Effect of P(i) on unloaded shortening velocity of slow and fast mammalian muscle fibers

Chemically skinned muscle fibers,prepared from the rat medial gastrocnemius and soleus, were subjectedto four sequential slack tests in Ca²⁺-activating solutionscontaining 0, 15, 30, and 0 mM added P_i. P_i (15 and 30 mM) had no effect on the unloaded shortening velocity (V_o) of fibers expressing type IIb myosin heavychain (MHC). For fibers expressing type I MHC, 15 mM P_i didnot alter V_o, whereas 30 mM P_ireduced V_o to 81 ± 1% of the original 0 mM P_i value. This effect was readily reversible whenP_i was lowered back to 0 mM. These results are notcompatible with current cross-bridge models, developed exclusively fromdata obtained from fast fibers, in which V_o isindependent of P_i. The response of the type I fibers at 30 mM P_i is most likely the result of increased internal drag opposing fiber shortening resulting from fiber type-specific effects ofP_i on cross bridges, the thin filament, or therate-limiting step of the cross-bridge cycle.

     

Mechanical signals and mechanosensitive modulation of intracellular [Ca(2+)] in smooth muscle

We testedthe hypothesis that strain is the primary mechanical signal in themechanosensitive modulation of intracellular Ca²⁺concentration ([Ca²⁺]_i) in airway smoothmuscle. We found that [Ca²⁺]_i wassignificantly correlated with muscle length during isotonic shorteningagainst 20% isometric force (F_iso). When the isotonic loadwas changed to 50% F_iso, data points from the 20 and 50% F_iso experiments overlapped in thelength-[Ca²⁺]_i relationship. Similarly, datapoints from the 80% F_iso experiments clustered near thosefrom the 50% F_iso experiments. Therefore, despite 2.5- and4-fold differences in external load, [Ca²⁺]_idid not deviate much from the length-[Ca²⁺]_irelation that fitted the 20% F_iso data. Maximal inhibition of sarcoplasmic reticular (SR) Ca²⁺ uptake by 10 µMcyclopiazonic acid (CPA) did not significantly change[Ca²⁺]_i in carbachol-induced isometriccontractions and isotonic shortening. CPA also did not significantlychange myosin light-chain phosphorylation or force redevelopment whencarbachol-activated muscle strips were quickly released from optimallength (L_o) to 0.5 L_o. These results are consistent with thehypothesis and suggest that SR Ca²⁺ uptake is not theunderlying mechanism.

     

Contractile apparatus and sarcoplasmic reticulum function: effects of fatigue, recovery, and elevated Ca2+

Williams, Jay H. Contractile apparatus and sarcoplasmicreticulum function: effects of fatigue, recovery, and elevated Ca²⁺. J. Appl.Physiol. 83(2): 444-450, 1997.This investigationtested the notion that fatiguing stimulation induces intrinsic changes in the contractile apparatus and sarcoplasmic reticulum (SR) and thatthese changes are initiated by elevated intracellularCa²⁺ concentration([Ca²⁺]_i).Immediately after stimulation of frog semitendinosus muscle, contractile apparatus and SR function were measured. Despite a largedecline in tetanic force (P_o),maximal Ca²⁺-activated force(F_max) of the contractileapparatus was not significantly altered. However,Ca²⁺ sensitivity was increased. Inconjunction, the rate constant ofCa²⁺ uptake by the SR wasdiminished, and the caffeine sensitivity ofCa²⁺ release was decreased. Duringrecovery, P_o, contractileapparatus, and SR function each returned to near-initial levels.Exposure of skinned fibers to 0.5 µM freeCa²⁺ for 5 min depressed bothF_max andCa²⁺ sensitivity of thecontractile apparatus. In addition, caffeine sensitivity ofCa²⁺ release was diminished.Results suggest that fatigue induces intrinsic alterations incontractile apparatus and SR function. Changes in contractile apparatusfunction do not appear to be mediated by increased[Ca²⁺]_i.However, a portion of the change in SRCa²⁺ release seems to be due toelevated[Ca²⁺]_i.

