the efflux of Ca during rest and the uptake of Ca induced by stimulation. These results are consistent with the conclusions drawn below, but they are dependent upon the interpretation that these extracellular Ca depletions are indicative of sarcoplasmic reticulum Ca movements. To examine further this process, contractures induced by rapid cooling to 0 degrees C were used as an independent assay of sarcoplasmic reticulum Ca content. These rapid cooling contractures were smaller after longer rest intervals (declining with a half time of 1.5 min). In the presence of ryanodine, the rapid cooling contracture immediately after a contraction was greater than that seen under control conditions. However, in the presence of ryanodine these rapid cooling contractures decline as a function of rest duration with a half time of about 1 s. These results suggest that in the presence of ryanodine the sarcoplasmic reticulum can still take up Ca, but that it also loses this Ca very rapidly at the onset of rest.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characteristics of functioning of electromechanical coupling in striated muscles of higher and lower vertebrates

A comparative pharmacological analysis of relative contributions of different signal transduction pathways in the activation of contraction (excitation-contraction coupling, ECC) in intact fast striated muscles of frog and lamprey was performed. It was found that the major mechanism responsible for the ECC in muscles of both animals is Ca2+ release from the sarcoplasmic reticulum through the ryanodine-sensitive channels. However, the ECC in lamprey muscle displays some important differences in the units of electromechanical coupling, which precede the calcium release from sarcoplasmic reticulum. The maximum contraction force in frog muscle develops during caffeine-induced contracture, which indicates that all Ca2+ stored in sarcoplasmic reticulum is released through ryanodine-sensitive channels. In contrast, in lamprey muscle, the maximum force develops not in response to high caffeine concentration, but in response to repetitive electrical stimulation. Hence, in addition to stores liberated by ryanodine-sensitive channels, some other sources of calcium ions should exist, which contribute to the contraction activation. A source of this additional Ca2+ ions can be external medium, because acetylcholine contracture is abolished in a calcium-free medium. In frog muscle, the acetylcholine contracture was abolished in a Na(+)-free solution. It was concluded that in frog muscle ECC can be triggered by changes in the transmembrane potential (depolarization-induced calcium release), while in lamprey muscle the entry of calcium ions into myoplasm as the trigger in ECC (calcium-induced calcium release). The lamprey muscle was found to be more resistant to tetrodotoxin and tetracaine, which is indicative of a role in the activation of contraction of tetrodotoxin-resistant Na+ and/or Ca2+ channels. It was concluded, that ECC mechanism in striated muscles of low vertebrates is not limited by the generally accepted scheme of depolarization-induced calcium release but can include some other schemes, which require the Ca2+ influx into the cell.     

Upregulation of Ca2+ removal in human skeletal muscle: a possible role for Ca2+-dependent priming of mitochondrial ATP synthesis

In muscle, ATP is required for the powerstroke of the myosin head, the detachment of actin and myosin filaments, and the reuptake of Ca²⁺ into the sarcoplasmic reticulum. During contraction-relaxation, large amounts of ATP are consumed at the sites of action of the myosin-ATPase and sarcoplasmic reticulum Ca²⁺-ATPase. The present study addresses the consequences of a reduction in mitochondrial ATP production capacity on sarcoplasmic Ca²⁺ handling. To this end, myotubes were cultured from patient quadriceps with a biochemically defined decrease in the maximal rate of mitochondrial ATP production and were loaded with indo 1 for imaging of sarcoplasmic Ca²⁺ changes in real time by confocal microscopy. Myotubes were field-stimulated with 10-ms pulses of 16 V to evoke transient rises in sarcoplasmic Ca²⁺ concentration ([Ca²⁺]_S). Three single pulses, two pulse trains (1 Hz), and one single pulse were applied in succession to mimic changing workloads. Control myotubes displayed [Ca²⁺]_S transients with an amplitude that was independent of the strength of the stimulus. Intriguingly, the rate of sarcoplasmic Ca²⁺ removal (CRR) was significantly upregulated during the second and subsequent transients. In myotubes with a reduced mitochondrial ATP production capacity, the amplitude of the [Ca²⁺]_S transients was markedly increased at higher stimulus intensities. Moreover, upregulation of the CRR was significantly decreased compared with control. Taken together, these results are in good agreement with a tight coupling between mitochondrial ATP production and sarcoplasmic Ca²⁺ handling. Moreover, they support the existence of a relatively long-lasting mitochondrial memory for sarcoplasmic [Ca²⁺] rises. This memory, which manifested itself as an increase in CRR upon recurrent stimulation, was impaired in patient myotubes with a reduced mitochondrial ATP production capacity. sarcoplasmic Ca²⁺ removal; video-rate imaging; indo 1; electrical stimulation; mitochondrial memory     

