Two novel types of calcium release from skeletal sarcoplasmic reticulum by phosphatidylinositol 4,5-biphosphate.

In both the heavy and light fractions of fragmented sarcoplasmic reticulum (SR) vesicles from the fast skeletal muscle, about 27 min after beginning the active Ca2+ uptake, the extravesicular Ca2+ concentration suddenly increased to reach a steady level (delayed Ca2+ release). Phosphatidylinositol 4,5-bisphosphate (PIP2) not only shortened the time to delayed Ca2+ release but also induced prompt Ca2+ release from the heavy fraction of SR. Delayed Ca2+ release and prompt Ca2+ release stimulated by 100 microM PIP2 were not modified by ruthenium red. PIP2 (>0.1 microM) markedly accelerated the rate of 45Ca2+ efflux from SR vesicles in a concentration-dependent manner. The PIP(2)-induced 45Ca2+ efflux was potentiated by ruthenium red but profoundly inhibited by La3+. The concentration-response curve for Ca2+ or Mg2+ in PIP2-induced 45Ca2+ release was clearly different from that in the Ca(2+)-induced Ca2+ release. PIP2 caused a concentration-dependent increase in Ca2+ release from SR of chemically skinned fibers from skeletal muscle. Furthermore, [3H]ryanodine or [3H]methyl-7-bromoeudistomin D (MBED) binding to SR was increased by PIP2 in a concentration-dependent manner. These observations present the first evidence that PIP2 most likely activates two types of SR Ca2+ release channels whose properties are entirely different from those of Ca(2+)-induced Ca2+ release channels (the ryanodine receptor 1).     

Dual effect of deafferentation on contractile characteristics and sarcoplasmic reticulum properties in rat soleus fibers.

The neural message is known to play a key role in muscle development and function. We analyzed the specific role of the afferent message on the functional regulation of two subcellular muscle components involved in the contractile mechanism: the contractile proteins and the sarcoplasmic reticulum (SR). Rats were submitted to bilateral deafferentation (DEAF group) by section of the dorsal roots L(3) to L(5) after laminectomy. Experiments were carried out in single skinned fibers of the soleus muscle. The maximal force developed by the contractile proteins was increased in the DEAF group compared with control, despite a decrease in muscle mass by 17%. The tension-pCa relationship was shifted toward lower calcium (Ca(2+)) concentrations. Different functional properties of the SR of DEAF soleus were examined by using caffeine-induced contractions. The caffeine sensitivity of the Ca(2+) release was decreased after deafferentation and ryanodine receptor 1 isoform was expressed at a lower level. The rate of Ca(2+) uptake was only slightly increased. The results underlined the dual effect of the afferent input on the functional regulation of both contractile proteins and SR.     

The Ca2+-release channel/ryanodine receptor is localized in junctional and corbular sarcoplasmic reticulum in cardiac muscle

The subcellular distribution of the Ca(2+)-release channel/ryanodine receptor in adult rat papillary myofibers has been determined by immunofluorescence and immunoelectron microscopical studies using affinity purified antibodies against the ryanodine receptor. The receptor is confined to the sarcoplasmic reticulum (SR) where it is localized to interior and peripheral junctional SR and the corbular SR, but it is absent from the network SR where the SR-Ca(2+)-ATPase and phospholamban are densely distributed. Immunofluorescence labeling of sheep Purkinje fibers show that the ryanodine receptor is confined to discrete foci while the SR-Ca(2+)-ATPase is distributed in a continuous network-like structure present at the periphery as well as throughout interior regions of these myofibers. Because Purkinje fibers lack T- tubules, these results indicate that the ryanodine receptor is localized not only to the peripheral junctional SR but also to corbular SR densely distributed in interfibrillar spaces of the I-band regions. We have previously identified both corbular SR and junctional SR in cardiac muscle as potential Ca(2+)-storage/Ca(2+)-release sites by demonstrating that the Ca2+ binding protein calsequestrin and calcium are very densely distributed in these two specialized domains of cardiac SR in situ. The results presented here provide strong evidence in support of the hypothesis that corbular SR is indeed a site of Ca(2+)-induced Ca2+ release via the ryanodine receptor during excitation contraction coupling in cardiac muscle. Furthermore, these results indicate that the function of the cardiac Ca(2+)-release channel/ryanodine receptor is not confined to junctional complexes between SR and the sarcolemma.     

