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)     

Effects of cyclopiazonic acid on Ca2+ regulation by the sarcoplasmic reticulum in saponin-permeabilized skeletal muscle fibres

Slices of lapine meniscus produced large amounts of nitric oxide after stimulation with interleukin-1, tumor necrosis factor alpha, or a mixture of lapine synovial cytokines known as chondrocyte-activating factors. Monolayer cultures of meniscal cells produced from the proteolysis of meniscal tissue contained a mixed population of chondrocytic and fibroblastic cells. These cultures also produced large amounts of nitric oxide in response to cytokines. Monolayer cultures of meniscal cells produced by the explant method, in contrast, were uniformly fibroblastic and did not produce nitric oxide in response to cytokines. We conclude that menisci contain two populations of cells, one fibroblastic and the other chondrocytic. The chondrocytic cells are responsible for generating most of the nitric oxide in response to cytokines. Endogenously generated nitric oxide suppressed the synthesis of collagen and proteoglycan by menisci but protected proteoglycan from the catabolic effects of interleukin-1. The inhibitory effect of nitric oxide on collagen synthesis occurred without greatly altering the abundance of mRNAs encoding the various collagen alpha chains. During further investigation, arginine was unexpectedly found to stimulate the synthesis of collagen and, to a lesser degree, of noncollagenous proteins but not of proteoglycans. Fragments of meniscus, but not meniscal cells in monolayer culture, increased their production of matrix metalloproteinases, lactate, and, especially, prostaglandin E2 in response to interleukin-1. Inhibition of nitric oxide production with NG-monomethyl-L-arginine enhanced production of matrix metalloproteinases but had little effect on the synthesis of lactate or prostaglandin E2.     

Calcium release from skeletal muscle sarcoplasmic reticulum: site of action of dantrolene sodium

The muscle relaxant dantrolene sodium acts directly and specifically on skeletal muscle, unlike other pharmacological agents which affect the central nervous system or act at the nueromuscular junction. Dantrolene sodium markedly suppresses the release of calcium previously sequestered by skeletal, but not cardiac, muscle sarcoplasmic reticulum. No effect in the total amount of calcium accumulated was found. In situ, the drug may reduce the amount of calcium necessary for muscle contraction.     

pH-modulation of chloride channels from the sarcoplasmic reticulum of skeletal muscle

Limited proteolysis of the NAD+-dependent DNA ligase from Bacillus stearothermophilus with thermolysin results in two fragments which were resistant to further proteolysis. These fragments were characterised by N-terminal protein sequencing and electrospray mass spectrometry. The larger, N-terminal fragment consists of the first 318 residues and the smaller, C-terminal fragment begins at residue 397 and runs to the C terminus. Both fragments were over-expressed in Escherichia coli and purified to homogeneity from this source. The large fragment retains the full self-adenylation activity of the intact enzyme, has minimal DNA binding activity and vastly reduced ligation activity. The small fragment lacks adenylation activity but binds to nicked DNA with a similar affinity to that of the intact enzyme. It is unable to stimulate the ligation activity of the large fragment. Atomic absorption spectroscopy showed that the intact protein and the small fragment bind a zinc ion but the large fragment does not. No evidence of any interaction between the two fragments could be obtained. Thus, we conclude that NAD+-dependent DNA ligases consist of at least two discrete functional domains: an N-terminal domain which is responsible for cofactor binding and self adenylation, and a C-terminal DNA-binding domain which contains a zinc binding site.     

Inhibition of skeletal muscle sarcoplasmic reticulum Ca2+-ATPase by nitric oxide

The effects of nitric oxide on the activities of thapsigargin-sensitive sarcoplasmic reticulum Ca2+-ATPase (SERCA) and Ca2+ uptake by sarcoplasmic reticulum (SR) membranes prepared from white skeletal muscle of rabbit femoral muscle were studied. Pretreatment of the SR preparations with nitric oxide at concentrations of up to 250 microM for 1 min decreased the SERCA activity concentration dependently, and also decreased their Ca2+ uptake. Both these effects of nitric oxide were reversible. Inhibitors of guanylyl cyclase and protein kinase G (PKG) had no significant effect on the nitric oxide-induced inhibitions of SERCA and Ca2+ uptake. Moreover, dithiothreitol did not reverse the inhibitory effects of nitric oxide on SERCA and Ca2+ uptake. These findings suggest that nitric oxide inhibits SERCA, mainly SERCA 1, of rabbit femoral skeletal muscle by an action independent of the cyclic GMP-PKG system or oxidation of thiols, and probably by a direct action on SERCA protein.     

