{micro}-Calpain and calpain-3 are not autolyzed with exhaustive exercise in humans

µ-calpain and calpain-3 are Ca²⁺-dependent proteases found in skeletal muscle. Autolysis of calpains is observed using Western blot analysis as the cleaving of the full-length proteins to shorter products. Biochemical assays suggest that µ-calpain becomes proteolytically active in the presence of 2–200 µM Ca²⁺. Although calpain-3 is poorly understood, autolysis is thought to result in its activation, which is widely thought to occur at lower intracellular Ca²⁺ concentration levels ([Ca²⁺]_i; 1 µM) than the levels at which µ-calpain activation occurs. We have demonstrated the Ca²⁺-dependent autolysis of the calpains in human muscle samples and rat extensor digitorum longus (EDL) muscles homogenized in solutions mimicking the intracellular environment at various [Ca²⁺] levels (0, 2.5, 10, and 25 µM). Autolysis of calpain-3 was found to occur across a [Ca²⁺] range similar to that for µ-calpain, and both calpains displayed a seemingly higher Ca²⁺ sensitivity in human than in rat muscle homogenates, with 15% autolysis observed after 1-min exposure to 2.5 µM Ca²⁺ in human muscle and almost none after 1- to 2-min exposure to the same [Ca²⁺]_i level in rat muscle. During muscle activity, [Ca²⁺]_i may transiently peak in the range found to autolyze µ-calpain and calpain-3, so we examined the effect of two types of exhaustive cycling exercise (30-s "all-out" cycling, n = 8; and 70% O_{2 peak} until fatigue, n = 3) on the amount of autolyzed µ-calpain or calpain-3 in human muscle. No significant autolysis of µ-calpain or calpain-3 occurred as a result of the exercise. These findings have shown that the time- and concentration-dependent changes in [Ca²⁺]_i that occurred during concentric exercise fall near but below the level necessary to cause autolysis of calpains in vivo. Ca²⁺-dependent proteases; proteolysis     

Purification and characterization of calpain and calpastatin from rainbow trout, Oncorhynchus mykiss

Although the calpain system has been studied extensively in mammalian animals, much less is known about the properties of μ-calpain, m-calpain, and calpastatin in lower vertebrates such as fish. These three proteins were isolated and partly characterized from rainbow trout, Oncorhynchus mykiss, muscle. Trout m-calpain contains an 80-kDa large subunit, but the  26-kDa small subunit from trout m-calpain is smaller than the 28-kDa small subunit from mammalian calpains. Trout μ-calpain and calpastatin were only partly purified; identity of trout μ-calpain was confirmed by labeling with antibodies to bovine skeletal muscle μ-calpain, and identity of trout calpastatin was confirmed by specific inhibition of bovine skeletal muscle μ- and m-calpain. Trout μ-calpain requires 4.4 ± 2.8 μM and trout m-calpain requires 585 ± 51 μM Ca²⁺ for half-maximal activity, similar to the Ca²⁺ requirements of μ- and m-calpain from mammalian tissues. Sequencing tryptic peptides indicated that the amino acid sequence of trout calpastatin shares little homology with the amino acid sequences of mammalian calpastatins. Screening a rainbow trout cDNA library identified three cDNAs encoding for the large subunit of a putative m-calpain. The amino acid sequence predicted by trout m-calpain cDNA was 65% identical to the human 80-kDa m-calpain sequence. Gene duplication and polyploidy occur in fish, and the amino acid sequence of the trout m-calpain 80-kDa subunit identified in this study was 83% identical to the sequence of a trout m-calpain 80-kDa subunit described earlier. This is the first report of two isoforms of m-calpain in a single species.     

