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.

     

Calcium transients in single fibers of low-frequency stimulated fast-twitch muscle of rat

Ca²⁺transients were investigated in single fibers isolated from ratextensor digitorum longus muscles exposed to chronic low-frequency stimulation for different time periods up to 10 days. Approximately 2.5-fold increases in resting Ca²⁺ concentration([Ca²⁺]) were observed 2 h after stimulationonset and persisted throughout the stimulation period. The elevated[Ca²⁺] levels were in the range characteristicof slow-twitch fibers from soleus muscle. In addition, we noticed atransitory elevation of the integral[Ca²⁺] per pulse with a maximum (~5-fold)after 1 day. Steep decreases in rate constant of[Ca²⁺] decay could be explained by an immediateimpairment of Ca²⁺ uptake and, with longer stimulationperiods, by an additional loss of cytosolic Ca²⁺ bindingcapacity resulting from a decay in parvalbumin content. A partialrecovery of the rate constant of [Ca²⁺] decayin 10-day stimulated muscle could be explained by an increasing mitochondrial contribution to Ca²⁺ sequestration.

     

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.

     

Effect of lactate on depolarization-induced Ca(2+) release in mechanically skinned skeletal muscle fibers

Itis unclear whether accumulation of lactate in skeletal muscle fibersduring intense activity contributes to muscle fatigue. Usingmechanically skinned fibers from rat and toad muscle, we were able toexamine the effect of L(+)-lactate onexcitation-contraction coupling independently of other metabolicchanges. We investigated the effects of lactate on the contractileapparatus, caffeine-induced Ca²⁺ release from thesarcoplasmic reticulum, and depolarization-induced Ca²⁺release. Lactate (15 or 30 mM) had only a small inhibitory effect directly on the contractile apparatus and caused appreciable(20-35%) inhibition of caffeine-induced Ca²⁺ release,seemingly by a direct effect on the Ca²⁺ release channels.However, 15 mM lactate had no detectable effect on Ca²⁺release when it was triggered by the normal voltage sensor mechanism, and 30 mM lactate reduced such release by only <10%. These results indicate that lactate has only a relatively small inhibitory effect onnormal excitation-contraction coupling, indicating that lactate accumulation per se is not a major factor in muscle fatigue.

     

Effects of a domain peptide of the ryanodine receptor on Ca2+ release in skinned skeletal muscle fibers

Mutations in the central domain of the skeletal muscle ryanodinereceptor (RyR) cause malignant hyperthermia (MH). A synthetic peptide(DP4) in this domain (Leu-2442-Pro-2477) produces enhanced ryanodine binding and sensitized Ca²⁺ release in isolatedsarcoplasmic reticulum, similar to the properties in MH, possiblybecause the peptide disrupts the normal interdomain interactions thatstabilize the closed state of the RyR (Yamamoto T, El-Hayek R, andIkemoto N. J Biol Chem 275: 11618-11625, 2000). Here, DP4 was applied to mechanically skinned fibers of rat muscle thathad the normal excitation-contraction coupling mechanism stillfunctional to determine whether muscle fiber responsiveness wasenhanced. DP4 (100 µM) substantially potentiated the Ca²⁺release and force response to caffeine (8 mM) and to low[Mg²⁺] (0.2 mM) in every fiber examined, with nosignificant effect on the properties of the contractile apparatus. DP4also potentiated the response to submaximal depolarization of thetransverse tubular system by ionic substitution. Importantly, DP4 didnot significantly alter the size of the twitch response elicited byaction potential stimulation. These results support the proposal thatDP4 causes an MH-like aberration in RyR function and are consistentwith the voltage sensor triggering Ca²⁺ release bydestabilizing the closed state of the RyRs.

