Regulation of sarcoplasmic reticulum Ca2+ reuptake in porcine airway smooth muscle

Regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) in airway smooth muscle (ASM) during agonist stimulation involves sarcoplasmic reticulum (SR) Ca(2+) release and reuptake. The sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) is key to replenishment of SR Ca(2+) stores. We examined regulation of SERCA in porcine ASM: our hypothesis was that the regulatory protein phospholamban (PLN) and the calmodulin (CaM)-CaM kinase (CaMKII) pathway (both of which are known to regulate SERCA in cardiac muscle) play a role. In porcine ASM microsomes, we examined the expression and extent of PLN phosphorylation after pharmacological inhibition of CaM (with W-7) vs. CaMKII (with KN-62/KN-93) and found that PLN is phosphorylated by CaMKII. In parallel experiments using enzymatically dissociated single ASM cells loaded with the Ca(2+) indicator fluo 3 and imaged using fluorescence microscopy, we measured the effects of PLN small interfering RNA, W-7, and KN-62 on [Ca(2+)](i) responses to ACh and direct SR stimulation. PLN small interfering RNA slowed the rate of fall of [Ca(2+)](i) transients to 1 microM ACh, as did W-7 and KN-62. The two inhibitors additionally slowed reuptake in the absence of PLN. In other cells, preexposure to W-7 or KN-62 did not prevent initiation of ACh-induced [Ca(2+)](i) oscillations (which were previously shown to result from repetitive SR Ca(2+) release/reuptake). However, when ACh-induced [Ca(2+)](i) oscillations reached steady state, subsequent exposure to W7 or KN-62 decreased oscillation frequency and amplitude and slowed the fall time of [Ca(2+)](i) transients, suggesting SERCA inhibition. Exposure to W-7 completely abolished ongoing ACh-induced [Ca(2+)](i) oscillations in some cells. Preexposure to W-7 or KN-62 did not affect caffeine-induced SR Ca(2+) release, indicating that ryanodine receptor channels were not directly inhibited. These data indicate that, in porcine ASM, the CaM-CaMKII pathway regulates SR Ca(2+) reuptake, potentially through altered PLN phosphorylation.     

Ca2+-induced sarcoplasmic reticulum Ca2+ release in myotubularin-deficient muscle fibers

Skeletal muscle deficiency in the 3-phosphoinositide (PtdInsP) phosphatase myotubularin (MTM1) causes myotubular myopathy which is associated with severe depression of voltage-activated sarcoplasmic reticulum Ca²⁺ release through ryanodine receptors. In the present study we aimed at further understanding how Ca²⁺ release is altered in MTM1-deficient muscle fibers, at rest and during activation. While in wild-type muscle fibers, SR Ca²⁺ release exhibits fast stereotyped kinetics of activation and decay throughout the voltage range of activation, Ca²⁺ release in MTM1-deficient muscle fibers exhibits slow and unconventional kinetics at intermediate voltages, suggestive of partial loss of the normal control of ryanodine receptor Ca²⁺ channel activity. In addition, the diseased muscle fibers at rest exhibit spontaneous elementary Ca²⁺ release events at a frequency 30 times greater than that of control fibers. Eighty percent of the events have spatiotemporal properties of archetypal Ca²⁺ sparks while the rest take either the form of lower amplitude, longer duration Ca²⁺ release events or of a combination thereof. The events occur at preferred locations in the fibers, indicating spatially uneven distribution of the parameters determining spontaneous ryanodine receptor 1 opening. Spatially large Ca²⁺ release sources were obviously involved in some of these events, suggesting that opening of ryanodine receptors in one cluster can activate opening of ryanodine receptors in a neighboring one. Overall results demonstrate that opening of Ca²⁺-activated ryanodine receptors is promoted both at rest and during excitation-contraction coupling in MTM1-deficient muscle fibers. Because access to this activation mode is denied to ryanodine receptors in healthy skeletal muscle, this may play an important role in the associated disease situation.     