Mechanism of anthraquinone-induced calcium release from skeletal muscle sarcoplasmic reticulum

The anthraquinones, doxorubicin, mitoxantrone, daunorubicin and rubidazone are shown to be potent stimulators of Ca2+ release from skeletal muscle sarcoplasmic reticulum (SR) vesicles and to trigger transient contractions in chemically skinned psoas muscle fibers. These effects of anthraquinones are the direct consequence of their specific interaction with the [3H] ryanodine receptor complex, which constitutes the Ca2+ release channel from the triadic junction. In the presence of adenine nucleotides and physiological Mg2+ concentrations (approximately 1.0 mM), channel activation by doxorubicin and daunorubicin exhibits a sharp dependence on submicromolar Ca2+ which is accompanied by a selective, dose-dependent increase in the apparent affinity of the ryanodine binding sites for Ca2+, in a manner similar to that previously reported with caffeine. Unlike caffeine, however, anthraquinones increase [3H]ryanodine receptor occupancy to the level observed in the presence of adenine nucleotides. A strong interaction between the anthraquinone and the caffeine binding sites on the Ca2+ release channel is also observed when monitoring Ca2+ fluxes across the SR. Millimolar caffeine both inhibits anthraquinone-stimulated Ca2+ release, and reduces anthraquinone-stimulated [3H]ryanodine receptor occupancy, without changing the effective binding constant of the anthraquinone for its binding site. The degree of cooperativity for daunorubicin activation of Ca2+ release from SR also increases in the presence of caffeine. These results demonstrate that the mechanism by which anthraquinones stimulate Ca2+ release is caused by a direct interaction with the [3H]ryanodine receptor complex, and by sensitization of the Ca2+ activator site for Ca2+.     

Mechanisms underlying the nitric oxide inhibitory effects in mouse ileal longitudinal muscle

We investigated the mechanisms involved in the nitric oxide (NO)-induced inhibitory effects on longitudinal smooth muscle of mouse ileum, using organ bath technique. Exogenously applied NO, delivered as sodium nitroprusside (SNP; 0.1-100 micromol/L) induced a concentration-dependent reduction of the ileal spontaneous contractions. 1H-[1,2,4]oxadiazolol[4,3,a]quinoxalin-1-one (ODQ; 1 micromol/L), a guanilyl cyclase inhibitor, reduced the SNP-induced effects. Tetraethylammonium chloride (20 mmol/L), a non-selective K+ channel blocker, and charybdotoxin (0.1 micromol/L), blocker of large conductance Ca2+-dependent K+ channels, significantly reduced SNP-induced inhibitory effects. In contrast, apamin (0.1 micromol/L), blocker of small conductance Ca2+-dependent K+ channels, was not able to affect the response to SNP. Ciclopiazonic acid (10 micromol/L) or thapsigargin (0.1 micromol/L), sarcoplasmatic reticulum Ca2+-ATPase inhibitors, decreased the SNP-inhibitory effects. Ryanodine (10 micromol/L), inhibitor of Ca2+ release from ryanodine-sensitive intracellular stores, significantly reduced the SNP inhibitory effects. The membrane permeable analogue of cGMP, 8-bromoguanosine 3',5'-cyclic monophosphate (100 micromol/L), also reduced spontaneous mechanical activity, and its effect was antagonized by ryanodine. The present study suggests that NO causes inhibitory effects on longitudinal smooth muscle of mouse ileum through cGMP which in turn would activate the large conductance Ca2+-dependent K+ channels, via localized ryanodine-sensitive Ca2+ release.     

