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1.
Westudied the interplay between matrix Ca2+ concentration([Ca2+]) and mitochondrial membrane potential() in regulation of the mitochondrial permeability transition(MPT) during anoxia and reoxygenation. Without Ca2+loading, anoxia caused near-synchronous dissipation,mitochondrial Ca2+ efflux, and matrix volume shrinkage whena critically low PO2 was reached, which wasrapidly reversible upon reoxygenation. These changes were related toelectron transport inhibition, not MPT. Cyclosporin A-sensitive MPT didoccur when extramitochondrial [Ca2+] was increased topromote significant Ca2+ uptake during anoxia, depending onthe Ca2+ load size and ability to maintain . However,when [Ca2+] was increased after complete dissipation, MPT did not occur until reoxygenation, at which timereactivation of electron transport led to partial regeneration.In the setting of elevated extramitochondrial Ca2+, thisenhanced matrix Ca2+ uptake while promoting MPT because ofless than full recovery of . The interplay between andmatrix [Ca2+] in accelerating or inhibiting MPT duringanoxia/reoxygenation has implications for preventing reoxygenationinjury associated with MPT.

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2.
HumanNa+-K+-ATPase11,21, and 31heterodimers were expressed individually in yeast, and ouabainbinding and ATP hydrolysis were measured in membrane fractions. Theouabain equilibrium dissociation constant was 13-17 nM for11 and 31at 37°C and 32 nM for 21, indicatingthat the human -subunit isoforms have a similar high affinity forcardiac glycosides. K0.5 values for antagonism of ouabain binding by K+ were ranked in order as follows:2 (6.3 ± 2.4 mM) > 3(1.6 ± 0.5 mM)  1 (0.9 ± 0.6 mM),and K0.5 values for Na+ antagonismof ouabain binding to all heterodimers were 9.5-13.8 mM. Themolecular turnover for ATP hydrolysis by11 (6,652 min1) was abouttwice as high as that by 31 (3,145 min1). These properties of the human heterodimersexpressed in yeast are in good agreement with properties of the humanNa+-K+-ATPase expressed in Xenopusoocytes (G Crambert, U Hasler, AT Beggah, C Yu, NN Modyanov, J-DHorisberger, L Lelievie, and K Geering. J Biol Chem275: 1976-1986, 2000). In contrast to Na+ pumpsexpressed in Xenopus oocytes, the21 complex in yeast membranes wassignificantly less stable than 11 or31, resulting in a lower functionalexpression level. The 21 complex was also more easily denatured by SDS than was the11 or the31 complex.

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3.
Toxin- (T)from the Brazilian scorpion Tityusserrulatus venom caused a concentration- andtime-dependent increase in the release of norepinephrine andepinephrine from bovine adrenal medullary chromaffin cells. T was~200-fold more potent than veratridine judged fromEC50 values, although the maximalsecretory efficacy of veratridine was 10-fold greater than that of T(1.2 vs. 12 µg/ml of catecholamine release). The combination of both toxins produced a synergistic effect that was particularly drastic at 5 mM extracellular Ca2+concentration([Ca2+]o),when 30 µM veratridine plus 0.45 µM T were used. T (0.45 µM) doubled the basal uptake of45Ca2+,whereas veratridine (100 µM) tripled it. Again, a drastic synergism in enhancing Ca2+ entry was seenwhen T and veratridine were combined; this was particularlypronounced at 5 mM[Ca2+]o.Veratridine induced oscillations of cytosolicCa2+ concentration([Ca2+]i)in single fura 2-loaded cells without elevation of basal levels. Incontrast, T elevated basal[Ca2+]ilevels, causing only small oscillations. When added together, T andveratridine elevated the basal levels of[Ca2+]iwithout causing large oscillations. T shifted the current-voltage (I-V) curve forNa+ channel current to the left.The combination of T with veratridine increased the shift of theI-V curve to the left, resulting in agreater recruitment of Na+channels at more hyperpolarizing potentials. This led to enhanced andmore rapid accumulation of Na+ inthe cell, causing cell depolarization, the opening of voltage-dependent Ca2+ channels, andCa2+ entry and secretion.