Properties of the sarcoplasmic reticulum Ca2+-pump in coronary artery skinned smooth muscle

Pig coronary artery cultured smooth muscle cells were skinned using saponin. In the presence of an ATP-regenerating system and oxalate, the skinned cells showed an ATP-dependent azide insensitive Ca²⁺-uptake which increased linearly with time for >1 h. The Ca²⁺-uptake occurred with K_m values of 0.20±0.03 M for Ca²⁺ and 400±34 M for MgATP^2–. Thapsigargin and cyclopiazonic acid inhibited this uptake with IC₅₀ values of 0.13±0.02 and 0.56±0.04 M, respectively. These properties of SR Ca²⁺-pump are similar to those reported for membrane fractions isolated from fresh smooth muscle of coronary artery and other arteries. However, optimum pH of the uptake in the skinned cells (6.2) was lower than that reported previously using isolated membranes (6.4–6.8).Abbreviations SR sarcoplasmic reticulum - ER endoplasmic reticulum - PM plasma membrane - CPA cyclopiazonic acid - DTT dithiothreitol     

The sarcoplasmic reticulum and sarcolemma together form a passive Ca2+ trap in colonic smooth muscle

In smooth muscle, active Ca(2+) uptake into regions of sarcoplasmic reticulum (SR) which are closely apposed to the sarcolemma has been proposed to substantially limit the increase in the cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) following Ca(2+) influx, i.e. the 'superficial buffer barrier hypothesis'. The present study has re-examined this proposal. The results suggest that the SR close to the sarcolemma acts as a passive barrier to Ca(2+) influx limiting [Ca(2+)](c) changes; for this, SR Ca(2+) pump activity is not required. In single voltage-clamped colonic myocytes, sustained opening of the ryanodine receptor (RyR) (and depletion of the SR) using ryanodine increased the amplitude of depolarisation-evoked Ca(2+) transients and accelerated the rate of [Ca(2+)](c) decline following depolarisation. These results could be explained by a reduction in the Ca(2+) buffer power of the cytosol taking place when RyR are opened (i.e. the SR is 'leaky'). Indeed, determination of the Ca(2+) buffer power confirmed it was reduced by approximately 40%. Inhibition of the SR Ca(2+) pump (with thapsigargin) also depleted the SR of Ca(2+) but did not reduce the Ca(2+) buffer power or increase depolarisation-evoked Ca(2+) transients and slowed (rather than accelerated) Ca(2+) removal. However, thapsigargin prevented the ryanodine-induced increase in [Ca(2+)](c) decline following depolarisation. Together, these results suggest that when the SR was rendered 'leaky' (a) more of the Ca(2+) entering the cell reached the bulk cytoplasm and (b) Ca(2+) was removed more quickly at the end of cell activation. Under physiological circumstances in the absence of blocking drugs, it is proposed that the SR limits the [Ca(2+)](c) increase following influx without the need for active Ca(2+) uptake. The SR and sarcolemma may form a passive physical barrier to Ca(2+) influx, a Ca(2+) trap, which limits the [Ca(2+)](c) rise occurring during depolarisation by about 50% and from which the ion only slowly escapes into the main part of the cytoplasm.     

The sarcoplasmic reticulum Ca2+-ATPase

Summary This review summarizes studies on the structural organization of Ca²⁺-ATPase in the sarcoplasmic reticulum membrane in relation to the function of the transport protein. Recent advances in this field have been made by a combination of protein-chemical, ultrastructural, and physicochemical techniques on membraneous and detergent solubilized ATPase. A particular feature of the ATPase (Part I) is the presence of a hydrophilic head, facing the cytoplasm, and a tail inserted in the membrane. In agreement with this view the protein is moderately hydrophobic, compared to many other integral membrane proteins, and the number of traverses of the 115 000 Dalton peptide chain through the lipid may be limited to 3–4.There is increasing evidence (Part II) that the ATPase is self-associated in the membrane in oligomeric form. This appears to be a common feature of many transport proteins. Each ATPase peptide seems to be able to perform the whole catalytic cycle of ATP hydrolysis and Ca²⁺ transport. Protein-protein interactions seem to have a modulatory effect on enzyme activity and to stabilize the enzyme against inactivation.Phospholipids (Part III) are not essential for the expression of enzyme activity which only requires the presence of flexible hydrocarbon chains that can be provided e.g. by polyoxyethylene glycol detergents. Perturbation of the lipid bilayer by the insertion of membrane protein leads to some immobilization of the lipid hydrocarbon chains, but not to the extent envisaged by the annulus hypothesis. Strong immobilization, whenever it occurs, may arise from steric hindrance due to protein-protein contacts. Recent studies suggest that breaks in Arrhenius plots of enzyme activity primarily reflect intrinsic properties of the protein rather than changes in the character of lipid motion as a function of temperature.     

