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.

     

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.

     

Coupling strength between localized Ca(2+) transients and K(+) channels is regulated by protein kinase C

Localized Ca²⁺ transients resulting from inositoltrisphosphate (IP₃)-dependent Ca²⁺ releasecouple to spontaneous transient outward currents (STOCs) in murinecolonic myocytes. Confocal microscopy and whole cell patch-clamptechniques were used to investigate coupling between localizedCa²⁺ transients and STOCs. Colonic myocytes were loadedwith fluo 3. Reduction in external Ca²⁺([Ca²⁺]_o) reduced localized Ca²⁺transients but increased STOC amplitude and frequency. Simultaneous recordings of Ca²⁺ transients and STOCs showed increasedcoupling strength between Ca²⁺ transients and STOCs when[Ca²⁺]_o was reduced. Gd³⁺ (10 µM) did not affect Ca²⁺ transients but increased STOCamplitude and frequency. Similarly, an inhibitor of Ca²⁺influx,1-2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1H-imidazole (SKF-96365), increased STOC amplitude and frequency. A protein kinase C(PKC) inhibitor, GF-109203X, also increased the amplitude and frequencyof STOCs but had no effect on Ca²⁺ transients. Phorbol12-myristate 13-acetate (1 µM) reduced STOC amplitude and frequencybut did not affect Ca²⁺ transients. 4-Phorbol (1 µM)had no effect on STOCs or Ca²⁺ transients. Single channelstudies indicated that large-conductance Ca²⁺-activatedK⁺ (BK) channels were inhibited by aCa²⁺-dependent PKC. In summary 1)Ca²⁺ release from IP₃ receptor-operated storesactivates Ca²⁺-activated K⁺ channels;2) Ca²⁺ influx through nonselective cationchannels facilitates activation of PKC; and 3) PKC reducesthe Ca²⁺ sensitivity of BK channels, reducing the couplingstrength between localized Ca²⁺ transients and BK channels.

     

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.

     

The Adenylyl Cyclase Inhibitor MDL-12,330A Potentiates Insulin Secretion via Blockade of Voltage-Dependent K+ Channels in Pancreatic Beta Cells

Objective

Adenylyl cyclases (ACs) play important role in regulating pancreatic beta cell growth, survival and secretion through the synthesis of cyclic AMP (cAMP). MDL-12,330A and SQ 22536 are two AC inhibitors used widely to establish the role of ACs. The goal of this study was to examine the effects of MDL-12,330A and SQ 22536 on insulin secretion and underlying mechanisms.

Methods

Patch-clamp recording, Ca²⁺ fluorescence imaging and radioimmunoassay were used to measure outward K⁺ currents, action potentials (APs), intracellular Ca²⁺ ([Ca²⁺]_i) and insulin secretion from rat pancreatic beta cells.

Results

MDL-12,330A (10 µmol/l) potentiated insulin secretion to 1.7 times of control in the presence of 8.3 mmol/l glucose, while SQ 22536 did not show significant effect on insulin secretion. MDL-12,330A prolonged AP durations (APDs) by inhibiting voltage-dependent K⁺ (K_V) channels, leading to an increase in [Ca²⁺]_i levels. It appeared that these effects induced by MDL-12,330A did not result from AC inhibition, since SQ 22536 did not show such effects. Furthermore, inhibition of the downstream effectors of AC/cAMP signaling by PKA inhibitor H89 and Epac inhibitor ESI-09, did not affect K_V channels and insulin secretion.

Conclusion

The putative AC inhibitor MDL-12,330A enhances [Ca²⁺]_i and insulin secretion via inhibition of K_V channels rather than AC antagonism in beta cells, suggesting that the non-specific effects is needed to be considered for the right interpretation of the experimental results using this agent in the analyses of the role of AC in cell function.     

