Serotonin Directly Increases a Calcium Current in Swim Motoneurons of Aplysia brasiliana

Muscle fibers in the swim appendages of the mollusk Aplysiabrasiliana are innervated by cholinergic motoneurons. Serotonin(5-HT) causes an increase in amplitude of junctional potentialsand muscle contractions at this neuromuscular synapse. We studiedmotoneurons with intracellular current-clamp recording and single-electrodevoltage-clamp analysis to determine the effects of 5-HT on somaticcurrents in these presynaptic neurons. Serotonin was found tohave no effect on action potential duration in motoneurons bathedin normal seawater, and no effect of 5-HT could be detectedon K⁺ currents, indicating that 5-HT does not indirectly enhancecalcium currents by prolonging the action potential. Calciumcurrents were isolated by replacing extracellular sodium withTEA and adding tetrodotoxin and other K⁺-channel blockers. Underthese conditions motoneuron action potentials were greatly prolongedand could be blocked with Co²⁺ or Cd²⁺. Addition of 5-HT increasedthe duration of these Ca²⁺ spikes by about 35%. In motoneuronsstudied with voltage clamp, the amine produced a 58% increasein total inward calcium current. Use of the calcium channelblockers nifedipine, nimodipine, -conotoxin GVIA, and -agatoxinTK revealed that motoneurons express varying amounts of L-,N- and P-like calcium channels, but only an agatoxin-sensitive,P-type channel is sensitive to 5-HT. It is concluded that 5-HTacts directly to increase a P-type Ca²⁺ current during a normalspike. The resulting increase in intracellular calcium couldcontribute to an increase in transmitter release and accountfor the increase in junctional potentials in swim muscles.     

Arachidonic acid reversibly enhances N-type calcium current at an extracellular site

We examined the effects of arachidonic acid(AA) on whole cell Ca²⁺ channel activity in rat superiorcervical ganglion neurons. Our companion paper (Liu L, Barrett CF, andRittenhouse AR. Am J Physiol Cell Physiol 280:C1293-C1305, 2001) demonstrates that AA induces severaleffects, including enhancement of current amplitude at negativevoltages, and increased activation kinetics. This study examines themechanisms underlying these effects. First, enhancement is rapidlyreversible by bath application of BSA. Second, enhancement appears tooccur extracellularly, since intracellular albumin was without effecton enhancement, and bath-applied arachidonoyl coenzyme A, anamphiphilic AA analog that cannot cross the cell membrane, mimickedenhancement. In addition, enhancement is voltage dependent, in thatcurrents were enhanced to the greatest degree at 10 mV, whereasvirtually no enhancement occurred positive of +30 mV. We alsodemonstrate that AA-induced increases in activation kinetics arecorrelated with enhancement of current amplitude. An observed increasein the voltage sensitivity may underlie these effects. Finally, themajority of enhancement is mediated through N-type current, thusproviding the first demonstration that this current type can beenhanced by AA.

     

Ca2+-activated Cl- current in cultured myenteric neurons from murine proximal colon

Whole cell patch-clamprecordings were made from cultured myenteric neurons taken from murineproximal colon. The micropipette contained Cs⁺ to removeK⁺ currents. Depolarization elicited a slowly activatingtime-dependent outward current (I_tdo), whereasrepolarization was followed by a slowly deactivating tail current(I_tail). I_tdo andI_tail were present in ~70% of neurons. Weidentified these currents as Cl currents(I_Cl), because changing the transmembraneCl gradient altered the measured reversal potential(E_rev) of both I_tdo andI_tail with that for I_tailshifted close to the calculated Cl equilibrium potential(E_Cl). I_Cl areCa²⁺-activated Cl current[I_Cl(Ca)] because they were Ca²⁺dependent. E_Cl, which was measured from theE_rev of I_Cl(Ca) using agramicidin perforated patch, was 33 mV. This value is more positivethan the resting membrane potential (56.3 ± 2.7 mV), suggestingmyenteric neurons accumulate intracellular Cl.-Conotoxin GIVA [0.3 µM; N-type Ca²⁺ channelblocker] and niflumic acid [10 µM; knownI_Cl(Ca) blocker], decreased theI_Cl(Ca). In conclusion, these neurons haveI_Cl(Ca) that are activated by Ca²⁺entry through N-type Ca²⁺ channels. These currents likelyregulate postspike frequency adaptation.