     

Evidence for Na+/Ca2+ exchange in intact single skeletal muscle fibers from the mouse

The myoplasmic free Ca²⁺concentration([Ca²⁺]_i)was measured in intact single fibers from mouse skeletal muscle withthe fluorescent Ca²⁺ indicatorindo 1. Some fibers were perfused in a solution in which theconcentration of Na⁺ was reducedfrom 145.4 to 0.4 mM (low-Na⁺solution) in an attempt to activate reverse-modeNa⁺/Ca²⁺exchange (Ca²⁺ entry in exchangefor Na⁺ leaving the cell). Undernormal resting conditions, application oflow-Na⁺ solution only increased[Ca²⁺]_iby 5.8 ± 1.8 nM from a mean resting[Ca²⁺]_iof 42 nM. In other fibers,[Ca²⁺]_iwas elevated by stimulating sarcoplasmic reticulum (SR)Ca²⁺ release with caffeine (10 mM)and by inhibiting SR Ca²⁺ uptakewith2,5-di(tert-butyl)-1,4-benzohydroquinone(TBQ; 0.5 µM) in an attempt to activate forward-modeNa⁺/Ca²⁺exchange (Ca²⁺ removal from thecell in exchange for Na⁺ influx).These two agents caused a large increase in[Ca²⁺]_i,which then declined to a plateau level approximately twice the baseline[Ca²⁺]_iover 20 min. If the cell was allowed to recover between exposures tocaffeine and TBQ in a solution in whichCa²⁺ had been removed, theincrease in[Ca²⁺]_iduring the second exposure was very low, suggesting thatCa²⁺ had left the cell during theinitial exposure. Application of caffeine and TBQ to a preparation inlow-Na⁺ solution produced a large,sustained increase in[Ca²⁺]_iof ~1 µM. However, when cells were exposed to caffeine and TBQ in alow-Na⁺ solution in whichCa²⁺ had been removed, a sustainedincrease in[Ca²⁺]_iwas not observed, although[Ca²⁺]_iremained higher and declined slower than in normalNa⁺ solution. This suggests thatforward-modeNa⁺/Ca²⁺exchange contributed to the fall of[Ca²⁺]_iin normal Na⁺ solution, but whenextracellular Na⁺ was low, aprolonged elevation of[Ca²⁺]_icould activate reverse-modeNa⁺/Ca²⁺exchange. The results provide evidence that skeletal muscle fibers possess aNa⁺/Ca²⁺exchange mechanism that becomes active in its forward mode when [Ca²⁺]_iis increased to levels similar to that obtained during contraction.

     

Lactate inhibits Ca2+-activated Ca2+-channel activity from skeletal muscle sarcoplasmic reticulum

Favero, Terence G., Anthony C. Zable, David Colter, andJonathan J. Abramson. Lactate inhibits Ca²⁺-activatedCa²⁺-channel activity from skeletal muscle sarcoplasmicreticulum. J. Appl. Physiol. 82(2): 447-452, 1997.Sarcoplasmic reticulum (SR) Ca²⁺-release channelfunction is modified by ligands that are generated during about ofexercise. We have examined the effects of lactate on Ca²⁺-and caffeine-stimulated Ca²⁺ release,[³H]ryanodine binding, and singleCa²⁺-release channel activity of SR isolated from rabbitwhite skeletal muscle. Lactate, at concentrations from 10 to 30 mM,inhibited Ca²⁺- and caffeine-stimulated[³H]ryanodine binding to and inhibited Ca²⁺-and caffeine-stimulated Ca²⁺ release from SR vesicles.Lactate also inhibited caffeine activation of single-channel activityin bilayer reconstitution experiments. These findings suggest thatintense muscle activity, which generates high concentrations oflactate, will disrupt excitation-contraction coupling. This may lead todecreases in Ca²⁺ transients promoting a decline in tensiondevelopment and contribute to muscle fatigue.

     

Heterogeneity of calcium stores and elementary release events in canine pulmonary arterial smooth muscle cells