Decreased conformational stability of the sarcoplasmic reticulum Ca-ATPase in aged skeletal muscle

Sarcoplasmic reticulum (SR) membranes purified from young adult (4-6 months) and aged (26-28 months) Fischer 344 male rat skeletal muscle were compared with respect to the functional and structural properties of the Ca-ATPase and its associated lipids. While we find no age-related alterations in (1) expression levels of Ca-ATPase protein, and (2) calcium transport and ATPase activities, the Ca-ATPase isolated from aged muscle exhibits more rapid inactivation during mild (37 degrees C) heat treatment relative to that from young muscle. Saturation-transfer EPR measurements of maleimide spin-labeled Ca-ATPase and parallel measurements of fatty acyl chain dynamics demonstrate that, accompanying heat inactivation, the Ca-ATPase from aged skeletal muscle more readily undergoes self-association to form inactive oligomeric species without initial age-related differences in association state of the protein. Neither age nor heat inactivation results in differences in acyl chain dynamics of the bilayer including those lipids at the lipid-protein interface. Initial rates of tryptic digestion associated with the Ca-ATPase in SR isolated from aged muscle are 16(+/- 2)% higher relative to that from young muscle. indicating more solvent exposure of a portion of the cytoplasmic domain. During heat inactivation these structural differences are amplified as a result of immediate and rapid further unfolding of the Ca-ATPase isolated from aged muscle relative to the delayed unfolding of the Ca-ATPase isolated from young muscle. Thus age-related alterations in the solvent exposure of cytoplasmic peptides of the Ca-ATPase are likely to be critical to the loss of conformational and functional stability.     

VDAC/porin is present in sarcoplasmic reticulum from skeletal muscle

Direct intraspinal injection of the catecholamines epinephrine and norepinephrine, and the alpha-adrenergic agents dexmedetomidine and clonidine, produced a dose-dependent elevation of pain thresholds in the Northern grass frog, Rana pipiens. Significant analgesic effects were noted for at least 4 h. The analgesic effect of intraspinal dexmedetomidine or epinephrine was blocked by systemic pretreatment with the alpha 2-adrenoceptor antagonists, yohimbine and atipamezole, but not with the alpha 1-adrenoceptor antagonist, prazosin. Dose-response analyses showed that dexmedetomidine, epinephrine, norepinephrine had similar analgesic potencies, but clonidine was significantly less potent. Analgesia was observed without accompanying motor or sedative effects. These results suggest that alpha 2-adrenoceptor mechanisms which mediate analgesia may have evolved early in vertebrate evolution and that descending epinephrine-containing fibers in the amphibian nervous system may be the source of endogenous catecholamines regulating nociceptive sensitivity in the amphibian spinal cord.     

Calcium additional to that bound to the transport sites is required for full activation of the sarcoplasmic reticulum Ca-ATPase from skeletal muscle

The sarcoplasmic reticulum Ca-ATPase is fully activated when approximately 1 microM [Ca2+] saturates the two transport sites; higher [Ca] inhibits the ATPase by competition of Ca-ATP with Mg-ATP as substrates. Here we describe a novel effect of EGTA and other chelators, raising the possibility of an additional activating effect of Ca in the sub- or low microM range. Sarcoplasmic reticulum membranes were isolated from rabbit skeletal muscles. The ATPase activity was measured after incubation at 37 degreesC in 3 mM ATP, 3 mM MgCl2, 50 mM MOPS-Tris (pH 7.2), 100 mM KCl, and variable CaCl2, EGTA and calcimycin. In the absence of added EGTA and Ca the ATPase activity is high due to contaminant Ca. The determination of the ATPase activity in the presence of increasing amounts of EGTA, without added Ca, yields a decreasing sigmoidal function. Ki ranged between 20 and 100 microM, depending on the enzyme concentration. Pi production is linear with time for several [EGTA] yielding suboptimal ATPase activities, which are inhibited by thapsigargin. These suboptimal Ca-ATPase activities are inhibited by preincubation of the enzyme in EGTA, at pH 7.2. This effect increases upon increasing EGTA concentration and preincubation time. The inhibitory effect of the previous exposure of the enzyme to EGTA is partially but significantly reverted by increasing [Ca2+] during incubations. Calcimycin and EDTA have similar effects as EGTA when added in preincubations. The effect of calcimycin is fully reverted by optimal [Ca2+] in incubations. The effects of EGTA, EDTA and calcimycin in preincubation are not additive. The results suggest that an additional calcium, lost during preincubations from a site with affinity near 1 microM, is necessary for full activation of the ATPase.     

