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.

     

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.

     

Effects of microtubules and microfilaments on [Ca(2+)](i) and contractility in a reconstituted fibroblast fiber

We used a reconstituted fiber formed when 3T3fibroblasts are grown in collagen to characterize nonmusclecontractility and Ca²⁺ signaling. Calf serum (CS) andthrombin elicited reversible contractures repeatable for >8 h. CSelicited dose-dependent increases in isometric force; 30% produced thelargest forces of 106 ± 12 µN (n = 30), whichis estimated to be 0.5 mN/mm² cell cross-sectionalarea. Half times for contraction and relaxation were 4.7 ± 0.3 and 3.1 ± 0.3 min at 37°C. With imposition of constant shortening velocities, force declined with time, yieldingtime-dependent force-velocity relations. Forces at 5 s fit thehyperbolic Hill equation; maximum velocity(V_max) was 0.035 ± 0.002 L_o/s.Compliance averaged 0.0076 ± 0.0006 L_o/F_o. Disruption of microtubules with nocodazole in a CS-contracted fiber had no net effects on force, V_max, or stiffness; force increased in 8, butdecreased in 13, fibers. Nocodazole did not affect baselineintracellular Ca²⁺ concentration([Ca²⁺]_i) but reduced (~30%) the[Ca²⁺]_i response to CS. The force afternocodazole treatment was the primary determinant of stiffness andV_max, suggesting that microtubules were not amajor component of fiber internal mechanical resistance. Cytochalasin Dhad major inhibitory effects on all contractile parameters measured butlittle effect on [Ca²⁺]_i.

     

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.

     

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.

     

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.

     

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.

     

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.

     

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.

     

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.

     

Cellular Responses to Mechanical Stress: Invited Review: Effects of flow on vascular endothelial intracellular calcium signaling of rat aortas ex vivo

To study the effects of flow on in situendothelial intracellular calcium concentration([Ca²⁺]_i) signaling, rat aortic rings wereloaded with fura 2, mounted on a tissue flow chamber, and divided intocontrol and flow-pretreated groups. The latter was perfused with bufferat a shear stress of 50 dyns/cm² for 1 h. Endothelial[Ca²⁺]_i responses to ACh or shear stresseswere determined by ratio image analysis. Moreover, ACh-induced[Ca²⁺]_i elevation responses were measured ina calcium-free buffer, or in the presence of SKF-96365, to elucidatethe role of calcium influx in the flow effects. Our results showed that1) ACh increased endothelial[Ca²⁺]_i in a dose-dependent manner, and theseresponses were incremented by flow-pretreatment; 2) thedifferences in ACh-induced [Ca²⁺]_i elevationbetween control and flow-pretreated groups were abolished by SKF-96365or by Ca²⁺-free buffer; and 3) in the presenceof 10⁵ M ATP, shear stress induced dose-dependent[Ca²⁺]_i elevation responses that were notaltered by flow-pretreatment. In conclusion, flow-pretreatment augmentsthe ACh-induced endothelial calcium influx in rat aortas ex vivo.

     

Effects of salbutamol on intracellular calcium oscillations in porcine airway smooth muscle

Prakash, Y. S., H. F. M. van der Heijden, M. S. Kannan, andG. C. Sieck. Effects of salbutamol on intracellular calcium oscillations in porcine airway smooth muscle. J. Appl.Physiol. 82(6): 1836-1843, 1997.Relaxation ofairway smooth muscle (ASM) by -adrenoceptor agonists involvesreduction of intracellular Ca²⁺concentration([Ca²⁺]_i).In porcine ASM cells, acetylcholine induces[Ca²⁺]_ioscillations that display frequency modulation by agonist concentration and basal[Ca²⁺]_i.We used real-time confocal microscopy to examine the effect ofsalbutamol (1 nM to 1 µM), a₂-adrenoceptor agonist, on[Ca²⁺]_ioscillations in freshly dissociated porcine ASM cells. Salbutamol decreased the frequency of[Ca²⁺]_ioscillations in a concentration-dependent fashion, completely inhibiting the oscillations at 1 µM. These effects were mimicked by acell-permeant analog of adenosine 3,5-cyclicmonophosphate. The inhibitory effect of salbutamol was partiallyreversed by BAY K 8644. Salbutamol reduced[Ca²⁺]_ieven when sarcoplasmic reticulum (SR)Ca²⁺ reuptake andCa²⁺ influx were blocked.Lanthanum blockade of Ca²⁺ effluxattenuated the inhibitory effect of salbutamol on[Ca²⁺]_i.The[Ca²⁺]_iresponse to caffeine was unaffected by salbutamol. On the basis ofthese results, we conclude that₂-adrenoceptor agonists have little effect on SR Ca²⁺ releasein ASM cells but reduce[Ca²⁺]_iby inhibiting Ca²⁺ influx throughvoltage-gated channels and by enhancingCa²⁺ efflux.