Intracellular Cl- fluxes play a novel role in Ca2+ handling in airway smooth muscle

Intracellular Ca(2+) is actively sequestered into the sarcoplasmic reticulum (SR), whereas the release of Ca(2+) from the SR can be triggered by activation of the inositol 1,4,5-trisphosphate and ryanodine receptors. Uptake and release of Ca(2+) across the SR membrane are electrogenic processes; accumulation of positive or negative charge across the SR membrane could electrostatically hinder the movement of Ca(2+) into or out of the SR, respectively. We hypothesized that the movement of intracellular Cl(-) (Cl(i)(-)) across the SR membrane neutralizes the accumulation of charge that accompanies uptake and release of Ca(2+). Thus inhibition of SR Cl(-) fluxes will reduce Ca(2+) sequestration and agonist-induced release. The Cl(-) channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 10(-4) M), previously shown to inhibit SR Cl(-) channels, significantly reduced the magnitude of successive acetylcholine-induced contractions of airway smooth muscle (ASM), suggesting a "run down" of sequestered Ca(2+) within the SR. Niflumic acid (10(-4) M), a structurally different Cl(-) channel blocker, had no such effect. Furthermore, NPPB significantly reduced caffeine-induced contraction and increases in intracellular Ca(2+) concentration ([Ca(2+)](i)). Depletion of Cl(i)(-), accomplished by bathing ASM strips in Cl(-)-free buffer, significantly reduced the magnitude of successive acetylcholine-induced contractions. In addition, Cl(-) depletion significantly reduced caffeine-induced increases in [Ca(2+)](i). Together these data suggest a novel role for Cl(i)(-) fluxes in Ca(2+) handling in smooth muscle. Because the release of sequestered Ca(2+) is the predominate source of Ca(2+) for contraction of ASM, targeting Cl(i)(-) fluxes may prove useful in the control of ASM hyperresponsiveness associated with asthma.     

Mice null for calsequestrin 1 exhibit deficits in functional performance and sarcoplasmic reticulum calcium handling

In skeletal muscle, the release of calcium (Ca(2+)) by ryanodine sensitive sarcoplasmic reticulum (SR) Ca(2+) release channels (i.e., ryanodine receptors; RyR1s) is the primary determinant of contractile filament activation. Much attention has been focused on calsequestrin (CASQ1) and its role in SR Ca(2+) buffering as well as its potential for modulating RyR1, the L-type Ca(2+) channel (dihydropyridine receptor, DHPR) and other sarcolemmal channels through sensing luminal [Ca(2+)]. The genetic ablation of CASQ1 expression results in significant alterations in SR Ca(2+) content and SR Ca(2+) release especially during prolonged activation. While these findings predict a significant loss-of-function phenotype in vivo, little information on functional status of CASQ1 null mice is available. We examined fast muscle in vivo and in vitro and identified significant deficits in functional performance that indicate an inability to sustain contractile activation. In single CASQ1 null skeletal myofibers we demonstrate a decrease in voltage dependent RyR Ca(2+) release with single action potentials and a collapse of the Ca(2+) release with repetitive trains. Under voltage clamp, SR Ca(2+) release flux and total SR Ca(2+) release are significantly reduced in CASQ1 null myofibers. The decrease in peak Ca(2+) release flux appears to be solely due to elimination of the slowly decaying component of SR Ca(2+) release, whereas the rapidly decaying component of SR Ca(2+) release is not altered in either amplitude or time course in CASQ1 null fibers. Finally, intra-SR [Ca(2+)] during ligand and voltage activation of RyR1 revealed a significant decrease in the SR[Ca(2+)](free) in intact CASQ1 null fibers and a increase in the release and uptake kinetics consistent with a depletion of intra-SR Ca(2+) buffering capacity. Taken together we have revealed that the genetic ablation of CASQ1 expression results in significant functional deficits consistent with a decrease in the slowly decaying component of SR Ca(2+) release.     