Ca2+ Sparks Act as Potent Regulators of Excitation-Contraction Coupling in Airway Smooth Muscle

Ca²⁺ sparks are short lived and localized Ca²⁺ transients resulting from the opening of ryanodine receptors in sarcoplasmic reticulum. These events relax certain types of smooth muscle by activating big conductance Ca²⁺-activated K⁺ channels to produce spontaneous transient outward currents (STOCs) and the resultant closure of voltage-dependent Ca²⁺ channels. But in many smooth muscles from a variety of organs, Ca²⁺ sparks can additionally activate Ca²⁺-activated Cl⁻ channels to generate spontaneous transient inward current (STICs). To date, the physiological roles of Ca²⁺ sparks in this latter group of smooth muscle remain elusive. Here, we show that in airway smooth muscle, Ca²⁺ sparks under physiological conditions, activating STOCs and STICs, induce biphasic membrane potential transients (BiMPTs), leading to membrane potential oscillations. Paradoxically, BiMPTs stabilize the membrane potential by clamping it within a negative range and prevent the generation of action potentials. Moreover, blocking either Ca²⁺ sparks or hyperpolarization components of BiMPTs activates voltage-dependent Ca²⁺ channels, resulting in an increase in global [Ca²⁺]_i and cell contraction. Therefore, Ca²⁺ sparks in smooth muscle presenting both STICs and STOCs act as a stabilizer of membrane potential, and altering the balance can profoundly alter the status of excitability and contractility. These results reveal a novel mechanism underlying the control of excitability and contractility in smooth muscle.     

Role of calcium mobilization in the regulation of spontaneous transient outward currents in porcine coronary artery myocytes

The purpose of the present study was to further study the characteristics and regulation of spontaneous transient outward currents (STOCs) in freshly isolated porcine coronary artery smooth muscle cells (ASMCs). STOCs were recorded using the perforated whole-cell patch-clamp configuration. STOCs were voltage-dependent and superimposed stochastically onto whole-cell Ca²⁺-activated-K⁺ (BK_Ca) currents. Charybdotoxin (ChTX, 200 nmol/L), a selective blocker of BK_Ca channels, completely inhibited STOCs within 10 min. STOCs activity was greatly suppressed when extracellular Ca²⁺ concentration decreased from 1.8 mmol/L to 200 nmol/L, further removal of Ca²⁺ abolished STOCs activity. Ca²⁺ ionophore A23187 (10 μmol/L) increased STOCs activity significantly. Verapamil (20 μmol/L) and CdCl₂ (200 μmol/L), two kinds of organic L-type voltage-dependent Ca²⁺ channels (L-VDCCs) antagonists, had little effect on STOCs. In addition, the ryanodine receptors (RyRs) agonist caffeine (5 mmol/L) significantly activated STOCs. Application of ryanodine (50 μmol/L) to block RyRs abolished STOCs, subsequent washout of ryanodine or application of caffeine failed to reproduce STOCs activity. Inhibition of inositol 1,4,5-trisphosphate receptors (IP₃Rs) by 2APB (40 μmol/L) greatly suppressed the activity of STOCs, application of caffeine (5 mmol/L) in the presence of 2APB caused a burst of outward currents followed by inhibition of STOCs. These results suggest that STOCs in porcine coronary ASMCs are mediated by BK_Ca channels. Extracellular Ca²⁺ is essential for STOCs activity, while Ca²⁺ entry through L-VDCCs has little effect on STOCs. Intracellular Ca²⁺ release induced by RyRs is responsible for the regulation of STOCs, whereas IP3Rs might also be involved.     

Role of calcium mobilization in the regulation of spontaneous transient outward currents in porcine coronary artery myocytes

The purpose of the present study was to further study the characteristics and regulation of spontaneous transient outward currents (STOCs) in freshly isolated porcine coronary artery smooth muscle cells (ASMCs). STOCs were recorded using the perforated whole-cell patch-clamp configuration. STOCs were voltage-dependent and superimposed stochastically onto whole-cell Ca2 -activated-K (BKCa) currents. Charybdotoxin (ChTX, 200 nmol/L), a selective blocker of BKCa channels, completely inhibited STOCs within 10 min. STOCs activity was greatly suppressed when extracellular Ca2 concentration decreased from 1.8 mmol/L to 200 nmol/L, further removal of Ca2 abolished STOCs activity. Ca2 ionophore A23187 (10 μmol/L) increased STOCs activity significantly. Verapamil (20 μmol/L) and CdCl2 (200 μmol/L), two kinds of organic L-type voltage-dependent Ca2 channels (L-VDCCs) antagonists, had little effect on STOCs. In addition, the ryanodine receptors (RyRs) agonist caffeine (5 mmol/L) significantly activated STOCs. Application of ryanodine (50 μmol/L) to block RyRs abolished STOCs, subsequent washout of ryanodine or application of caffeine failed to reproduce STOCs activity. Inhibition of inositol 1,4,5-trisphosphate receptors (IP3Rs) by 2APB (40 μmol/L) greatly suppressed the activity of STOCs, application of caffeine (5 mmol/L) in the presence of 2APB caused a burst of outward currents followed by inhibition of STOCs. These results suggest that STOCs in porcine coronary ASMCs are mediated by BKCa channels. Extracellular Ca2 is essential for STOCs activity, while Ca2 entry through L-VDCCs has little effect on STOCs. Intracellular Ca2 release induced by RyRs is responsible for the regulation of STOCs, whereas IP3Rs might also be involved.     