To examine the natureof inositol 1,4,5-trisphosphate (IP₃)-sensitive andryanodine (Ryn)-sensitive Ca²⁺ stores in isolated caninepulmonary arterial smooth cells (PASMC), agonist-induced changes inglobal intracellular Ca²⁺ concentration([Ca²⁺]_i) were measured using fura2-AM fluorescence. Properties of elementary local Ca²⁺release events were characterized using fluo 3-AM or fluo 4-AM, incombination with confocal laser scanning microscopy. In PASMC, depletion of sarcoplasmic reticulum Ca²⁺ stores with Ryn(300 µM) and caffeine (Caf; 10 mM) eliminated subsequent Caf-inducedintracellular Ca²⁺ transients but had little or no effecton the initial IP₃-mediated intracellular Ca²⁺transient induced by ANG II (1 µM). Cyclopiazonic acid (CPA; 10 µM) abolished IP₃-induced intracellularCa²⁺ transients but failed to attenuate the initialCaf-induced intracellular Ca²⁺ transient. These resultssuggest that in canine PASMC, IP₃-, and Ryn-sensitiveCa²⁺ stores are organized into spatially distinctcompartments while similar experiments in canine renal arterial smoothmuscle cells (RASMC) reveal that these Ca²⁺ stores arespatially conjoined. In PASMC, spontaneous local intracellular Ca²⁺ transients sensitive to modulation by Caf and Ryn weredetected, exhibiting spatial-temporal characteristics similar to thosepreviously described for "Ca²⁺ sparks" in cardiac andother types of smooth muscle cells. After depletion of Ryn-sensitiveCa²⁺ stores, ANG II (8 nM) induced slow, sustained[Ca²⁺]_i increases originating at sites nearthe cell surface, which were abolished by depleting IP₃stores. Discrete quantal-like events expected due to the coordinatedopening of IP₃ receptor clusters ("Ca²⁺puffs") were not observed. These data provide new information regarding the functional properties and organization of intracellular Ca²⁺ stores and elementary Ca²⁺ release eventsin isolated PASMC.

     

Differential modulation of caffeine- and IP3-induced calcium release in cultured arterial tissue

To investigatethe Ca²⁺-dependent plasticity ofsarcoplasmic reticulum (SR) function in vascular smooth muscle,transient responses to agents releasing intracellularCa²⁺ by either ryanodine(caffeine) orD-myo-inositol1,4,5-trisphosphate [IP₃;produced in response to norepinephrine (NE),5-hydroxytryptamine (5-HT), arginine vasopressin (AVP)] receptorsin rat tail arterial rings were evaluated after 4 days of organculture. Force transients induced by all agents were increased comparedwith those induced in fresh rings. Stimulation by 10% FCSduring culture further potentiated the force andCa²⁺ responses to caffeine (20 mM)but not to NE (10 µM), 5-HT (10 µM), or AVP (0.1 µM). The effectwas persistent, and SR capacity was not altered after reversibledepletion of stores with cyclopiazonic acid. The effects of serum couldbe mimicked by culture in depolarizing medium (30 mMK⁺) and blocked by the additionof verapamil (1 µM) or EGTA (1 mM) to the medium, loweringintracellular Ca²⁺ concentration([Ca²⁺]_i)during culture. These results show that modulation of SR function canoccur in vitro by a mechanism dependent on long-term levels of basal[Ca²⁺]_iand involving ryanodine- but notIP₃ receptor-mediatedCa²⁺release.     

All three ryanodine receptor isoforms generate rapid cooling responses in muscle cells

The rapid cooling (RC) response in muscle is an increase in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) that is probably caused by Ca²⁺ release from the sarcoplasmic reticulum (SR). However, the molecular bases of this response have not been completely elucidated. Three different isoforms of the SR Ca²⁺ release channels, or ryanodine receptors (RyRs), have been isolated (RyR1, RyR2, and RyR3). In the current investigation, the RC response was studied in RyR-null muscle cells (1B5) before and after transduction with HSV-1 virions containing the cDNAs encoding for RyR1, RyR2, or RyR3. Cells were loaded with fluo 4-AM to monitor changes in [Ca²⁺]_i and perfused with either cold (0°C), room temperature (RT), or RT buffer containing 40 mM caffeine. Control cells showed no significant response to cold or caffeine, whereas robust Ca²⁺ transients were recorded in response to both RC and caffeine in transduced cells expressing any one of the three RyR isoforms. Our data demonstrate directly that RyRs are responsible for the RC response and that all three isoforms respond in a similar manner. Ca²⁺ release from RyRs is likely caused by a RC-induced conformational change of the channel from the closed to the open state. calcium release channel; sarcoplasmic reticulum; excitation-contraction coupling     

Muscarinic stimulation increases basal Ca(2+) and inhibits spontaneous Ca(2+) transients in murine colonic myocytes

