Activators of protein kinase C decrease Ca2+ spark frequency in smooth muscle cells from cerebral arteries

LocalCa²⁺ transients("Ca²⁺ sparks") caused bythe opening of one or the coordinated opening of a number of tightlyclustered ryanodine-sensitiveCa²⁺-release (RyR) channels in thesarcoplasmic reticulum (SR) activate nearbyCa²⁺-dependentK⁺(K_Ca) channels to cause anoutward current [referred to as a "spontaneous transientoutward current" (STOC)]. TheseK_Ca currents cause membranepotential hyperpolarization of arterial myocytes, which would lead tovasodilation through decreasingCa²⁺ entry throughvoltage-dependent Ca²⁺ channels.Therefore, modulation of Ca²⁺spark frequency should be a means to regulation ofK_Ca channel currents and hencemembrane potential. We examined the frequency modulation ofCa²⁺ sparks and STOCs byactivation of protein kinase C (PKC). The PKC activators, phorbol12-myristate 13-acetate (PMA; 10 nM) and 1,2-dioctanoyl-sn-glycerol (1 µM),decreased Ca²⁺ spark frequency by72% and 60%, respectively, and PMA reduced STOC frequency by 83%.PMA also decreased STOC amplitude by 22%, which could be explained byan observed reduction (29%) inK_Ca channel open probability inthe absence of Ca²⁺ sparks. Thereduction in STOC frequency occurred in the presence of an inorganicblocker (Cd²⁺) ofvoltage-dependent Ca²⁺ channels.The reduction in Ca²⁺ sparkfrequency did not result from SRCa²⁺ depletion, sincecaffeine-induced Ca²⁺ transientsdid not decrease in the presence of PMA. These results suggest thatactivators of PKC can modulate the frequency ofCa²⁺ sparks, through an effect onthe RyR channel, which would decrease STOC frequency (i.e.,K_Ca channel activity).

     

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

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

     

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

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

Ca2+ dependence and pharmacology of large-conductance K+ channels in nonlabor and labor human uterine myocytes

Two populations,Ca²⁺-dependent(BK_Ca) andCa²⁺-independentK⁺ (BK) channels of largeconductance were identified in inside-out patches of nonlabor and laborfreshly dispersed human pregnant myometrial cells, respectively.Cell-attached recordings from nonlabor myometrial cells frequentlydisplayed BK_Ca channel openings characterized by a relatively low open-state probability, whereas similar recordings from labor tissue displayed either no channel openings or consistently high levels of channel activity that oftenexhibited clear, oscillatory activity. In inside-out patch recordings,Ba²⁺ (2-10 mM),4-aminopyridine (0.1-1 mM), andShaker B inactivating peptide("ball peptide") blocked theBK_Ca channel but were much lesseffective on BK channels. Application of tetraethylammonium toinside-out membrane patches reduced unitary current amplitude ofBK_Ca and BK channels, withdissociation constants of 46 mM and 53 µM, respectively.Tetraethylammonium applied to outside-out patches decreased the unitaryconductance of BK_Ca and BKchannels, with dissociation constants of 423 and 395 µM,respectively. These results demonstrate that the properties of humanmyometrial large-conductance K⁺channels in myocytes isolated from laboring patients are significantly different from those isolated from nonlaboring patients.

     

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.

     

Purinergic activation of spontaneous transient outward currents in guinea pig taenia colonic myocytes

Spontaneous transient outward currents(STOCs) were recorded from smooth muscle cells of theguinea pig taenia coli using the whole cell patch-clamp technique.STOCs were resolved at potentials positive to 50 mV. Treatingcells with caffeine (1 mM) caused a burst of outward currentsfollowed by inhibition of STOCs. Replacing extracellularCa²⁺ with equimolarMn²⁺ caused STOCs to "rundown." Iberiotoxin (200 nM) or charybdotoxin (ChTX; 200 nM)inhibited large-amplitude STOCs, but small-amplitude "mini-STOCs"remained in the presence of these drugs. Mini-STOCs were reduced byapamin (500 nM), an inhibitor of small-conductance Ca²⁺-activatedK⁺ channels (SK channels).Application of ATP or 2-methylthioadenosine 5'-triphosphate(2-MeS-ATP) increased the frequency of STOCs. The effects of 2-MeS-ATPpersisted in the presence of charybdotoxin but were blocked bycombination of ChTX (200 nM) and apamin (500 nM). 2-MeS-ATP did notincrease STOCs in the presence of pyridoxal phosphate6-azophenyl-2',4'-disulfonic acid, aP₂ receptor blocker. Similarly,pretreatment of cells with U-73122 (1 µM), an inhibitor ofphospholipase C (PLC), abolished the effects of 2-MeS-ATP. XestosponginC, an inositol 1,4,5-trisphosphate(IP₃) receptor blocker,attenuated STOCs, but these events were not affected by ryanodine. Thedata suggest that purinergic activation through P_2Y receptors results in localizedCa²⁺ release via PLC- andIP₃-dependent mechanisms. Releaseof Ca²⁺ is coupled to STOCs, whichare composed of currents mediated by large-conductanceCa²⁺-activatedK⁺ channels and SK channels. Thelatter are thought to mediate hyperpolarization and relaxationresponses of gastrointestinal muscles to inhibitory purinergic stimulation.

