Methods for demonstration of enzyme activity in muscle fibres at the muscle/bone interface in demineralized tissue

A method for demonstration of activity for ATPase and various oxidative enzymes (succinic dehydrogenase, alpha-glycerophosphate dehydrogenase, and lactic dehydrogenase) in muscle/bone sections of fixed and demineralized tissue has been developed. It was found that it is possible to preserve considerable amounts of the above mentioned enzymes in the muscle fibres at the muscle/bone interfaces. The best results were obtained after 20 min fixation, and 2-3 weeks of storage in MgNa2EDTA containing media. As the same technique previously has been used to describe patterns of resorption and deposition with the aid of a mapping of presence of phosphomonoesterases on bone surfaces, the method may be used to study possible biochemical interactions between bone and muscle tissue at the muscle/bone interface.     

Reduction in Gh protein expression is associated with cytodifferentiation of vascular smooth muscle cells

G_h, a high molecular weight GTP-binding protein that couples ₁-adrenoceptors in heart and liver to phosphatidylinositol (Ptdlns)-specific phospholipase C (PLC), has recently been shown to be a tissue transglutaminase type 11. Transglutaminases have been suggested to play a role in the maintenance of blood vessel structure, and therefore it is possible that changes in their expression may accompany pathological states which involve phenotypic modulation of smooth muscle. Hence, we investigated the expression of G_h during differentiation of rat aortic smooth muscle cells in culture. G_h content was reduced markedly in cultured smooth muscle cells compared to freshly isolated cells as determined by Western blotting using a G_h-specific monoclonal antibody. In contrast, the level of G_q, a heterotrimeric G-protein that couples ₁-adrenoceptors to PLC, was maintained throughout the culture period. These findings indicate that changes in G, expression accompany phenotypic modulation of vascular smooth muscle cells. These changes in G_h protein expression may be important in the altered responsiveness of vessels in pathological disease states.     

Further enzyme histochemical observations on the segmentation of the proximal tubules in the kidney of the male rat

Summary The segmentation of the proximal tubules of the male rat kidney was studied by means of enzyme histochemical reactions. Soluble oxidoreductases (glucose 6-phosphate dehydrogenase, -glycerophosphate dehydrogenase, 3-hydroxysteroid dehydrogenase, NAD- and NADP-dependent isocitrate dehydrogenases, NAD-dependent malate dehydrogenase, NADP-dependent, decarboxylating malate dehydrogenase, uridine diphosphate glucose dehydrogenase) were demonstrated using methods which reduce enzyme diffusion (incubating in presence of polyvinyl alcohol) and eliminate interference from tissue tetrazolium reductases. Less soluble or insoluble enzymes (glucose 6-phosphatase, -hydroxybutyrate dehydrogenase, succinate dehydrogenase and tetrazolium reductases) were demonstrated by incubation in conventional watery media.Segmental differences were observed in respect to all enzymes studied, and most reactions clearly visualized the three segments known to exist from ultrastructural as well as previous histochemical studies: The pars convoluta includes the first (P₁) and most of the second (P₂) segment. The transition to the third segment (P₃) is in the beginning of the pars recta. Also these reactions revealed a difference between the first part of the P₃, which runs through the cortex in the medullary rays, and the terminal part transversing the outer stripe of the medulla. In most instances intensity of reaction decreased in the last portion of the P₃.A number of the enzymes studied were mainly or solely localized to the P₃ (glucose 6-phosphate dehydrogenase, -glycerophosphate dehydrogenases, -hydroxybutyrate dehydrogenase, 3-hydroxysteroid dehydrogenase, decarboxylating malate dehydrogenase and uridine diphosphate glucose dehydrogenase). Some possible functional implications of the findings are discussed.Supported by grants from Fonden til Lægevidenskabens Fremme and the Danish Medical Research Council. — Mr. Kaj L. Pedersen is thanked for valuable photographic assistance.     

Comparative levels of muscle glycolytic enzymes in mammals, fish, echinoderm and molluscs.