Inhibition of Ca(2+) sparks by ruthenium red in permeabilized rat ventricular myocytes

We have compared the effects of the sarcoplasmic reticulum (SR) Ca(2+) release inhibitor, ruthenium red (RR), on single ryanodine receptor (RyR) channels in lipid bilayers, and on Ca(2+) sparks in permeabilized rat ventricular myocytes. Ruthenium red at 5 microM inhibited the open probability (P(o)) of RyRs approximately 20-50-fold, without significantly affecting the conductance or mean open time of the channel. At the same concentration, RR inhibited the frequency of Ca(2+) sparks in permeabilized myocytes by approximately 10-fold, and reduced the amplitude of large amplitude events (with most probable localization on the line scan) by approximately 3-fold. According to our theoretical simulations, performed with a numerical model of Ca(2+) spark formation, this reduction in Ca(2+) spark amplitude corresponds to an approximately 4-fold decrease in Ca(2+) release flux underlying Ca(2+) sparks. Ruthenium red (5 microM) increased the SR Ca(2+) content by approximately 2-fold (from 151 to 312 micromol/l cytosol). Considering the degree of inhibition of local Ca(2+) release events, the increase in SR Ca(2+) load by RR, and the lack of effects of RR on single RyR open time and conductance, we have estimated that Ca(2+) sparks under normal conditions are generated by openings of at least 10 single RyRs.     

Reconstitution of purified cardiac muscle calcium release channel (ryanodine receptor) in planar bilayers

The purified ryanodine receptor of heart sarcoplasmic reticulum (SR) has been reconstituted into planar phospholipid bilayers and found to form Ca2+-specific channels. The channels are strongly activated by Ca2+ (10 nM) in the presence of ATP (1 mM) and ryanodine, and inactivated by Mg2+ (3 mM) or ruthenium red (30 microM). These characteristics are diagnostic of calcium release from heart SR. The cardiac ryanodine receptor, which has previously been identified as the foot structure, is now identified as the calcium release channel. A similar identity of the calcium release channel has recently been reported for skeletal muscle. The characteristics of the calcium release channel from skeletal muscle and heart are similar in that they: 1) consist of an oligomer of a single high molecular weight polypeptide (Mr 360,000 for skeletal muscle and 340,000 for heart); 2) exist morphologically as the foot structure; 3) are activated (ATP, Ca2+, ryanodine) and inhibited (ruthenium red and Mg2+) by a number of the same ligands. Important differences include: 1) Ca2+ activation at lower concentration of Ca2+ for the heart; 2) more dramatic stabilization by ryanodine of the open state for the skeletal muscle channel; and 3) different relative permeabilities (PCa/PK).     

Hg2+-induced contracture in mechanically skinned fibers of frog skeletal muscle

In mechanically skinned fibers of the semitendinosus muscle of bullfrogs, we examined the role of membrane sulfhydryl groups on Ca2+ release from the sarcoplasmic reticulum (SR). Hg2+, a sulfhydryl reagent (20-100 microM), induced a repetitive contracture of skinned fibers, and this contracture did not occur in skinned fibers in which the SR had been disrupted by treatment with a detergent (Brij 58). Procaine (10 mM), Mg2+ (5 mM), or dithiothreitol (1 mM) blocked the Hg2+-induced contracture. Ag+ or p-chloromercuribenzenesulfonic acid produced similar contractures to that induced by Hg2+. We conclude that Hg2+ releases Ca2+ from SR of a skinned fiber by modifying sulfhydryl groups on the SR membrane, and suggest that the Ca2+ released by Hg2+ may trigger a greater release of Ca2+ from SR to develop tension.     

Regulation of slow wave frequency by IP(3)-sensitive calcium release in the murine small intestine

Slow waves determine frequency and propagation characteristics of contractions in the small intestine, yet little is known about mechanisms of slow wave regulation. We propose a role for intracellular Ca(2+), inositol 1,4,5,-trisphosphate (IP(3))-sensitive Ca(2+) release, and sarcoplasmic reticulum (SR) Ca(2+) content in the regulation of slow wave frequency because 1) 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, a cytosolic Ca(2+) chelator, reduced the frequency or abolished the slow waves; 2) thapsigargin and cyclopiazonic acid (CPA), inhibitors of SR Ca(2+)-ATPase, decreased slow wave frequency; 3) xestospongin C, a reversible, membrane-permeable blocker of IP(3)-induced Ca(2+) release, abolished slow wave activity; 4) caffeine and phospholipase C inhibitors (U-73122, neomycin, and 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate) inhibited slow wave frequency; 5) in the presence of CPA or thapsigargin, stimulation of IP(3) synthesis with carbachol, norepinephrine, or phenylephrine acting on alpha(1)-adrenoceptors initially increased slow wave frequency but thereafter increased the rate of frequency decline, 6) thimerosal, a sensitizing agent of IP(3) receptors increased slow wave frequency, and 7) ryanodine, a selective modulator of Ca(2+)-induced Ca(2+) release, had no effect on slow wave frequency. In summary, these data are consistent with a role of IP(3)-sensitive Ca(2+) release and the rate of SR Ca(2+) refilling in regulation of intestinal slow wave frequency.     