     

{Omega}-3 and {omega}-6 polyunsaturated fatty acids block HERG channels

Dietary polyunsaturated fatty acids (PUFAs) have been reported to exhibit antiarrhythmic properties, which have been attributed to their availability to modulate Na⁺, Ca²⁺, and several K⁺ channels. However, their effects on human ether-a-go-go-related gene (HERG) channels are unknown. In this study we have analyzed the effects of arachidonic acid (AA, -6) and docosahexaenoic acid (DHA, -3) on HERG channels stably expressed in Chinese hamster ovary cells by using the whole cell patch-clamp technique. At 10 µM, AA and DHA blocked HERG channels, at the end of 5-s pulses to –10 mV, to a similar extent (37.7 ± 2.4% vs. 50.2 ± 8.1%, n = 7–10, P > 0.05). 5,6,11,14-Eicosatetrayenoic acid, a nonmetabolizable AA analog, induced effects similar to those of AA on HERG current. Both PUFAs shifted the midpoint of activation curves of HERG channels by –5.1 ± 1.8 mV (n = 10, P < 0.05) and –11.2 ± 1.1 mV (n = 7, P < 0.01). Also, AA and DHA shifted the midpoint of inactivation curves by +12.0 ± 3.9 mV (n = 4; P < 0.05) and +15.8 ± 4.3 mV (n = 4; P < 0.05), respectively. DHA and AA accelerated the deactivation kinetics and slowed the inactivation kinetics at potentials positive to +40 mV. Block induced by DHA, but not that produced by AA, was higher when measured after applying a pulse to –120 mV (IO). Finally, both AA and DHA induced a use-dependent inhibition of HERG channels. In summary, block induced by AA and DHA was time, voltage, and use dependent. The results obtained suggest that both PUFAs bind preferentially to the open state of the channel, although an interaction with inactivated HERG channels cannot be ruled out for AA. K⁺ channel; membrane currents; ion channels; arrhythmia; antiarrhythmics     

Spontaneous acetylcholine release in mammalian neuromuscular junctions

Spontaneous secretion of the neurotransmitter acetylcholine inmammalian neuromuscular synapsis depends on theCa²⁺ content of nerve terminals.The Ca²⁺ electrochemical gradientfavors the entry of this cation. We investigated the possibleinvolvement of three voltage-dependent Ca²⁺ channels (VDCC) (L-, N-, andP/Q-types) on spontaneous transmitter release at the rat neuromuscularjunction. Miniature end-plate potential (MEPP) frequency was clearlyreduced by 5 µM nifedipine, a blocker of the L-type VDCC, and to alesser extent by the N-type VDCC blocker, -conotoxin GVIA (-CgTx,5 µM). On the other hand, nifedipine and -CgTx had no effect onK⁺-induced transmitter secretion.-Agatoxin IVA (100 nM), a P/Q-type VDCC blocker, preventsacetylcholine release induced byK⁺ depolarization but failed toaffect MEPP frequency in basal conditions. These results suggest thatin the mammalian neuromuscular junction Ca²⁺ enters nerve terminalsthrough at least three different channels, two of them (L- and N-types)mainly related to spontaneous acetylcholine release and the other(P/Q-type) mostly involved in depolarization-induced neurotransmitterrelease. Ca²⁺-bindingmolecule-related spontaneous release apparently binds Ca²⁺ very rapidly and wouldprobably be located very close toCa²⁺ channels, since the fastCa²⁺ chelator (BAPTA-AM)significantly reduced MEPP frequency, whereas EGTA-AM, exhibitingslower kinetics, had a lower effect. The increase in MEPP frequencyinduced by exposing the preparation to hypertonic solutions wasaffected by neither external Ca²⁺concentration nor L-, N-, and P/Q-type VDCC blockers, indicating thatextracellular Ca²⁺ is notnecessary to produce hyperosmotic neurosecretion. On the other hand,MEPP frequency was diminished by BAPTA-AM and EGTA-AM to the sameextent, supporting the view that hypertonic response is promoted by"bulk" intracellular Ca²⁺concentration increases.