     

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.

     

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.     

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.

     

Histamine-mediated increases in cytosolic [Ca2+] involve different mechanisms in human pulmonary artery smooth muscle and endothelial cells

Agonist stimulation of human pulmonary artery smooth muscle cells (PASMC) and endothelial cells (PAEC) with histamine showed similar spatiotemporal patterns of Ca²⁺ release. Both sustained elevation and oscillatory patterns of changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) were observed in the absence of extracellular Ca²⁺. Capacitative Ca²⁺ entry (CCE) was induced in PASMC and PAEC by passive depletion of intracellular Ca²⁺ stores with 10 µM cyclopiazonic acid (CPA; 15–30 min). The pyrazole derivative BTP2 inhibited CPA-activated Ca²⁺ influx, suggesting that depletion of CPA-sensitive internal stores is sufficient to induce CCE in both PASMC and PAEC. The recourse of histamine-mediated Ca²⁺ release was examined after exposure of cells to CPA, thapsigargin, caffeine, ryanodine, FCCP, or bafilomycin. In PASMC bathed in Ca²⁺-free solution, treatment with CPA almost abolished histamine-induced rises in [Ca²⁺]_cyt. In PAEC bathed in Ca²⁺-free solution, however, treatment with CPA eliminated histamine-induced sustained and oscillatory rises in [Ca²⁺]_cyt but did not affect initial transient increase in [Ca²⁺]_cyt. Furthermore, treatment of PAEC with a combination of CPA (or thapsigargin) and caffeine (and ryanodine), FCCP, or bafilomycin did not abolish histamine-induced transient [Ca²⁺]_cyt increases. These observations indicate that 1) depletion of CPA-sensitive stores is sufficient to cause CCE in both PASMC and PAEC; 2) induction of CCE in PAEC does not require depletion of all internal Ca²⁺ stores; 3) the histamine-releasable internal stores in PASMC are mainly CPA-sensitive stores; 4) PAEC, in addition to a CPA-sensitive functional pool, contain other stores insensitive to CPA, thapsigargin, caffeine, ryanodine, FCCP, and bafilomycin; and 5) although the CPA-insensitive stores in PAEC may not contribute to CCE, they contribute to histamine-mediated Ca²⁺ release. intracellular calcium stores; oscillations; pulmonary hypertension     

Metabolic inhibition with cyanide induces calcium release in pulmonary artery myocytes and Xenopus oocytes

We examined the effectsof metabolic inhibition on intracellular Ca²⁺ release insingle pulmonary arterial smooth muscle cells (PASMCs). Severemetabolic inhibition with cyanide (CN, 10 mM) increased intracellularcalcium concentration ([Ca²⁺]_i) and activatedCa²⁺-activated Cl currents[I_Cl(Ca)] in PASMCs, responses that were greatlyinhibited by BAPTA-AM or caffeine. Mild metabolic inhibition with CN (1 mM) increased spontaneous transient inward currents andCa²⁺ sparks in PASMCs. In Xenopus oocytes, CNalso induced Ca²⁺ release and activatedI_Cl(Ca), and these responses were inhibited by thapsigarginand cyclopiazonic acid to deplete sarcoplasmic reticulum (SR)Ca²⁺, whereas neither heparin nor anti-inositol1,4,5-trisphosphate receptor (IP₃R) antibodies affected CNresponses. In both PASMCs and oocytes, CN-evoked Ca²⁺release was inhibited by carbonyl cyanidem-chlorophenylhydrazone (CCCP) and oligomycin or CCCP andthapsigargin. Whereas hypoxic stimuli resulted in Ca²⁺release in pulmonary but not mesenteric artery myocytes, CN induced release in both cell types. We conclude that metabolic inhibition withCN increases [Ca²⁺]_i in both pulmonary andsystemic artery myocytes by stimulating Ca²⁺ release fromthe SR and mitochondria.