Molecular regulation of cardiac ryanodine receptor ion channel

The release of Ca(2+) ions from intracellular stores is a key step in a wide variety of cellular functions. In striated muscle, the release of Ca(2+) from the sarcoplasmic reticulum (SR) leads to muscle contraction. Ca(2+) release occurs through large, high-conductance Ca(2+) release channels, also known as ryanodine receptors (RyRs) because they bind the plant alkaloid ryanodine with high affinity and specificity. The RyRs are isolated as 30S protein complexes comprised of four 560 kDa RyR2 subunits and four 12 kDa FK506 binding protein (FKBP12) subunits. Multiple endogenous effector molecules and posttranslational modifications regulate the RyRs. This review focuses on current research toward understanding the control of the isolated cardiac Ca(2+) release channel/ryanodine receptor (RyR2) by Ca(2+), calmodulin, thiol oxidation/reduction and nitrosylation, and protein phosphorylation.     

Role of extracellular Ca2+ in diaphragmatic contraction: effects of ouabain, monensin, and ryanodine.

The effects of zero extracellular Ca2+ on the contractility of rat diaphragmatic strips in vitro were studied in conjunction with various pharmacological agents known to influence the intracellular Ca2+ concentration: the Na+ ionophore, monensin, and the Na(+)-K+ pump inhibitor, ouabain, which enhance [Ca2+]i, caffeine, which induces Ca2+ release from the sarcoplasmic reticulum (SR), and ryanodine, which prevents Ca2+ retention by the SR. The effect of increasing [Ca2+]i on diaphragmatic contraction was assessed by comparing contractions induced by 120 mM K+ in the small muscle strips before and after the addition of ouabain or monensin. Monensin (20 microM) and ouabain (1-100 microM) augmented contractions up to threefold. Treatment of diaphragm strips with 3 nM ryanodine increased baseline tension 360% above the original resting tension but only if the diaphragm was electrically stimulated concurrently; 100 microM ryanodine induced contracture in quiescent tissue. High K+ contractures were of greater magnitude in the presence of ryanodine compared with control, and relaxation time was prolonged by greater than 200%. Ca(2+)-free conditions ameliorated these actions of ryanodine. Ryanodine reduced contractions induced by 10 mM caffeine and nearly abolished them in Ca(2+)-free solution. The data demonstrate that extracellular Ca2+ is important in certain types of contractile responses of the diaphragm and suggest that the processes necessary to utilize extracellular Ca2+ are present in the diaphragm.     

Effects of ryanodine in skinned cardiac cells

Ryanodine (1 X 10(-5) M) did not affect the Ca2+ sensitivity of the myofilaments of skinned (sarcolemma removed by microdissection) cardiac cells from the rat ventricle. Ryanodine (1 X 10(-5) M) inhibited three types of Ca2+ release from the sarcoplasmic reticulum (SR), which have different mechanisms: 1) Ca2+-induced release of Ca2+ triggered by a rapid and transient increase of [free Ca2+] at the outer surface of the SR; 2) caffeine-induced release of Ca2+; 3) spontaneous cyclic release of Ca2+ occurring in the continuous presence of a [free Ca2+] sufficient to overload the SR. These results suggest that the three types of Ca2+ release are through the same channel across the SR membrane, although the gating mechanisms are different for the three types. Ryanodine also diminished the rate of Ca2+ accumulation into the SR. Even in the presence of 1 X 10(-5) M ryanodine the SR accumulated Ca2+ that could be released when the SR was sufficiently overloaded with Ca2+. Thus, ryanodine pretreatment did not permit the direct activation of the myofilaments by externally applied Ca2+. The approximately 1000-fold difference in the effective concentrations of ryanodine in intact vs. skinned cardiac cells suggests that low concentrations of ryanodine act in the intact cardiac tissues through processes or on structures that are destroyed by the skinning procedure. No significant differences were observed in the effects of ryanodine in skinned cardiac cells from different adult mammalian species.     