Localized Ca²⁺ transients inisolated murine colonic myocytes depend on Ca²⁺ releasefrom inositol 1,4,5-trisphosphate (IP₃) receptors.Localized Ca²⁺ transients couple to spontaneous transientoutward currents (STOCs) and mediate hyperpolarization responses inthese cells. We used confocal microscopy and whole cell patch-clamprecording to investigate how muscarinic stimulation, which causesformation of IP₃, can suppress Ca²⁺ transientsand STOCs that might override the excitatory nature of cholinergicresponses. ACh (10 µM) reduced localized Ca²⁺ transientsand STOCs, and these effects were associated with a rise in basalcytosolic Ca²⁺. These effects of ACh were mimicked bygeneralized rises in basal Ca²⁺ caused by ionomycin(250-500 nM) or elevated external Ca²⁺ (6 mM).Atropine (10 µM) abolished the effects of ACh. Pretreatment of cellswith nicardipine (1 µM), or Cd²⁺ (200 µM) had no effecton responses to ACh. An inhibitor of phospholipase C, U-73122, blockedCa²⁺ transients and STOCs but did not affect the increasein basal Ca²⁺ after ACh stimulation. Xestospongin C (Xe-C;5 µM), a membrane-permeable antagonist of IP₃ receptors,blocked spontaneous Ca²⁺ transients but did not prevent theincrease of basal Ca²⁺ in response to ACh. Gd³⁺(10 µM), a nonselective cation channel inhibitor, prevented the increase in basal Ca²⁺ after ACh and increased thefrequency and amplitude of Ca²⁺ transients and waves.Another inhibitor of receptor-mediated Ca²⁺ influxchannels, SKF-96365, also prevented the rise in basal Ca²⁺after ACh and increased Ca²⁺ transients and development ofCa²⁺ waves. FK-506, an inhibitor ofFKBP12/IP₃ receptor interactions, had no effect onthe rise in basal Ca²⁺ but blocked the inhibitory effectsof increased basal Ca²⁺ and ACh on Ca²⁺transients. These results suggest that the rise in basalCa²⁺ that accompanies muscarinic stimulation of colonicmuscles inhibits localized Ca²⁺ transients that couldcouple to activation of Ca²⁺-activated K⁺channels and reduce the excitatory effects of ACh.

     

Effects of lactic acid and catecholamines on contractility in fast-twitch muscles exposed to hyperkalemia

Intensive exercise is associated with a pronounced increase in extracellular K⁺ ([K⁺]_o). Because of the ensuing depolarization and loss of excitability, this contributes to muscle fatigue. Intensive exercise also increases the level of circulating catecholamines and lactic acid, which both have been shown to alleviate the depressing effect of hyperkalemia in slow-twitch muscles. Because of their larger exercise-induced loss of K⁺, fast-twitch muscles are more prone to fatigue caused by increased [K⁺]_o than slow-twitch muscles. Fast-twitch muscles also produce more lactic acid. We therefore compared the effects of catecholamines and lactic acid on the maintenance of contractility in rat fast-twitch [extensor digitorum longus (EDL)] and slow-twitch (soleus) muscles. Intact muscles were mounted on force transducers and stimulated electrically to evoke short isometric tetani. Elevated [K⁺]_o (11 and 13 mM) was used to reduce force to 20% of control force at 4 mM K⁺. In EDL, the ₂-agonist salbutamol (10^–5 M) restored tetanic force to 83 ± 2% of control force, whereas in soleus salbutamol restored tetanic force to 93 ± 1%. In both muscles, salbutamol induced hyperpolarization (5–8 mV), reduced intracellular Na⁺ content and increased Na⁺-K⁺ pump activity, leading to an increased K⁺ tolerance. Lactic acid (24 mM) restored force from 22 ± 4% to 58 ± 2% of control force in EDL, an effect that was significantly lower than in soleus muscle. These results amplify and generalize the concept that the exercise-induced acidification and increase in plasma catecholamines counterbalance fatigue arising from rundown of Na⁺ and K⁺ gradients. muscle fatigue; Na⁺-K⁺ pump; membrane potential     

Calcium phosphate precipitation in the sarcoplasmic reticulum reduces action potential-mediated Ca2+ release in mammalian skeletal muscle