1. Levels of glycolytic enzymes were determined in terms of units of enzyme/mg protein in rat striated muscle, carp lateral muscle, holothuria longitudinal muscle of the body wall, and a snail foot muscle. 2. An attempt has been made to correlate levels of glycolytic enzymes as a parameter to establish a "biochemical distance" at molecular level and correlate this with the phylogenetic position in animals sufficiently separated in the animal tree of evolution. 3. The possibility of a peculiar kinetic behaviour of the glycolytic pathway in each muscle tissue studied, has been analyzed as the profiles of the ratios of pairs of enzymes bearing a substrate-product dependence. 4. A possible "futile synthesis" of some glycolytic enzymes, such as FDP-aldolase in the case of fish muscle, is proposed.     

Control of mitochondrial respiration in muscle

Summary Control of mitochondrial respiration depends on ADP availability to the F₁ATPase. An electrochemical gradient of ADP and ATP across the mitochondrial inner membrane is maintained by the adenine nucleotide translocase which provides ADP to the matrix for ATP synthesis and ATP for energy-dependent processes in the cytosol. Mitochondrial respiration is responsive to the cytosolic phosphorylation potential, ATP/ADP · P_i which is in apparent equilibrium with the first two sites in the electron transport chain. Conventional measures of free adenine nucleotides is a confounding issue in determining cytosolic and mitochondrial phosphorylation potentials. The advent of phosphorus-31 nuclear magnetic resonance (P-31 NMR) allows the determination of intracellular free concentrations of ATP, creatine-P and Pi in perfused muscle in situ. In the glucose-perfused heart, there is an absence of correlation between the cytosolic phosphorylation potential as determined by P-31 NMR and cardiac oxygen consumption over a range of work loads. These data suggest that contractile work leads to increased generation of mitochondrial NADH so that ATP production keeps pace with myosin ATPase activity. The mechanism of increased ATP synthesis is referred to as stimulusre-sponse-metabolism coupling. In muscle, increased contractility is a result of interventions which increase cytosolic free Ca²⁺ concentrations. The Ca^2- signal thus generated increases glycogen breakdown and myosin ATPase in the cytosol. This signal is concomitantly transmitted to the mitochondria which respond to small increases in matrix Ca²⁺ by activation of Ca²⁺-sensitive dehydrogenases. The Ca²⁺-activated dehydrogenase activities are key rate-controlling enzymes in tricarboxylic acid cycle flux, and their activation by Ca^2- leads to increased pyridine nucleotide reduction and oxidative phosphorylation. These observations which have been consistent in preparations both in vitro and in situ do not obviate a role for ADP control of muscle respiration, but do explain, in part, the lack of dramatic fluctuations in the cytosolic phosphorylation potential over a large range of contractile activities.     

Reconstruction of rat skeletal muscle glycerophosphate shuttle

Summary The activity of -glycerophosphate shuttle in homogenate and in a reconstructed system (isolated mitochondria and cytoplasm) from rat skeletal muscle is presented. The influence of some inhibitors of mitochondria) -glycerosphate dehydrogenase on the activity -glycerophosphate shuttle is demonstrated. The possible operativity of -glycerophosphate shuttle in skeletal muscle in vivo is discussed.Abbreviations used CCCP carbonyl cyanide mchlorophenyl hydrazone - DHAP dihydroxyacetone phosphate - F-1,6 P₂ fructose 1,6 diphosphate - -GP -glycerophosphate - GAP glyceraldehyde 3-phosphate - PEP phosphoenolpyruvate - 3-PG 3-phosphoglycerate - PYR pyruvate - PAL-CoA palmitoyl CoA - MIT mitochondria - CYT cytoplasm - PAL-CARN palmitoyl carnitine     

Localization in cardiac muscle of some enzymes related to glutamate metabolism.