Effects of high myoplasmic L-lactate concentration on E-C coupling in mammalian skeletal muscle.

The effects of high myoplasmic L-lactate concentrations (20-40 mM) at constant pH (7.1) were investigated on contractile protein function, voltage-dependent Ca(2+) release, and passive Ca(2+) leak from the sarcoplasmic reticulum (SR) in mechanically skinned fast-twitch (extensor digitorum longus; EDL) and slow-twitch (soleus) fibers of the rat. L-Lactate (20 mM) significantly reduced maximum Ca(2+)-activated force by 4 +/- 0.5% (n = 5, P < 0.05) and 5 +/- 0.4% (n = 6, P < 0.05) for EDL and soleus, respectively. The Ca(2+) sensitivity was also significantly decreased by 0.06 +/- 0. 002 (n = 5, P < 0.05) and 0.13 +/- 0.01 (n = 6, P < 0.001) pCa units, respectively. Exposure to L-lactate (20 mM) for 30 s reduced depolarization-induced force responses by ChCl substitution by 7 +/- 3% (n = 17, P < 0.05). This inhibition was not obviously affected by the presence of the lactate transport blocker quercetin (10 microM), or the chloride channel blocker anthracene-9-carboxylic acid (100 microM). L-Lactate (20 mM) increased passive Ca(2+) leak from the SR in EDL fibers (the integral of the response to caffeine was reduced by 16 +/- 5%, n = 9, P < 0.05) with no apparent effect in soleus fibers (100 +/- 2%, n = 3). These results indicate that the L-lactate ion per se has negligible effects on either voltage-dependent Ca(2+) release or SR Ca(2+) handling and exerts only a modest inhibitory effect on muscle contractility at the level of the contractile proteins.     

Phosphatidylinositol 4,5-bisphosphate enhances calcium release from sarcoplasmic reticulum of skeletal muscle

In the course of our study on the function of sarcoplasmic reticulum (SR) in skeletal muscle, the stimulatory action of phosphatidylinositol 4,5-bisphosphate (PIP2) on the Ca2+ release from SR was demonstrated by using chemically skinned fibers and fragmented SR vesicles. PIP2 induced a tension spike followed by sustained contraction in skinned fibers. PIP2 enhanced the caffeine-induced Ca2+ release from SR vesicles at low concentrations and triggered Ca2+ release by itself at high concentrations. PIP2 also enhanced 45Ca2+ efflux from SR vesicles. However, inositol 1,4,5-triphosphate never produced these effects. The Ca2+-releasing action of PIP2 was only weakly affected by ruthenium red or procaine. These observations suggest that PIP2 activates an SR Ca2+ release channel whose properties are different from those of the Ca2+-induced Ca2+ release channel.     

Muscle weakness in Ryr1I4895T/WT knock-in mice as a result of reduced ryanodine receptor Ca2+ ion permeation and release from the sarcoplasmic reticulum

The type 1 isoform of the ryanodine receptor (RYR1) is the Ca(2+) release channel of the sarcoplasmic reticulum (SR) that is activated during skeletal muscle excitation-contraction (EC) coupling. Mutations in the RYR1 gene cause several rare inherited skeletal muscle disorders, including malignant hyperthermia and central core disease (CCD). The human RYR1(I4898T) mutation is one of the most common CCD mutations. To elucidate the mechanism by which RYR1 function is altered by this mutation, we characterized in vivo muscle strength, EC coupling, SR Ca(2+) content, and RYR1 Ca(2+) release channel function using adult heterozygous Ryr1(I4895T/+) knock-in mice (IT/+). Compared with age-matched wild-type (WT) mice, IT/+ mice exhibited significantly reduced upper body and grip strength. In spite of normal total SR Ca(2+) content, both electrically evoked and 4-chloro-m-cresol-induced Ca(2+) release were significantly reduced and slowed in single intact flexor digitorum brevis fibers isolated from 4-6-mo-old IT/+ mice. The sensitivity of the SR Ca(2+) release mechanism to activation was not enhanced in fibers of IT/+ mice. Single-channel measurements of purified recombinant channels incorporated in planar lipid bilayers revealed that Ca(2+) permeation was abolished for homotetrameric IT channels and significantly reduced for heterotetrameric WT:IT channels. Collectively, these findings indicate that in vivo muscle weakness observed in IT/+ knock-in mice arises from a reduction in the magnitude and rate of RYR1 Ca(2+) release during EC coupling that results from the mutation producing a dominant-negative suppression of RYR1 channel Ca(2+) ion permeation.     

