Differential regulation of Ca(2+) sparks and Ca(2+) waves by UTP in rat cerebral artery smooth muscle cells

Uridine 5'-triphosphate (UTP), a potent vasoconstrictor that activatesphospholipase C, shifted Ca²⁺ signaling from sparks towaves in the smooth muscle cells of rat cerebral arteries. UTPdecreased the frequency of Ca²⁺ sparks and transientCa²⁺-activated K⁺ (K_Ca) currentsand increased the frequency of Ca²⁺ waves. The UTP-inducedreduction in Ca²⁺ spark frequency did not reflect adecrease in global cytoplasmic Ca²⁺, Ca²⁺influx through voltage-dependent Ca²⁺ channels (VDCC), orCa²⁺ load of the sarcoplasmic reticulum (SR), since globalCa²⁺ was elevated, blocking VDCC did not prevent theeffect, and SR Ca²⁺ load did not decrease. However,blocking protein kinase C (PKC) with bisindolylmaleimide I did preventUTP reduction of Ca²⁺ sparks and transient K_Cacurrents. UTP decreased the effectiveness of caffeine, which increasesthe Ca²⁺ sensitivity of ryanodine-sensitiveCa²⁺ release (RyR) channels, to activate transientK_Ca currents. This work supports the concept thatvasoconstrictors shift Ca²⁺ signaling modalities fromCa²⁺ sparks to Ca²⁺ waves through the concertedactions of PKC on the Ca²⁺ sensitivity of RyR channels,which cause Ca²⁺ sparks, and of inositol trisphosphate(IP₃) on IP₃ receptors to generateCa²⁺ waves.

     

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.

     

Hypoxia reduces KCa channel activity by inducing Ca2+ spark uncoupling in cerebral artery smooth muscle cells

Arterial smooth muscle cell large-conductance Ca²⁺-activated potassium (K_Ca) channels have been implicated in modulating hypoxic dilation of systemic arteries, although this is controversial. K_Ca channel activity in arterial smooth muscle cells is controlled by localized intracellular Ca²⁺ transients, termed Ca²⁺ sparks, but hypoxic regulation of Ca²⁺ sparks and K_Ca channel activation by Ca²⁺ sparks has not been investigated. We report here that in voltage-clamped (–40 mV) cerebral artery smooth muscle cells, a reduction in dissolved O₂ partial pressure from 150 to 15 mmHg reversibly decreased Ca²⁺ spark-induced transient K_Ca current frequency and amplitude to 61% and 76% of control, respectively. In contrast, hypoxia did not alter Ca²⁺ spark frequency, amplitude, global intracellular Ca²⁺ concentration, or sarcoplasmic reticulum Ca²⁺ load. Hypoxia reduced transient K_Ca current frequency by decreasing the percentage of Ca²⁺ sparks that activated a transient K_Ca current from 89% to 63%. Hypoxia reduced transient K_Ca current amplitude by attenuating the amplitude relationship between Ca²⁺ sparks that remained coupled and the evoked transient K_Ca currents. Consistent with these data, in inside-out patches at –40 mV hypoxia reduced K_Ca channel apparent Ca²⁺ sensitivity and increased the K_d for Ca²⁺ from 17 to 32 µM, but did not alter single-channel amplitude. In summary, data indicate that hypoxia reduces K_Ca channel apparent Ca²⁺ sensitivity via a mechanism that is independent of cytosolic signaling messengers, and this leads to uncoupling of K_Ca channels from Ca²⁺ sparks. Transient K_Ca current inhibition due to uncoupling would oppose hypoxic cerebrovascular dilation. transient calcium-activated potassium current     