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.

     

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).

     

Intermittent hypoxia protects cardiomyocytes against ischemia-reperfusion injury-induced alterations in Ca2+ homeostasis and contraction via the sarcoplasmic reticulum and Na+/Ca2+ exchange mechanisms

We have previously demonstrated that intermittent high-altitude (IHA) hypoxia significantly attenuates ischemia-reperfusion (I/R) injury-induced excessive increase in resting intracellular Ca²⁺ concentrations ([Ca²⁺]_i). Because the sarcoplasmic reticulum (SR) and Na⁺/Ca²⁺ exchanger (NCX) play crucial roles in regulating [Ca²⁺]_i and both are dysfunctional during I/R, we tested the hypothesis that IHA hypoxia may prevent I/R-induced Ca²⁺ overload by maintaining Ca²⁺ homeostasis via SR and NCX mechanisms. We thus determined the dynamics of Ca²⁺ transients and cell shortening during preischemia and I/R injury in ventricular cardiomyocytes from normoxic and IHA hypoxic rats. IHA hypoxia did not affect the preischemic dynamics of Ca²⁺ transients and cell shortening, but it significantly suppressed the I/R-induced increase in resting [Ca²⁺]_i levels and attenuated the depression of the Ca²⁺ transients and cell shortening during reperfusion. Moreover, IHA hypoxia significantly attenuated I/R-induced depression of the protein contents of SR Ca²⁺ release channels and/or ryanodine receptors (RyRs) and SR Ca²⁺ pump ATPase (SERCA2) and SR Ca²⁺ release and uptake. In addition, a delayed decay rate time constant of Ca²⁺ transients and cell shortening of Ca²⁺ transients observed during ischemia was accompanied by markedly inhibited NCX currents, which were prevented by IHA hypoxia. These findings indicate that IHA hypoxia may preserve Ca²⁺ homeostasis and contraction by preserving RyRs and SERCA2 proteins as well as NCX activity during I/R. intracellular Ca²⁺ concentration; Ca²⁺ transients; Ca²⁺ transporters; myofilament Ca²⁺ sensitivity     

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     

Effect of beta -adrenoceptor activation on [Ca2+]i regulation in murine skeletal myotubes

The presentstudy used real-time confocal microscopy to examine the effects of the₂-adrenoceptor agonistsalbutamol on regulation of intracellularCa²⁺ concentration([Ca²⁺]_i)in myotubes derived from neonatal mouse limb muscles.Immunocytochemical staining for ryanodine receptors and skeletal musclemyosin confirmed the presence of sarcomeres. The myotubes displayedboth spontaneous and ACh-induced rapid (<2-ms rise time)[Ca²⁺]_itransients. The[Ca²⁺]_itransients were frequency modulated by both low and high concentrations of salbutamol. Exposure to -bungarotoxin and tetrodotoxin inhibited ACh-induced[Ca²⁺]_itransients and the response to low concentrations of salbutamol but notthe response to higher concentrations. Preexposure to caffeineinhibited the subsequent[Ca²⁺]_iresponse to lower concentrations of salbutamol and significantly blunted the response to higher concentrations. Preexposure to salbutamol diminished the[Ca²⁺]_iresponse to caffeine. Inhibition of dihydropyridine-sensitive Ca²⁺ channels with nifedipine orPN-200-110 did not prevent[Ca²⁺]_ielevations induced by higher concentrations of salbutamol. The effectsof salbutamol were mimicked by the membrane-permeant analog dibutyryladenosine 3',5'-cyclic monophosphate. Thesedata indicate that salbutamol effects in skeletal muscle predominantly involve enhanced sarcoplasmic reticulumCa²⁺ release.     