     

Ca2+ sensitivity of BK channels in GH3 cells involves cytosolic phospholipase A2

To test thehypothesis that intracellular Ca²⁺activation of large-conductanceCa²⁺-activatedK⁺ (BK) channels involves thecytosolic form of phospholipase A₂ (cPLA₂), we first inhibited theexpression of cPLA₂ by treating GH₃ cells with antisenseoligonucleotides directed at the two possible translation start siteson cPLA₂. Western blot analysis and a biochemical assay of cPLA₂activity showed marked inhibition of the expression ofcPLA₂ in antisense-treated cells.We then examined the effects of intracellularCa²⁺ concentration([Ca²⁺]_i)on single BK channels from these cells. Open channel probability (P_o) for thecells exposed to cPLA₂ antisenseoligonucleotides in 0.1 µM intracellularCa²⁺ was significantly lower thanin untreated or sense oligonucleotide-treated cells, but the voltagesensitivity did not change (measured as the slope of theP_o-voltagerelationship). In fact, a 1,000-fold increase in[Ca²⁺]_ifrom 0.1 to 100 µM did not significantly increaseP_oin these cells, whereas BK channels from cells in the other treatmentgroups showed a normalP_o-[Ca²⁺]_iresponse. Finally, we examined the effect of exogenous arachidonic acidon theP_oof BK channels from antisense-treated cells. Although arachidonic aciddid significantly increaseP_o,it did so without restoring the[Ca²⁺]_isensitivity observed in untreated cells. We conclude that although [Ca²⁺]_idoes impart some basal activity to BK channels inGH₃ cells, the steepP_o-[Ca²⁺]_irelationship that is characteristic of these channels involves cPLA₂.

     

E-C coupling failure in mouse EDL muscle after in vivo eccentric contractions

The objectives of this research were to determine thecontribution of excitation-contraction (E-C) coupling failure to the decrement in maximal isometric tetanic force(P_o) in mouse extensor digitorumlongus (EDL) muscles after eccentric contractions and to elucidatepossible mechanisms. The left anterior crural muscles of femaleICR mice (n = 164) wereinjured in vivo with 150 eccentric contractions.P_o, caffeine-,4-chloro-m-cresol-, andK⁺-induced contracture forces,sarcoplasmic reticulum (SR) Ca²⁺release and uptake rates, and intracellularCa²⁺ concentration([Ca²⁺]_i)were then measured in vitro in injured and contralateral control EDLmuscles at various times after injury up to 14 days. On the basis ofthe disproportional reduction inP_o (~51%) compared with caffeine-induced force (~11-21%), we estimate that E-C coupling failure can explain 57-75% of theP_o decrement from 0 to 5 days postinjury. Comparable reductions inP_o andK⁺-induced force (51%), and minorreductions (0-6%) in the maximal SRCa²⁺ release rate, suggest thatthe E-C coupling defect site is located at the t tubule-SR interfaceimmediately after injury. Confocal laser scanning microscopy indicatedthat resting[Ca²⁺]_iwas elevated and peak tetanic[Ca²⁺]_iwas reduced, whereas peak4-chloro-m-cresol-induced[Ca²⁺]_iwas unchanged immediately after injury. By 3 days postinjury, 4-chloro-m-cresol-induced[Ca²⁺]_ibecame depressed, probably because of decreased SRCa²⁺ release and uptake rates(17-31%). These data indicate that the decrease inP_o during the first several daysafter injury primarily stems from a failure in the E-C couplingprocess.

     

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.