Voltage dependence of Ca2+ sparks in intact cerebral arteries

Ca²⁺ sparks have beenpreviously described in isolated smooth muscle cells. Here we presentthe first measurements of local Ca²⁺ transients("Ca²⁺ sparks") in an intactsmooth muscle preparation. Ca²⁺sparks appear to result from the opening of ryanodine-sensitive Ca²⁺ release (RyR) channels in thesarcoplasmic reticulum (SR). Intracellular Ca²⁺ concentration([Ca²⁺]_i)was measured in intact cerebral arteries (40-150 µm in diameter) from rats, using the fluorescentCa²⁺ indicator fluo 3 and a laserscanning confocal microscope. Membrane potential depolarization byelevation of external K⁺ from 6 to30 mM increased Ca²⁺ sparkfrequency (4.3-fold) and amplitude (~2-fold) as well as globalarterial wall[Ca²⁺]_i(~1.7-fold). The half time of decay (~50 ms) was not affected bymembrane potential depolarization. Ryanodine (10 µM), which inhibitsRyR channels and Ca²⁺ sparks inisolated cells, and thapsigargin (100 nM), which indirectly inhibitsRyR channels by blocking the SRCa²⁺-ATPase, completely inhibitedCa²⁺ sparks in intact cerebralarteries. Diltiazem, an inhibitor of voltage-dependentCa²⁺ channels, lowered global[Ca²⁺]_iand Ca²⁺ spark frequency andamplitude in intact cerebral arteries in a concentration-dependentmanner. The frequency of Ca²⁺sparks (<1s¹ · cell¹),even under conditions of steady depolarization, was too low tocontribute significant amounts ofCa²⁺ to globalCa²⁺ in intact arteries. Theseresults provide direct evidence that Ca²⁺ sparks exist in quiescentsmooth muscle cells in intact arteries and that changes of membranepotential that would simulate physiological changes modulate bothCa²⁺ spark frequency and amplitudein arterial smooth muscle.

     

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.

     

Ca2+ sparks are initiated by Ca2+ entry in embryonic mouse skeletal muscle and decrease in frequency postnatally

"Spontaneous" Ca²⁺ sparks and ryanodine receptor type 3 (RyR3) expression are readily detected in embryonic mammalian skeletal muscle but not in adult mammalian muscle, which rarely exhibits Ca²⁺ sparks and expresses predominantly RyR1. We have used confocal fluorescence imaging and systematic sampling of enzymatically dissociated single striated muscle fibers containing the Ca²⁺ indicator dye fluo 4 to show that the frequency of spontaneous Ca²⁺ sparks decreases dramatically from embryonic day 18 (E18) to postnatal day 14 (P14) in mouse diaphragm and from P1 to P14 in mouse extensor digitorum longus fibers. In contrast, the relative levels of RyR3 to RyR1 protein remained constant in diaphragm muscles from E18 to P14, indicating that changes in relative levels of RyR isoform expression did not cause the decline in Ca²⁺ spark frequency. E18 diaphragm fibers were used to investigate possible mechanisms underlying spark initiation in embryonic fibers. Spark frequency increased or decreased, respectively, when E18 diaphragm fibers were exposed to 8 or 0 mM Ca²⁺ in the extracellular Ringer solution, with no change in either the average resting fiber fluo 4 fluorescence or the average properties of the sparks. Either CoCl₂ (5 mM) or nifedipine (30 µM) markedly decreased spark frequency in E18 diaphragm fibers. These results indicate that Ca²⁺ sparks may be triggered by locally elevated [Ca²⁺] due to Ca²⁺ influx via dihydropyridine receptor L-type Ca²⁺ channels in embryonic mammalian skeletal muscle. calcium; ryanodine receptor; dihydropyridine receptor; muscle development     

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     

Role of phospholamban in the modulation of arterial Ca(2+) sparks and Ca(2+)-activated K(+) channels by cAMP