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4.
A reduction in angiotensinII (ANG II) in vivo by treatment of rabbits with theangiotensin-converting enzyme inhibitor, captopril, increasesNa+-K+ pump current (Ip)of cardiac myocytes. This increase is abolished by exposure of myocytesto ANG II in vitro. Because ANG II induces translocation of the-isoform of protein kinase C (PKC), we examined whether thisisozyme regulates the pump. We treated rabbits with captopril, isolatedmyocytes, and measured Ip of myocytes voltageclamped with wide-tipped patch pipettes. Ip ofmyocytes from captopril-treated rabbits was larger thanIp of myocytes from controls. ANG II superfusionof myocytes from captopril-treated rabbits decreasedIp to levels similar to controls. Inclusion ofPKC-specific blocking peptide in pipette solutions used to perfusethe intracellular compartment abolished the effect of ANG II. Inclusionof RACK, a PKC-specific activating peptide, in pipettesolutions had an effect on Ip that was similarto that of ANG II. There was no additive effect of ANG II andRACK. We conclude that PKC regulates the sarcolemmalNa+-K+ pump.

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5.
Neuronal7 nicotinic acetylcholine receptors (nAChRs) arepermeable to Ca2+ and other divalent cations. Wecharacterized the modulation of the pharmacological properties ofnondesensitizing mutant (L247T andS240T/L247T) 7 nAChRs bypermeant (Ca2+, Ba2+, and Sr2+) andimpermeant (Cd2+ and Zn2+) divalent cations.7 receptors were expressed in Xenopus oocytes and studied with two-electrode voltage clamp. Extracellular permeant divalent cations increased the potency and maximal efficacy of ACh,whereas impermeant divalent cations decreased potency and maximalefficacy. The antagonist dihydro--erythroidine (DHE) was a strongpartial agonist of L247T andS240T/L247T 7 receptors in thepresence of divalent cations but was a weak partial agonist in thepresence of impermeant divalent cations. Mutation of the"intermediate ring" glutamates (E237A) inL247T 7 nAChRs eliminated Ca2+conductance but did not alter the Ca2+-dependent increasein ACh potency, suggesting that site(s) required for modulation are onthe extracellular side of the intermediate ring. The difference betweenpermeant and impermeant divalent cations suggests that sites within thepore are important for modulation by divalent cations.

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6.
Role of caveolae in signal-transducing function of cardiac Na+/K+-ATPase   总被引:2,自引:0,他引:2  
Ouabain binding toNa+/K+-ATPase activates Src/epidermal growthfactor receptor (EGFR) to initiate multiple signal pathways thatregulate growth. In cardiac myocytes and the intact heart, the earlyouabain-induced pathways that cause rapid activations of ERK1/2 alsoregulate intracellular Ca2+ concentration([Ca2+]i) and contractility. The goal of thisstudy was to explore the role of caveolae in these early signalingevents. Subunits of Na+/K+-ATPase were detectedby immunoblot analysis in caveolae isolated from cardiac myocytes,cardiac ventricles, kidney cell lines, and kidney outer medulla byestablished detergent-free procedures. Isolated rat cardiac caveolaecontained Src, EGFR, ERK1/2, and 20-30% of cellular contents of1- and 2-isoforms ofNa+/K+-ATPase, along with nearly all ofcellular caveolin-3. Immunofluorescence microscopy of adult cardiacmyocytes showed the presence of caveolin-3 and -isoforms inperipheral sarcolemma and T tubules and suggested their partialcolocalization. Exposure of contracting isolated rat hearts to apositive inotropic dose of ouabain and analysis of isolated cardiaccaveolae showed that ouabain caused 1) no change in totalcaveolar ERK1/2, but a two- to threefold increase in caveolarphosphorylated/activated ERK1/2; 2) no change in caveolar 1-isoform and caveolin-3; and 3) 50-60%increases in caveolar Src and 2-isoform. These findings,in conjunction with previous observations, show that components of thepathways that link Na+/K+-ATPase to ERK1/2 and[Ca2+]i are organized within cardiac caveolaemicrodomains. They also suggest that ouabain-induced recruitments ofSrc and 2-isoform to caveolae are involved in themanifestation of the positive inotropic effect of ouabain.

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7.
We investigated the effects ofclinically relevant ethanol concentrations (5-20 mM) on thesingle-channel kinetics of bovine aortic smooth muscle maxi-K channelsreconstituted in lipid bilayers (1:1palmitoyl-oleoyl-phosphatidylethanolamine:palmitoyl-oleoyl-phosphatidylcholine). Ethanol at 10 and 20 mMdecreased the channel open probability (Po) by75 ± 20.3% mainly by increasing the mean closed time (+82 to+960%, n = 7). In some instances, ethanol alsodecreased the mean open time (40.8 ± 22.5%). ThePo-voltage relation in the presence of 20 mMethanol exhibited a rightward shift in the midpoint of voltageactivation (V1/2  17 mV), a slightlysteeper relationship (change in slope factor, k,  2.5 mV), and a decreased maximum Po (from~0.82 to ~0.47). Interestingly, channels inhibited by ethanol atlow Ca2+ concentrations (2.5 µM) were veryresistant to ethanol in the presence of increased Ca2+ ( 20 µM). Alcohol consumption in clinically relevant amounts may alterthe contribution of maxi-K channels to the regulation of arterial tone.