     

Two-photon molecular excitation imaging of Ca2+ transients in Langendorff-perfused mouse hearts

The ability to image calciumsignals at subcellular levels within the intact depolarizing heartcould provide valuable information toward a more integratedunderstanding of cardiac function. Accordingly, a system combiningtwo-photon excitation with laser-scanning microscopy was developed tomonitor electrically evoked [Ca²⁺]_itransients in individual cardiomyocytes within noncontracting Langendorff-perfused mouse hearts. [Ca²⁺]_itransients were recorded at depths 100 µm from the epicardial surface with the fluorescent indicators rhod-2 or fura-2 in the presence of the excitation-contraction uncoupler cytochalasin D. Evoked[Ca²⁺]_i transients were highly synchronizedamong neighboring cardiomyocytes. At 1 Hz, the times from 90 to 50%(t_90-50%) and from 50 to 10%(t_50-10%) of the peak[Ca²⁺]_i were (means ± SE) 73 ± 4 and 126 ± 10 ms, respectively, and at 2 Hz, 62 ± 3 and94 ± 6 ms (n = 19, P < 0.05 vs.1 Hz) in rhod-2-loaded cardiomyocytes.[Ca²⁺]_i decay was markedly slower infura-2-loaded hearts (t_90-50% at 1 Hz,128 ± 9 ms and at 2 Hz, 88 ± 5 ms;t_50-10% at 1 Hz, 214 ± 18 ms and at2 Hz, 163 ± 7 ms; n = 19, P < 0.05 vs. rhod-2). Fura-2-induced deceleration of[Ca²⁺]_i decline resulted from increasedcytosolic Ca²⁺ buffering, because the kinetics of rhod-2decay resembled those obtained with fura-2 after incorporation of theCa²⁺ chelator BAPTA. Propagating calcium waves and[Ca²⁺]_i amplitude alternans were readilydetected in paced hearts. This approach should be of general utility tomonitor the consequences of genetic and/or functional heterogeneity incellular calcium signaling within whole mouse hearts at tissue depthsthat have been inaccessible to single-photon imaging.

     

Ca2+ depolarizes adrenal cortical cells through selective inhibition of an ATP-activated K+ current

Bovine adrenalzona fasciculata cells (AZF) express a noninactivatingK⁺ current(I_AC) whoseinhibition by adrenocorticotropic hormone and ANG II may be coupled tomembrane depolarization andCa²⁺-dependentcortisol secretion. We studiedI_ACinhibition byCa²⁺ and theCa²⁺ionophore ionomycin in whole cell and single-channel patch-clamp recordings of AZF. In whole cell recordings with intracellular (pipette)Ca²⁺concentration([Ca²⁺]_i)buffered to 0.02 µM,I_AC reachedmaximum current density of 25.0 ± 5.1 pA/pF(n = 16); raising[Ca²⁺]_ito 2.0 µM reduced it 76%. In inside-out patches, elevated[Ca²⁺]_idramatically reducedI_AC channelactivity. Ionomycin inhibited I_AC by 88 ± 4% (n = 14) without altering rapidlyinactivating A-type K⁺ current.Inhibition of I_ACby ionomycin was unaltered by adding calmodulin inhibitory peptide tothe pipette or replacing ATP with its nonhydrolyzable analog5'-adenylylimidodiphosphate.I_AC inhibition byionomycin was associated with membrane depolarization. When[Ca²⁺]_iwas buffered to 0.02 µM with 2 and 11 mM1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), ionomycin inhibitedI_AC by 89.6 ± 3.5 and 25.6 ± 14.6% and depolarized the same AZF by 47 ± 8 and 8 ± 3 mV, respectively (n = 4). ANG II inhibitedI_AC significantlymore effectively when pipette BAPTA was reduced from 11 to 2 mM. Raising[Ca²⁺]_iinhibits I_ACthrough a mechanism not requiring calmodulin or protein kinases,suggesting direct interaction withI_AC channels. ANGII may inhibitI_AC anddepolarize AZF by activating parallel signaling pathways, one of whichuses Ca²⁺ asa mediator.