Calcium sparks activate calcium-dependent Cl- current in rat corpus cavernosum smooth muscle cells

Spontaneous transient currents, due to activation of Ca²⁺-dependent K⁺ and Cl^– channels, occur in corpus cavernosum smooth muscle cells (CCSMC) of the penis. The Ca²⁺ events responsible for triggering Ca²⁺-dependent Cl^– channels have never been identified in vascular muscle. We used high-speed fluorescence imaging combined with patch-clamp electrophysiology to provide the first characterization of Ca²⁺ events underlying these currents. Freshly isolated rat CCSMC loaded with fluo-4 exhibited localized, spontaneous elevations of intracellular Ca²⁺ (Ca²⁺ sparks) in 57% of cells. There was an average of 6.4 ± 0.5 release sites/cell with a frequency of 0.9 ± 1 Hz/cell and peak amplitude F/F_o of 67 ± 10%. We addressed the controversy of whether these events are mediated by ryanodine or inositol 1,4,5 trisphosphate (IP₃) receptors. Caffeine caused either a global Ca²⁺ rise at high concentrations or an increase in spark frequency at lower concentrations, whereas ryanodine dramatically reduced the amplitude and frequency of sparks. 2-Aminoethoxydiphenyl borate, an inhibitor of IP₃ receptors, had no effect on spark frequency. Combined imaging and electrophysiological recording revealed strong coupling between Ca²⁺ sparks and biphasic transient currents, a relationship never before shown in vascular muscle. Moreover, spark frequency increased on depolarization, an effect abolished with the blockade of Ca²⁺ channels, consistent with Ca²⁺ influx regulating Ca²⁺ release from stores. We establish for the first time that Ca²⁺ sparks occur in CCSMC and arise from Ca²⁺ release through ryanodine receptors. Moreover, the voltage dependence of spark frequency demonstrated here provides novel functional evidence for voltage-dependent Ca²⁺ influx in CCSMC. calcium signaling; potassium and chloride channels; ryanodine receptors     

NAADP as a second messenger: neither CD38 nor base-exchange reaction are necessary for in vivo generation of NAADP in myometrial cells

Nicotinic acid adenine dinucleotide phosphate (NAADP) has recently been shown to act as a second messenger controlling intracellular Ca²⁺ responses in mammalian cells. Many questions remain regarding this signaling pathway, including the role of the ryanodine receptor (RyR) in NAADP-induced Ca²⁺ transients. Furthermore, the exact metabolic pathway responsible for the synthesis of NAADP in vivo has not been determined. Here, we demonstrate that the NAADP mediated Ca²⁺ release system is present in human myometrial cells. We also demonstrate that human myometrial cells use the NAADP second messenger system to generate intracellular Ca²⁺ transients in response to histamine. It has been proposed in the past that the NAADP system in mammalian cells is dependent on the presence of functional RyRs. Here, we observed that the histamine-induced Ca²⁺ transients are dependent on both the NAADP and inositol 1,4,5-trisphosphate signaling pathways but are independent of RyRs. The enzyme CD38 has been shown to catalyze the synthesis of NAADP in vitro by the base-exchange reaction. Furthermore, it has been proposed that this enzyme is responsible for the intracellular generation of NAADP in vivo. Using CD38 knockout mice, we observed that both the basal and histamine stimulated levels of NAADP are independent of CD38 and the base-exchange reaction. Our group is the first to demonstrate that NAADP is a second messenger for histamine-elicited Ca²⁺ transients in human myometrial cells. Furthermore, the NAADP mediated mechanism in mammalian cells can be independent of RyRs and CD38. Our data provides novel insights into the understanding of the mechanism of action and metabolism of this new second messenger system. cADP ribose; inositol 1,4,5-trisphosphate; endoplasmic reticulum; ryanodine channel; nicotinic acid adenine dinucleotide phosphate; CD38; base-exchange reaction     

Norepinephrine-induced Ca2+ waves depend on InsP3 and ryanodine receptor activation in vascular myocytes