Sodium/calcium exchange in amphibian skeletal muscle fibers and isolated transverse tubules

TheNa⁺/Ca²⁺ exchanger participates inCa²⁺ homeostasis in a variety of cells and has a key rolein cardiac muscle physiology. We studied in this work the exchanger ofamphibian skeletal muscle, using both isolated inside-out transversetubule vesicles and single muscle fibers. In vesicles, increasingextravesicular (intracellular) Na⁺ concentrationcooperatively stimulated Ca²⁺ efflux (reverse mode), withthe Hill number equal to 2.8. In contrast to the stimulation of thecardiac exchanger, increasing extravesicular (cytoplasmic)Ca²⁺ concentration ([Ca²⁺]) inhibited thisreverse activity with an IC₅₀ of 91 nM. Exchanger-mediated currents were measured at 15°C in single fibers voltage clamped at90 mV. Photolysis of a cytoplasmic caged Ca²⁺ compoundactivated an inward current (forward mode) of 23 ± 10 nA(n = 3), with an average current density of 0.6 µA/µF. External Na⁺ withdrawal generated an outwardcurrent (reverse mode) with an average current density of 0.36 ± 0.17 µA/µF (n = 6) but produced a minimal increasein cytosolic [Ca²⁺]. These results suggest that, inskeletal muscle, the main function of the exchanger is to removeCa²⁺ from the cells after stimulation.

     

Imaging caffeine-induced Ca2+ transients in individual fast-twitch and slow-twitch rat skeletal muscle fibers

Fast-twitch and slow-twitch rat skeletal muscles producedissimilar contractures with caffeine. We used digital imagingmicroscopy to monitor Ca²⁺ (withfluo 3-acetoxymethyl ester) and sarcomere motion in intact, unrestrained rat muscle fibers to study this difference. Changes inCa²⁺ in individual fibers weremarkedly different from average responses of a population. All fibersshowed discrete, nonpropagated, local Ca²⁺ transients occurring randomlyin spots about one sarcomere apart. Caffeine increased localCa²⁺ transients and sarcomeremotion initially at 4 mM in soleus and 8 mM in extensor digitorumlongus (EDL; ~23°C). Ca²⁺release subsequently adapted or inactivated; this was surmounted byhigher doses. Motion also adapted but was not surmounted. Prolonged exposure to caffeine evidently suppressed myofilament interaction inboth types of fiber. In EDL fibers, 16 mM caffeine moderately increasedlocal Ca²⁺ transients. In soleusfibers, 16 mM caffeine greatly increased Ca²⁺ release and producedpropagated waves of Ca²⁺(~1.5-2.5 µm/s). Ca²⁺waves in slow-twitch fibers reflect the caffeine-sensitive mechanism ofCa²⁺-inducedCa²⁺ release. Fast-twitch fiberspossibly lack this mechanism, which could account for their lowersensitivity to caffeine.

     

Effect of clenbuterol on sarcoplasmic reticulum function in single skinned mammalian skeletal muscle fibers

We examined the effect of the₂-agonist clenbuterol (50 µM)on depolarization-induced force responses and sarcoplasmic reticulum (SR) function in muscle fibers of the rat (Rattusnorvegicus; killed by halothane overdose) that had beenmechanically skinned, rendering the₂-agonist pathway inoperable.Clenbuterol decreased the peak of depolarization-induced forceresponses in the extensor digitorum longus (EDL) and soleus fibers to77.2 ± 9.0 and 55.6 ± 5.4%, respectively, ofcontrols. The soleus fibers did not recover. Clenbuterol significantlyand reversibly reduced SR Ca²⁺loading in EDL and soleus fibers to 81.5 ± 2.8 and 78.7 ± 4.0%, respectively, of controls. Clenbuterol also producedan ~25% increase in passive leak ofCa²⁺ from the SR of the EDL andsoleus fibers. These results indicate that clenbuterol has directeffects on fast- and slow-twitch skeletal muscle, in the absence of the₂-agonist pathway. Theincreased Ca²⁺ leak in the triadregion may lead to excitation-contraction coupling damage in the soleusfibers and could also contribute to the anabolic effect of clenbuterolin vivo.