     

Age-related differences in Na+-dependent Ca2+ accumulation in rabbit hearts exposed to hypoxia and acidification

In this study, we test the hypothesisthat in newborn hearts (as in adults) hypoxia and acidificationstimulate increased Na⁺ uptake, in part via pH-regulatoryNa⁺/H⁺ exchange. Resulting increases inintracellular Na⁺ (Na_i) alter the force drivingthe Na⁺/Ca²⁺ exchanger and lead to increasedintracellular Ca²⁺. NMR spectroscopy measuredNa_i and cytosolic Ca²⁺ concentration([Ca²⁺]_i) and pH (pH_i) inisolated, Langendorff-perfused 4- to 7-day-old rabbit hearts. AfterNa⁺/K⁺ ATPase inhibition, hypoxic hearts gainedNa⁺, whereas normoxic controls did not [19 ± 3.4 to139 ± 14.6 vs. 22 ± 1.9 to 22 ± 2.5 (SE) meq/kg drywt, respectively]. In normoxic hearts acidified using theNH₄Cl prepulse, pH_i fell rapidly and recovered,whereas Na_i rose from 31 ± 18.2 to 117.7 ± 20.5 meq/kg dry wt. Both protocols caused increases in [Ca]_i;however, [Ca]_i increased less in newborn hearts than inadults (P < 0.05). Increases in Na_i and[Ca]_i were inhibited by theNa⁺/H⁺ exchange inhibitormethylisobutylamiloride (MIA, 40 µM; P < 0.05), aswell as by increasing perfusate osmolarity (+30 mosM) immediately before and during hypoxia (P < 0.05). The data supportthe hypothesis that in newborn hearts, like adults, increases inNa_i and [Ca]_i during hypoxia and afternormoxic acidification are in large part the result of increased uptakevia Na⁺/H⁺ and Na⁺/Ca²⁺exchange, respectively. However, for similar hypoxia and acidification protocols, this increase in [Ca]_i is less in newborn thanadult hearts.

     

L-type voltage-dependent Ca2+ channels in cerebral microvascular endothelial cells and ET-1 biosynthesis

We investigatedthe role of intracellular calcium concentration([Ca²⁺]_i) in endothelin-1 (ET-1) production,the effects of potential vasospastic agents on[Ca²⁺]_i, and the presence of L-typevoltage-dependent Ca²⁺ channels in cerebral microvascularendothelial cells. Primary cultures of endothelial cells isolated frompiglet cerebral microvessels were used. Confluent cells were exposed toeither the thromboxane receptor agonist U-46619 (1 µM),5-hydroxytryptamine (5-HT; 0.1 mM), or lysophosphatidic acid (LPA; 1 µM) alone or after pretreatment with the Ca²⁺-chelatingagent EDTA (100 mM), the L-type Ca²⁺ channel blockerverapamil (10 µM), or the antagonist of receptor-operated Ca²⁺ channel SKF-96365 HCl (10 µM) for 15 min. ET-1production increased from 1.2 (control) to 8.2 (U-46619), 4.9 (5-HT),or 3.9 (LPA) fmol/µg protein, respectively. Such elevated ET-1biosynthesis was attenuated by verapamil, EDTA, or SKF-96365 HCl. Toinvestigate the presence of L-type voltage-dependent Ca²⁺channels in endothelial cells, the [Ca²⁺]_isignal was determined fluorometrically by using fura 2-AM. Superfusionof confluent endothelial cells with U-46619, 5-HT, or LPA significantlyincreased [Ca²⁺]_i. Pretreatment ofendothelial cells with high K⁺ (60 mM) or nifedipine (4 µM) diminished increases in [Ca²⁺]_i inducedby the vasoactive agents. These results indicate that 1)elevated [Ca²⁺]_i signals are involved in ET-1biosynthesis induced by specific spasmogenic agents, 2) theincreases in [Ca²⁺]_i induced by thevasoactive agents tested involve receptor as well as L-typevoltage-dependent Ca²⁺ channels, and 3) primarycultures of cerebral microvascular endothelial cells express L-typevoltage-dependent Ca²⁺ channels.