During vigorous exercise, P_i concentration levels within the cytoplasm of fast-twitch muscle fibers may reach 30 mM. Cytoplasmic P_i may enter the sarcoplasmic reticulum (SR) and bind to Ca²⁺ to form a precipitate (CaP_i), thus reducing the amount of releasable Ca²⁺. Using mechanically skinned rat fast-twitch muscle fibers, which retain the normal action potential-mediated Ca²⁺ release mechanism, we investigated the consequences of P_i exposure on normal excitation-contraction coupling. The total amount of Ca²⁺ released from the SR by a combined caffeine/low-Mg²⁺ concentration stimulus was reduced by 20%, and the initial rate of force development slowed after 2-min exposure to 30 mM P_i (with or without the presence creatine phosphate). Peak (50 Hz) tetanic force was also reduced (by 25% and 45% after 10 and 30 mM P_i exposure, respectively). Tetanic force responses produced after 30 mM P_i exposure were nearly identical to those observed in the same fiber after depletion of total SR Ca²⁺ by 35%. Ca²⁺ content assays revealed that the total amount of Ca²⁺ in the SR was not detectably changed by exposure to 30 mM P_i, indicating that Ca²⁺ had not leaked from the SR but instead formed a precipitate with the P_i, reducing the amount of available Ca²⁺ for rapid release. These results suggest that CaP_i precipitation that occurs within the SR could contribute to the failure of Ca²⁺ release observed in the later stages of metabolic muscle fatigue. They also demonstrate that the total amount of Ca²⁺ stored in the SR cannot drop substantially below the normal endogenous level without reducing tetanic force responses. muscle fatigue; excitation-contraction coupling     

Inositol 1,4,5-trisphosphate receptors modulate Ca2+ sparks and Ca2+ store content in vas deferens myocytes

Spontaneous Ca²⁺ sparks were observed in fluo 4-loaded myocytes from guinea pig vas deferens with line-scan confocal imaging. They were abolished by ryanodine (100 µM), but the inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R) blockers 2-aminoethoxydiphenyl borate (2-APB; 100 µM) and intracellular heparin (5 mg/ml) increased spark frequency, rise time, duration, and spread. Very prolonged Ca²⁺ release events were also observed in 20% of cells treated with IP₃R blockers but not under control conditions. 2-APB and heparin abolished norepinephrine (10 µM; 0 Ca²⁺)-evoked Ca²⁺ transients but increased caffeine (10 mM; 0 Ca²⁺) transients in fura 2-loaded myocytes. Transients evoked by ionomycin (25 µM; 0 Ca²⁺) were also enhanced by 2-APB. Ca²⁺ sparks and transients evoked by norepinephrine and caffeine were abolished by thimerosal (100 µM), which sensitizes the IP₃R to IP₃. In cells voltage clamped at –40 mV, spontaneous transient outward currents (STOCs) were increased in frequency, amplitude, and duration in the presence of 2-APB. These data are consistent with a model in which the Ca²⁺ store content in smooth muscle is limited by tonic release of Ca²⁺ via an IP₃-dependent pathway. Blockade of IP₃Rs elevates sarcoplasmic reticulum store content, promoting Ca²⁺ sparks and STOC activity. calcium ion release; calcium ion transients; smooth muscle     

Shortening and [Ca2+] dynamics of left ventricular myocytes isolated from exercise-trained rats

The effects of run endurance training and fura 2 loading on the contractile function andCa²⁺ regulation of rat leftventricular myocytes were examined. In myocytes not loaded with fura 2, the maximal extent of myocyte shortening was reduced with trainingunder our pacing conditions [0.5 Hz at 2.0 and 0.75 mM externalCa²⁺ concentration([Ca²⁺]_o)], although training had noeffect on the temporal characteristics. The "light" loading ofmyocytes with fura 2 markedly suppressed (~50%) maximal shorteningin the sedentary and trained groups, although the temporalcharacteristics of myocyte shortening were significantly prolonged inthe trained group. No discernible differences in the dynamiccharacteristics of the intracellularCa²⁺ concentration([Ca²⁺]) transientwere detected at 2.0 mM[Ca²⁺]_o, althoughpeak [Ca²⁺] and rateof [Ca²⁺] rise duringcaffeine contracture were greater in the trained state at 0.75 mM[Ca²⁺]_o. We concludethat training induced a diminished myocyte contractile function underthe conditions studied here and a more effective coupling of inwardCa²⁺ current to sarcoplasmicreticulum Ca²⁺ release at low[Ca²⁺]_o,and that fura 2 and its loading vehicle DMSO significantly alter theintrinsic characteristics of myocyte contractile function andCa²⁺ regulation.