A tetrazolium staining medium incorporated in a gel has been used in a histochemical study of enzymes in thin sections of heart muscle. Formazan distribution patterns given by mitochondrial enzymes were inconsistent with the location of these enzymes revealed by the extraction of whole tissue. Similar stain distributions were given by lactate dehydrogenase, glutamate oxaloacetate transaminase and glutamate dehydrogenase. The distribution given by succinate dehydrogenase was not the same as that given by cytochrome oxidase stained by a different technique. Alcohol dehydrogenase added to the tissue assumed a distribution which suggested some adsorption of the enzyme to the tissue. But experiments suggested that this enzyme was not firmly bound to muscle proteins in the manner of some glycolytic enzymes.     

Impact of Lead and Cadmium on Enzyme of Citric Acid Cycle in Germinating Pea Seeds

The present investigations were conducted to ascertain the influence of Pb₂₊ and Cd₂₊ both individually and in combination on selected enzymes of tricarboxylic acid (TCA) cycle. All the enzymes of TCA cycle examined (-ketoglutarate dehydrogenase, isocitrate dehydrogenase, succinate dehydrogenase, malate dehydrogenase) were affected deleteriously by Pb₂₊ as well as Cd₂₊ and these metals in combination gave more or less an additive effect.     

Age-related effects in enzyme catalysis

Summary Rat muscle glyceraldehyde-3-phosphate dehydrogenase is one of several enzymes which have been found to undergo age-related modifications. While the amount of this enzyme in muscle tissue does not change with age, both its specific activity and affinity towards its co-enzyme are significantly reduced in the old tissue.Age-related structural changes were found to exist in the nicotinamide binding site of the enzyme and the reactions leading to the activity loss in old glyceraldehyde-3-phosphate dehydrogenase were shown to involve a reversible modification of the essential cysteine-149 residue at the active site of the enzyme. The aging effects were simulated by a controlled oxidation of cys-149 in samples of young glyceraldehyde-3-phosphate dehydrogenase and subsequent reduction of this residue by 2-mercaptoethanol. The enzyme modified in this way closely resembles native old glyceraldehyde-3-phosphate dehydrogenase, indicating that the structural modifications in the latter enzyme are indeed introduced by a post-translational process. The mechanism for aging of glyceraldehyde-3-phosphate dehydrogenase which is proposed, based on these observations, thus assumes an oxidation of cys-149 as its first step followed by irreversible conformational changes in the enzyme molecule. The aging of glyceraldehyde-3-phosphate dehydrogenase may thus be triggered by the reduced ability of old muscle tissue to protect its constituents against oxidation.Abbreviations CPL circular polarization of luminescence - DTNB 5,5-dithiobis (2-nitrobenzoic acid) - GPDH D-glyceraldehyde-3-phosphate dehydrogenase - ENAD⁺ nicotinamide 1,N⁶-ethenoadenine dinucleotide     

Relation between growth rate and metabolic organization of white muscle,liver and digestive tract in cod,Gadus morhua

To determine whether the aerobic capacity of tissues required for growth specifically reflects growth rates, we monitored the activities of key enzymes of oxidative, glycolytic and amino acid metabolism in muscle, liver and intestine of Atlantic cod (Gadus morhua) growing at different rates. Fish were maintained individually in small tanks at 10°C and fed on rations that allowed growth rates ranging from-0.6 to 1.6% per day. The correlation between growth rate and muscle enzyme activity was pronounced for the glycolytic enzymes (LDH, PFK and PK). The activities of glycolytic enzymes were more than four times higher for fish having higher growth rates compared to those that did not grow. Mitochondrial enzyme (cytochrome c oxidase, citrate synthase and -hydroxyacyl-CoA dehydrogenase) activities remained unchanged in fish with positive growth. The liver seems to respond to requirements of growth by an increase in size. In the liver, the activities of the enzymes of amino acid metabolism expressed as units · g DNA^-1 specifically increases with growth rate. In contrast to the two other tissues, the specific activities of mitochondrial enzymes in the intestine increased with growth rate while the relative mass of the intestine remained constant. Intestinal cytochrome c oxidase activity increased from a minimum of about 2 to more than 8 units · g intestine^-1. Cytochrome c oxidase activity increased in parallel with the food conversion efficiency. This suggests that the aerobic capacity of the intestine may initially limit the rates of digestion and growth in this species.Abbreviations AA amino acid(s) - BM body mass - CCO cytochrome c oxydase - CS citrate synthase - DTNB 5,5 dithiobis-2-nitrobenzoic acid - GDH glutamate dehydrogenase - GOT glutamate oxalacetate transaminase - GPT glutamate pyruvate transaminase - GR growth rate(s) - HOAD -hydroxyacyl-CoA dehydrogenase - HSI hepatosomatic index - LDH lactate dehydrogenase - MR metabolic rate(s) - PCA perchloric acid - PFK phosphofructokinase - PK pyruvate kinase - PMSF phenylmethylsulphonyl fluoride; TRIS     