Mechanism of Ca-ATPase inhibition by melittin in skeletal sarcoplasmic reticulum

We have previously shown that the basic, amphipathic peptide melittin inhibits the Ca-ATPase of the sarcoplasmic reticulum membrane by inducing large-scale aggregation of the enzyme via electrostatic cross-linking. To better understand the physical mechanism by which melittin-induced Ca-ATPase aggregation inhibits the enzyme, we have performed time-resolved phosphorescence anisotropy (TPA) and steady-state fluorescence experiments in combination with enzyme kinetic assays, utilizing (1) native and charge-modified melittin in order to characterize the peptide charge dependence of the melittin-SR interaction, and (2) various calcium levels in order to define the effect of melittin on the enzyme's E1 and E2 conformational equilibrium. TPA results showed that decreasing melittin's positive charge dramatically decreases the ability of the peptide to aggregate the enzyme, which correlates with a reduced potency of the modified peptide to inhibit enzymatic activity. Steady-state fluorescence of fluorescein isothiocyanate-labeled Ca-ATPase showed that melittin reduces Ca-ATPase affinity for calcium by shifting the enzyme's E1-E2 conformational equilibrium toward E2, but increasing calcium progressively reverses this shift. Kinetic experiments showed that melittin does not prevent ATP-dependent enzyme phosphorylation, but it completely inhibits Pi-dependent EP formation and substantially slows Pi release during steady-state cycling. We conclude that melittin-induced aggregation of the Ca-ATPase depends on the electrostatic interaction of the peptide with cytoplasmic Ca(2+)-dependent sites on the enzyme, and that enforced Ca-ATPase protein-protein interactions inhibit the conformational transitions that facilitate phosphoenzyme hydrolysis.     

Direct monitoring of the calcium concentration in the sarcoplasmic and endoplasmic reticulum of skeletal muscle myotubes

Direct monitoring of the free Ca2+ concentration in the sarcoplasmic reticulum (SR) was carried out in rat skeletal myotubes transfected with a specifically targeted aequorin chimera (srAEQ). Myotubes were also transfected with a chimeric aequorin (erAEQ) that we have demonstrated previously is retained in the endoplasmic reticulum (ER). Immunolocalization analysis showed that although both recombinant proteins are distributed in an endomembrane network identifiable with immature SR, the erAEQ protein was retained also in the perinuclear membrane. The difficulty of measuring [Ca2+] in 100-1000 microM range was overcome with the use of the synthetic coelenterazine analogue, coelenterazine n. We demonstrate that the steady state levels of [Ca2+] measured with srAEQ is around 300 microM, whereas that measured with erAEQ is significantly lower, i.e. around 200 microM. The effects of caffeine, high KCl, and nicotinic receptor stimulation, in the presence or absence of external calcium or after blockade of the Ca-ATPase, were investigated with both chimeras. The kinetics of [Ca2+] changes revealed by the erAEQ were similar, but not identical, neither quantitatively nor qualitatively, to those monitored with the srAEQ, indicating that at this stage of muscle development, differences exist between SR and ER in their mechanisms of Ca2+ handling. The functional implications of these findings are discussed.     

Ca2+ release from the sarcoplasmic reticulum compared in amphibian and mammalian skeletal muscle

This paper describes and evaluates a method for quantifying the amounts of specific plasma proteins adsorbed to biomaterial surfaces. In particular, it demonstrates that macroscopic images ('stains'), that assess the spatial distribution of albumin, IgG, fibrinogen, and HMK (high molecular weight kininogen), can be obtained over areas of at least 12 cm2 using immunospecific adhesion of dyed polystyrene beads. Stain intensities, measured with a scanner and an image analysis system, were found to quantify the amount of specific protein in the solution used to coat the surfaces. Results obtained with the proposed method produced single protein isotherms for albumin, immunoglobulin G (IgG) and fibrinogen that followed Langmuir-like adsorption behavior and were similar to previously published isotherms. The HMK isotherm also exhibited Langmuir-like adsorption behavior. The proposed method also detected the presence of an expected maximum in the adsorption of fibrinogen onto glass as a function of plasma dilution. Adsorption of fibrinogen out of 6.4% plasma onto glass from a separated flow produced results indicating the quantity as well as the location of fibrinogen at the boundary of the separated region. This result confirmed the utility of the proposed method for detecting spatial distributions of specific proteins adsorbed from plasma in practical devices.     