The effects of sphingosine on sarcoplasmic reticulum membrane calcium release.

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

Calcium source diversity in feline lower esophageal sphincter circular and sling muscle

Within muscular equivalents of cat lower esophageal sphincter (LES), the circular muscle develops greater spontaneous tone, whereas the sling muscle is more responsive to cholinergic stimulation. Smooth muscle contraction involves a combination of calcium release from stores and of calcium entry via several pathways. We hypothesized that there are differences in the sources of Ca(2+) used for contraction in sling and circular muscles and that these differences could contribute to functional asymmetry observed within LES. Contraction of muscle strips from circular and sling regions of LES was assessed in the presence of TTX. In Ca(2+)-free Krebs, tone was inhibited to a greater degree in circular than sling muscle. L-type Ca(2+) channel blockade with nifedipine or verapamil inhibited tone in LES circular but not sling muscle. Sarcoplasmic reticulum (SR) Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA) caused greater increase in tone in sling than in circular muscle. The phospholipase C inhibitor U-73122 and the SR inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] receptor blocker 2-aminoethoxydiphenyl borate (2-APB) inhibited tone in circular and sling muscles, demonstrating that continuous release of Ca(2+) from Ins(1,4,5)P(3)-sensitive stores is important in tone generation in both muscles. In Ca(2+)-free Krebs, ACh-induced contractions (AChC) were inhibited to a greater degree in sling than circular muscles. However, nifedipine and verapamil greatly inhibited AChC in the circular but not sling muscle. Depletion of SR Ca(2+) stores with CPA or inhibition of Ins(1,4,5)P(3)-mediated store release with either U-73122 or 2-APB inhibited AChC in both muscles. We demonstrate that LES circular and sling muscles 1) use intracellular and extracellular Ca(2+) sources to different degrees in the generation of spontaneous tone and AChC and 2) use different Ca(2+) entry pathways. These differences hold the potential for selective modulation of LES tone in health and disease.     

Complex effects of ryanodine on the sarcoplasmic reticulum Ca2+ levels in smooth muscle cells

We have studied the effects of ryanodine and inhibition of the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) with thapsigargin, on both [Ca(2+)](i) and the sarcoplasmic reticulum (SR) Ca(2+) level during caffeine-induced Ca(2+) release in single smooth muscle cells. Incubation with 10 microM ryanodine did not inhibit the first caffeine-induced [Ca(2+)](i) response, although it abolished the [Ca(2+)](i) response to a second application of caffeine. To assess whether ryanodine was inducing a permanent depletion of the internal Ca(2+) stores, we measured the SR Ca(2+) level with Mag-Fura-2. The magnitude of the caffeine-induced reduction in the SR Ca(2+) level was not augmented by incubating cells with 1 microM ryanodine. Moreover, on removal of caffeine, the SR Ca(2+) levels partially recovered in 61% of the cells due to the activity of thapsigargin-sensitive SERCA pumps. Unexpectedly, 10 microM ryanodine instead of inducing complete depletion of SR Ca(2+) stores markedly reduced the caffeine-induced SR Ca(2+) response. It was necessary to previously inhibit SERCA pumps with thapsigargin for ryanodine to be able to induce caffeine-triggered permanent depletion of SR Ca(2+) stores. These data suggest that the effect of ryanodine on smooth muscle SR Ca(2+) stores was markedly affected by the activity of SERCA pumps. Our data highlight the importance of directly measuring SR Ca(2+) levels to determine the effect of ryanodine on the internal Ca(2+) stores.