Changes in skeletal muscle oxygenation during incremental exercise measured with near infrared spectroscopy

To determine the change in muscle oxygenation in response to progressively increasing work rate exercise, muscle oxyhemoglobin + oxymyoglobin saturation was measured transcutaneously with near infrared spectroscopy in the vastus lateralis muscle during cycle ergometry. Studies were done in 11 subjects while gas exchange was measured breath-by-breath. As work rate was increased, tissue oxygenation initially either remained constant near resting levels or, more usually, decreased. Near the work rate and metabolic rate where significant lactic acidosis was detected by excess CO₂ production (lactic acidosis threshold, LAT), muscle oxygenation decreased more steeply. As maximum oxygen uptake ( ) was approached, the rate of desaturation slowed. In 8 of the 11 subjects, tissue O₂ saturation reached a minimum which was sustained for 1–3 min before was reached. The LAT correlated with both the (r = 0.95,P < 0.0001) and the work rate (r = 0.94,P < 0.0001) at which the rate of tissue O₂ desaturation accelerated. These results describe a consistent pattern in the rate of decrease in muscle oxygenation, slowly decreasing over the lower work rate range, decreasing more rapidly in the work rate range of the LAT and then slowing at about 80% of , approaching or reaching a minimum saturation at .     

A histoenzymatic study of rat intrafusal muscle fibres

Summary The histochemical activities of myofibrillar adenosine triphosphatase (ATPase), succinic dehydrogenase (SDH) and alpha glycerophosphate dehydrogenase (-GPD) were studied in intrafusal muscle fibres of rat fast and slow muscles. The ATPase reaction was carried out after the three standard acid preincubations. The cold K₂-EDTA preincubated ATPase reaction product was similar to that seen following the regular or alkalipreincubated ATPase reaction, except that the intermediate bag fibres exhibited much higher activity after cold K₂-EDTA preincubation. Following either acetic acid solution or cold and room temperature K₂-EDTA-preincubation, followed by the ATPase reaction, chain fibres of the fast muscles vastus lateralis and extensor digitorum longus exhibited a very low amount of reaction product as compared with those of the slow soleus. Veronal acetate and K₂-EDTA preincubations (and equally preincubation in acetic acid solution) resulted in acid stable ATPase activity along the entire length of the typical bag fibres but only in the polar regions of the intermediate bag fibres. On the basis of differing -GPD reaction, two sub populations of nuclear chain fibres were discovered in one spindle. It is a matter of conjecture, to what extent the histochemical differences of intrafusal fibres from fast and slow muscles reflects functional distinctions in the response to stretch of muscle spindles from fast and slow muscles.     