Adenosine reduces the reverse mode of the Na+/Ca(2+) exchanger in ferret cardiac fibres

The aim of this study was to investigate the effects of adenosine on reverse mode Na+/Ca(2+) exchange. In intact ferret cardiac trabeculae, Na+-free contractures were investigated after treating preparations with ryanodine, a sarcoplasmic reticulum Ca(2+) -channel inhibitor, and thapsigargin, a sarcoplasmic reticulum Ca(2+) -pump inhibitor added to suppress the sarcoplasmic reticulum function. The effects of adenosine (50-100 nmol/L), adenosine deaminase (ADA, 0.1-0.5 U/L), the A1 and A2A receptor agonists CCPA (3-100 nmol/L) and CGS 21680 (25-100 nmol/L), and the A1 and A2A receptor antagonists DPCPX (25 nmol/L) and ZM 241385 (25 nmol/L) were tested on Na+-free contractures. The application of adenosine (50-100 nmol/L) had no significant effect on the characteristics of the Na+-free contractures. However, the results show that treatment with ADA (0.3 U/L), adenosine (> or =50 nmol/L) and CCPA, a specific A1 receptor agonist (3-100 nmol/L), all reduced the Na+-free contracture amplitude. In the presence of ADA, the effects of adenosine and CCPA were also reduced by a specific antagonist of A1 receptors (DPCPX, 25 nmol/L). Furthermore, adenosine, ADA, and CCPA did not affect the properties of the contractile apparatus in Triton-skinned fibres. It is therefore proposed that endogenous adenosine reduced the reverse mode of the Na+/Ca(2+) exchanger by acting on A1 receptors present in the sarcolemmal membrane.     

Localization of Ca2+ + Mg2+-ATPase of the sarcoplasmic reticulum in adult rat papillary muscle

Localization of the Ca2+ + Mg2+-ATPase of the sarcoplasmic reticulum in rat papillary muscle was determined by indirect immunofluorescence and immunoferritin labeling of cryostat and ultracryotomy sections, respectively. The Ca2+ + Mg2+-ATPase was found to be rather uniformly distributed in the free sarcoplasmic reticulum membrane but to be absent from both peripheral and interior junctional sarcoplasmic reticulum membrane, transverse tubules, sarcolemma, and mitochondria. This suggests that the Ca2+ + Mg2+-ATPase of the sarcoplasmic reticulum is antigenically unrelated to the Ca2+ + Mg2+-ATPase of the sarcolemma. These results are in agreement with the idea that the sites of interior and peripheral coupling between sarcoplasmic reticulum membrane and transverse tubules and between sarcoplasmic reticulum and sarcolemmal membranes play the same functional role in the excitation-contraction coupling in cardiac muscle.     

Optical probe responses on sarcoplasmic reticulum: oxacarbocyanines as probes of membrane potential.

The relationship between Ca2+ fluxes and the ion diffusion potential was analyzed on sarcoplasmic reticulum membranes using oxacarbocyanine dyes as optical probes for membrane potential. 3.3'-Diethyloxodicarbocyanine responds to ATP-induced Ca2+ uptake by isolated sarcoplasmic reticulum vesicles with a decrease in absorbance at 600 nm. The optical change is reversed during Ca2+ release from sarcoplasmic reticulum induced by KCl or by ADP and inorganic phosphate. The absorbance changes are largely attributable to the binding of accumulated Ca2+ to the membrane. There is no indication that sustained changes in membrane diffusion potential would accompany pump-mediated Ca2+ fluxes. A large change in the absorbance of 3,3'-diethyloxodicarbocyanine was observed on sarcoplasmic reticulum vesicles under the influence of membrane potential generated by valinomycin in the presence of a K+ gradient or by ionophore A23187 in the presence of a Ca2+ gradient. The maximum of the potential-dependent absorbance change is at 575--580 nm. The potentials generated by valinomycin or ionophore A23187 are short-lived due to the high permeability of sarcoplasmic reticulum membranes for cations and anions. There is no correlation between the direction and magnitude of the artifically imposed membrane potential and the rate of Ca2+ uptake or release by isolated sarcoplasmic reticulum vesicles.     