     

Peak power output is maintained in rabbit psoas and rat soleus single muscle fibers when CTP replaces ATP

The chemomechanicalcoupling mechanism in striated muscle contraction was examined bychanging the nucleotide substrate from ATP to CTP. Maximum shorteningvelocity [extrapolation to zero force from force-velocity relation(V_max) andslope of slack test plots (V₀)], maximumisometric force (P_o), power, andthe curvature of the force-velocity curve[a/P_o(dimensionless parameter inversely related to the curvature)] weredetermined during maximumCa²⁺-activated isotoniccontractions of fibers from fast rabbit psoas and slow rat soleusmuscles by using 0.2 mM MgATP, 4 mM MgATP, 4 mM MgCTP, or 10 mM MgCTPas the nucleotide substrate. In addition to a decrease in the maximumCa²⁺-activated force in both fibertypes, a change from 4 mM ATP to 10 mM CTP resulted in a decrease inV_max in psoasfibers from 3.26 to 1.87 muscle length/s. In soleus fibers,V_max was reduced from 1.94 to 0.90 muscle length/s by this change in nucleotide. Surprisingly, peak power was unaffected in either fiber type by thechange in nucleotide as the result of a three- to fourfold decrease inthe curvature of the force-velocity relationship. The results areinterpreted in terms of the Huxley model of muscle contraction as anincrease in f₁and g₁ coupled toa decrease in g₂(where f₁ is therate of cross-bridge attachment and g₁ andg₂ are rates ofdetachment) when CTP replaces ATP. This adequately accounts for theobserved changes in P_o,a/P_o,and V_max.However, the two-state Huxley model does not explicitly reveal thecross-bridge transitions that determine curvature of the force-velocityrelationship. We hypothesize that a nucleotide-sensitive transitionamong strong-binding cross-bridge states followingP_i release, but before the release of the nucleotide diphosphate, underlies the alterations ina/P_o reported here.

     

Gender-specific reduction in contractility and [Ca(2+)](i) in vascular smooth muscle cells of female rat

The hypothesisthat vascular protection in females and its absence in males reflectsgender differences in [Ca²⁺]_i andCa²⁺ mobilization mechanisms of vascular smooth musclecontraction was tested in fura 2-loaded aortic smooth muscle cellsisolated from intact and gonadectomized male and female Wistar-Kyoto(WKY) and spontaneously hypertensive (SHR) rats. In WKY cells incubated in Hanks' solution (1 mM Ca²⁺), the resting length and[Ca²⁺]_i were significantlydifferent in intact males (64.5 ± 1.2 µm and 83 ± 3 nM) than inintact females (76.5 ± 1.5 µm and 64 ± 7 nM). In intact male WKY,phenylephrine (Phe, 10⁵ M) caused transient increasein [Ca²⁺]_i to 428 ± 13 nMfollowed by maintained increase to 201 ± 8 nM and 32% cellcontraction. In intact female WKY, the Phe-induced [Ca²⁺]_i transient was notsignificantly different, but the maintained [Ca²⁺]_i (159 ± 7 nM) and cellcontraction (26%) were significantly less than in intact male WKY. InCa²⁺-free (2 mM EGTA) Hanks', Phe and caffeine (10 mM)caused transient increases in[Ca²⁺]_i and contraction that werenot significantly different between males and females. Membranedepolarization by 51 mM KCl caused 31% cell contraction and increased[Ca²⁺]_i to 259 ± 9 nM in intactmale WKY, which were significantly greater than a 24% contraction and214 ± 8 nM [Ca²⁺]_i in intactfemale WKY. Maintained Phe- and KCl-stimulated cell contraction and[Ca²⁺]_i were significantly greaterin SHR than WKY in all groups of rats. Reduction in cell contractionand [Ca²⁺]_i in intact femalescompared with intact males was significantly greater in SHR (~30%)than WKY (~20%). No significant differences in cell contraction or[Ca²⁺]_i were observed betweencastrated males, ovariectomized (OVX) females, and intact males, orbetween OVX females with 17-estradiol implants and intact females.Exogenous application of 17-estradiol (10⁸ M) tocells from OVX females caused greater reduction in Phe- and KCl-inducedcontraction and [Ca²⁺]_i in SHR thanWKY. Thus the basal, maintained Phe- and depolarization-induced [Ca²⁺]_i and contraction of vascularsmooth muscle triggered by Ca²⁺ entry from theextracellular space exhibit differences depending on gender and thepresence or absence of female gonads. Cell contraction and[Ca²⁺]_i due to Ca²⁺release from the intracellular stores are not affected by gender or gonadectomy. Gender-specific reduction in contractility and [Ca²⁺]_i in vascular smoothmuscle of female rats is greater in SHR than WKY rats.