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8.
Thecalcium-binding protein parvalbumin (PV) occurs at high concentrationsin fast-contracting vertebrate muscle fibers. Its putative role infacilitating the rapid relaxation of mammalian fast-twitch musclefibers by acting as a temporary buffer for Ca2+ is still controversial. Wegenerated knockout mice for PV (PV /) and compared theCa2+ transients and the dynamicsof contraction of their muscles with those from heterozygous (PV+/) and wild-type (WT) mice. In the muscles of PV-deficientmice, the decay of intracellularCa2+ concentration([Ca2+]i)after 20-ms stimulation was slower compared with WT mice and led to aprolongation of the time required to attain peak twitch tension and toan extension of the half-relaxation time. The integral [Ca2+]iin muscle fibers of PV / mice was higher and consequently the force generated during a single twitch was ~40% greater than inPV +/ and WT animals. Acceleration of the contraction-relaxation cycle of fast-twitch muscle fibers by PV may confer an advantage in theperformance of rapid, phasic movements.  相似文献   

9.
The effectsof -adrenoceptor stimulation with isoproterenol on electricallyinduced contraction and intracellular calcium ([Ca2+]i) transient, and cAMP inmyocytes from both hypertrophied right and nonhypertrophied leftventricles of rats exposed to 10% oxygen for 4 wk, were significantlyattenuated. The increased [Ca2+]i transientin response to cholera toxin was abolished, whereas increased cAMPafter NaF significantly attenuated. The biologically activeisoform, Gs-small (45 kDa), was reduced while thebiologically inactive isoform, Gs-large (52 kDa),increased. The increased electrically induced[Ca2+]i transient and cAMP with 10-100µM forskolin were significantly attenuated in chronically hypoxicrats. The content of Gi2, the predominantisoform of Gi protein in the heart, was unchanged. Resultsindicate that impaired functions of Gs protein and adenylyl cyclase cause -adrenoceptor desensitization. The impaired function of the Gs protein may be due to reducedGs-small and/or increased Gs-large, whichdoes not result from changes in Gi protein. Responses toall treatments were the same for right and left ventricles, indicatingthat the impaired cardiac functions are not secondary to cardiac hypertrophy.

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10.
Polyaminesare essential for early mucosal restitution that occurs by epithelialcell migration to reseal superficial wounds after injury. Normalintestinal epithelial cells are tightly bound in sheets, but they needto be rapidly disassembled during restitution. -Catenin is involvedin cell-cell adhesion, and its tyrosine phosphorylation causesdisassembly of adhesion junctions, enhancing the spreading of cells.The current study determined whether polyamines are required for thestimulation of epithelial cell migration by altering -catenintyrosine phosphorylation. Migration of intestinal epithelial cells(IEC-6 line) after wounding was associated with an increase in-catenin tyrosine phosphorylation, which decreased the bindingactivity of -catenin to -catenin. Polyamine depletion by-difluoromethylornithine reduced cytoplasmic free Ca2+concentration ([Ca2+]cyt), preventedinduction of -catenin phosphorylation, and decreased cell migration.Elevation of [Ca2+]cyt induced by theCa2+ ionophore ionomycin restored -cateninphosphorylation and promoted migration in polyamine-deficient cells.Decreased -catenin phosphorylation through the tyrosine kinaseinhibitor herbimycin-A or genistein blocked cell migration, which wasaccompanied by reorganization of cytoskeletal proteins. These resultsindicate that -catenin tyrosine phosphorylation plays a criticalrole in polyamine-dependent cell migration and that polyamines induce-catenin tyrosine phosphorylation at least partially through[Ca2+]cyt.