ATP-sensitive voltage- and calcium-dependent chloride channels in sarcoplasmic reticulum vesicles from rabbit skeletal muscle

In addition to its role in olfaction and as a primary epileptogenic site, the anterior piriform cortex has been suggested to play a role in neuroperception of deficiencies or imbalances in physiologically essential amino acids. In recent studies, amino acid deficient diets were shown to induce expression of c-fos in the anterior piriform cortex within the rapid time frame associated with the normal anorectic response to such diets. It became important to examine the neurocytochemical architecture of this region for clues as to how and more precisely where dietary amino acid deficiency or imbalance might be monitored. The relationships of neuropeptide Y-, somatostatin-, and cholecystokinin-containing neurons were of particular interest because ongoing studies indicate that those peptides administered to the anterior piriform cortex alter intake of diets deficient in essential amino acids. The neuropeptides were endogenous to intrinsic neurons only; none resembled pyramidal projection neurons. Peptidergic neurons and fibers were concentrated most heavily in layer III of the paleocortex. The cytoarchitecture suggests that neuropeptide Y-, somatostatin-, and cholecystokin-containing neurons of the anterior piriform cortex may relate synaptically or multisynaptically to local circuit neurons during electrical activity, modulation of olfactory information, and neuroperception of essential amino acids.     

Uridine triphosphate-sensitive pathway of Ca2+ release from the sarcoplasmic reticulum of rat skeletal muscle fibers

The pyrimidine nucleotide, uridine triphosphate (UTP), was tested with skinned skeletal muscle fibers in order to investigate the UTP-sensitive pathway of Ca2+ release from the sarcoplasmic reticulum. The presence of ryanodine (200 microM), ruthenium red (10 microM) or heparin (2.5 mg/ml) did not affect the tension elicited in the presence of UTP, demonstrating that the UTP-induced Ca2+ release involved neither ryanodine nor inositol triphosphate-sensitive channels. Drugs such as compound 48/80 or cyclopiazonic acid used to inhibit Ca2+-ATPase in its reverse function appeared to be, respectively, non-specific or without any inhibitory effect on the tension induced by UTP. Finally, the UTP-induced tension as well as the trifluoperazine-induced tension were abolished in the presence of spermidine (50 mM), supporting the hypothesis that the UTP-sensitive pathway of the SR Ca2+ release might occur through the uncoupled calcium ATPase.     

Contraction and relaxation in the absence of a sarcoplasmic reticulum: muscle fibres in the small pelagic tunicate Doliolum

PURPOSE: The purpose of the study was to evaluate the biomicroscopic, light microscopic, and electron microscopic effects of ultraviolet-B (UV-B) exposure on the outcome of photorefractive keratectomy (PRK). METHODS: A total of 24 pigmented rabbits were used in the study. One eye of 16 rabbits received a 193-nm, 45-micron deep (-5.0 diopter) excimer laser PRK. Twenty-one days after PRK, eight of the laser-treated eyes were exposed to 100 mJ/cm2 UV-B (280-315 nm) UV radiation by placing the rabbits in a standard clinically used dermatologic chamber for 7 minutes. Eight PRK-treated rabbits received no further treatment. The remaining eight non-PRK-treated rabbits received 100 mJ/cm2 UV-B only to one eye. Subepithelial haze was assessed before and after UV irradiation. Corneal morphology was assessed 4, 8, 12, and 16 weeks after UV-B exposure, using light microscopic and transmission electron microscopic (TEM) techniques. RESULTS: Untreated eyes exposed to 100 mJ/cm2 UV-B only exhibited photokeratitis for 2 days, but showed no haze and were normal histologically at all intervals. The PRK-treated UV-B irradiated eyes exhibited a significant increase of stromal haze compared to eyes receiving PRK alone. Histologically, the main difference between the UV-B irradiated and nonirradiated post-PRK eyes was the presence of anterior stromal extracellular vacuolization in the UV-B-exposed eyes. The vacuolated foci were confined to the PRK treatment area and showed increased keratocyte density and disorganization of normal collagen lamellae. TEM showed activated keratocytes containing abundant rough endoplasmic reticulum, prominent Golgi zones, and extracellular vacuoles filled with amorphous material. The haze and morphologic changes showed a tendency to incomplete resolution over the period of 16 weeks. CONCLUSIONS: The UV-B exposure during post-PRK stromal healing exacerbates and prolongs the stromal healing response and is manifest biomicroscopically by augmentation of subepithelial haze. The findings suggest that excessive ocular UV-B exposure should be avoided during the period of post-PRK stromal repair and that UV-B may modulate the response of tissues to 193-nm excimer laser and perhaps other laser energy in general.     