     

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

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

     

Frequency modulation of Ca2+ sparks is involved in regulation of arterial diameter by cyclic nucleotides

Forskolin, which elevates cAMP levels, and sodium nitroprusside(SNP) and nicorandil, which elevate cGMP levels, increased, by two- tothreefold, the frequency of subcellularCa²⁺ release("Ca²⁺ sparks") throughryanodine-sensitive Ca²⁺ release(RyR) channels in the sarcoplasmic reticulum (SR) of myocytes isolatedfrom cerebral and coronary arteries of rats. Forskolin, SNP,nicorandil, dibutyryl-cAMP, and adenosine increased the frequency ofCa²⁺-sensitiveK⁺(K_Ca) currents["spontaneous transient outward currents" (STOCs)] bytwo- to threefold, consistent withCa²⁺ sparks activating STOCs.These agents also increased the mean amplitude of STOCs by 1.3-fold, aneffect that could be explained by activation ofK_Ca channels, independent ofeffects on Ca²⁺ sparks. To testthe hypothesis that cAMP could act to dilate arteries throughactivation of the Ca²⁺sparkK_Ca channel pathway,the effects of blockers of K_Cachannels (iberiotoxin) and of Ca²⁺sparks (ryanodine) on forskolin-induced dilations of pressurized cerebral arteries were examined. Forskolin-induced dilations were partially inhibited by iberiotoxin and ryanodine (with no additive effects) and were entirely prevented by elevating externalK⁺. Forskolin lowered averageCa²⁺ in pressurized arteries whileincreasing ryanodine-sensitive, caffeine-inducedCa²⁺ transients. These experimentssuggest a new mechanism for cyclic nucleotide-mediated dilationsthrough an increase in Ca²⁺ sparkfrequency, caused by effects on SRCa²⁺ load and possibly on the RyRchannel, which leads to increased STOC frequency, membrane potentialhyperpolarization, closure of voltage-dependentCa²⁺ channels, decrease inarterial wall Ca²⁺, and,ultimately, vasodilation.

     

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.

     

Micromolar Ca2+ from sparks activates Ca2+-sensitive K+ channels in rat cerebral artery smooth muscle

The goal of the present study was to testthe hypothesis that local Ca²⁺ release events(Ca²⁺ sparks) deliver high local Ca²⁺concentration to activate nearby Ca²⁺-sensitiveK⁺ (BK) channels in the cell membrane of arterial smoothmuscle cells. Ca²⁺ sparks and BK channels were examined inisolated myocytes from rat cerebral arteries with laser scanningconfocal microscopy and patch-clamp techniques. BK channels had anapparent dissociation constant for Ca²⁺ of 19 µM and aHill coefficient of 2.9 at 40 mV. At near-physiological intracellularCa²⁺ concentration ([Ca²⁺]_i; 100 nM) and membrane potential (40 mV), the open probability of a singleBK channel was low (1.2 × 10⁶). A Ca²⁺spark increased BK channel activity to 18. Assuming that 1-100% of the BK channels are activated by a single Ca²⁺ spark, BKchannel activity increases 6 × 10⁵-fold to 6 × 10³-fold, which corresponds to ~30 µM to 4 µM sparkCa²⁺ concentration.1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acidacetoxymethyl ester caused the disappearance of all Ca²⁺sparks while leaving the transient BK currents unchanged. Our resultssupport the idea that Ca²⁺ spark sites are in closeproximity to the BK channels and that local[Ca²⁺]_i reaches micromolar levels to activateBK channels.

     

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.

     

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.     