In rat portal veinmyocytes, Ca²⁺ signals can begenerated by inositol 1,4,5-trisphosphate(InsP₃)- and ryanodine-sensitive Ca²⁺ release channels, which arelocated on the same intracellular store. Using a laser scanningconfocal microscope associated with the patch-clamp technique, weshowed that propagated Ca²⁺ wavesevoked by norepinephrine (in the continuous presence of oxodipine) werecompletely blocked after internal application of ananti-InsP₃ receptor antibody.These propagated Ca²⁺ waves werealso reduced by ~50% and transformed in homogenous Ca²⁺ responses after applicationof an anti-ryanodine receptor antibody or ryanodine. All-or-noneCa²⁺ waves obtained withincreasing concentrations of norepinephrine were transformed in adose-response relationship with a Hill coefficient close to unity afterryanodine receptor inhibition. Similar effects of the ryanodinereceptor inhibition were observed on the norepinephrine- andACh-induced Ca²⁺ responses innon-voltage-clamped portal vein and duodenal myocytes and on thenorepinephrine-induced contraction. Taken together, these results showthat ryanodine-sensitive Ca²⁺release channels are responsible for the fast propagation of Ca²⁺ responses evoked by variousneurotransmitters producing InsP₃ in vascular and visceral myocytes.

     

Functional Coupling of Ryanodine Receptors to KCa Channels in Smooth Muscle Cells from Rat Cerebral Arteries

The relationship between Ca²⁺ release (“Ca²⁺ sparks”) through ryanodine-sensitive Ca²⁺ release channels in the sarcoplasmic reticulum and K_Ca channels was examined in smooth muscle cells from rat cerebral arteries. Whole cell potassium currents at physiological membrane potentials (−40 mV) and intracellular Ca²⁺ were measured simultaneously, using the perforated patch clamp technique and a laser two-dimensional (x–y) scanning confocal microscope and the fluorescent Ca²⁺ indicator, fluo-3. Virtually all (96%) detectable Ca²⁺ sparks were associated with the activation of a spontaneous transient outward current (STOC) through K_Ca channels. A small number of sparks (5 of 128) were associated with currents smaller than 6 pA (mean amplitude, 4.7 pA, at −40 mV). Approximately 41% of STOCs occurred without a detectable Ca²⁺ spark. The amplitudes of the Ca²⁺ sparks correlated with the amplitudes of the STOCs (regression coefficient 0.8; P < 0.05). The half time of decay of Ca²⁺ sparks (56 ms) was longer than the associated STOCs (9 ms). The mean amplitude of the STOCs, which were associated with Ca²⁺ sparks, was 33 pA at −40 mV. The mean amplitude of the “sparkless” STOCs was smaller, 16 pA. The very significant increase in K_Ca channel open probability (>10⁴-fold) during a Ca²⁺ spark is consistent with local Ca²⁺ during a spark being in the order of 1–100 μM. Therefore, the increase in fractional fluorescence (F/F_o) measured during a Ca²⁺ spark (mean 2.04 F/F_o or ∼310 nM Ca²⁺) appears to significantly underestimate the local Ca²⁺ that activates K_Ca channels. These results indicate that the majority of ryanodine receptors that cause Ca²⁺ sparks are functionally coupled to K_Ca channels in the surface membrane, providing direct support for the idea that Ca²⁺ sparks cause STOCs.     

Presynaptic NMDA receptors act as local high‐gain glutamate detector in developing cerebellar molecular layer interneurons

In the classical view, NMDA receptors (NMDARs) are located postsynaptically and play a pivotal role in excitatory transmission and synaptic plasticity. In developing cerebellar molecular layer interneurons (MLIs) however, NMDARs are known to be solely extra‐ or presynaptic and somewhat poorly expressed. Somatodendritic NMDARs are exclusively activated by glutamate spillover from adjacent synapses, but the mode of activation of axonal NMDARs remains unclear. Our data suggest that a volume transmission is likely to stimulate presynaptic NMDARs (preNMDARs) since NMDA puffs directed to the axon led to inward currents and Ca²⁺ transients restricted to axonal varicosities. Using local glutamate photoliberation, we show that pre‐ and post‐synaptic NMDARs share the same voltage dependence indicating their containing NR2A/B subunits. Ca²⁺ transients elicited by NMDA puffs are eventually followed by delayed events reminding of the spontaneous Ca²⁺ transients (ScaTs) described at the basket cell/Purkinje cell terminals. Moreover, the presence of Ca²⁺ transients at varicosities located more than 5 μm away from the uncaging site indicates that the activation of preNMDARs sensitizes the Ca²⁺ stores in adjacent varicosities, a process that is abolished in the presence of a high concentration of ryanodine. Altogether, the data demonstrate that preNMDARs act as high‐gain glutamate detectors.     