     

Elevated growth hormone increases the Ca2+ sensitivity of slow- and fast-twitch skeletal muscle of female rats

To determine theeffect of plasma growth hormone (GH) on skeletal muscle function, wemeasured the free Ca²⁺concentration-tension relationship of slow-twitch (soleus) and fast-twitch (peroneus longus) muscles isolated from rats undergoing acromegaly in response to implanted, GH-secreting tumors. Muscles fromadult (9 mo) and aged rats (24 mo) were studied after the tumor-bearingrats weighed over 50% more than their age-matched controls.Ca²⁺-activated isometric tensionwas recorded from skinned muscle fibers. For soleus muscles, the freeCa²⁺ concentration producing 50%of maximal tension([Ca²⁺]₅₀)was 2.0 µM for rats with tumors and 3.4-3.6 µM for controls. For peroneus longus fibers,[Ca²⁺]₅₀shifted from 6.1-6.7 µM in controls to 3.5 µM after tumors were introduced into either adult or aged rats. Soleus muscle fibersfrom neonatal rats (14 days) were less sensitive toCa²⁺ than those isolated fromadult rats, having a[Ca²⁺]₅₀of 7.3 µM. The Ca²⁺ sensitivityof peroneus longus fibers did not change with age. We conclude thatsignificant increases in myofibrillarCa²⁺ sensitivity occur in skeletalmuscles undergoing rapid growth induced by GH-secreting tumors.

     

Cyclopiazonic acid-induced changes in the contraction and Ca2+ transient of frog fast-twitch skeletal muscle

The effects ofcyclopiazonic acid (CPA) were investigated on isolated skeletal musclefibers of frog semitendinosus muscle. CPA (0.5-10 µM) enhancedisometric twitch but produced little change in resting tension. Athigher concentrations (10-50 µM), CPA depressed twitch andinduced sustained contracture without affecting resting and actionpotentials. In Triton-skinned fibers, CPA had no significant effect onmyofibrillar Ca²⁺ sensitivity butdecreased maximal activated force at concentrations >5 µM. Inintact cells loaded with the Ca²⁺fluorescence indicator indo 1, CPA (2 µM) induced an increase inCa²⁺-transient amplitude (10 ± 2.5%), which was associated with an increase in time to peak and inthe time constant of decay. Consequently, peak force was increased by35 ± 4%, and both time to peak and the time constant of relaxationwere prolonged. It is concluded that CPA effects, at a concentration ofup to 2 µM, were associated with specific inhibition of sarcoplasmicreticulumCa²⁺-adenosinetriphosphatase inintact skeletal muscle and that inhibition of the pump directlyaffected the handling of intracellularCa²⁺ and force production.

     

Effect of intracellular pH on acetylcholine-induced Ca2+ waves in mouse pancreatic acinar cells

We have used fluo3-loaded mouse pancreatic acinar cells to investigate the relationshipbetween Ca²⁺ mobilization andintracellular pH (pH_i). TheCa²⁺-mobilizing agonist ACh (500 nM) induced a Ca²⁺ release in theluminal cell pole followed by spreading of the Ca²⁺ signal toward the basolateralside with a mean speed of 16.1 ± 0.3 µm/s. In the presence of anacidic pH_i, achieved by blockade of theNa⁺/H⁺exchanger or by incubation of the cells in aNa⁺-free buffer, a slowerspreading of ACh-evoked Ca²⁺ waveswas observed (7.2 ± 0.6 µm/s and 7.5 ± 0.3 µm/s,respectively). The effects of cytosolic acidification on thepropagation rate of ACh-evokedCa²⁺ waves were largely reversibleand were not dependent on the presence of extracellularCa²⁺. A reduction in the spreadingspeed of Ca²⁺ waves could also beobserved by inhibition of the vacuolarH⁺-ATPase with bafilomycinA₁ (11.1 ± 0.6 µm/s), whichdid not lead to cytosolic acidification. In contrast, inhibition of theendoplasmic reticulum Ca²⁺-ATPaseby 2,5-di-tert-butylhydroquinone ledto faster spreading of the ACh-evokedCa²⁺ signals (25.6 ± 1.8 µm/s), which was also reduced by cytosolic acidification or treatmentof the cells with bafilomycin A₁.Cytosolic alkalinization had no effect on the spreading speed of theCa²⁺ signals. The data suggestthat the propagation rate of ACh-induced Ca²⁺ waves is decreased byinhibition of Ca²⁺ release fromintracellular stores due to cytosolic acidification or toCa²⁺ pool alkalinizationand/or to a decrease in the proton gradient directed from theinositol 1,4,5-trisphosphate-sensitiveCa²⁺ pool to the cytosol.