Fructose 1,6-diphosphatase in striated muscle

1. The occurrence of fructose diphosphatase in muscle tissue was investigated with reference to the question whether lactate can be converted into glycogen in muscle, as postulated by Meyerhof (1930), fructose diphosphatase being one of the enzymes required for this conversion. 2. Fructose diphosphatase was found in skeletal muscle of man, dog, cat, rat, mouse, rabbit, guinea pig, cattle, sheep, pigeon, fowl and frog. Under the test conditions between 5 and 60 μmoles of substrate were split/g. fresh wt./hr. at 22°. 3. Like liver fructose diphosphatase, the muscle enzyme is inhibited by substrate concentrations above 0·1 mm, by AMP and by trace quantities of Zn²⁺, Fe²⁺ and Fe³⁺; it is `activated' by EDTA. Inhibitions by the above agents may account for the failure of previous authors to detect the enzyme. 4. Heart muscle of several vertebrate species and the smooth muscle of pigeon and fowl gizzard had no measurable activity. 5. The presence of fructose diphosphatase and the virtual absence of the enzyme systems converting pyruvate into phosphopyruvate means that lactate and pyruvate cannot be converted into glycogen in muscle, whereas the phosphorylated C₃ compounds can. The reconversion into carbohydrate of lactate (which readily diffuses out of muscle) occurs in liver and kidney only. The reconversion of phosphorylated C₃ intermediates (which cannot diffuse out of the tissue) can occur only within the muscle. 6. α-Glycerophosphate is probably the main intermediate requiring conversion into glycogen. The possible role of α-glycerophosphate formation in vertebrate muscle, already well established in insect muscle, is discussed.     

The role of phospholipase A2 in calcium-induced damage in cardiac and skeletal muscle

Summary This study compares the action of inhibitors of the eicosanoid cascade on calcium-induced myofilament damage in cardiac muscle of the perfused frog heart and incubated frog ventricle slices, and in skeletal muscle of incubated mammalian diaphragm and isolated and saponin-skinned amphibian pectoris cutaneous muscle. Mepacrine (10^-5M) and indomethacin (3×10^-6M) protected completely against myofilament damage induced by entry of calcium in the calcium-paradox in frog heart. However, inhibition of phospholipase A₂ (PLA₂) (with chlorpromazine, 2×10^-4M, or mepacrine, 10^-5M, 5x10^-5M), of cyclo-oxygenase enzymes (with indomethacin, 3x10^-6M to 10^-5M or BW755C, 3.8x10^-4M), or of lipoxygenase enzymes (with BW755C, 3.8x10^-4M or nordihydroguaiaretic acid, 2x10^-6M or 5x10^-6M) all failed in intact cardiac or skeletal muscle cells to prevent the myofilament damage that is rapidly triggered by 10^-2M caffeine, 6x10^-6M ruthenium red, 10^-4M DNP or 5 g ml^-1 A23187. These agents also failed completely to protect against myofilament damage in saponin-skinned amphibian skeletal muscle when [Ca]_i was raised to 8x10^-6M. Thus, inhibition of PLA₂ does not protect the myofilament apparatus against calcium released intracellularly, and it is suggested that mepacrine and indomethacin can block entry of calcium in the calcium-paradox in the amphibian heart. Chlorpromazine (2x10^-4M) and mepacrine (10^-3M) at zero [Ca] caused severe myofilament damage in skinned muscle, possibly due to an effect on membranes. Since inhibitors of PLA₂ and of lipoxygenases prevent efflux of creatine kinase and sarcolemma damage in mammalian skeletal muscle, it is evident that experimentally-induced rises in [Ca]_i (by caffeine or A23187) can trigger two separate pathways: (i) PLA₂ and the arachidonic acid cascade which culminate in membrane damage, and (ii) a different, Ca-activated system that causes rapid damage of myofilaments.     

Virtual cooperativity in myoglobin oxygen saturation curve in skeletal muscle <Emphasis Type="Italic">in vivo</Emphasis>

Background

Myoglobin (Mb) is the simplest monomeric hemoprotein and its physicochemical properties including reversible oxygen (O₂)binding in aqueous solution are well known. Unexpectedly, however, its physiological role in intact muscle has not yet been established in spite of the fact that the role of the more complex tetrameric hemoprotein, hemoglobin (Hb), in red cells is well established. Here, I report my new findings on an overlooked property of skeletal Mb.