Pituitary Adenylate Cyclase-Activating Polypeptide Causes Ca2+ Release from Ryanodine/Caffeine Stores Through a Novel Pathway Independent of Both Inositol Trisphosphates and Cyclic AMP in Bovine Adrenal Medullary Cells

Abstract: Pituitary adenylate cyclase-activating polypeptide (PACAP) causes both Ca²⁺ release and Ca²⁺ influx in bovine adrenal chromaffin cells. To elucidate the mechanisms of PACAP-induced Ca²⁺ release, we investigated expression of PACAP receptors and measured inositol trisphosphates (IP₃), cyclic AMP, and the intracellular Ca²⁺ concentration in bovine adrenal medullary cells maintained in primary culture. RT-PCR analysis revealed that bovine adrenal medullary cells express the PACAP receptor hop, which is known to couple with both IP₃ and cyclic AMP pathways. The two naturally occurring forms of PACAP, PACAP38 and PACAP27, both increased cyclic AMP and IP₃, and PACAP38 was more potent than PACAP27 in both effects. Despite the effects of PACAP on IP₃ production, the Ca²⁺ release induced by PACAP38 or by PACAP27 was unaffected by cinnarizine, a blocker of IP₃ channels. The potencies of the peptides to cause Ca²⁺ release in the presence of cinnarizine were similar. The Ca²⁺ release induced by PACAP38 or by PACAP27 was strongly inhibited by ryanodine and caffeine. In the presence of ryanodine and caffeine, PACAP38 was more potent than PACAP27. PACAP-induced Ca²⁺ release was unaffected by Rp-adenosine 3′,5′-cyclic monophosphothioate, an inhibitor of protein kinase A. Ca²⁺ release induced by bradykinin and angiotensin II was also inhibited by ryanodine and caffeine, but unaffected by cinnarizine. Although IP₃ production stimulated by PACAP38 or bradykinin was abolished by the phospholipase C inhibitor, U-73122, Ca²⁺ release in response to the peptides was unaffected by U-73122. These results suggest that PACAP induces Ca²⁺ release from ryanodine/caffeine stores through a novel intracellular mechanism independent of both IP₃ and cyclic AMP and that the mechanism may be the common pathway through which peptides release Ca²⁺ in adrenal chromaffin cells.     

Effects of ATP, Mg2+, and redox agents on the Ca2+ dependence of RyR channels from rat brain cortex

Despite their relevance for neuronal Ca²⁺-induced Ca²⁺ release (CICR), activation by Ca²⁺ of ryanodine receptor (RyR) channels of brain endoplasmic reticulum at the [ATP], [Mg²⁺], and redox conditions present in neurons has not been reported. Here, we studied the effects of varying cis-(cytoplasmic) free ATP concentration ([ATP]), [Mg²⁺], and RyR redox state on the Ca²⁺ dependence of endoplasmic reticulum RyR channels from rat brain cortex. At pCa 4.9 and 0.5 mM adenylylimidodiphosphate (AMP-PNP), increasing free [Mg²⁺] up to 1 mM inhibited vesicular [³H]ryanodine binding; incubation with thimerosal or dithiothreitol decreased or enhanced Mg²⁺ inhibition, respectively. Single RyR channels incorporated into lipid bilayers displayed three different Ca²⁺ dependencies, defined by low, moderate, or high maximal fractional open time (P_o), that depend on RyR redox state, as we have previously reported. In all cases, cis-ATP addition (3 mM) decreased threshold [Ca²⁺] for activation, increased maximal P_o, and shifted channel inhibition to higher [Ca²⁺]. Conversely, at pCa 4.5 and 3 mM ATP, increasing cis-[Mg²⁺] up to 1 mM inhibited low activity channels more than moderate activity channels but barely modified high activity channels. Addition of 0.5 mM free [ATP] plus 0.8 mM free [Mg²⁺] induced a right shift in Ca²⁺ dependence for all channels so that [Ca²⁺] <30 µM activated only high activity channels. These results strongly suggest that channel redox state determines RyR activation by Ca²⁺ at physiological [ATP] and [Mg²⁺]. If RyR behave similarly in living neurons, cellular redox state should affect RyR-mediated CICR. Ca²⁺-induced Ca²⁺ release; Ca²⁺ release channels; endoplasmic reticulum; thimerosal; 2,4-dithiothreitol; ryanodine receptor