     

Ca(2+)-dependent activation of Cl(-) currents in Xenopus oocytes is modulated by voltage

Ca²⁺-activatedCl currents (I_Cl,Ca) wereexamined using fluorescence confocal microscopy to monitorintracellular Ca²⁺ liberation evoked by flash photolysis ofcaged inositol 1,4,5-trisphosphate (InsP₃) involtage-clamped Xenopus oocytes. Currents at +40 mV exhibited asteep dependence on InsP₃ concentration([InsP₃]), whereas currents at140 mV exhibited a higher threshold and more graded relationshipwith [InsP₃]. Ca²⁺ levelsrequired to half-maximally activate I_Cl,Ca wereabout 50% larger at 140 mV than at +40 mV, and currents evokedby small Ca²⁺ elevations were reduced >25-fold. Thehalf-decay time of Ca²⁺ signals shortened at increasinglypositive potentials, whereas the decay of I_Cl,Calengthened. The steady-state current-voltage (I-V) relationshipfor I_Cl,Ca exhibited outward rectification withweak photolysis flashes but became more linear with stronger stimuli.Instantaneous I-V relationships were linear with both strongand weak stimuli. Current relaxations following voltage steps duringactivation of I_Cl,Ca decayed with half-times that shortened from about 100 ms at +10 mV to 20 ms at 160 mV. We conclude that InsP₃-mediated Ca²⁺liberation activates a single population of Clchannels, which exhibit voltage-dependent Ca²⁺ activationand voltage-independent instantaneous conductance.

     

Modulation of K+ channels by arachidonic acid in T84 cells. I.Inhibition of the Ca2+-dependent K+ channel

TheCl secretory response ofcolonic cells to Ca²⁺-mediatedagonists is transient despite a sustained elevation of intracellular Ca²⁺. We evaluated the effects ofsecond messengers proposed to limit Ca²⁺-mediatedCl secretion on thebasolateral membrane,Ca²⁺-dependentK⁺ channel(K_Ca) in colonic secretorycells, T84. Neither protein kinase C (PKC) nor inositoltetrakisphosphate (1,3,4,5 or 3,4,5,6 form) affectedK_Ca in excised inside-out patches.In contrast, arachidonic acid (AA; 3 µM) potently inhibitedK_Ca, reducingNP_o, the productof number of channels and channel open probability, by 95%. Theapparent inhibition constant for this AA effect was 425 nM. AAinhibited K_Ca in the presence ofboth indomethacin and nordihydroguaiaretic acid, blockers of thecyclooxygenase and lipoxygenase pathways. In the presence of albumin,the effect of AA on K_Ca wasreversed. A similar effect of AA was observed onK_Ca during outside-out recording.We determined also the effect of thecis-unsaturated fatty acid linoleate,the trans-unsaturated fatty acidelaidate, and the saturated fatty acid myristate. At 3 µM, all ofthese fatty acids inhibited K_Ca,reducing NP_o by 72-86%. Finally, the effect of the cytosolic phospholipaseA₂ inhibitorarachidonyltrifluoromethyl ketone(AACOCF₃) on thecarbachol-induced short-circuit current(I_sc) responsewas determined. In the presence ofAACOCF₃, the peakcarbachol-inducedI_sc response wasincreased ~2.5-fold. Our results suggest that AA generation inducedby Ca²⁺-mediated agonists maycontribute to the dissociation observed between the rise inintracellular Ca²⁺ evoked by theseagonists and the associatedCl secretory response.