Characteristics of phosphate-induced Ca(2+) efflux from the SR in mechanically skinned rat skeletal muscle fibers

ATP is a candidate enteric inhibitory neurotransmitterin visceral smooth muscles. ATP hyperpolarizes visceral muscles via activation of small-conductance, Ca²⁺-activatedK⁺ (SK) channels. Coupling between ATP stimulation and SKchannels may be mediated by localized Ca²⁺ release.Isolated myocytes of the murine colon produced spontaneous, localizedCa²⁺ release events. These events corresponded tospontaneous transient outward currents (STOCs) consisting ofcharybdotoxin (ChTX)-sensitive and -insensitive events.ChTX-insensitive STOCs were inhibited by apamin. LocalizedCa²⁺ transients were not blocked by ryanodine, but theseevents were reduced in magnitude and frequency by xestospongin C(Xe-C), a blocker of inositol 1,4,5-trisphosphate receptors. Thus wehave termed the localized Ca²⁺ events in colonic myocytes"Ca²⁺ puffs." The P_2Y receptor agonist2-methylthio-ATP (2-MeS-ATP) increased the intensity and frequency ofCa²⁺ puffs. 2-MeS-ATP also increased STOCs in associationwith the increase in Ca²⁺ puffs.Pyridoxal-phospate-6-azophenyl-2',4'-disculfonic acid tetrasodium, aP₂ receptor inhibitor, blocked responses to 2-MeS-ATP. Spontaneous Ca²⁺ transients and the effects of 2-MeS-ATP onCa²⁺ puffs and STOCs were blocked by U-73122, an inhibitorof phospholipase C. Xe-C and ryanodine also blocked responses to2-MeS-ATP, suggesting that, in addition to release from IP₃receptor-operated stores, ryanodine receptors may be recruited duringagonist stimulation to amplify release of Ca²⁺. These datasuggest that localized Ca²⁺ release modulatesCa²⁺-dependent ionic conductances in the plasma membrane.Localized Ca²⁺ release may contribute to the electricalresponses resulting from purinergic stimulation.

     

Reduced Ca2+ uptake by mitochondria in pyruvate dehydrogenase-deficient human diploid fibroblasts

Physiological and pathologicalCa²⁺ loads are thought to be takenup by mitochondria via a process dependent on aerobic metabolism. Wesought to determine whether human diploid fibroblasts from a patientwith an inherited defect in pyruvate dehydrogenase (PDH) exhibit adecreased ability to sequester cytosolicCa²⁺ into mitochondria.Mobilization of Ca²⁺ stores withbradykinin (BK) increased the cytosolicCa²⁺ concentration([Ca²⁺]_c)to comparable levels in control and PDH-deficient fibroblasts. Innormal fibroblasts transfected with plasmid DNA encodingmitochondrion-targeted apoaequorin, BK elicited an increase inCa²⁺-dependent aequorinluminescence corresponding to an increase in the mitochondrialCa²⁺ concentration([Ca²⁺]_mt)of 2.0 ± 0.2 µM. The mitochondrial uncoupling agent carbonyl cyanidep-(trifluoromethoxy)phenylhydrazoneblocked the BK-induced [Ca²⁺]_mtincrease, although it did not affect the[Ca²⁺]_ctransient. Basal[Ca²⁺]_cand[Ca²⁺]_mtin control and PDH-deficient cells were similar. However, confocalimaging of the potential-sensitive dye JC-1 indicated that thepercentage of highly polarized mitochondria was reduced from 30 ± 1% in normal cells to 19 ± 2% in the PDH-deficient fibroblasts. BK-elicited[Ca²⁺]_mttransients in PDH-deficient cells were reduced to 4% of control, indicating that PDH-deficient mitochondria have a decreased ability totake up cytosolic Ca²⁺. Thus cellswith compromised aerobic metabolism have a reduced capacity tosequester Ca²⁺.

     

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.

     

Intracellular Ca(2+) stores in chemoreceptor cells of the rabbit carotid body: significance for chemoreception

Thenotion that intracellular Ca²⁺ (Ca_i²⁺)stores play a significant role in the chemoreception process inchemoreceptor cells of the carotid body (CB) appears in the literaturein a recurrent manner. However, the structural identity of theCa²⁺ stores and their real significance in the function ofchemoreceptor cells are unknown. To assess the functional significanceof Ca_i²⁺ stores in chemoreceptor cells, we havemonitored 1) the release of catecholamines (CA) from thecells using an in vitro preparation of intact rabbit CB and2) the intracellular Ca²⁺ concentration([Ca²⁺]_i) using isolated chemoreceptor cells;both parameters were measured in the absence or the presence of agentsinterfering with the storage of Ca²⁺. We found thatthreshold [Ca²⁺]_i for high extracellularK⁺ (K_e⁺) to elicit a release response is250 nM. Caffeine (10-40 mM), ryanodine (0.5 µM), thapsigargin(0.05-1 µM), and cyclopiazonic acid (10 µM) did not alter thebasal or the stimulus (hypoxia, high K_e⁺)-inducedrelease of CA. The same agents produced Ca_i²⁺transients of amplitude below secretory threshold; ryanodine (0.5 µM), thapsigargin (1 µM), and cyclopiazonic acid (10 µM) did notalter the magnitude or time course of the Ca_i²⁺responses elicited by high K_e⁺. Several potentialactivators of the phospholipase C system (bethanechol, ATP, andbradykinin), and thereby of inositol 1,4,5-trisphosphate receptors,produced minimal or no changes in [Ca²⁺]_i anddid not affect the basal release of CA. It is concluded that, in therabbit CB chemoreceptor cells, Ca_i²⁺ stores do not playa significant role in the instant-to-instant chemoreception process.