     

Specific knockdown of m-calpain blocks myogenesis with cDNA deduced from the corresponding RNAi

Fusion of mononuclear myoblast to multinucleated myotubes is crucial for myogenesis. Both µ- and m-calpain are ubiquitously expressed in most cells and are particularly abundant in muscle cells. Knockout of calpain-1 (catalytic subunit of µ-calpain) induced moderate platelet dysaggregation, preserving the normal development and growth, although knockout of calpain-2 (m-calpain) is lethal in mice. Therefore, there should be muscle-specific function of m-calpain per se. Previous methods lack direct evidence for the involvement of m-calpain, because the specific inhibitor to m-calpain has not been developed yet and the inhibition was less potent. Here, we show that screened RNA interference (RNAi) specifically blocked the m-calpain expression by 95% at both the protein and the activity levels. After transfection of adenovirus vector-mediated cDNA corresponding to the RNAi-induced short hairpin RNA, m-calpain in C₂C₁₂ myoblasts was knocked down with no compensatory overexpression of µ-calpain or calpain-3. The specific knockdown strongly inhibited the fusion to multinucleated myotubes. In addition, the knockdown modestly blocked ubiquitous effects, including cell migration, cell spreading, and alignment of central stress fiberlike structures. These results may indicate that m-calpain requiring millimolar Ca²⁺ level for the full activation plays specific roles in myogenesis, independent of µ-calpain, and leave us challenging problems in the future. RNA interference; muscle cell development; fusion; adenovirus vector     

Cadmium-induced ceramide formation triggers calpain-dependent apoptosis in cultured kidney proximal tubule cells

A major target of cadmium (Cd²⁺) toxicity is the kidney proximal tubule (PT) cell. Cd²⁺-induced apoptosis of PT cells is mediated by sequential activation of calpains at 3–6 h and caspases-9 and -3 after 24-h exposure. Calpains also partly contribute to caspase activation, which emphasizes the importance of calpains for PT apoptosis by Cd²⁺. Upstream processes underlying Cd²⁺-induced calpain activation remain unclear. We describe for the first time that 10–50 µM Cd²⁺ causes a significant increase in ceramide formation by 22% (3 h) and 72% (24 h), as measured by diacylglycerol kinase assay. Inhibition of ceramide synthase with fumonisin B₁ (3 µM) prevents ceramide formation at 3 h and abolishes calpain activation at 6 h, which is associated with significant attenuation of apoptosis at 3–6 h with Hoechst 33342 nuclear staining and/or 3(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) death assays. This indicates that Cd²⁺ enhances de novo ceramide synthesis and that calpains are a downstream target of ceramides in apoptosis execution. Moreover, addition of C₆-ceramide to PT cells increases cytosolic Ca²⁺ and activates calpains. Apoptosis mediated by C₆-ceramide at 24 h is significantly reduced by caspase-3 inhibition, which supports cross talk between calpain- and caspase-dependent apoptotic pathways. We conclude that Cd²⁺-induced apoptosis of PT cells entails endogenous ceramide elevation and subsequent Ca²⁺-dependent calpain activation, which propagates kidney damage by Cd²⁺. nephrotoxicity; cell signaling; cell biology and structure     

Long-lasting muscle fatigue: partial disruption of excitation-contraction coupling by elevated cytosolic Ca2+ concentration during contractions