     

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 extracellular calcium and potassium on the sodium pump of rat adrenal glomerulosa cells

Because the activity of thesodium pump (Na-K-ATPase) influences the secretion of aldosterone, wedetermined how extracellular potassium (K_o) and calciumaffect sodium pump activity in rat adrenal glomerulosa cells. Sodiumpump activity was measured as ouabain-sensitive ⁸⁶Rb uptakein freshly dispersed cells containing 20 mM sodium as measured withsodium-binding benzofluran isophthalate. Increasing K_o from4 to 10 mM in the presence of 1.8 mM extracellular calcium (Ca_o) stimulated sodium pump activity up to 165% andincreased intracellular free calcium as measured with fura 2. Increasing K_o from 4 to 10 mM in the absence ofCa_o stimulated the sodium pump ~30% and did not increaseintracellular free calcium concentration ([Ca²⁺]_i). In some experiments, addition of1.8 mM Ca_o in the presence of 4 mM K_o increased[Ca²⁺]_i above the levels observed in theabsence of Ca_o and stimulated the sodium pump up to 100%.Ca-dependent stimulation of the sodium pump by K_o andCa_o was inhibited by isradipine (10 µM), a blocker of L-and T-type calcium channels, by compound 48/80 (40 µg/ml) andcalmidizolium (10 µM), which inhibits calmodulin (CaM), and by KN-62(10 µM), which blocks some forms of Ca/CaM kinase II (CaMKII).Staurosporine (1 µM), which effectively blocks most forms of proteinkinase C, had no effect. In the presence of A-23187, a calciumionophore, the addition of 0.1 mM Ca_o increased[Ca²⁺]_i to the level observed in the presenceof 10 mM K_o and 1.8 mM Ca_o and stimulated thesodium pump 100%. Ca-dependent stimulation by A-23187 and 0.1 mMCa_o was not reduced by isradipine but was blocked by KN-62.Thus, under the conditions that K_o stimulates aldosteronesecretion, it stimulates the sodium pump by two mechanisms: directbinding to the pump and by increasing calcium influx, which isdependent on Ca_o. The resulting increase in[Ca²⁺]_i may stimulate the sodium pump byactivating CaM and/or CaMKII.

     

Regulation of intracellular Ca2+ release in corpus cavernosum smooth muscle: synergism between nitric oxide and cGMP