     

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

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

     

Neuropeptide Y inhibition of calcium channels in PC-12 pheochromocytoma cells

We previouslydemonstrated, using rat PC-12 pheochromocytoma cells differentiated toa sympathetic neuronal phenotype with nerve growth factor (NGF), thatneuropeptide Y (NPY) inhibits catecholamine synthesis as well asrelease. Inquiry into the mechanisms of these inhibitions implicateddistinct pathways involving reduction ofCa²⁺ influx throughvoltage-activated Ca²⁺ channels.In the present investigation the effects of NPY on whole cellBa²⁺ currents were examined toobtain direct evidence supporting the mechanisms suggested by thosestudies. NPY was found to inhibit the voltage-activatedBa²⁺ current in NGF-differentiatedPC-12 cells in a reversible fashion with anEC₅₀ of 13 nM. This inhibition waspertussis toxin sensitive and resulted from NPY modulation of L- andN-type Ca²⁺ channels. Theinhibition of L-type channels was not seen with <1 nM freeintracellular Ca²⁺ or when proteinkinase C (PKC) was inhibited by chelerythrine or PKC-(1931).Furthermore, the effect of NPY on L-type channels was mimicked by thePKC activator phorbol 12-myristate 13-acetate. These studiesdemonstrate that, in addition to inhibition of N-type Ca²⁺ channels, inNGF-differentiated PC-12 cells NPY inhibits L-type Ca²⁺ channels via an intracellularCa²⁺- and PKC-dependent pathway.

     

L-Arginine inhibits vasopressin-stimulated mesangial cell Ca2+

L-Arginine (L-Arg) affects variousparameters that modulate the progression of renal disease. These samefactors [e.g., glomerular filtration rate, changes in mesangialcell (MC) tension, and production of NO] are all controlled atleast in part by changes in MC intracellular Ca²⁺concentration([Ca²⁺]_i). Wetherefore evaluated the effect of L-Arg on MC[Ca²⁺]_i. We found thatL-Arg inhibits the vasopressin-stimulated rise in MC[Ca²⁺]_i both in rat andmurine cell cultures. This effect does not appear to be due tometabolism of L-Arg to either NO or L-ornithine (L-Orn). Blocking the metabolism of L-Arg withN-monomethyl-L-arginine, an NOsynthase inhibitor, or with 20 mM L-valine(L-Val), an inhibitor of Orn formation,does not reverse the inhibition. However, other cationic amino acids,as well guanidine, the functional group ofL-Arg, all inhibit thevasopressin-stimulated rise in[Ca²⁺]_i,consistent with a structural basis for this effect. We conclude that1)L-Arg inhibitsvasopressin-stimulated murine and rat MC [Ca²⁺]_irise, 2) this inhibition is notmediated by metabolism of L-Arg to either NO or L-Orn, and3) the effect ofL-Arg is due to its cationicfunctional group, guanidine.

     