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11.
The role of the Na+ pump2-subunit in Ca2+ signaling was examined inprimary cultured astrocytes from wild-type(2+/+ = WT) mouse fetuses and thosewith a null mutation in one [2+/ = heterozygote (Het)] or both [2/ = knockout (KO)] 2 genes. Na+ pump catalytic() subunit expression was measured by immunoblot; cytosol[Na+] ([Na+]cyt) and[Ca2+] ([Ca2+]cyt) weremeasured with sodium-binding benzofuran isophthalate and fura 2 byusing digital imaging. Astrocytes express Na+ pumpswith both 1- (80% of total ) and2- (20% of total ) subunits. Het astrocytesexpress 50% of normal 2; those from KO express none.Expression of 1 is normal in both Het and KO cells.Resting [Na+]cyt = 6.5 mM in WT, 6.8 mMin Het (P > 0.05 vs. WT), and 8.0 mM in KO cells(P < 0.001); 500 nM ouabain (inhibits only2) equalized [Na+]cyt at 8 mMin all three cell types. Resting[Ca2+]cyt = 132 nM in WT, 162 nM in Het,and 196 nM in KO cells (both P < 0.001 vs. WT).Cyclopiazonic acid (CPA), which inhibits endoplasmic reticulum (ER)Ca2+ pumps and unloads the ER, induces transient (inCa2+-free media) or sustained (in Ca2+-repletemedia) elevation of [Ca2+]cyt. TheseCa2+ responses to 10 µM CPA were augmented in Het as wellas KO cells. When CPA was applied in Ca2+-free media, thereintroduction of Ca2+ induced significantly largertransient rises in [Ca2+]cyt (due toCa2+ entry through store-operated channels) in Het and KOcells than in WT cells. These results correlate with published evidencethat 2 Na+ pumps andNa+/Ca2+ exchangers are confined to plasmamembrane microdomains that overlie the ER. The data suggest thatselective reduction of 2 Na+ pump activitycan elevate local [Na+] and, viaNa+/Ca2+ exchange, [Ca2+] in thetiny volume of cytosol between the plasma membrane and ER. This, inturn, augments adjacent ER Ca2+ stores and therebyamplifies Ca2+ signaling without elevating bulk[Na+]cyt.

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12.
Effects of HCO3 on protein kinase C (PKC)-and protein kinase A (PKA)-induced anion conductances were investigatedin Necturus gallbladder epithelial cells. InHCO3-free media, activation of PKC via12-O-tetradecanoylphorbol 13-acetate (TPA) depolarizedapical membrane potential (Va) and decreased fractional apical voltage ratio (FR). These effects wereblocked by mucosal 5-nitro-2-(3-phenylpropylamino) benzoic acid(NPPB), a Cl channel blocker. In HCO3media, TPA induced significantly greater changes inVa and FR. These effects wereblocked only when NPPB was present in both mucosal and basolateralcompartments. The data suggest that TPA activates NPPB-sensitive apicalCl conductance (gCla) in theabsence of HCO3; in its presence, TPA stimulated bothNPPB-sensitive gCla and basolateralCl conductance (gClb).Activation of PKA via 3-isobutyl-1-methylxanthine (IBMX) also decreased Va and FR; however, thesechanges were not affected by external HCO3. Weconclude that HCO3 modulates the effects of PKC ongClb. In HCO3 medium, TPAand IBMX also induced an initial transient hyperpolarization andincrease in intracellular pH. Because these changes were independent ofmucosal Na+ and Cl, it is suggested that TPAand IBMX induce a transient increase in apical HCO3 conductance.

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13.
The possibility that membrane depolarization of synovialfibroblasts caused by interleukin-1 (IL-1) was mediated byprotein kinase C (PKC) and Ca2+influx was studied using inhibitor and activator analysis. The effectof IL-1 was blocked by bisindolylmaleimide I, an inhibitor of PKC,and by the Ca2+ channel blockersnifedipine and verapamil. In other experiments, PKC was activated usingphorbol 12-myristate 13-acetate, andCa2+ influx was increased by meansof a Ca2+ ionophore. Simultaneousapplication of phorbol ester andCa2+ ionophore in the absence ofIL-1 mimicked the depolarization caused by IL-1. The results wereconsistent with the hypothesis that, under the conditions studied,activation of PKC and Ca2+ influxare necessary and sufficient processes in the transduction of IL-1by synovial cells leading to membrane depolarization. Theessential role of protein phosphorylation andCa2+ influx in the earlyelectrophysiological response of synovial fibroblasts to IL-1 wastherefore established. The role of IL-1-induced depolarization inregulating protein expression by the cells remains to be determined,but the results reported here, taken together with observations thatprotein phosphorylation and Ca2+influx also mediate the effect of IL-1 on protease production (1, 2), suggest that electrophysiological changes are actually part of thepathway for expression of proteases in response to IL-1.