Influence of ryanodine receptor agonist and antagonist on development of potassium contracture in phasic (twitch) and tonic fibers of frog skeletal muscle

We compared the influence of external calcium and the inhibitor (dantrolene) and activator (4-chloro-m-cresol) of ryanodine-sensitive Ca channels of the sarcoplasmic reticulum on the characteristics of potassium contracture in phasic and tonic frog skeletal muscle fibers. The duration of contracture in tonic fibers, as contrasted to the phasic ones, is not limited by the presence of Ca²⁺. The tonic contractile response is virtually indifferent to dantrolene and is much less sensitive to chlorocresol than the phasic one (1 mM vs. 0.25 mM). In phasic fibers, the K⁺ contracture on the chlorocresol background is quite similar in amplitude and dynamics to that in control, whereas tonic fibers exhibit response summation without relaxation upon removal of excessive K⁺. One can suggest that in phasic fibers the Ca²⁺ influx can directly create a level sufficient to sustain contraction, while in tonic fibers its effect is mediated by Ca-dependent activation of the beta isoform of the ryanodine-sensitive channel.     

The Effect of Quinidine on Calcium Accumulation by Isolated Sarcoplasmic Reticulum of Skeletal and Cardiac Muscle

Quinidine potentiates twitch tension and (at higher concentrations) causes contracture of skeletal muscle whereas the same drug reduces tension development of cardiac muscle. To gain insight into the possible differences in the excitation-contraction coupling mechanism of the two types of muscle the effect of quinidine on calcium accumulation by isolated sarcoplasmic reticulum from skeletal and cardiac muscle was investigated. In a medium containing ATP, Mg⁺⁺, oxalate, and ⁴⁵Ca, pharmacologically active concentrations of the drug inhibited calcium accumulation by both skeletal and cardiac sarcoplasmic reticulum. The inhibition of the rates of calcium, uptake by the skeletal muscle preparation ranged from 11% with 10^-4 M quinidine to 90% with 10^-3 M quinidine. With the cardiac muscle preparation the inhibition ranged from 16% with 3 x 10^-6 M quinidine to 100% with 10^-3 M quinidine. With both preparations the inhibition of calcium transport was accompanied by an inhibition of the Ca⁺⁺-activated ATPase activity of the sarcoplasmic reticulum. The effect of quinidine on the skeletal sarcoplasmic reticulum supports the hypothesis that this compound produces twitch potentiation and contracture by interfering with intracellular calcium, sequestration. Its effect on cardiac sarcoplasmic reticulum. has been interpreted in terms of the hypothesis that cardiac contractility is a function of the amount of calcium released from the sarcoplasmic reticulum which is in turn dependent upon the absolute calcium content of the reticulum. Hence, following inhibition of calcium transport there would be less calcium available for coupling.     

Chloride and osmotic contractures in skinned frog muscle fibers

Summary Single contractures were elicited in segments of skinned frog muscle fibers when the segments were moved from relaxing-loading solutions to various test solutions. The effective test solutions produced an increase in the concentration of chloride ions in the myofilament space, [Cl] _ms , and/or presumably caused the sarcoplasmic reticulum to undergo a change in volume. The contractures were quantified in terms of their maximum tension and time-integral. Two outer segments from each fiber underwent a contracture in a control solution (chloride ions were substituted for all of the methanesulfonate ions in the relaxing solution). The mean values of tension and area in the control contractures of each fiber were divided into the corresponding values from a test contracture obtained in the central segment of the same fiber. Test contractures obtained upon increasing [Cl] _ms and increasing the product, [K] _ms ×[Cl] _ms , were compared to contractures that were obtained by increasing [Cl] _ms while keeping [K] _ms ×[Cl] _ms constant. The former contractures were greater in magnitude for a given [Cl] _ms . Whereas the former solutions may have caused an increase in the volume of the sarcoplasmic reticulum and altered the electrical potential across the membranes of the sarcoplasmic reticulum as well, only a change in potential was presumed to have occurred in the latter solutions. Other types of contractures were investigated to show that both swelling of the sarcoplasmic reticulum and changes in the electrical potential of its membranes can cause release of calcium ions and elicit contractures in skinned fibers.     