Voltage-gated Ca2+ currents in rat gastric enterochromaffin-like cells

Enterochromaffin-like (ECL) cells are histamine-containingendocrine cells in the gastric mucosa that maintain a negative membranepotential of about 50 mV, largely due to voltage-gated K⁺ currents [D. F. Loo, G. Sachs, and C. Prinz. Am. J. Physiol. 270 (Gastrointest Liver Physiol. 33):G739-G745, 1996]. The current study investigated thepresence of voltage-gated Ca²⁺channels in single ECL cells. ECL cells were isolated from rat fundicmucosa by elutriation, density gradient centrifugation, and primaryculture to a purity >90%. Voltage-gatedCa²⁺ currents were measured insingle ECL cells using the whole cell configuration of the patch-clamptechnique. Depolarization-activated currents were recorded in thepresence of Na⁺ orK⁺ blocking solutions and additionof 20 mM extracellular Ca²⁺. ECLcells showed inward currents in response to voltage steps that wereactivated at a test potential of around 20 mV with maximalinward currents observed at +20 mV and 20 mM extracellular Ca²⁺. The inactivation rate of thecurrent decreased with increasingly negative holding potentials and wastotally abolished at a holding potential of 30 mV. Addition ofextracellular 20 mM Ba²⁺ insteadof 20 mM Ca²⁺ increased thedepolarization-induced current and decreased the inactivation rate. Theinward current was fully inhibited by the specific L-typeCa²⁺ channel inhibitor verapamil(0.2 mM) and was augmented by the L-typeCa²⁺ channel activator BAY K 8644 (0.07 mM). We conclude that depolarization activateshigh-voltage-activated Ca²⁺channels in ECL cells. Activation characteristics,Ba²⁺ effects, and pharmacologicalresults imply the presence of L-type Ca²⁺ channels, whereasinactivation kinetics suggest the presence of additional N-typechannels in rat gastric ECL cells.

     

Activation of Ca2+-dependent K+ channels by cyanide in guinea pig adrenal chromaffin cells

The effects ofcyanide (CN) on whole cell current measured with the perforated-patchmethod were studied in adrenal medullary cells. Application of CNproduced initially inward and then outward currents at 52 mV ormore negative. As the membrane potential was hyperpolarized, amplitudeand latency of the outward current (I_o) by CNbecame small and long, respectively. A decrease in the externalNa⁺ concentration did not affectthe latency for CN-inducedI_o but enhancedthe amplitude markedly. The CNI_o reversedpolarity at 85 mV, close to the Nernst potential forK⁺, and was suppressed by theK⁺ channel blockers curare andapamin but not by glibenclamide, suggesting thatI_o is due to theactivation of Ca²⁺-dependentK⁺ channels. Consistent with thisnotion, the Ca²⁺-mobilizingagents, muscarine and caffeine, also producedI_o. Exposure toCN in a Ca²⁺-deficient medium for4 min abolished caffeine- or muscarine-induced I_o withoutdevelopment ofI_o, and additionof Ca²⁺ to the CN-containingsolution inducedI_o. We concludethat exposure to CN producesCa²⁺-dependentK⁺ currents in an externalCa²⁺-dependent manner, probablyvia facilitation of Ca²⁺ influx.

     

Inhibition of muscle carbonic anhydrase slows the Ca(2+) transient in rat skeletal muscle fibers

A countertransport ofH⁺ is coupled to Ca²⁺ transport across thesarcoplasmic reticulum (SR) membrane. We propose that SR carbonic anhydrase (CA) accelerates the CO₂-HCO reaction so that H⁺ ions, which are exchanged forCa²⁺ ions, are produced or buffered in the SR at sufficientrates. Inhibition of this SR-CA is expected to reduce the rate ofH⁺ fluxes, which then will retard the kinetics ofCa²⁺ transport. Fura 2 signals and isometric force weresimultaneously recorded in fiber bundles of the soleus (SOL) andextensor digitorum longus (EDL) from rats in the absence and presenceof the lipophilic CA inhibitors L-645151, chlorzolamide (CLZ), andethoxzolamide (ETZ), as well as the hydrophilic inhibitor acetazolamide(ACTZ). Fura 2 and force signals were analyzed for time to peak (TTP), 50% decay time (t₅₀), and their amplitudes.L-645151, CLZ, and ETZ significantly increased TTP of fura 2 by10-25 ms in SOL and by 5-7 ms in EDL and TTP of force by6-30 ms in both muscles. L-645151 and ETZ significantly prolongedt₅₀ of fura 2 and force by 20-55 and40-160 ms, respectively, in SOL and EDL. L-645151, CLZ, and ETZalso increased peak force of single twitches and amplitudes of furafluorescence ratio (R_340/380) at an excitation wavelengthof 340 to 380 nm. All effects of CA inhibitors on fura 2 and forcesignals could be reversed. ACTZ did not affect TTP, t₅₀, and amplitudes of fura 2 signals or force.L-645151, CLZ, and ETZ had no effects on myosin-, Ca²⁺-,and Na⁺-K⁺-ATPase activities, nor did theyaffect the amplitude and half-width of action potentials. We concludethat inhibition of SR-CA by impairing H⁺ countertransportis responsible for deceleration of intracellular Ca²⁺transients and contraction times.