Methods

I directly observed the oxygenation of Mb in perfused rat skeletal muscle under various states of tissue respiration. A computer-controlled rapid scanning spectrophotometer was used to measure the oxygenation of Mb in the transmission mode. The light beam was focused on the thigh (quadriceps) through a 5-mm-diameter light guide. The transmitted light was conducted to the spectrophotometer through another 5-mm-diameter light guide. Visible difference spectra in the range of 500–650 nm were recorded when O₂ uptake in the hindlimb muscle reached a constant value after every stepwise change in the O₂ concentration of the buffer.

Results

The O₂ dissociation curve (ODC) of Mb, when the effluent buffer O₂ pressure was used as the abscissa, was of a sigmoid shape under normal and increased respiratory conditions whereas it was of rectangular hyperbolic shape under a suppressed respiratory condition. The dissociation curve was shifted toward the right and became more sigmoid with an increase in tissue respiration activity. These observations indicate that an increase in O₂ demand in tissues makes the O₂ saturation of Mb more sensitive to O₂ pressure change in the capillaries and enhances the Mb-mediated O₂ transfer from Hb to cytochrome oxidase (Cyt. aa₃), especially under heavy O₂ demands.

Conclusion

The virtual cooperativity and O₂ demand-dependent shifts of the ODC may provide a basis for explaining why Mb has been preserved as monomer during molecular evolution.

     

Deficiency of the mitochondrial electron transport chain in muscle does not cause insulin resistance

Background

It has been proposed that muscle insulin resistance in type 2 diabetes is due to a selective decrease in the components of the mitochondrial electron transport chain and results from accumulation of toxic products of incomplete fat oxidation. The purpose of the present study was to test this hypothesis.

Methodology/Principal Findings

Rats were made severely iron deficient, by means of an iron-deficient diet. Iron deficiency results in decreases of the iron containing mitochondrial respiratory chain proteins without affecting the enzymes of the fatty acid oxidation pathway. Insulin resistance was induced by feeding iron-deficient and control rats a high fat diet. Skeletal muscle insulin resistance was evaluated by measuring glucose transport activity in soleus muscle strips. Mitochondrial proteins were measured by Western blot. Iron deficiency resulted in a decrease in expression of iron containing proteins of the mitochondrial respiratory chain in muscle. Citrate synthase, a non-iron containing citrate cycle enzyme, and long chain acyl-CoA dehydrogenase (LCAD), used as a marker for the fatty acid oxidation pathway, were unaffected by the iron deficiency. Oleate oxidation by muscle homogenates was increased by high fat feeding and decreased by iron deficiency despite high fat feeding. The high fat diet caused severe insulin resistance of muscle glucose transport. Iron deficiency completely protected against the high fat diet-induced muscle insulin resistance.

Conclusions/Significance

The results of the study argue against the hypothesis that a deficiency of the electron transport chain (ETC), and imbalance between the ETC and β-oxidation pathways, causes muscle insulin resistance.     

Histochemical fiber types characteristics in the normal and the persistent levator ani muscle of the rat

Summary Morphological features of the persistent levator ani (LA) muscle of the female rat normally undergoing involution but maintained by application of testosterone propionate to newborn animals were compared with that of the normal LA muscle of untreated male rat. The two muscles differ in number and size of muscle fibers.Using histochemical methods for myofibrillar adenosine triphosphatase (ATPase), mitochondrial -glycerophosphate dehydrogenase (-GPDH) and succinic dehydrogenase (SDH) the distribution of these enzymes in individual muscle fiber types was studied.ATPase and -GPDH activities show a homogenously positive reaction in the muscle fibers of the male rat, whereas a small portion of fibers with low activity is found in the persistent muscle of female rat.The most pronounced difference concerns SDH activity, i.e. two kinds of fibre types are barely discernible with prolonged incubation in LA muscle of male rat, but three basic fibre types (white, intermediate and red) are clearly distinguished in LA muscle of the female rat.The results are discussed in relation to neural and hormonal influences on histochemical features in cross-striated muscle.This work was supported by a grant from the Muscular Dystrophy Association of America to the Institute of General Pathology, University of Padua, Italy.     