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14.
Thickening of airway mucus and lungdysfunction in cystic fibrosis (CF) results, at least in part, fromabnormal secretion of Cl and HCO3across the tracheal epithelium. The mechanism of the defect in HCO3 secretion is ill defined; however, a lack ofapical Cl/HCO3 exchange may exist inCF. To test this hypothesis, we examined the expression ofCl/HCO3 exchangers in trachealepithelial cells exhibiting physiological features prototypical ofcystic fibrosis [CFT-1 cells, lacking a functional cystic fibrosistransmembrane conductance regulator (CFTR)] or normal trachea (CFT-1cells transfected with functional wild-type CFTR, termed CFT-WT). Cellswere grown on coverslips and were loaded with the pH-sensitive dye2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, andintracellular pH was monitored. Cl/HCO3exchange activity increased by ~300% in cells transfected with functional CFTR, with activities increasing from 0.034 pH/min in CFT-1cells to 0.11 in CFT-WT cells (P < 0.001, n = 8). This activity was significantly inhibited byDIDS. The mRNA expression of the ubiquitous basolateral AE-2Cl/HCO3 exchanger remained unchanged.However, mRNA encoding DRA, recently shown to be aCl/HCO3 exchanger (Melvin JE, Park K,Richardson L, Schultheis PJ, and Shull GE. J Biol Chem 274:22855-22861, 1999.) was abundantly expressed in cells expressingfunctional CFTR but not in cells that lacked CFTR or that expressedmutant CFTR. In conclusion, CFTR induces the mRNA expression of"downregulated in adenoma" (DRA) and, as a result, upregulates theapical Cl/HCO3 exchanger activity intracheal cells. We propose that the tracheal HCO3secretion defect in patients with CF is partly due to thedownregulation of the apical Cl/HCO3exchange activity mediated by DRA.

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15.
Using a novel pharmacological tool with125I-echistatin to detect integrins on the cell, we haveobserved that cardiac fibroblasts harbor five different RGD-bindingintegrins: 81,31, 51, v1, and v3.Stimulation of cardiac fibroblasts by angiotensin II (ANG II) ortransforming growth factor-1 (TGF-1) resulted in an increase ofprotein and heightening by 50% of the receptor density of81-integrin. The effect of ANG II wasblocked by an AT1, but not an AT2, receptorantagonist, or by an anti-TGF-1 antibody. ANG II and TGF-1increased fibronectin secretion, smooth muscle -actin synthesis, andformation of actin stress fibers and enhanced attachment of fibroblaststo a fibronectin matrix. The 8- and1-subunits were colocalized by immunocytochemistry with vinculin or 3-integrin at focal adhesion sites.These results indicate that 81-integrinis an abundant integrin on rat cardiac fibroblasts. Its positivemodulation by ANG II and TGF-1 in a myofibroblast-likephenotype suggests the involvement of81-integrin in extracellularmatrix protein deposition and cardiac fibroblast adhesion.

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16.
We studied the effects of protein kinase C (PKC) activation onendothelial cell surface expression and function of the proteolytically activated thrombin receptor 1 (PAR-1). Cell surface PAR-1 expression was assessed by immunofluorescence (using anti-PAR-1 monoclonal antibody), and receptor activation was assessed by measuring increases in cytosolic Ca2+ concentration inhuman dermal microvascular endothelial cells (HMEC) exposed to-thrombin or phorbol ester,12-O-tetradecanoylphorbol-13-acetate (TPA).Immunofluorescence showed that thrombin and TPA reduced the cellsurface expression of PAR-1. Prior exposure of HMEC to thrombin for 5 min desensitized the cells to thrombin, indicating homologous PAR-1desensitization. In contrast, prior activation of PKC with TPA produceddesensitization to thrombin and histamine, indicatingheterologous PAR-1 desensitization. Treatment of cells withstaurosporine, a PKC inhibitor, fully prevented heterologous desensitization, whereas thrombin-induced homologous desensitization persisted. Depletion of PKC isozymes(PKCI andPKCII) by transducing cellswith antisense cDNA of PKCIprevented the TPA-induced decrease in cell surface PAR-1 expression andrestored ~60% of the cytosolic Ca2+ signal in response tothrombin. In contrast, depletion of PKC isozymes did not affect theloss of cell surface PAR-1 and induction of homologous PAR-1desensitization by thrombin. Therefore, homologous PAR-1desensitization by thrombin occurs independently of PKC isozymes,whereas the PKC-activated pathway is important in signaling heterologous PAR-1 desensitization in endothelial cells.