Tonic contraction of corpus cavernosum smooth muscle cells (SMCs) maintains the flaccid state of the penis, and relaxation is initiated by nitric oxide (NO), leading to erection. Our aim was to investigate the effect of NO on the smooth muscle cellular response to adrenergic stimulation in corpus cavernosum. Fura-2 fluorescence was used to record intracellular Ca²⁺ concentration ([Ca²⁺]_i) from freshly isolated SMCs from rat and human. Phenylephrine (PE) transiently elevated [Ca²⁺]_i in the presence and absence of extracellular Ca²⁺, indicating release from intracellular stores. Whereas the NO donor S-nitroso-N-acetylpenicillamine (SNAP) with sildenafil citrate (SIL) caused no change in basal [Ca²⁺]_i, the PE-induced rise of [Ca²⁺]_i was reversibly inhibited by 27 ± 7% (n = 21, P < 0.005) in rat and by 55 ± 15% (n = 9, P < 0.01) in human SMCs. SNAP and SIL also reduced the contractile response to PE. To investigate the mechanism, we applied mediators alone or in combination. The soluble guanylyl cyclase inhibitor ODQ reduced the effect of SNAP and SIL. SIL, cGMP analogs, and NO donors without SIL did not reduce the PE-induced rise of [Ca²⁺]_i. However, the combination of 8-bromo-cGMP with SNAP reduced the Ca²⁺ peak by 42 ± 9% (n = 22, P < 0.01). Our results demonstrate that NO and cGMP act synergistically to reduce Ca²⁺ release from intracellular stores. Reduction of intracellular Ca²⁺ release may contribute to relaxation of the corpus cavernosum, leading to erection. calcium stores; nitric oxide; sildenafil citrate; inositol 1,4,5-trisphosphate receptor     

Enhanced response to caffeine and 4-chloro-m-cresol in malignant hyperthermia-susceptible muscle is related in part to chronically elevated resting [Ca2+]i

Malignant hyperthermia (MH) is a potentially fatal pharmacogenetic syndrome caused by exposure to halogenated volatile anesthetics and/or depolarizing muscle relaxants. We have measured intracellular Ca²⁺ concentration ([Ca²⁺]_i) using double-barreled, Ca²⁺-selective microelectrodes in myoballs prepared from skeletal muscle of MH-susceptible (MHS) and MH-nonsusceptible (MHN) swine. Resting [Ca²⁺]_i was approximately twofold in MHS compared with MHN quiescent myoballs (232 ± 35 vs. 112 ± 11 nM). Treatment of myoballs with caffeine or 4-chloro-m-cresol (4-CmC) produced an elevation in [Ca²⁺]_i in both groups; however, the concentration required to cause a rise in [Ca²⁺]_i elevation was four times lower in MHS than in MHN skeletal muscle cells. Incubation of MHS cells with the fast-complexing Ca²⁺ buffer BAPTA reduced [Ca²⁺]_i, raised the concentration of caffeine and 4-CmC required to cause an elevation of [Ca²⁺]_i, and reduced the amount of Ca²⁺ release associated with exposure to any given concentration of caffeine or 4-CmC to MHN levels. These results suggest that the differences in the response of MHS skeletal myoballs to caffeine and 4-CmC may be mediated at least in part by the chronic high resting [Ca²⁺]_i levels in these cells. calcium homeostasis; 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid     

Comparative effects of a low-carbohydrate diet and exercise plus a low-carbohydrate diet on muscle sarcoplasmic reticulum responses in males

We employed a glycogen-depleting session of exercise followed by a low-carbohydrate (CHO) diet to investigate modifications that occur in muscle sarcoplasmic reticulum (SR) Ca²⁺-cycling properties compared with low-CHO diet alone. SR properties were assessed in nine untrained males [peak aerobic power (O_{2 peak}) = 43.6 ± 2.6 (SE) ml·kg^–1·min^–1] during prolonged cycle exercise to fatigue performed at 58% O_{2 peak} after 4 days of low-CHO diet (Lo CHO) and after glycogen-depleting exercise plus 4 days of low-CHO (Ex+Lo CHO). Compared with Lo CHO, Ex+Lo CHO resulted in 12% lower (P < 0.05) resting maximal Ca²⁺-ATPase activity (V_max = 174 ± 12 vs. 153 ± 10 µmol·g protein^–1·min^–1) and smaller reduction in V_max induced during exercise. A similar effect was observed for Ca²⁺ uptake. The Hill coefficient, defined as slope of the relationship between cytosolic free Ca²⁺ concentration and Ca²⁺-ATPase activity, was higher (P < 0.05) at rest (2.07 ± 0.15 vs. 1.90 ± 0.10) with Ex+Lo CHO, an effect that persisted throughout the exercise. The coupling ratio, defined as the ratio of Ca²⁺ uptake to V_max, was 23–30% elevated (P < 0.05) at rest and during the first 60 min of exercise with Ex+Lo CHO. The 27 and 34% reductions (P < 0.05) in phase 1 and phase 2 Ca²⁺ release, respectively, observed during exercise with Lo CHO were not altered by Ex+Lo CHO. These results indicate that when prolonged exercise precedes a short-term Lo CHO diet, Ca²⁺ sequestration properties and efficiency are improved compared with those during Lo CHO alone. calcium cycling; vastus lateralis; contractile activity; glycogen; phosphorylation potential     