     

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

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

     

VIP and PACAP regulate localized Ca2+ transients via cAMP-dependent mechanism

Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) have been suggested as participants in enteric inhibitory neural regulation of gastrointestinal motility. These peptides cause a variety of postjunctional responses including membrane hyperpolarization and inhibition of contraction. Neuropeptides released from enteric motor neurons can elicit responses by direct stimulation of smooth muscle cells as opposed to other transmitters that rely on synapses between motor nerve terminals and interstitial cells of Cajal. Therefore, we studied the responses of murine colonic smooth muscle cells to VIP and PACAP(1–38) with confocal microscopy and patch-clamp technique. Localized Ca²⁺ transients (Ca²⁺ puffs) were observed in colonic myocytes, and these events coupled to spontaneous transient outward currents (STOCs). VIP and PACAP increased Ca²⁺ transients and STOC frequency and amplitude. Application of dibutyryl cAMP had similar effects. The adenylyl cyclase blocker MDL-12,330A alone did not affect spontaneous Ca²⁺ puffs and STOCs but prevented responses to VIP. Disruption of A-kinase-anchoring protein (AKAP) associations by application of AKAP St-Ht31 inhibitory peptide had effects similar to those of MDL-12,330A. Inhibition of ryanodine receptor channels did not block spontaneous Ca²⁺ puffs and STOCs but prevented the effects of dibutyryl cAMP. These findings suggest that regulation of Ca²⁺ transients (which couple to activation of STOCs) may contribute to the inhibitory effects of VIP and PACAP. Regulation of Ca²⁺ transients by VIP and PACAP occurs via adenylyl cyclase, increased synthesis of cAMP, and PKA-dependent regulation of ryanodine receptor channels. calcium puffs; ryanodine receptor channels; enteric nervous system; gastrointestinal motility     

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

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

     

Nitric oxide induces [Ca2+]i oscillations in pituitary GH3 cells: involvement of IDR and ERG K+ currents

The role of nitric oxide (NO) in the occurrence of intracellular Ca²⁺ concentration ([Ca²⁺]_i) oscillations in pituitary GH₃ cells was evaluated by studying the effect of increasing or decreasing endogenous NO synthesis with L-arginine and nitro-L-arginine methyl ester (L-NAME), respectively. When NO synthesis was blocked with L-NAME (1 mM) [Ca²⁺]_i, oscillations disappeared in 68% of spontaneously active cells, whereas 41% of the quiescent cells showed [Ca²⁺]_i oscillations in response to the NO synthase (NOS) substrate L-arginine (10 mM). This effect was reproduced by the NO donors NOC-18 and S-nitroso-N-acetylpenicillamine (SNAP). NOC-18 was ineffective in the presence of the L-type voltage-dependent Ca²⁺ channels (VDCC) blocker nimodipine (1 µM) or in Ca²⁺-free medium. Conversely, its effect was preserved when Ca²⁺ release from intracellular Ca²⁺ stores was inhibited either with the ryanodine-receptor blocker ryanodine (500 µM) or with the inositol 1,4,5-trisphosphate receptor blocker xestospongin C (3 µM). These results suggest that NO induces the appearance of [Ca²⁺]_i oscillations by determining Ca²⁺ influx. Patch-clamp experiments excluded that NO acted directly on VDCC but suggested that NO determined membrane depolarization because of the inhibition of voltage-gated K⁺ channels. NOC-18 and SNAP caused a decrease in the amplitude of slow-inactivating (I_DR) and ether-à-go-go-related gene (ERG) hyperpolarization-evoked, deactivating K⁺ currents. Similar results were obtained when GH₃ cells were treated with L-arginine. The present study suggests that in GH₃ cells, endogenous NO plays a permissive role for the occurrence of spontaneous [Ca²⁺]_i oscillations through an inhibitory effect on I_DR and on I_ERG. voltage-gated potassium channels; ether-à-go-go-related gene potassium channels; slow-inactivating outward currents; fast-inactivating outward currents