Phospholipid asymmetry in the isolated sarcoplasmic reticulum membrane

The total phospholipid content and distribution of phospholipid species between the outer and inner monolayers of the isolated sarcoplasmic reticulum membrane was measured by phospholipase A2 activities and neutron diffraction. Phospholipase measurements showed that specific phospholipid species were asymmetric in their distribution between the outer and inner monolayers of the sarcoplasmic reticulum lipid bilayer; phosphatidylcholine (PC) was distributed 48/52 +/- 2% between the outer and inner monolayer of the sarcoplasmic reticulum bilayer, 69% of the phosphatidyl-ethanolamine (PE) resided mainly in the outer monolayer of the bilayer, 85% of the phosphatidylserine (PS) and 88% of the phosphatidylinositol (PI) were localized predominantly in the inner monolayer. The total phospholipid distribution determined by these measurements was 48/52 +/- 2% for the outer/inner monolayer of the sarcoplasmic reticulum lipid bilayer. Sarcoplasmic reticulum phospholipids were biosynthetically deuterated and exchanged into isolated vesicles with both a specific lecithin and a general exchange protein. Neutron diffraction measurements directly provided lipid distribution profiles for both PC and the total lipid content in the intact sarcoplasmic reticulum membrane. The outer/inner monolayer distribution for PC was 47/53 +/- 1%, in agreement with phospholipase measurements, while that for the total lipid was 46/54 +/- 1%, similar to the phospholipase measurements. These neutron diffraction results regarding the sarcoplasmic reticulum membrane bilayer were used in model calculations for decomposing the electron-density profile structure (10 A resolution) of isolated sarcoplasmic reticulum previously determined by X-ray diffraction into structures for the separate membrane components. These structure studies showed that the protein profile structure within the membrane lipid bilayer was asymmetric, complementary to the asymmetric lipid structure. Thus, the total phospholipid asymmetry obtained by two independent methods was small but consistent with a complementary asymmetric protein structure, and may be related to the highly vectorial functional properties of the calcium pump ATPase protein in the sarcoplasmic reticulum membrane.     

Caffeine contracture in the cultured chick myotube

A possible function of Ca store site in cultured chick myotubes was examined by recording contraction of the myotube with special reference to the effect of caffeine. Caffeine at low concentrations (below 1 mM), applied focally on the myotube through a micropipette with a pressure pulse, elicited focal contraction without membrane potential changes. Procaine inhibited the caffeine contracture. Deuterium oxide also inhibited the caffeine contracture at low concentrations, but enhanced the maximal contracture. These observations are similar to those in the mature frog muscle fiber in which the sarcoplasmic reticulum (SR) is a main site of caffeine action. On the basis of these similarities, it was considered that caffeine acts on SR to elicit contracture in the myotube. The ability of SR to accumulate and release Ca ion seemed to be low, because caffeine contracture decreased or disappeared in a Ca-free solution in many myotubes.     

Effect of calcium on the interactions between Ca2+-ATPase molecules in sarcoplasmic reticulum

The interaction between Ca2+-ATPase molecules in the native sarcoplasmic reticulum membrane and in detergent solutions was analyzed by chemical crosslinking, high performance liquid chromatography (HPLC), and by the polarization of fluorescence of fluorescein 5'-isothiocyanate (FITC) covalently attached to the Ca2+-ATPase. Reaction of sarcoplasmic reticulum vesicles with glutaraldehyde causes the crosslinking of Ca2+-ATPase molecules with the formation of dimers, tetramers and higher oligomers. At moderate concentrations of glutaraldehyde solubilization of sarcoplasmic reticulum by C12 E8 or Brij 36T (approximately equal to 4 mg/mg protein) decreased the formation of higher oligomers without significant interference with the appearance of crosslinked ATPase dimers. These observations are consistent with the existence of Ca2+-ATPase dimers in detergent-solubilized sarcoplasmic reticulum. Ca2+ (2-20 mM) and glycerol (10-20%) increased the degree of crosslinking at pH 6.0 both in vesicular and in solubilized sarcoplasmic reticulum, presumably by promoting interactions between ATPase molecules; at pH 7.5 the effect of Ca2+ was less pronounced. In agreement with these observations, high performance liquid chromatography of sarcoplasmic reticulum proteins solubilized by Brij 36T or C12 E10 revealed the presence of components with the expected elution characteristics of Ca2+-ATPase oligomers. The polarization of fluorescence of FITC covalently attached to the Ca2+-ATPase is low in the native sarcoplasmic reticulum due to energy transfer, consistent with the existence of ATPase oligomers (Highsmith, S. and Cohen, J.A. (1987) Biochemistry 26, 154-161); upon solubilization of the sarcoplasmic reticulum by detergents, the polarization of fluorescence increased due to dissociation of ATPase oligomers. Based on its effects on the fluorescence of FITC-ATPase, Ca2+ promoted the interaction between ATPase molecules, both in the native membrane and in detergent solutions.     