     

Effect of creatine on contractile force and sensitivity in mechanically skinned single fibers from rat skeletal muscle

Increasing the intramuscular stores of total creatine [TCr = creatine (Cr) + creatine phosphate (CrP)] can result in improved muscle performance during certain types of exercise in humans. Initial uptake of Cr is accompanied by an increase in cellular water to maintain osmotic balance, resulting in a decrease in myoplasmic ionic strength. Mechanically skinned single fibers from rat soleus (SOL) and extensor digitorum longus (EDL) muscles were used to examine the direct effects on the contractile apparatus of increasing [Cr], increasing [Cr] plus decreasing ionic strength, and increasing [Cr] and [CrP] with no change in ionic strength. Increasing [Cr] from 19 to 32 mM, accompanied by appropriate increases in water to maintain osmolality, had appreciable beneficial effects on contractile apparatus performance. Compared with control conditions, both SOL and EDL fibers showed increases in Ca²⁺ sensitivity (+0.061 ± 0.004 and +0.049 ± 0.009 pCa units, respectively) and maximum Ca²⁺-activated force (to 104 ± 1 and 105 ± 1%, respectively). In contrast, increasing [Cr] alone had a small inhibitory effect. When both [Cr] and [CrP] were increased, there was virtually no change in Ca²⁺ sensitivity of the contractile apparatus, and maximum Ca²⁺-activated force was 106 ± 1% compared with control conditions in both SOL and EDL fibers. These results suggest that the initial improvement in performance observed with Cr supplementation is likely due in large part to direct effects of the accompanying decrease in myoplasmic ionic strength on the properties of the contractile apparatus. ergogenic aid; muscle contraction; fatigue     

Changes in intracellular Ca2+ concentration induced by L-type Ca2+ channel current in guinea pig gastric myocytes

We investigatedthe relationship between voltage-operatedCa²⁺ channel current and thecorresponding intracellular Ca²⁺concentration([Ca²⁺]_i)change (Ca²⁺ transient) in guineapig gastric myocytes. Fluorescence microspectroscopy was combined withconventional whole cell patch-clamp technique, and fura 2 (80 µM) wasadded to CsCl-rich pipette solution. Step depolarization to 0 mVinduced inward Ca²⁺ current(I_Ca) andconcomitantly raised[Ca²⁺]_i.Both responses were suppressed by nicardipine, an L-typeCa²⁺ channel blocker, and thevoltage dependence of Ca²⁺transient was similar to the current-voltage relation ofI_Ca. When pulseduration was increased by up to 900 ms, peakCa²⁺ transient increased andreached a steady state when stimulation was for longer. The calculatedfast Ca²⁺ buffering capacity(B value), determined as the ratio ofthe time integral ofI_Ca divided bythe amplitude of Ca²⁺ transient,was not significantly increased after depletion of Ca²⁺ stores by the cyclicapplication of caffeine (10 mM) in the presence of ryanodine (4 µM).The addition of cyclopiazonic acid (CPA, 10 µM), a sarco(endo)plasmicreticulum Ca²⁺-ATPase inhibitor,decreased B value by ~20% in areversible manner. When KCl pipette solution was used,Ca²⁺-activatedK⁺ current[I_K(Ca)]was also recorded during step depolarization. CPA sensitivelysuppressed the initial peak and oscillations of I_K(Ca) withirregular effects on Ca²⁺transients. The above results suggest that, in guinea pig gastric myocyte, Ca²⁺ transient is tightlycoupled to I_Caduring depolarization, and global[Ca²⁺]_iis not significantly affected byCa²⁺-inducedCa²⁺ release from sarcoplasmicreticulum during depolarization.