     

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.

     

Myogenic NOS in canine lower esophageal sphincter: enzyme activation, substrate recycling, and product actions

Depolarization elicited outwardK⁺ currents from canine loweresophageal sphincter (LES) muscle cells, primarily through iberiotoxin (IbTX)- and tetraethylammonium-sensitiveCa²⁺-dependentK⁺ channels. Current magnitudesvaried with pipette Ca²⁺concentration (EC₅₀ = 108.5 nM).N^G-nitro-L-arginine(L-NNA,10⁴ M), IbTX(10⁸ M), or bufferingintracellular Ca²⁺ to 8 nMdecreased outward currents >80%. Sodium nitroprusside (NaNP,10⁴ M) restoredL-NNA-inhibited or lowintracellular Ca²⁺ concentration(not IbTX)-inhibited currents.L-NNA or IbTXapplication depolarized LES cells from 43 to 35 mV. NaNPrestored the membrane potential to 46 mV afterL-NNA but not after IbTXapplication. Nifedipine (30 µM) reduced outward currents andabolished or reduced L-NNA orNaNP effects, respectively. Immunocytochemistry revealed the presenceof both argininosuccinate synthetase and argininosuccinate lyase in LESmuscle cells. L-Citrulline, likeL-arginine, reversed L-NNA inhibition of outwardcurrents; only L-argininereversed inhibition of outward currents by an antibody toargininosuccinate synthetase. Therefore, endogenous nitric oxideproduction, activated by Ca²⁺entrance involving L-typeCa²⁺ channels, may continuouslyenhance outward currents to modulate LES muscle cell membrane potentialand excitability.

     

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     

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     

Interaction of reactive oxygen species with ion transport mechanisms

The use ofelectrophysiological and molecular biology techniques has shed light onreactive oxygen species (ROS)-induced impairment of surface andinternal membranes that control cellular signaling. These deleteriouseffects of ROS are due to their interaction with various ion transportproteins underlying the transmembrane signal transduction, namely,1) ion channels, such asCa²⁺ channels (includingvoltage-sensitive L-type Ca²⁺currents, dihydropyridine receptor voltage sensors, ryanodine receptorCa²⁺-release channels, andD-myo-inositol1,4,5-trisphosphate receptor Ca²⁺-release channels),K⁺ channels (such asCa²⁺-activatedK⁺ channels, inward and outwardK⁺ currents, and ATP-sensitiveK⁺ channels),Na⁺ channels, andCl channels;2) ion pumps, such as sarcoplasmicreticulum and sarcolemmal Ca²⁺pumps,Na⁺-K⁺-ATPase(Na⁺ pump), andH⁺-ATPase(H⁺ pump);3) ion exchangers such as theNa⁺/Ca²⁺exchanger andNa⁺/H⁺exchanger; and 4) ion cotransporterssuch asK⁺-Cl,Na⁺-K⁺-Cl,andP_i-Na⁺cotransporters. The mechanism of ROS-induced modificationsin ion transport pathways involves1) oxidation of sulfhydryl groups located on the ion transport proteins,2) peroxidation of membrane phospholipids, and 3) inhibition ofmembrane-bound regulatory enzymes and modification of the oxidativephosphorylation and ATP levels. Alterations in the ion transportmechanisms lead to changes in a second messenger system, primarilyCa²⁺ homeostasis, which furtheraugment the abnormal electrical activity and distortion of signaltransduction, causing cell dysfunction, which underlies pathologicalconditions.