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17.
These experiments were performed to determine the effects ofreducing Ca2+ influx(Cain) onK+ currents(IK) inmyocytes from rat small mesenteric arteries by1) adding externalCd2+ or2) lowering externalCa2+ to 0.2 mM. When measured froma holding potential (HP) of 20 mV(IK20),decreasing Cain decreasedIK at voltageswhere it was active (>0 mV). When measured from a HP of 60 mV(IK60),decreasing Cain increasedIK at voltagesbetween 30 and +20 mV but decreased IK at voltagesabove +40 mV. Difference currents(IK) weredetermined by digital subtraction of currents recorded under controlconditions from those obtained whenCain was decreased. At testvoltages up to 0 mV,IK60 exhibitedkinetics similar to controlIK60, with rapidactivation to a peak followed by slow inactivation. At 0 mV, peakIK60 averaged75 ± 13 pA (n = 8) withCd2+ and 120 ± 20 pA(n = 9) with lowCa2+ concentration. At testvoltages from 0 to +60 mV,IK60 always had an early positive peak phase, but its apparent "inactivation" increased with voltage and its steady value became negative above +20mV. At +60 mV, the initial peakIK60 averaged115 ± 18 pA with Cd2+ and 187 ± 34 pA with low Ca2+. With 10 mM pipette BAPTA, Cd2+ produced asmall inhibition ofIK20 but stillincreased IK60 between 30 and +10 mV. InCa2+-free external solution,Cd2+ only decreased bothIK20 andIK60. In thepresence of iberiotoxin (100 nM) to inhibitCa2+-activatedK+ channels(KCa),Cd2+ increasedIK60 at allvoltages positive to 30 mV while BAY K 8644 (1 µM) decreasedIK60. Theseresults suggest that Cain, through L-type Ca2+ channels and perhapsother pathways, increases KCa(i.e., IK20) and decreases voltage-dependent K+currents in this tissue. This effect could contribute to membrane depolarization and force maintenance.

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18.
Earlier studies demonstrated that deprivation of growth hormone(GH) for 3 h decreased basal and maximally stimulated cytosolic Ca2+ in rat adipocytes andsuggested that membrane Ca2+channels might be decreased. Measurement of L-typeCa2+ channels in purified plasmamembranes by immunoassay or dihydropyridine binding indicated a two- tofourfold decrease after 3 h of incubation without GH. No such decreasewas seen in unfractionated adipocyte membrane preparations. Thedecrease in plasma membrane channel content was largely accounted forby redistribution of channels to a light microsomal membrane fraction.Immunoassay of 1-,2/-, and -channelsubunits in membrane fractions indicated that the channelsredistributed as intact complexes. Addition of GH during the 1st h ofincubation prevented channel redistribution, and addition of GH after 3 h restored channel distribution to the GH-replete state of freshlyisolated adipocytes. The studies suggest that GH may regulate theabundance of Ca2+ channels in theadipocyte plasma membrane and thereby modulate sensitivity to signals,the expression of which is Ca2+dependent.

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19.
An HEK-293 cell line stably expressing the humanrecombinant ClC-2 Cl channel was used in patch-clampstudies to study its regulation. The relative permeabilityPx/PCl calculated fromreversal potentials was I > Cl = NO3 = SCNBr. Theabsolute permeability calculated from conductance ratios wasCl = Br = NO3  SCN > I. The channel was activatedby cAMP-dependent protein kinase (PKA), reduced extracellular pH, oleicacid (C:18 cis9), elaidic acid (C:18trans9), arachidonic acid (AA; C:20cis5,8,11,14), and by inhibitors of AA metabolism,5,8,11,14-eicosatetraynoic acid (ETYA; C:20trans5,8,11,14),-methyl-4-(2-methylpropyl)benzeneacetic acid (ibuprofen), and2-phenyl-1,2-benzisoselenazol-3-[2H]-one (PZ51, ebselen). ClC-2Cl channels were activated by a combination of forskolinplus IBMX and were inhibited by the cell-permeant myristoylated PKAinhibitor (mPKI). Channel activation by reduction of bath pH wasincreased by PKA and prevented by mPKI. AA activation of the ClC-2Cl channel was not inhibited by mPKI or staurosporine andwas therefore independent of PKA or protein kinase C activation.

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20.
Ca2+-activatedCl currents (ICl,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 ICl,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 ICl,Calengthened. The steady-state current-voltage (I-V) relationshipfor ICl,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 ICl,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.

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