Mechanisms of P(i) regulation of the skeletal muscle SR Ca(2+) release channel

Inorganic phosphate(P_i) accumulates in the fibers of actively working musclewhere it acts at various sites to modulate contraction. To characterizethe role of P_i as a regulator of the sarcoplasmic reticulum(SR) calcium (Ca²⁺) release channel, we examined the actionof P_i on purified SR Ca²⁺ release channels,isolated SR vesicles, and skinned skeletal muscle fibers. In singlechannel studies, addition of P_i to the cis chamberincreased single channel open probability (P_o;0.079 ± 0.020 in 0 P_i, 0.157 ± 0.034 in 20 mMP_i) by decreasing mean channel closed time; mean channelopen times were unaffected. In contrast, the ATP analog,,-methyleneadenosine 5'-triphosphate (AMP-PCP), enhancedP_o by increasing single channel open time anddecreasing channel closed time. P_i stimulation of[³H]ryanodine binding by SR vesicles wassimilar at all concentrations of AMP-PCP, suggesting P_i andadenine nucleotides act via independent sites. In skinned musclefibers, 40 mM P_i enhanced Ca²⁺-inducedCa²⁺ release, suggesting an in situ stimulation ofthe release channel by high concentrations of P_i. Ourresults support the hypothesis that P_i may be an importantendogenous modulator of the skeletal muscle SR Ca²⁺ releasechannel under fatiguing conditions in vivo, acting via a mechanismdistinct from adenine nucleotides.

     

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.

     

Intravascular pressure regulates local and global Ca2+ signaling in cerebral artery smooth muscle cells

The regulationof intracellular Ca²⁺ signals in smooth muscle cells andarterial diameter by intravascular pressure was investigated in ratcerebral arteries (~150 µm) using a laser scanning confocal microscope and the fluorescent Ca²⁺ indicator fluo 3. Elevation of pressure from 10 to 60 mmHg increased Ca²⁺spark frequency 2.6-fold, Ca²⁺ wave frequency 1.9-fold, andglobal intracellular Ca²⁺ concentration([Ca²⁺]_i) 1.4-fold in smooth muscle cells,and constricted arteries. Ryanodine (10 µM), an inhibitor ofryanodine-sensitive Ca²⁺ release channels, or thapsigargin(100 nM), an inhibitor of the sarcoplasmic reticulumCa²⁺-ATPase, abolished sparks and waves, elevated global[Ca²⁺]_i, and constricted pressurized (60 mmHg) arteries. Diltiazem (25 µM), a voltage-dependentCa²⁺ channel (VDCC) blocker, significantly reduced sparks,waves, and global [Ca²⁺]_i, and dilatedpressurized (60 mmHg) arteries. Steady membrane depolarization elevatedCa²⁺ signaling similar to pressure and increased transientCa²⁺-sensitive K⁺ channel current frequencye-fold for ~7 mV, and these effects were prevented by VDCCblockers. Data are consistent with the hypothesis that pressure inducesa steady membrane depolarization that activates VDCCs, leading to anelevation of spark frequency, wave frequency, and global[Ca²⁺]_i. In addition, pressure inducescontraction via an elevation of global[Ca²⁺]_i, whereas the net effect of sparks andwaves, which do not significantly contribute to global[Ca²⁺]_i in arteries pressurized to between 10 and 60 mmHg, is to oppose contraction.