The influence of ionic strength upon relaxation from rigor induced by flash photolysis of caged-ATP in skinned murine skeletal muscle fibres

The influence of ionic strength upon relaxation kinetics from rigor in skinned murine extensor digitorum longus (EDL) skeletal muscle fibres was examined using photolysis of caged-ATP at low Ca2+. The ionic strength was adjusted with either KMeSO3 or ethylene glycolbis-(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid, dipotassium salt (K2EGTA) in the range of tau /2 = 65-215mM, or I.E. 49-194mM, where I.E. denotes ionic equivalent. Following rigor development at a tau /2 of 165-215mM (I.E. 144-194mM), the liberation of approximately 0.5mM ATP resulted in an initial 6-to 10-ms detachment phase with a decline in force of approximately 10-20% followed by a 10-to 30-ms reattachment with up to a 60% increase compared to the corresponding rigor level and a final detachment leading to complete relaxation. Interestingly, when similar ATP concentrations were liberated at lower ionic strengths between a tau /2 of 65mM and 110mM (I.E. 60-100mM), the initial detachment phase was shortened and force decreased by only approximately 5-10%, while the following reattachment phase was lengthened and led to an increased steady-state force of approximately 20-80% without final relaxation. ATP-induced detachment and subsequent reattachment were mainly determined by the currently present ionic strength and were relatively independent of the preceding rigor state which had been developed at higher or lower ionic strengths. The effects of phosphate and apyrase on the force transient suggest that reattachment of ADP- binding crossbridges may contribute to the increase in tension at high and even more at low ionic strengths. The study shows that the kinetics of initial fast relaxation and subsequent redevelopment of force following flash photolysis of similar ATP concentrations are markedly modified by the ionic strength in the narrow range of between 65mM and 215mM.     

Dantrolene inhibition of sarcoplasmic reticulum Ca2+ release by direct and specific action at skeletal muscle ryanodine receptors

The skeletal muscle relaxant dantrolene inhibits the release of Ca2+ from the sarcoplasmic reticulum during excitation-contraction coupling and suppresses the uncontrolled Ca2+ release that underlies the skeletal muscle pharmacogenetic disorder malignant hyperthermia; however, the molecular mechanism by which dantrolene selectively affects skeletal muscle Ca2+ regulation remains to be defined. Here we provide evidence of a high-affinity, monophasic inhibition by dantrolene of ryanodine receptor Ca2+ channel function in isolated sarcoplasmic reticulum vesicles prepared from malignant hyperthermia-susceptible and normal pig skeletal muscle. In media simulating resting myoplasm, dantrolene increased the half-time for 45Ca2+ release from both malignant hyperthermia and normal vesicles approximately 3.5-fold and inhibited sarcoplasmic reticulum vesicle [3H]ryanodine binding (Ki approximately 150 nM for both malignant hyperthermia and normal). Inhibition of vesicle [3H]ryanodine binding by dantrolene was associated with a decrease in the extent of activation by both calmodulin and Ca2+. Dantrolene also inhibited [3H]ryanodine binding to purified skeletal muscle ryanodine receptor protein reconstituted into liposomes. In contrast, cardiac sarcoplasmic reticulum vesicle 45Ca2+ release and [3H]ryanodine binding were unaffected by dantrolene. Together, these results demonstrate selective effects of dantrolene on skeletal muscle ryanodine receptors that are consistent with the actions of dantrolene in vivo and suggest a mechanism of action in which dantrolene may act directly at the skeletal muscle ryanodine receptor complex to limit its activation by calmodulin and Ca2+. The potential implications of these results for understanding how dantrolene and malignant hyperthermia mutations may affect the voltage-dependent activation of Ca2+ release in intact skeletal muscle are discussed.     

Force-velocity properties and myosin light chain isoform composition of an identified type of skinned fibres from rat skeletal muscle

Force-velocity relations, myosin heavy chain (MHC) and myosin light chain (MLC) isoform composition of single skinned fibres from rat plantaris muscle were determined. In fibres containing the same (2X) isoform of myosin heavy chain, several parameters derived from the force-velocity relation and isometric force (Po) were tested for relation with the fibre content in alkali myosin light chain (MLC) isoforms. Whereas maximum shortening velocity was found to be proportional to the relative content in the 3f isoform of alkali MLC, velocity of shortening at 5% relative load, maximum power output, and Po were not. These results strengthen the idea that, in mammalian skeletal fibres, alkali MLC isoforms modulate shortening velocity at zero load, but suggest that they do not control the contractile behaviour at loads higher than zero.