The repeated elevation of cytosolic Ca²⁺ concentration ([Ca²⁺]_i) above resting levels during contractile activity has been associated with long-lasting muscle fatigue. The mechanism underlying this fatigue appears to involve elevated [Ca²⁺]_i levels that induce disruption of the excitation-contraction (E-C) coupling process at the triad junction. Unclear, however, are which aspects of the activity-related [Ca²⁺]_i changes are responsible for the deleterious effects, in particular whether they depend primarily on the peak [Ca²⁺]_i reached locally at particular sites or on the temporal summation of the increased [Ca²⁺] in the cytoplasm as a whole. In this study, we used mechanically skinned fibers from rat extensor digitorum longus muscle, in which the normal E-C coupling process remains intact. The [Ca²⁺]_i was raised either by applying a set elevated [Ca²⁺] throughout the fiber or by using action potential stimulation to induce the release of sarcoplasmic reticulum Ca²⁺ by the normal E-C coupling system with or without augmentation by caffeine or buffering with BAPTA. Herein we show that elevating [Ca²⁺]_i in the physiological range of 2–20 µM irreversibly disrupts E-C coupling in a concentration-dependent manner but requires exposure for a relatively long time (1–3 min) to cause substantial uncoupling. The effectiveness of Ca²⁺ released via the endogenous system in disrupting E-C coupling indicates that the relatively high [Ca²⁺]_i attained close to the release site at the triad junction is a more important factor than the increase in bulk [Ca²⁺]_i. Our results suggest that during prolonged vigorous activity, the many repeated episodes of relatively high triadic [Ca²⁺] can disrupt E-C coupling and lead to long-lasting fatigue. skeletal muscle; low-frequency fatigue; ryanodine receptor; skinned fiber     

cGMP-mediated Ca2+ release from IP3-insensitive Ca2+ stores in smooth muscle

Recent studies on the role of nitric oxide (NO) ingastrointestinal smooth muscle have raised the possibility thatNO-stimulated cGMP could, in the absence of cGMP-dependent proteinkinase (PKG) activity, act as aCa²⁺-mobilizing messenger[K. S. Murthy, K.-M. Zhang, J.-G. Jin, J. T. Grider, and G. M. Makhlouf. Am. J. Physiol. 265 (Gastrointest. Liver Physiol. 28):G660-G671, 1993]. This notion was examined indispersed gastric smooth muscle cells with 8-bromo-cGMP (8-BrcGMP) andwith NO and vasoactive intestinal peptide (VIP), which stimulate endogenous cGMP. In muscle cells treated with cAMP-dependent protein kinase (PKA) and PKG inhibitors (H-89 and KT-5823), 8-BrcGMP (10 µM),NO (1 µM), and VIP (1 µM) stimulated⁴⁵Ca²⁺release (21 ± 3 to 30 ± 1% decrease in⁴⁵Ca²⁺cell content); Ca²⁺ releasestimulated by 8-BrcGMP was concentration dependent with anEC₅₀ of 0.4 ± 0.1 µM and athreshold of 10 nM. 8-BrcGMP and NO increased cytosolic freeCa²⁺ concentration([Ca²⁺]_i)and induced contraction; both responses were abolished after Ca²⁺ stores were depleted withthapsigargin. With VIP, which normally increases[Ca²⁺]_iby stimulating Ca²⁺ influx,treatment with PKA and PKG inhibitors caused a further increase in[Ca²⁺]_ithat reverted to control levels in cells pretreated with thapsigargin. Neither Ca²⁺ release norcontraction induced by cGMP and NO in permeabilized muscle cells wasaffected by heparin or ruthenium red.Ca²⁺ release induced by maximallyeffective concentrations of cGMP and inositol 1,4,5-trisphosphate(IP₃) was additive, independent of which agent was applied first. We conclude that, in the absence ofPKA and PKG activity, cGMP stimulatesCa²⁺ release from anIP₃-insensitive store and that itseffect is additive to that of IP₃.

     

Effects of dihydropyridine receptor II-III loop peptides on Ca(2+) release in skinned skeletal muscle fibers