Phospholamban (PLB) inhibits the sarcoplasmic reticulum (SR)Ca²⁺-ATPase, and this inhibition is relieved bycAMP-dependent protein kinase (PKA)-mediated phosphorylation. The roleof PLB in regulating Ca²⁺ release throughryanodine-sensitive Ca²⁺ release channels, measured asCa²⁺ sparks, was examined using smooth muscle cells ofcerebral arteries from PLB-deficient ("knockout") mice(PLB-KO). Ca²⁺ sparks were monitored opticallyusing the fluorescent Ca²⁺ indicator fluo 3 or electricallyby measuring transient large-conductance Ca²⁺-activatedK⁺ (BK) channel currents activated by Ca²⁺sparks. Basal Ca²⁺ spark and transient BK current frequencywere elevated in cerebral artery myocytes of PLB-KO mice. Forskolin, anactivator of adenylyl cyclase, increased the frequency ofCa²⁺ sparks and transient BK currents in cerebral arteriesfrom control mice. However, forskolin had little effect on thefrequency of Ca²⁺ sparks and transient BK currents fromPLB-KO cerebral arteries. Forskolin or PLB-KO increased SRCa²⁺ load, as measured by caffeine-induced Ca²⁺transients. This study provides the first evidence that PLB is criticalfor frequency modulation of Ca²⁺ sparks and associated BKcurrents by PKA in smooth muscle.

     

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.

     

Single Ryanodine Receptor Channel Basis of Caffeine's Action on Ca Sparks

Caffeine (1, 3, 7-trimethylxanthine) is a widely used pharmacological agonist of the cardiac ryanodine receptor (RyR2) Ca²⁺ release channel. It is also a well-known stimulant that can produce adverse side effects, including arrhythmias. Here, the action of caffeine on single RyR2 channels in bilayers and Ca²⁺ sparks in permeabilized ventricular cardiomyocytes is defined. Single RyR2 caffeine activation depended on the free Ca²⁺ level on both sides of the channel. Cytosolic Ca²⁺ enhanced RyR2 caffeine affinity, whereas luminal Ca²⁺ essentially scaled maximal caffeine activation. Caffeine activated single RyR2 channels in diastolic quasi-cell-like solutions (cytosolic MgATP, pCa 7) with an EC₅₀ of 9.0 ± 0.4 mM. Low-dose caffeine (0.15 mM) increased Ca²⁺ spark frequency ∼75% and single RyR2 opening frequency ∼150%. This implies that not all spontaneous RyR2 openings during diastole are associated with Ca²⁺ sparks. Assuming that only the longest openings evoke sparks, our data suggest that a spark may result only when a spontaneous single RyR2 opening lasts >6 ms.     

Large-conductance calcium-activated potassium channel activity is absent in human and mouse neutrophils and is not required for innate immunity

Large-conductance Ca²⁺-activated K⁺ (BK) channels are reported to be essential for NADPH oxidase-dependent microbial killing and innate immunity in leukocytes. Using human peripheral blood and mouse bone marrow neutrophils, pharmacological targeting, and BK channel gene-deficient (BK^–/–) mice, we stimulated NADPH oxidase activity with 12-O-tetradecanoylphorbol-13-acetate (PMA) and performed patch-clamp recordings on isolated neutrophils. Although PMA stimulated NADPH oxidase activity as assessed by O₂^– and H₂O₂ production, our patch-clamp experiments failed to show PMA-activated BK channel currents in neutrophils. In our studies, PMA induced slowly activating currents, which were insensitive to the BK channel inhibitor iberiotoxin. Instead, the currents were blocked by Zn²⁺, which indicates activation of proton channel currents. BK channels are gated by elevated intracellular Ca²⁺ and membrane depolarization. We did not observe BK channel currents, even during extreme depolarization to +140 mV and after elevation of intracellular Ca²⁺ by N-formyl-L-methionyl-L-leucyl-phenylalanine. As a control, we examined BK channel currents in cerebral and tibial artery smooth muscle cells, which showed characteristic BK channel current pharmacology. Iberiotoxin did not block killing of Staphylococcus aureus or Candida albicans. Moreover, we addressed the role of BK channels in a systemic S. aureus and Yersinia enterocolitica mouse infection model. After 3 and 5 days of infection, we found no differences in the number of bacteria in spleen and kidney between BK^–/– and BK^+/+ mice. In conclusion, our experiments failed to identify functional BK channels in neutrophils. We therefore conclude that BK channels are not essential for innate immunity. killing assay; reactive oxygen species; BK-deficient mice; mice infection     