Ionomycin releases calcium from the sarcoplasmic reticulum and activates Na+/Ca2+ exchange in vascular smooth muscle cells

Ionomycin (1 microM) produced a large spike in cytosolic free Ca2+ [( Ca2+]i). The ionophore had no effect on [Ca2+]i if the sarcoplasmic reticulum had previously been Ca2+ depleted by stimulating neurohormone receptors. Ionomycin markedly increased 45Ca2+ efflux and decreased total cell Ca2+ by 60 to 70% in 1 min. Replacing extracellular Na+ [( Na+]o) with choline or N-methyl-D-glucamine strongly inhibited the effects of ionomycin on 45Ca2+ efflux and total Ca2+. Ionomycin caused similar peak increases in [Ca2+]i in the presence and absence of [Na+]o, but the exponential fall from the peak was faster in the presence of [Na+]o. Dimethylbenzamil, a potent blocker of Na+/Ca2+ exchange in these cells, strongly inhibited the effects of ionomycin on 45Ca2+ efflux and total cell Ca2+. We conclude that the increase in cytosolic free Ca2+ produced by ionomycin may be sufficient to activate the plasma membrane Na+/Ca2+ exchanger which removes Ca2+ from the cytosol and helps restore basal [Ca2+]i.     

Relationship between Ca2+ sparklets and sarcoplasmic reticulum Ca2+ load and release in rat cerebral arterial smooth muscle

Ca(+) sparklets are subcellular Ca(2+) signals produced by the opening of sarcolemmal L-type Ca(2+) channels. Ca(2+) sparklet activity varies within the sarcolemma of arterial myocytes. In this study, we examined the relationship between Ca(2+) sparklet activity and sarcoplasmic reticulum (SR) Ca(2+) accumulation and release in cerebral arterial myocytes. Our data indicate that the SR is a vast organelle with multiple regions near the sarcolemma of these cells. Ca(2+) sparklet sites were located at or <0.2 μm from SR-sarcolemmal junctions. We found that while Ca(2+) sparklets increase the rate of SR Ca(2+) refilling in arterial myocytes, their activity did not induce regional variations in SR Ca(2+) content or Ca(2+) spark activity. In arterial myocytes, L-type Ca(2+) channel activity was independent of SR Ca(2+) load. This ruled out a potential feedback mechanism whereby SR Ca(2+) load regulates the activity of these channels. Together, our data suggest a model in which Ca(2+) sparklets contribute Ca(2+) influx into a cytosolic Ca(2+) pool from which sarco(endo)plasmic reticulum Ca(2+)-ATPase pumps Ca(2+) into the SR, indirectly regulating SR function.     

Chloride channel blockers inhibit Ca2+ uptake by the smooth muscle sarcoplasmic reticulum.

Despite the fact that Ca2+ transport into the sarcoplasmic reticulum (SR) of muscle cells is electrogenic, a potential difference is not maintained across the SR membrane. To achieve electroneutrality, compensatory charge movement must occur during Ca2+ uptake. To examine the role of Cl- in this charge movement in smooth muscle cells, Ca2+ transport into the SR of saponin-permeabilized smooth muscle cells was measured in the presence of various Cl- channel blockers or when I-, Br-, or SO42- was substituted for Cl-. Calcium uptake was inhibited in a dose-dependent manner by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and by indanyloxyacetic acid 94 (R(+)-IAA-94), but not by niflumic acid or 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS). Smooth muscle SR Ca2+ uptake was also partially inhibited by the substitution of SO42- for Cl-, but not when Cl- was replaced by I- or Br-. Neither NPPB nor R(+)-IAA-94 inhibited Ca2+ uptake into cardiac muscle SR vesicles at concentrations that maximally inhibited uptake in smooth muscle cells. These results indicate that Cl- movement is important for charge compensation in smooth muscle cells and that the Cl- channel or channels involved are different in smooth and cardiac muscle cells.     