     

Regulation of intracellular calcium in human esophageal smooth muscles

We have investigated sources ofCa²⁺ contributing to excitation ofhuman esophageal smooth muscle, using fura 2 to study cytosolic freeCa²⁺ concentration([Ca²⁺]_i)in dispersed cells and contraction of intact muscles. Acetylcholine (ACh) caused an initial peak rise of[Ca²⁺]_ifollowed by a plateau accompanied by reversible contraction. Removal ofextracellular Ca²⁺ or addition ofdihydropyridine Ca²⁺ channelblockers reduced the plateau phase but did not prevent contraction.Caffeine also caused elevation of[Ca²⁺]_iand blocked responses to ACh. Undershoots of[Ca²⁺]_iwere apparent after ACh or caffeine. Blockade of the sarcoplasmic reticular Ca²⁺-ATPase bycyclopiazonic acid (CPA) reduced the ACh-evoked increase of[Ca²⁺]_iand abolished the undershoot, indicating involvement ofCa²⁺ stores. When contraction wasstudied in intact muscles, removal ofCa²⁺ or addition of nifedipinereduced, but did not abolish, carbachol (CCh)-induced contraction.Elevation of extracellular K⁺caused contraction that was inhibited by nifedipine, although CCh stillelicited contraction. CPA caused contraction and suppressed theCCh-induced contraction, whereas ryanodine reduced CCh-induced contraction. Our studies provide evidence that muscarinic excitation ofhuman esophagus involves both release ofCa²⁺ from intracellular stores andinflux of Ca²⁺.

     

Differing temporal roles of Ca2+ and cAMP in nicotine-elicited elevation of tyrosine hydroxylase mRNA

The involvement of cAMP- andCa²⁺-mediated pathways in theactivation of tyrosine hydroxylase (TH) gene expression by nicotine wasexamined in PC-12 cells. ExtracellularCa²⁺ and elevations inintracellular Ca²⁺ concentration([Ca²⁺]_i)were required for nicotine to increase TH mRNA. The nicotine-elicited rapid rise in[Ca²⁺]_iwas inhibited by blockers of either L-type or N-type, and to a lesserextent P/Q-, but not T-type, voltage-gatedCa²⁺ channels. With continualnicotine treatment,[Ca²⁺]_ireturned to basal levels within 3-4 min. After a lag of~5-10 min, there was a smaller elevation in[Ca²⁺]_ithat persisted for 6 h and displayed different responsiveness toCa²⁺ channel blockers. This secondphase of elevated[Ca²⁺]_iwas blocked by an inhibitor of store-operatedCa²⁺ channels, consistent with theobserved generation of inositol trisphosphate.1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (BAPTA-AM), when added before or 2 h after nicotine,prevented elevation of TH mRNA. Nicotine treatment significantly raised cAMP levels. Addition of the adenylyl cyclase inhibitor2',5'-dideoxyadenosine (DDA) prevented thenicotine-elicited phosphorylation of cAMP response element bindingprotein. DDA also blocked the elevation of TH mRNA only when addedafter the initial transient rise in [Ca²⁺]_iand not after 1 h. This study reveals that several temporal phases areinvolved in the induction of TH gene expression by nicotine, each ofthem with differing requirements forCa²⁺ and cAMP.

     

Regulation of intracellular calcium in N1E-115 neuroblastoma cells: the role of Na(+)/Ca(2+) exchange

In fura 2-loaded N1E-115 cells, regulationof intracellular Ca²⁺ concentration([Ca²⁺]_i) following a Ca²⁺ loadinduced by 1 µM thapsigargin and 10 µM carbonylcyanidep-trifluoromethyoxyphenylhydrazone (FCCP) wasNa⁺ dependent and inhibited by 5 mM Ni²⁺. Incells with normal intracellular Na⁺ concentration([Na⁺]_i), removal of bath Na⁺,which should result in reversal of Na⁺/Ca²⁺exchange, did not increase [Ca²⁺]_i unlesscell Ca²⁺ buffer capacity was reduced. When N1E-115 cellswere Na⁺ loaded using 100 µM veratridine and 4 µg/mlscorpion venom, the rate of the reverse mode of theNa⁺/Ca²⁺ exchanger was apparently enhanced,since an ~4- to 6-fold increase in [Ca²⁺]_ioccurred despite normal cell Ca²⁺ buffering. In SBFI-loadedcells, we were able to demonstrate forward operation of theNa⁺/Ca²⁺ exchanger (net efflux ofCa²⁺) by observing increases (~ 6 mM) in[Na⁺]_i. These Ni²⁺ (5 mM)-inhibited increases in [Na⁺]_i could onlybe observed when a continuous ionomycin-induced influx ofCa²⁺ occurred. The voltage-sensitive dyebis-(1,3-diethylthiobarbituric acid) trimethine oxonol was used tomeasure changes in membrane potential. Ionomycin (1 µM) depolarizedN1E-115 cells (~25 mV). This depolarization was Na⁺dependent and blocked by 5 mM Ni²⁺ and 250-500 µMbenzamil. These data provide evidence for the presence of anelectrogenic Na⁺/Ca²⁺ exchanger that is capableof regulating [Ca²⁺]_i after release ofCa²⁺ from cell stores.