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

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

     

A probable role of dihydropyridine receptors in repression of Ca2+ sparks demonstrated in cultured mammalian muscle

To activate skeletal muscle contraction, action potentials must be sensed by dihydropyridine receptors (DHPRs) in the T tubule, which signal the Ca²⁺ release channels or ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) to open. We demonstrate here an inhibitory effect of the T tubule on the production of sparks of Ca²⁺ release. Murine primary cultures were confocally imaged for Ca²⁺ detection and T tubule visualization. After 72 h of differentiation, T tubules extended from the periphery for less than one-third of the myotube radius. Spontaneous Ca²⁺ sparks were found away from the region of cells where tubules were found. Immunostaining showed RyR1 and RyR3 isoforms in all areas, implying inhibition of both isoforms by a T tubule component. To test for a role of DHPRs in this inhibition, we imaged myotubes from dysgenic mice (mdg) that lack DHPRs. These exhibited T tubule development similar to that of normal myotubes, but produced few sparks, even in regions where tubules were absent. To increase spark frequency, a high-Ca²⁺ saline with 1 mM caffeine was used. Wild-type cells in this saline plus 50 µM nifedipine retained the topographic suppression pattern of sparks, but dysgenic cells in high-Ca²⁺ saline did not. Shifted excitation and emission ratios of indo-1 in the cytosol or mag-indo-1 in the SR were used to image [Ca²⁺] in these compartments. Under the conditions of interest, wild-type and mdg cells had similar levels of free [Ca²⁺] in cytosol and SR. These data suggest that DHPRs play a critical role in reducing the rate of spontaneous opening of Ca²⁺ release channels and/or their susceptibility to Ca²⁺-induced activation, thereby suppressing the production of Ca²⁺ sparks. excitation-contraction coupling; sarcoplasmic reticulum; ryanodine receptors; Ca²⁺ imaging     

PKC activity modulates availability and long openings of L-type Ca2+ channels in A7r5 cells

The possibility that protein kinase C (PKC) could control theactivity of L-type Ca²⁺ channelsin A7r5 vascular smooth muscle-derived cells in the absence of agoniststimulation was investigated using the patch-clamp technique.Consistent with the possibility that L-typeCa²⁺ channels are maximallyphosphorylated by PKC under these conditions, we show that1) activation of PKC with thephorbol ester phorbol 12,13-dibutyrate was ineffective in modulatingwhole cell and single-channel currents, 2) inhibition of PKC activity with staurosporine orchelerythrine inhibited channel activity,3) inhibition of proteinphosphatases by intracellular dialysis of okadaic acid did not affectwhole cell currents, and 4) theinhibitory effect of staurosporine was absent in the presence ofokadaic acid. The inhibition ofCa²⁺ currents by PKC inhibitorswas due to a decrease in channel availability and long open events,whereas the voltage dependence of the open probability and thesingle-channel conductance were not affected. The evidence suggeststhat in resting, nonstimulated A7r5 cells there is a high level of PKCactivity that modulates the gating of L-typeCa²⁺ channels.

     

Hypotonic swelling stimulates L-type Ca2+ channel activity in vascular smooth muscle cells through PKC

It has been suggested that L-type Ca²⁺ channels play an important role in cell swelling-induced vasoconstriction. However, there is no direct evidence that Ca²⁺ channels in vascular smooth muscle are modulated by cell swelling. We tested the hypothesis that L-type Ca²⁺ channels in rabbit portal vein myocytes are modulated by hypotonic cell swelling via protein kinase activation. Ba²⁺ currents (I_Ba) through L-type Ca²⁺ channels were recorded in smooth muscle cells freshly isolated from rabbit portal vein with the conventional whole cell patch-clamp technique. Superfusion of cells with hypotonic solution reversibly enhanced Ca²⁺ channel activity but did not alter the voltage-dependent characteristics of Ca²⁺ channels. Bath application of selective inhibitors of protein kinase C (PKC), Ro-31–8425 or Go-6983, prevented I_Ba enhancement by hypotonic swelling, whereas the specific protein kinase A (PKA) inhibitor KT-5720 had no effect. Bath application of phorbol 12,13-dibutyrate (PDBu) significantly increased I_Ba under isotonic conditions and prevented current stimulation by hypotonic swelling. However, PDBu did not have any effect on I_Ba when cells were first exposed to hypotonic solution. Furthermore, downregulation of endogenous PKC by overnight treatment of cells with PDBu prevented current enhancement by hypotonic swelling. These data suggest that hypotonic cell swelling can enhance Ca²⁺ channel activity in rabbit portal vein smooth muscle cells through activation of PKC. cell swelling; protein kinases; calcium current     