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.     

A single luminally continuous sarcoplasmic reticulum with apparently separate Ca2+ stores in smooth muscle

Whether or not the sarcoplasmic reticulum (SR) is a continuous, interconnected network surrounding a single lumen or comprises multiple, separate Ca2+ pools was investigated in voltage-clamped single smooth muscle cells using local photolysis of caged compounds and Ca2+ imaging. The entire SR could be depleted or refilled from one small site via either inositol 1,4,5-trisphosphate receptors (IP3R) or ryanodine receptors (RyR) suggesting the SR is luminally continuous and that Ca2+ may diffuse freely throughout. Notwithstanding, regulation of the opening of RyR and IP3R, by the [Ca2+] within the SR, may create several apparent SR elements with various receptor arrangements. IP3R and RyR may appear to exist entirely on a single store, and there may seem to be additional SR elements that express either only RyR or only IP3R. The various SR receptor arrangements and apparently separate Ca2+ storage elements exist in a single luminally continuous SR entity.     

Effects of peroxynitrite on sarcoplasmic reticulum Ca2+ pump in pig coronary artery smooth muscle

Peroxynitritegenerated in arteries from superoxide and NO may damageCa²⁺ pumps. Here, we report the effects of peroxynitrite onATP-dependent azide-insensitive uptake of Ca²⁺ into pigcoronary artery vesicular membrane fractions F2 [enriched in plasmamembrane (PM)] and F3 [enriched in sarcoplasmic reticulum (SR)].Membranes were pretreated with peroxynitrite and then with DTT toquench this agent. This pretreatment inhibited Ca²⁺ uptakein a peroxynitrite concentration-dependent manner, but the effect wasmore severe in F3 than in F2. The inhibition was thus not overcome byexcess DTT used to quench peroxynitrite and was not affected ifcatalase, SOD, or mannitol was added along with peroxynitrite. Suchdamage to the pump protein would be difficult to repair if producedduring ischemia-reperfusion. The acylphosphates formed with ATPin F3 corresponded mainly to the SR Ca²⁺ pump (110 kDa),but in F2 both PM (140 kDa) and 110-kDa bands were observed.Peroxynitrite treatment of F2 inhibited only the 110-kDa band.Inhibition of Ca²⁺ uptake and acylphosphate formation fromATP correlated well in peroxynitrite-treated F3 samples. However,inhibition of acylphosphates from orthophosphate (reverse reaction ofthe pump) was slightly poorer. Peroxynitrite treatment also covalentlycross-linked the pump protein, yielding no dimers but only largeroligomers. In contrast, cross-linking of the SR Ca²⁺ pumpin skeletal and cardiac muscles gives dimers as the first oligomers.Therefore, we speculate that SERCA2 has a different quaternarystructure in the coronary artery smooth muscle.

     

GTP requirement for inositol-1,4,5-trisphosphate-induced Ca2+ release from sarcoplasmic reticulum in smooth muscle

We have examined inositol-1,4,5-trisphosphate (IP3)-induced Ca2+ release from the sarcoplasmic reticulum (SR) in the skinned vascular smooth muscle. The amount of Ca2+ in the SR was estimated indirectly by caffeine-induced contraction of the skinned preparation. The Ca2+ release from the SR by IP3 required GTP. A non-hydrolyzable analogue of GTP, guanosine 5'-(beta gamma-imido) triphosphate (GppNHp) could substitute for GTP in the IP3-induced Ca2+ release. These results suggest an involvement of GTP-binding protein in the mechanism of Ca2+ release from the SR by IP3 in smooth muscle.     