     

Effects of chronic run training on Na+-dependent Ca2+ efflux from rat left ventricular myocytes

The effects ofendurance run training onNa⁺-dependentCa²⁺ regulation in rat leftventricular myocytes were examined. Myocytes were isolated fromsedentary and trained rats and loaded with fura 2. Contractile dynamicsand fluorescence ratio transients were recorded during electricalpacing at 0.5 Hz, 2 mM extracellular Ca²⁺ concentration, and 29°C.Resting and peak cytosolic Ca²⁺concentration([Ca²⁺]_c)did not change with exercise training. However, resting and peak[Ca²⁺]_cincreased significantly in both groups during 5 min of continuous pacing, although diastolic[Ca²⁺]_cin the trained group was less susceptible to this elevation ofintracellular Ca²⁺. Run trainingalso significantly reduced the rate of[Ca²⁺]_cdecay during relaxation. Myocytes were then exposed to 10 mM caffeinein the absence of external Na⁺ orCa²⁺ to trigger sarcoplasmicreticular Ca²⁺ release and tosuppress cellular Ca²⁺ efflux.This maneuver elicited an elevated steady-state[Ca²⁺]_c.External Na⁺ was then added, andthe rate of[Ca²⁺]_cclearance was determined. Run training significantly reduced the rateof Na⁺-dependent clearance of[Ca²⁺]_cduring the caffeine-induced contractures. These data demonstrate thatthe removal of cytosolic Ca²⁺ wasdepressed with exercise training under these experimental conditionsand may be specifically reflective of a training-induced decrease inthe rate of cytosolic Ca²⁺ removalviaNa⁺/Ca²⁺exchange and/or in the amount ofCa²⁺ moved across the sarcolemmaduring a contraction.     

Probing the extracellular release site of the plasma membrane calcium pump

Theplasma membrane Ca²⁺ pump is known to mediateCa²⁺/H⁺ exchange. Extracellular protonsactivated ⁴⁵Ca²⁺ efflux from human red bloodcells with a half-maximal inhibition constant of 2 nM when theintracellular pH was fixed. An increase in pH from 7.2 to 8.2 decreasedthe IC₅₀ for extracellular Ca²⁺ from ~33 to~6 mM. Changing the membrane potential by >54 mV had no effect onthe IC₅₀ for extracellular Ca²⁺. This arguesagainst Ca²⁺ release through a high-field access channel.Extracellular Ni²⁺ inhibited Ca²⁺ efflux withan IC₅₀ of 11 mM. Extracellular Cd²⁺ inhibitedwith an IC₅₀ of 1.5 mM, >10 times better thanCa²⁺. The Cd²⁺ IC₅₀ also decreasedwhen the pH was raised from 7.1 to 8.2, consistent withCa²⁺, Cd²⁺, and H⁺ competing forthe same site. The higher affinity for inhibition by Ni²⁺and Cd²⁺ is consistent with a histidine or cysteine as partof the release site. The cysteine reagent 2-(trimethylammonium)ethylmethanethiosulfonate did not inhibit Ca²⁺ efflux. Ourresults are consistent with the notion that the release site contains a histidine.

     

Role of extracellular [Ca2+] in fatigue of isolated mammalian skeletal muscle

The possiblerole of altered extracellular Ca²⁺concentration([Ca²⁺]_o)in skeletal muscle fatigue was tested on isolated slow-twitch soleusand fast-twitch extensor digitorum longus muscles of the mouse. Thefollowing findings were made. 1) Achange from the control solution (1.3 mM[Ca²⁺]_o)to 10 mM[Ca²⁺]_o,or to nominally Ca²⁺-freesolutions, had little effect on tetanic force in nonfatigued muscle.2) Almost complete restoration oftetanic force was induced by 10 mM[Ca²⁺]_oin severely K⁺-depressed muscle(extracellular K⁺ concentration of10-12 mM). This effect was attributed to a 5-mV reversal of theK⁺-induced depolarization andsubsequent restoration of ability to generate action potentials(inferred by using the twitch force-stimulation strength relationship).3) Tetanic force depressed bylowered extracellular Na⁺concentration (40 mM) was further reduced with 10 mM[Ca²⁺]_o.4) Tetanic force loss at elevatedextracellular K⁺ concentration (8 mM) and lowered extracellular Na⁺concentration (100 mM) was partially reversed with 10 mM[Ca²⁺]_oor markedly exacerbated with low[Ca²⁺]_o.5) Fatigue induced by using repeatedtetani in soleus was attenuated at 10 mM[Ca²⁺]_o(due to increased resting and evoked forces) and exacerbated at low[Ca²⁺]_o.These combined results suggest, first, that raised[Ca²⁺]_oprotects against fatigue rather than inducing it and, second, that aconsiderable depletion of[Ca²⁺]_oin the transverse tubules may contribute to fatigue.