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     

Stimulation of unitary T-type Ca(2+) channel currents by calmodulin-dependent protein kinase II

The effect ofCa²⁺/calmodulin-dependent protein kinase II (CaMKII)stimulation on unitary low voltage-activated (LVA) T-type Ca²⁺ channel currents in isolated bovine adrenalglomerulosa (AG) cells was measured using the patch-clamp technique. Incell-attached and inside-out patches, LVA channel activity wasidentified by voltage-dependent inactivation and a single-channelconductance of ~9 pS in 110 mM BaCl₂ orCaCl₂. In the cell-attached patch, elevation of bathCa²⁺ from 150 nM to 1 µM raised intracellularCa²⁺ in K⁺-depolarized (140 mM) cells andevoked an increase in the LVA Ca²⁺ channel probability ofopening (NP_o) by two- to sixfold. This augmentation was associated with an increase in the number of nonblanksweeps, a rise in the frequency of channel opening in nonblank sweeps,and a 30% reduction in first latency. No apparent changes in thesingle-channel open-time distribution, burst lengths, or openings/burstwere apparent. Preincubation of AG cells with lipophilic or peptideinhibitors of CaMKII in the cell-attached or excised (inside-out)configurations prevented the rise in NP_o elicited by elevated Ca²⁺ concentration.Furthermore, administration of a mutant recombinant CaMKIIexhibiting cofactor-independent activity in the absence of elevatedCa²⁺ produced a threefold elevation in LVA channelNP_o. These data indicate that CaMKII activity isboth necessary and sufficient for LVA channel activation byCa²⁺.

     

Elevated resting [Ca(2+)](i) in myotubes expressing malignant hyperthermia RyR1 cDNAs is partially restored by modulation of passive calcium leak from the SR

Malignant hyperthermia (MH) is a pharmacogenetic disorder of skeletal muscle triggered in susceptible individuals by inhalation anesthetics and depolarizing skeletal muscle relaxants. This syndrome has been linked to a missense mutation in the type 1 ryanodine receptor (RyR1) in more than 50% of cases studied to date. Using double-barreled Ca²⁺ microelectrodes in myotubes expressing wild-type RyR1 (_WTRyR1) or RyR1 with one of four common MH mutations (_MHRyR1), we measured resting intracellular Ca²⁺ concentration ([Ca²⁺]_i). Changes in resting [Ca²⁺]_i produced by several drugs known to modulate the RyR1 channel complex were investigated. We found that myotubes expressing any of the _MHRyR1s had a 2.0- to 3.7-fold higher resting [Ca²⁺]_i than those expressing _WTRyR1. Exposure of myotubes expressing _MHRyR1s to ryanodine (500 µM) or (2,6-dichloro-4-aminophenyl)isopropylamine (FLA 365; 20 µM) had no effects on their resting [Ca²⁺]_i. However, when myotubes were exposed to bastadin 5 alone or to a combination of ryanodine and bastadin 5, the resting [Ca²⁺]_i was significantly reduced (P < 0.01). Interestingly, the percent decrease in resting [Ca²⁺]_i in myotubes expressing _MHRyR1s was significantly greater than that for _WTRyR1. From these data, we propose that the high resting myoplasmic [Ca²⁺]_i in _MHRyR1 expressing myotubes is due in part to a related structural conformation of _MHRyR1s that favors "passive" calcium leak from the sarcoplasmic reticulum. ryanodine; FLA 365; bastadin 5; resting intracellular calcium concentration; sarcoplasmic reticulum     