Characterization of Ca2+ release from heterogeneous Ca2+ stores in sarcoplasmic reticulum isolated from arterial and gastric smooth muscle

Ca2+ transport was investigated in vesicles of sarcoplasmic reticulum subfractionated from bovine main pulmonary artery and porcine gastric antrum using digitonin binding and zonal density gradient centrifugation. Gradient fractions recovered at 15-33% sucrose were studied as the sarcoplasmic reticulum component using Fluo-3 fluorescence or 45Ca2+ Millipore filtration. Thapsigargin blocked active Ca2+ uptake and induced a slow Ca2+ release from actively loaded vesicles. Unidirectional 45Ca2+ efflux from passively loaded vesicles showed multicompartmental kinetics. The time course of an initial fast component could not be quantitatively measured with the sampling method. The slow release had a half-time of several minutes. Both components were inhibited by 20 microM ruthenium red and 10 mM Mg2+. Caffeine, inositol 1,4,5-trisphosphate, ATP, and diltiazem accelerated the slow component. A Ca2+ release component activated by ryanodine or cyclic adenosine diphosphate ribose was resolved with Fluo-3. Comparison of tissue responses showed that the fast Ca2+ release was significantly smaller and more sensitive to inhibition by Mg2+ and ruthenium red in arterial vesicles. They released more Ca2+ in response to inositol 1,4,5-trisphosphate and were more sensitive to activation by cyclic adenosine diphosphate ribose. Ryanodine and caffeine, in contrast, were more effective in gastric antrum. In each tissue, the fraction of the Ca2+ store released by sequential application of caffeine and inositol 1,4,5-trisphosphate depended on the order applied and was additive. The results indicate that sarcoplasmic reticulum purified from arterial and gastric smooth muscle represents vesicle subpopulations that retain functional Ca2+ channels that reflect tissue-specific pharmacological modulation. The relationship of these differences to physiological responses has not been determined.     

Ca2+ Modulation of sarcoplasmic reticulum Ca2+ release in rat skeletal muscle fibers

Ca²⁺ transients and the rate of Ca²⁺ release (dCa_REL/dt) from the sarcoplasmic reticulum (SR) in voltage-clamped, fast-twitch skeletal muscle fibers from the rat were studied with the double Vaseline gap technique and using mag-fura-2 and fura-2 as Ca²⁺ indicators. Single pulse experiments with different returning potentials showed that Ca²⁺ removal from the myoplasm is voltage independent. Thus, the myoplasmic Ca²⁺ removal (dCa_REM/dt) was studied by fitting the decaying phase of the Ca²⁺ transient (Melzer, Ríos & Schneider, 1986) and dCa_REL/dt was calculated as the difference between dCa/dt and dCa_REM/dt. The fast Ca²⁺ release decayed as a consequence of Ca²⁺ inactivation of Ca²⁺ release. Double pulse experiments showed inactivation of the fast Ca²⁺ release depending on the prepulse duration. At constant interpulse interval, long prepulses (200 msec) induced greater inactivation of the fast Ca²⁺ release than shorter depolarizations (20 msec). The correlation (r) between the myoplasmic [Ca²⁺]_i and the inhibited amount of Ca²⁺ release was 0.98. The [Ca²⁺]_i for 50% inactivation of dCa_REL/dt was 0.25 m, and the minimum number of sites occupied by Ca²⁺ to inactivate the Ca²⁺ release channel was 3.0. These data support Ca²⁺ binding and inactivation of SR Ca²⁺ release.This work was supported by Grant-in-Aid from the American Heart Association (National) and Muscular Dystrophy Association (USA). Part of this work was developed in Dr. Stefani's laboratory at Baylor College of Medicine.     

Ca2+ regulation of vascular smooth muscle

Regulation of intracellular free Ca2+ concentrations in vascular smooth muscle is accomplished mainly by Ca2+ channels and ATP-dependent Ca2+ pumps in the plasmalemma and sarcoplasmic reticulum (SR). Ca2+ entry through the plasmalemma is apparently mediated by four different pathways: leak; receptor-operated Ca2+ channels; potential sensitive Ca2+ channels; and stretch-activated channels. The agonist releasable intracellular Ca2+ store appears to be identical with the SR. Evidence for the involvement of Ca2+-induced Ca2+ release and inositol-1,4,5-trisphosphate in the release of SR Ca2+ is discussed. Smooth muscle contractions induced by certain agonists may be further enhanced by inhibition of Ca2+ uptake by the SR and of active Ca2+ extrusion across the plasmalemma. At the moment it is not clear from a consideration of the Ca2+ regulatory mechanisms present in vascular smooth muscle how dietary Ca2+ affects vascular tone. The increased Ca2+ permeation through smooth muscle cell membranes of resistance arteries taken from spontaneously hypertensive rats may be relevant to this problem.     