Inskeletal muscle fibers, the intracellular loop between domains II andIII of the ₁-subunit of the dihydropyridine receptor (DHPR) may directly activate the adjacent Ca²⁺ releasechannel in the sarcoplasmic reticulum. We examined the effects ofsynthetic peptide segments of this loop on Ca²⁺ release inmechanically skinned skeletal muscle fibers with functional excitation-contraction coupling. In rat fibers at physiological Mg²⁺ concentration ([Mg²⁺]; 1 mM), a20-residue skeletal muscle DHPR peptide[A_S(20);Thr⁶⁷¹-Leu⁶⁹⁰; 30 µM], shown previously toinduce Ca²⁺ release in a triad preparation, caused onlysmall spontaneous force responses in ~40% of fibers, although itpotentiated responses to depolarization and caffeine in all fibers. TheCOOH-terminal half of A_S(20)[A_S(10)] induced much larger spontaneousresponses but also caused substantial inhibition of Ca²⁺release to both depolarization and caffeine. Both peptides induced orpotentiated Ca²⁺ release even when the voltage sensors wereinactivated, indicating direct action on the Ca²⁺ releasechannels. The corresponding 20-residue cardiac DHPR peptide [A_C(20);Thr⁷⁹³-Ala⁸¹²] was ineffective, but itsCOOH-terminal half [A_C(10)] had effects similar to A_S(20). In the presence of lower[Mg²⁺] (0.2 mM), exposure to eitherA_S(20) or A_C(10) (30 µM) induced substantial Ca²⁺ release. PeptideC_S (100 µM), a loop segment reported to inhibit Ca²⁺ release in triads, caused partial inhibition ofdepolarization-induced Ca²⁺ release. In toad fibers, eachof the A peptides had effects similar to or greater than those in ratfibers. These findings suggest that the A and C regions of the skeletalDHPR II-III loop may have important roles in vivo.

     

Differential Ca(2+) sensitivity of skeletal and cardiac muscle ryanodine receptors in the presence of calmodulin

Calmodulin (CaM) activates the skeletal muscle ryanodine receptorCa²⁺ release channel (RyR1) in the presence of nanomolarCa²⁺ concentrations. However, the role of CaM activation inthe mechanisms that control Ca²⁺ release from thesarcoplasmic reticulum (SR) in skeletal muscle and in the heart remainsunclear. In media that contained 100 nM Ca²⁺, the rate of⁴⁵Ca²⁺ release from porcine skeletal muscle SRvesicles was increased approximately threefold in the presence of CaM(1 µM). In contrast, cardiac SR vesicle⁴⁵Ca²⁺ release was unaffected by CaM,suggesting that CaM activated the skeletal RyR1 but not the cardiacRyR2 channel isoform. The activation of RyR1 by CaM was associated withan approximately sixfold increase in the Ca²⁺ sensitivityof [³H]ryanodine binding to skeletal muscle SR, whereasthe Ca²⁺ sensitivity of cardiac SR[³H]ryanodine binding was similar in the absence andpresence of CaM. Cross-linking experiments identified both RyR1 andRyR2 as predominant CaM binding proteins in skeletal and cardiac SR,respectively, and [³⁵S]CaM binding determinations furtherindicated comparable CaM binding to the two isoforms in the presence ofmicromolar Ca²⁺. In nanomolar Ca²⁺, however,the affinity and stoichiometry of RyR2 [³⁵S]CaM bindingwas reduced compared with that of RyR1. Together, our results indicatethat CaM activates RyR1 by increasing the Ca²⁺ sensitivityof the channel, and further suggest differences in CaM's functionalinteractions with the RyR1 and RyR2 isoforms that may potentiallycontribute to differences in the Ca²⁺ dependence of channelactivation in skeletal and cardiac muscle.

     

Stretch-induced Ca(2+) release via an IP(3)-insensitive Ca(2+) channel

Various mechanicalstimuli increase the intracellular Ca²⁺ concentration([Ca²⁺]_i) in vascular smooth muscle cells(VSMC). A part of the increase in [Ca²⁺]_i isdue to the release of Ca²⁺ from intracellular stores. Wehave investigated the effect of mechanical stimulation produced bycyclical stretch on the release of Ca²⁺ from theintracellular stores. Permeabilized VSMC loaded with ⁴⁵Ca²⁺ were subjected to 7.5% average (15%maximal) cyclical stretch. This resulted in an increase in⁴⁵Ca²⁺ rate constant by 0.126 ± 0.0035. Inhibition of inositol 1,4,5-trisphosphate (IP₃),ryanodine, and nicotinic acid adenine dinucleotide phosphate channels(NAADP) with 50 µg/ml heparin, 50 µM ruthenium red, and 25 µMthio-NADP, respectively, did not block the increase in⁴⁵Ca²⁺ efflux in response to cyclical stretch.However, 10 µM lanthanum, 10 µM gadolinium, and 10 µMcytochalasin D but not 10 µM nocodazole inhibited the increase in⁴⁵Ca²⁺ efflux. This supports the existence of anovel stretch-sensitive intracellular Ca²⁺ store in VSMCthat is distinct from the IP₃-, ryanodine-, and NAADP-sensitive stores.