Ouabain potentiation and Ca release from sarcoplasmic reticulum in cardiac and skeletal muscle cells

In this article, we describe a possible mechanism of ouabain potentiation in heart based on the following findings in cardiac and skeletal muscles of various species. (1) In heart ventricle muscles of frog and guinea pig, the ouabain potentiation is produced without an effect on Ca influx. In both frog and cat heart ventricle muscles, ouabain potentiates the rapid cooling contracture with or without caffeine in a Ca-deprived medium. It follows, therefore, that the ouabain potentiation is produced by an "intracellular" mechanism. (2) In crab single muscle fibers, contractile responses such as twitch, potassium-induced contracture, caffeine-induced contracture, and water-induced contracture are remarkably potentiated if ouabain is present within the fibers by microinjection, whereas the situation is reversed if the drug is given extracellularly. (3) The ouabain potentiated the Ca release from fragmented sarcoplasmic reticulum (FSR) isolated from cat, guinea pig, and frog heart and from skeletal muscles as a result of the procedures used, such as changing the ionic environment. (4) In frog, cat, and guinea pig heart ventricle muscles, a reduction of contractility as a result of pretreatment with urea--Ringer's was completely cancelled by ouabain almost without influencing the membrane depolarization. Based on these findings and others, the deduction was made that the positive inotropic effect of cardiac glycosides on the heart is brought about by potentiation of contraction - Ca release from the intracellular store sites, namely the sarcoplasmic reticulum.     

Analysis of the arrangement of protein components in the sarcomplasmic reticulum of rat skeletal muscle.

The nature of the protein components and their location in the sarcoplasmic reticulum membrane were studied using sarcoplasmic reticulum vesicles isolated from rat skeletal muscle and purified by a density gradient centrifugation system. On the basis of analysis by means of sodium dodecyl sulfate gel electrophoresis, the protein components appear to be similar if not identical with those reported by others for rabbit sarcoplasmic reticulum, and the relative amount of each component is also similar to that found with rabbit sarcoplasmic reticulum. Evidence is presented that radioiodine-labeled diazotized diiodosulfanilic acid is a nonpermeant labeling agent of the protein components of sarcoplasmic reticulum vesicles; this agent minimally disturbs the functional activities of these membranes. By means of this labeling agent and perturbing agents, it is concluded that the protein components with molecular weights greater than 120,000 and the (Ca2+ + Mg2+)-adenosine triphosphatase partially or totally reside on or at the external surface of the sarcoplasmic reticulum vesicles. In the case of the adenosine triphosphatase, highly controlled trypsin treatment cleaves the molecule into two products, a 65,000 molecular weight fragment and a 56,000 molecular weight fragment. The evidence indicates that the 65,000 molecular weight component of the (Ca2+ + Mg2+)-adenosine triphosphatase is located in a more exposed fashion on the external surface of the vesicles than the 56,000 molecular weight compoenet and that some adenosine triphosphatase molecules have a more exposed position on the external surface of the vesicle than others. The protein components designated by MacLennan (MacLennan, D. H. (1975) Can. J. Biochem. 53, 251-261) as "calsequestrin" and "high affinity Ca2+ binding protein" are shown not to be on the external surface of the rat sarcoplasmic reticulum vesicle but rather to reside either within the core of the membrane or on the inside surface of the vesicle. The results of this study are in agreement with the model for the organization of the protein components of the sarcoplasmic reticulum membrene recently proposed by MacLennan (MacLennan, D. H. (1975) Can. J. Biochem. 53, 251-261).     

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.     

Calculating psychoacoustic parameters of amplitude modulated narrow noise bands

A simple model is suggested enabling the comprehensive calculation of pschoacoustic parameters (absolute modulation thresholds, modulation difference limens, and the subjective magnitude of supraliminal modulation differences) for amplitude modulated narrow noise bands. A comparison of experimental and model data reveals good agreement.