     

Ca2+ mediates the effect of inhibition of Na+-K+-ATPase on the basolateral K+ channels in the rat CCD

We investigated the effect ofinhibiting Na⁺-K⁺-ATPase on the basolateral18-pS K⁺ channel in the cortical collecting duct (CCD) ofthe rat kidney. Inhibiting Na⁺-K⁺-ATPase withstrophanthidin decreased the activity of the 18-pS K⁺channel and increased the intracellular Ca²⁺ to 420 nM.Removal of extracellular Ca²⁺ abolished the effect ofstrophanthidin. When intracellular Ca²⁺ was raised with 5 µM ionomycin or A-23187 to 300, 400, and 500 nM, the activity of the18-pS K⁺ channel in cell-attached patches fell by 40, 85, and 96%, respectively. To explore the mechanism ofCa²⁺-induced inhibition, the effect of 400 nMCa²⁺ on channel activity was studied in the presence ofcalphostin C, an inhibitor of protein kinase C, or KN-93 and KN-62,inhibitors of calmodulin-dependent kinase II. Addition of calphostin Cor KN-93 or KN-62 failed to block the inhibitory effect of highconcentrations of Ca²⁺. This suggested that the inhibitoryeffect of high concentrations of Ca²⁺ was not mediated byprotein kinase C or calmodulin-dependent kinase II pathways. To examinethe possibility that the inhibitory effect of high concentrations ofCa²⁺ was mediated by the interaction of nitric oxide withsuperoxide, we investigated the effect of 400 nM Ca²⁺ onchannel activity in the presence of 4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron) orN-nitro-L-arginine methyl ester.Pretreatment of the tubules with 4,5-dihydroxy-1,3-benzenedisulfonicacid or N-nitro-L-arginine methylester completely abolished the inhibitory effect of 400 nMCa²⁺ on channel activity. Moreover, application of4,5-dihydroxy-1,3-benzenedisulfonic acid reversed the inhibitory effectof strophanthidin. We conclude that the effect of inhibitingNa⁺-K⁺-ATPase is mediated by intracellularCa²⁺ and the inhibitory effect of high concentrations ofCa²⁺ is the result of interaction of nitric oxide with superoxide.

     

Signal Transduction in Smooth Muscle: Selected Contribution: NO released to flow reduces myogenic tone of skeletal muscle arterioles by decreasing smooth muscle Ca2+ sensitivity

To clarify the contribution of intracellularCa²⁺ concentration([Ca²⁺]_i)-dependent and -independentsignaling mechanisms in arteriolar smooth muscle (aSM) to modulation ofarteriolar myogenic tone by nitric oxide (NO), released in response toincreases in intraluminal flow from the endothelium, changes in aSM[Ca²⁺]_i and diameter of isolated rat gracilismuscle arterioles (pretreated with indomethacin) were studied byfluorescent videomicroscopy. At an intraluminal pressure of 80 mmHg, [Ca²⁺]_i significantly increased andmyogenic tone developed in response to elevations of extracellularCa²⁺ concentration. The Ca²⁺ channelinhibitor nimodipine substantially decreased[Ca²⁺]_i and completely inhibited myogenictone. Dilations to intraluminal flow (that were inhibited byN-nitro-L-arginine methyl ester)or dilations to the NO donorS-nitroso-N-acetyl-DL-penicillamine (that were inhibited by the guanylate cyclase inhibitor1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) were notaccompanied by substantial decreases in aSM[Ca²⁺]_i. 8-Bromoguanosine cGMP and thecGMP-specific phosphodiesterase inhibitor zaprinast significantlydilated arterioles yet elicited only minimal decreases in[Ca²⁺]_i. Thus flow-induced endothelialrelease of NO elicits relaxation of arteriolar smooth muscle by acGMP-dependent decrease of the Ca²⁺ sensitivity of thecontractile apparatus without substantial changes in thepressure-induced level of [Ca²⁺]_i.