     

Effects of elevated physiological temperatures on sarcoplasmic reticulum function in mechanically skinned muscle fibers of the rat

Properties of the sarcoplasmic reticulum (SR) with respect to Ca²⁺ loading and release were measured in mechanically skinned fiber preparations from isolated extensor digitorum longus (EDL) muscles of the rat that were either kept at room temperature (23°C) or exposed to temperatures in the upper physiological range for mammalian skeletal muscle (30 min at 40 or 43°C). The ability of the SR to accumulate Ca²⁺ was significantly reduced by a factor of 1.9–2.1 after the temperature treatments due to a marked increase in SR Ca²⁺ leak, which persisted for at least 3 h after treatment. Results with blockers of Ca²⁺ release channels (ruthenium red) and SR Ca²⁺ pumps [2,5-di(tert-butyl)-1,4-hydroquinone] indicate that the increased Ca²⁺ leak was not through the SR Ca²⁺ release channel or the SR Ca²⁺ pump, although it is possible that the leak pathway was via oligomerized Ca²⁺ pump molecules. No significant change in the maximum SR Ca²⁺-ATPase activity was observed after the temperature treatment, although there was a tendency for a decrease in the SR Ca²⁺-ATPase. The observed changes in SR properties were fully prevented by the superoxide (O₂^–) scavenger Tiron (20 mM), indicating that the production of O₂^– at elevated temperatures is responsible for the increase in SR Ca²⁺ leak. Results show that physiologically relevant elevated temperatures 1) induce lasting changes in SR properties with respect to Ca²⁺ handling that contribute to a marked increase in the SR Ca²⁺ leak and, consequently, to the reduction in the average coupling ratio between Ca²⁺ transport and SR Ca²⁺-ATPase and muscle performance, and 2) that these changes are mediated by temperature-induced O₂^– production. skeletal muscle; calcium ion leak; superoxide; skinned fibers     

Pacemaker activity in urethral interstitial cells is not dependent on capacitative calcium entry

The aim of the present study was to investigate the properties and role of capacitative Ca²⁺ entry (CCE) in interstitial cells (IC) isolated from the rabbit urethra. Ca²⁺ entry in IC was larger in cells with depleted intracellular Ca²⁺ stores compared with controls, consistent with influx via a CCE pathway. The nonselective Ca²⁺ entry blockers Gd³⁺ (10 µM), La³⁺ (10 µM), and Ni²⁺ (100 µM) reduced CCE by 67% (n = 14), 65% (n = 11), and 55% (n = 9), respectively. These agents did not inhibit Ca²⁺ entry when stores were not depleted. Conversely, CCE in IC was resistant to SKF-96365 (10 µM), wortmannin (10 µM), and nifedipine (1 µM). Spontaneous transient inward currents were recorded from IC voltage-clamped at –60 mV. These events were not significantly affected by Gd³⁺ (10 µM) or La³⁺ (10 µM) and were only slightly decreased in amplitude by 100 µM Ni²⁺. The results from this study demonstrate that freshly dispersed IC from the rabbit urethra possess a CCE pathway. However, influx via this pathway does not appear to contribute to spontaneous activity in these cells. smooth muscle; patch clamp; spontaneous transient inward currents     

Hypotonic swelling-induced Ca2+ release by an IP3-insensitive Ca2+ store

Hypotonicswelling increases the intracellular Ca²⁺ concentration([Ca²⁺]_i) in vascular smooth muscle cells(VSMC). The source of this Ca²⁺ is not clear. To study thesource of increase in [Ca²⁺]_i in response tohypotonic swelling, we measured [Ca²⁺]_i infura 2-loaded cultured VSMC (A7r5 cells). Hypotonic swelling produced a40.7-nM increase in [Ca²⁺]_i that was notinhibited by EGTA but was inhibited by 1 µM thapsigargin. Priordepletion of inositol 1,4,5-trisphosphate (IP₃)-sensitive Ca²⁺ stores with vasopressin did not inhibit the increasein [Ca²⁺]_i in response to hypotonic swelling.Exposure of ⁴⁵Ca²⁺-loaded intracellular storesto hypotonic swelling in permeabilized VSMC produced an increase in⁴⁵Ca²⁺ efflux, which was inhibited by 1 µMthapsigargin but not by 50 µg/ml heparin, 50 µM ruthenium red, or25 µM thio-NADP. Thus hypotonic swelling of VSMC causes a release ofCa²⁺ from the intracellular stores from a novel sitedistinct from the IP₃-, ryanodine-, and nicotinic acidadenine dinucleotide phosphate-sensitive stores.