The creatine kinase system is essential for optimal refill of the sarcoplasmic reticulum Ca2+ store in skeletal muscle.

Muscle function depends on an adequate ATP supply to sustain the energy consumption associated with Ca(2+) cycling and actomyosin sliding during contraction. In this regulation of energy homeostasis, the creatine kinase (CK) circuit for high energy phosphoryl transfer between ATP and phosphocreatine plays an important role. We earlier established a functional connection between the activity of the CK system and Ca(2+) homeostasis during depolarization and contractile activity of muscle. Here, we show how CK activity is coupled to the kinetics of spontaneous and electrically induced Ca(2+) transients in the sarcoplasm of myotubes. Using the UV ratiometric Ca(2+) probe Indo-1 and video-rate confocal microscopy in CK-proficient and -deficient cultured cells, we found that spontaneous and electrically induced transients were dependent on ryanodine-sensitive Ca(2+) release channels, sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase pumps, extracellular calcium, and functional mitochondria in both cell types. However, at increasing sarcoplasmic Ca(2+) load (induced by electrical stimulation at 0.1, 1, and 10 Hz), the Ca(2+) removal rate and the amount of Ca(2+) released per transient were gradually reduced in CK-deficient (but not wild-type) myotubes. We conclude that the CK/phosphocreatine circuit is essential for efficient delivery of ATP to the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase pumps and thereby directly influences sarcoplasmic reticulum refilling and the kinetics of the sarcoplasmic Ca(2+) signals.     

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.

     

Minor role of a Ca2+-depleted sarcoplasmic reticulum in heterologous desensitization of smooth muscle to K+

Exposure ofporcine carotid artery smooth muscle (PCASM) to histamine was followedby a large reduction in the rate of force generation in response to 40 mM KCl. This was shown to be a manifestation of slow attainment of asteady-state myoplasmic Ca²⁺concentration([Ca²⁺]_i).We hypothesized that if net transsarcolemmalCa²⁺ flux into the depolarizedPCASM cells is the same before and after a desensitizing histaminetreatment, then the transient attenuation of the increase in[Ca²⁺]_imay be due to accelerated uptake ofCa²⁺ by a partially depletedsarcoplasmic reticulum (SR) acting as aCa²⁺ sink or superficial bufferbarrier. We tested this hypothesis by eliciting responses of"desensitized PCASM" to 40 mM KCl in the presence ofcyclopiazonic acid (CPA), an SRCa²⁺-ATPase inhibitor.Contractions of CPA-treated tissues were attenuated less than those oftissues not treated with CPA, but they were not abolished.CPA-insensitive mechanism(s) dominated the desensitization. We concludethat histamine pretreatment reduced net transsarcolemmal Ca²⁺ flux into PCASM in responseto 40 mM KCl.

     

Ca2+ regulation in vascular smooth muscle.

Regulation of aorta smooth muscle contraction by Ca ion requires the collaboration of the 80,000 dalton factor and tropomyosin. A method for preparing pure actin from aorta smooth muscle is described.     

The effect of phenothiazines on Ca2+ fluxes in skeletal muscle sarcoplasmic reticulum

The effect of phenothiazines (trifluoperazine, chlorpromazine, methochlorpromazine, and imipramine) on Ca2+ fluxes in light and heavy sarcoplasmic reticulum (SR) isolated from rabbit fast-twitch skeletal muscle was investigated. These drugs inhibited Ca2+ loading and (Ca2+,Mg2+)-ATPase activity, but had no effect on unidirectional Ca2+ efflux from vesicles loaded either actively or passively with Ca2+. Chlorpromazine, which is membrane permeable, and its quaternary analog, methochlorpromazine, which is membrane impermeable, gave identical results. It is concluded that (a) the enhancement of net Ca2+ release by phenothiazines is due to inhibition of Ca2+ influx mediated by the Ca2+ pump rather than to the opening of a Ca2+ channel; and (b) phenothiazines act at the outer (myoplasmic) face of the SR membrane.