Effects of osmotic shrinkage on voltage-gated Ca2+ channel currents in rat anterior pituitary cells

Increased extracellular osmolarity ([Os]_e) suppresses stimulated hormone secretion from anterior pituitary cells. Ca²⁺ influx may mediate this effect. We show that increase in [Os]_e (by 18–125%) differentially suppresses L-type and T-type Ca²⁺ channel currents (I_L and I_T, respectively); I_L was more sensitive than I_T. Hyperosmotic suppression of I_L depended on the magnitude of increase in [Os]_e and was correlated with the percent decrease in pituitary cell volume, suggesting that pituitary cell shrinkage can modulate L-type currents. The hyperosmotic suppression of I_L and I_T persisted after incubation of pituitary cells either with the actin-disrupter cytochalasin D or with the actin stabilizer phalloidin, suggesting that the actin cytoskeleton is not involved in this modulation. The hyperosmotic suppression of Ca²⁺ influx was not correlated with changes in reversal potential, membrane capacitance, and access resistance. Together, these results suggest that the hyperosmotic suppression of Ca²⁺ influx involves Ca²⁺ channel proteins. We therefore recorded the activity of L-type Ca²⁺ channels from cell-attached patches while exposing the cell outside the patch pipette to hyperosmotic media. Increased [Os]_e reduced the activity of Ca²⁺ channels but did not change single-channel conductance. This hyperosmotic suppression of Ca²⁺ currents may therefore contribute to the previously reported hyperosmotic suppression of hormone secretion. L-type Ca²⁺ channels; osmosensitivity; mechanosensitivity; osmolarity; hyperosmolarity     

Ca2+ entry-independent effects of L-type Ca2+ channel modulators on Ca2+ sparks in ventricular myocytes

During the cardiac action potential, Ca²⁺ entry through dyhidropyridine receptor L-type Ca²⁺ channels (DHPRs) activates ryanodine receptors (RyRs) Ca²⁺-release channels, resulting in massive Ca²⁺ mobilization from the sarcoplasmic reticulum (SR). This global Ca²⁺ release arises from spatiotemporal summation of many localized elementary Ca²⁺-release events, Ca²⁺ sparks. We tested whether DHPRs modulate Ca²⁺sparks in a Ca²⁺ entry-independent manner. Negative modulation by DHPR of RyRs via physical interactions is accepted in resting skeletal muscle but remains controversial in the heart. Ca²⁺ sparks were studied in cat cardiac myocytes permeabilized with saponin or internally perfused via a patch pipette. Bathing and pipette solutions contained low Ca²⁺ (100 nM). Under these conditions, Ca²⁺ sparks were detected with a stable frequency of 3–5 sparks·s^–1·100 µm^–1. The DHPR blockers nifedipine, nimodipine, FS-2, and calciseptine decreased spark frequency, whereas the DHPR agonists Bay-K8644 and FPL-64176 increased it. None of these agents altered the spatiotemporal characteristics of Ca²⁺ sparks. The DHPR modulators were also without effect on SR Ca²⁺ load (caffeine-induced Ca²⁺ transients) or sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) activity (Ca²⁺ loading rates of isolated SR microsomes) and did not change cardiac RyR channel gating (planar lipid bilayer experiments). In summary, DHPR modulators affected spark frequency in the absence of DHPR-mediated Ca²⁺ entry. This action could not be attributed to a direct action of DHPR modulators on SERCA or RyRs. Our results suggest that the activity of RyR Ca²⁺-release units in ventricular myocytes is modulated by Ca²⁺ entry-independent conformational changes in neighboring DHPRs. exitation-contraction coupling; ryanodine receptor; sarco(endo)plasmic reticulum Ca²⁺-ATPase; dihydropyridine receptor; sarcoplasmic reticulum