     

Ryanodine receptor expression is associated with intracellular Ca2+ release in rat parotid acinar cells

The ryanodinereceptor mediates intracellularCa²⁺ mobilization in muscle andnerve, but its physiological role in nonexcitable cells is less welldefined. Like adenosine 3',5'-cyclic monophosphate andinositol 1,4,5-trisphosphate, cyclic ADP-ribose (0.3-5 µM) andADP (1-25 µM) produced a concentration-dependent rise incytosolic Ca²⁺ in permeabilizedrat parotid acinar cells. Adenosine and AMP were less effective.Ryanodine markedly depressed theCa²⁺-mobilizing action of theadenine nucleotides and forskolin in permeabilized cells and waslikewise effective in depressing the action of forskolin in intactcells. Cyclic ADP-ribose-evoked Ca²⁺ release was enhanced bycalmodulin and depressed by W-7, a calmodulin inhibitor. Afluorescently labeled ligand,4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3,4-diaza-s-indacene-3-propionic acid-glycyl ryanodine, was synthesized to detect the expression anddistribution of ryanodine receptors. In addition, ryanodine receptorexpression was detected in rat parotid cells with a sequence highlyhomologous to a rat skeletal muscle type 1 and a novel brain type 1 ryanodine receptor. These findings demonstrate the presence of aryanodine-sensitive intracellularCa²⁺ store in rat parotid cellsthat shares many of the characteristics of stores in muscle and nerveand may mediate Ca²⁺-inducedCa²⁺ release or a modified form ofthis process.

     

Aortic smooth muscle and endothelial plasma membrane Ca2+ pump isoforms are inhibited differently by the extracellular inhibitor caloxin 1b1

Plasma membrane Ca²⁺ pumps (PMCA) that expel Ca²⁺ from cells are encoded by four genes (PMCA1–4). In this study, we show that aortic endothelium and smooth muscle differ in their PMCA isoform mRNA expression: endothelium expressed predominantly PMCA1, and smooth muscle expressed PMCA4 and a lower level of PMCA1. In this study, we report a novel peptide (caloxin 1b1, obtained by screening for binding to extracellular domain 1 of PMCA4), which inhibited PMCA extracellularly, selectively, and had a higher affinity for PMCA4 than PMCA1. It inhibited the PMCA Ca²⁺-Mg²⁺-ATPase activity in leaky erythrocyte ghosts (mainly PMCA4) with a K_i value of 46 ± 5 µM, making it 10x more potent than the previously reported caloxin 2a1. It was isoform selective because it inhibited the PMCA1 Ca²⁺-Mg²⁺-ATPase in human embryonic kidney-293 cells with a higher K_i value (105 ± 11 µM) than for PMCA4. Caloxin 1b1 was selective in that it did not inhibit other ATPases. Because caloxin 1b1 had been selected to bind to an extracellular domain of PMCA, it could be added directly to cells and tissues to examine its effects on smooth muscle and endothelium. In deendothelialized aortic rings, caloxin 1b1 (200 µM) produced a contraction. It also increased the force of contraction produced by a submaximum concentration of phenylephrine. In aortic rings with endothelium intact, precontracted with phenylephrine and relaxed partially with a submaximum concentration of carbachol, caloxin 1b1 increased the force of contraction rather than potentiating the endothelium-dependent relaxation. In cultured cells, caloxin 1b1 increased the cytosolic [Ca²⁺] more in arterial smooth muscle cells than in endothelial cells. Thus caloxin 1b1 is the first highly selective extracellular PMCA inhibitor that works better on vascular smooth muscle than on endothelium. coronary artery; rat aorta; smooth muscle; endothelium