Evaluation of functional erythropoietin receptor status in skeletal muscle in vivo: acute and prolonged studies in healthy human subjects

Background

Erythropoietin receptors have been identified in human skeletal muscle tissue, but downstream signal transduction has not been investigated. We therefore studied in vivo effects of systemic erythropoietin exposure in human skeletal muscle.

Methodology/Principal Findings

The protocols involved 1) acute effects of a single bolus injection of erythropoietin followed by consecutive muscle biopsies for 1–10 hours, and 2) a separate study with prolonged administration for 16 days with biopsies obtained before and after. The presence of erythropoietin receptors in muscle tissue as well as activation of Epo signalling pathways (STAT5, MAPK, Akt, IKK) were analysed by western blotting. Changes in muscle protein profiles after prolonged erythropoietin treatment were evaluated by 2D gel-electrophoresis and mass spectrometry. The presence of the erythropoietin receptor in skeletal muscle was confirmed, by the M20 but not the C20 antibody. However, no significant changes in phosphorylation of the Epo-R, STAT5, MAPK, Akt, Lyn, IKK, and p70S6K after erythropoietin administration were detected. The level of 8 protein spots were significantly altered after 16 days of rHuEpo treatment; one isoform of myosin light chain 3 and one of desmin/actin were decreased, while three isoforms of creatine kinase and two of glyceraldehyd-3-phosphate dehydrogenase were increased.

Conclusions/Significance

Acute exposure to recombinant human erythropoietin is not associated by detectable activation of the Epo-R or downstream signalling targets in human skeletal muscle in the resting situation, whereas more prolonged exposure induces significant changes in the skeletal muscle proteome. The absence of functional Epo receptor activity in human skeletal muscle indicates that the long-term effects are indirect and probably related to an increased oxidative capacity in this tissue.     

The role of phosphoinositide metabolism in Ca2+ signalling of skeletal muscle cells

• 1.1. The mobilization of Ca²⁺ from intracellular stores by d-myo-inositol 1,4,5-triphosphate[Ins(1,4,5)P₃] is now widely accepted as the primary link between plasma membrane receptors that stimulate phospholipase C and the subsequent increase in intracellular free Ca²⁺ that occurs when such receptors are activated (Berridge, 1993). Since the observations of VoIpe et al. (1985) which showed that Ins(1,4,5)P₃ could induce Ca²⁺ release from isolated terminal cisternae membranes and elicit contracture of chemically skinned muscle fibres, research has focused on the role of Ins(1,4,5)P₃ in the generation of SR Ca²⁺ transients and in the mechanism of excitation-contraction coupling (EC-coupling).
• 2.2. The mechanism of signal transduction at the triadic junction during EC-coupling is unknown. Asymmetric charge movement and mechanical coupling between highly specialized triadic proteins has been proposed as the primary mechanism for voltage-activated generation of SR Ca²⁺ signals and subsequent contraction. Ins(1,4,5)P₃ has also been proposed as the major signal transduction molecule for the generation of the primary Ca²⁺ transient produced during EC-coupling.
• 3.3. Investigations on the generation of Ca²⁺ transients by Ins(1,4,5)P₃ have been conducted on ion channels incorporated into lipid bilayers, skinned and intact fibres and isolated membrane vesicles. Ins(1,4,5)P₃ induces SR Ca²⁺ release and the enzymes responsible for its synthesis and degradation are present in muscle tissue. However, the sensitivity of the Ca²⁺ release mechanism to Ins(l,4,5)P₃ is highly dependent on experimental conditions and on membrane potential.
• 4.4. While Ins(1,4,5)P₃ may not be the major signal transduction molecule for the generation of the primary Ca²⁺ signal produced during voltage-activated contraction, this inositol polyphosphate may play a functional role as a modulator of EC-coupling and/or of the processes of myoplasmic Ca²⁺ regulation occurring on a time scale of seconds, during the events of contraction.