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1.
To study the effects of -opioid receptor stimulation onintracellular Ca2+ concentration([Ca2+]i)homeostasis during extracellular acidosis, we determined the effects of-opioid receptor stimulation on[Ca2+]iresponses during extracellular acidosis in isolated single ratventricular myocytes, by a spectrofluorometric method. U-50488H (10-30 µM), a selective -opioid receptor agonist, dosedependently decreased the electrically induced[Ca2+]itransient, which results from the influx ofCa2+ and the subsequentmobilization of Ca2+ from thesarcoplasmic reticulum (SR). U-50488H (30 µM) also increased theresting[Ca2+]iand inhibited the[Ca2+]itransient induced by caffeine, which mobilizesCa2+ from the SR, indicating thatthe effects of the -opioid receptor agonist involved mobilization ofCa2+ from its intracellular poolinto the cytoplasm. The Ca2+responses to 30 µM U-50488H were abolished by 5 µMnor-binaltorphimine, a selective -opioid receptorantagonist, indicating that the event was mediated by the -opioidreceptor. The effects of the agonist on[Ca2+]iand the electrically induced[Ca2+]itransient were significantly attenuated when the extracellular pH(pHe) was loweredto 6.8, which itself reduced intracellular pH(pHi) and increased[Ca2+]i.The inhibitory effects of U-50488H were restored during extracellular acidosis in the presence of 10 µM ethylisopropyl amiloride, a potentNa+/H+exchange blocker, or 0.2 mM Ni2+,a putativeNa+/Ca2+exchange blocker. The observations indicate that acidosismay antagonize the effects of -opioid receptor stimulation viaNa+/H+andNa+/Ca2+exchanges. When glucose at 50 mM, known to activate theNa+/H+exchange, was added, both the resting[Ca2+]iand pHi increased. Interestingly,the effects of U-50488H on [Ca2+]iand the electrically induced[Ca2+]itransient during superfusion with glucose were significantly attenuated; this mimicked the responses during extracellular acidosis. When a high-Ca2+ (3 mM) solutionwas superfused, the resting[Ca2+]iincreased; the increase was abolished by 0.2 mMNi2+, but thepHi remained unchanged. Like theresponses to superfusion with high-concentration glucose andextracellular acidosis, the responses of the[Ca2+]iand electrically induced[Ca2+]itransients to 30 µM U-50488H were also significantly attenuated. Results from the present study demonstrated for the first time thatextracellular acidosis antagonizes the effects of -opioid receptorstimulation on the mobilization ofCa2+ from SR. Activation of bothNa+/H+andNa+/Ca2+exchanges, leading to an elevation of[Ca2+]i,may be responsible for the antagonistic action of extracellular acidosis against -opioid receptor stimulation.

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2.
In the presentstudy, we examined the ability of adenosine 3',5'-cyclicmonophosphate (cAMP) to reduce elevated levels of cytosolicCa2+ concentration([Ca2+]i)in pancreatic -cells.[Ca2+]iand reduced pyridine nucleotide, NAD(P)H, were measured in rat single-cells by fura 2 and autofluorescence microfluorometry. Sustained[Ca2+]ielevation, induced by high KCl (25 mM) at a basal glucose concentration (2.8 mM), was substantially reduced by cAMP-increasing agents, dibutyryl cAMP (DBcAMP, 5 mM), an adenylyl cyclase activatorforskolin (10 µM), and an incretin glucagon-likepeptide-1-(7-36) amide (109 M), as well as byglucose (16.7 mM). The[Ca2+]i-reducingeffects of cAMP were greater at elevated glucose (8.3-16.7 mM)than at basal glucose (2.8 mM). An inhibitor of protein kinase A (PKA),H-89, counteracted[Ca2+]i-reducingeffects of cAMP but not those of glucose. Okadaic acid, a phosphataseinhibitor, at 10-100 nM also reduced sustained [Ca2+]ielevation in a concentration-dependent manner. Glucose, but not DBcAMP,increased NAD(P)H in -cells.[Ca2+]i-reducingeffects of cAMP were inhibited by 0.3 µM thapsigargin, an inhibitorof the endoplasmic reticulum (ER)Ca2+ pump. In contrast,[Ca2+]i-reducingeffects of cAMP were not altered by ryanodine, an ERCa2+-release inhibitor,Na+-free conditions, or diazoxide,an ATP-sensitive K+ channelopener. In conclusion, the cAMP-PKA pathway reduces[Ca2+]ielevation by sequestering Ca2+ inthapsigargin-sensitive stores. This process does not involve, but ispotentiated by, activation of -cell metabolism. Together with theknown[Ca2+]i-increasingaction of cAMP, our results reveal dual regulation of -cell[Ca2+]iby the cAMP-signaling pathway and by a physiological incretin.

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3.
The hypothesisthat vascular protection in females and its absence in males reflectsgender differences in [Ca2+]i andCa2+ mobilization mechanisms of vascular smooth musclecontraction was tested in fura 2-loaded aortic smooth muscle cellsisolated from intact and gonadectomized male and female Wistar-Kyoto(WKY) and spontaneously hypertensive (SHR) rats. In WKY cells incubated in Hanks' solution (1 mM Ca2+), the resting length and[Ca2+]i were significantlydifferent in intact males (64.5 ± 1.2 µm and 83 ± 3 nM) than inintact females (76.5 ± 1.5 µm and 64 ± 7 nM). In intact male WKY,phenylephrine (Phe, 105 M) caused transient increasein [Ca2+]i to 428 ± 13 nMfollowed by maintained increase to 201 ± 8 nM and 32% cellcontraction. In intact female WKY, the Phe-induced [Ca2+]i transient was notsignificantly different, but the maintained [Ca2+]i (159 ± 7 nM) and cellcontraction (26%) were significantly less than in intact male WKY. InCa2+-free (2 mM EGTA) Hanks', Phe and caffeine (10 mM)caused transient increases in[Ca2+]i and contraction that werenot significantly different between males and females. Membranedepolarization by 51 mM KCl caused 31% cell contraction and increased[Ca2+]i to 259 ± 9 nM in intactmale WKY, which were significantly greater than a 24% contraction and214 ± 8 nM [Ca2+]i in intactfemale WKY. Maintained Phe- and KCl-stimulated cell contraction and[Ca2+]i were significantly greaterin SHR than WKY in all groups of rats. Reduction in cell contractionand [Ca2+]i in intact femalescompared with intact males was significantly greater in SHR (~30%)than WKY (~20%). No significant differences in cell contraction or[Ca2+]i were observed betweencastrated males, ovariectomized (OVX) females, and intact males, orbetween OVX females with 17-estradiol implants and intact females.Exogenous application of 17-estradiol (108 M) tocells from OVX females caused greater reduction in Phe- and KCl-inducedcontraction and [Ca2+]i in SHR thanWKY. Thus the basal, maintained Phe- and depolarization-induced [Ca2+]i and contraction of vascularsmooth muscle triggered by Ca2+ entry from theextracellular space exhibit differences depending on gender and thepresence or absence of female gonads. Cell contraction and[Ca2+]i due to Ca2+release from the intracellular stores are not affected by gender or gonadectomy. Gender-specific reduction in contractility and [Ca2+]i in vascular smoothmuscle of female rats is greater in SHR than WKY rats.

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4.
In the ratsphincter pupillae, as in other smooth muscles, the primary signaltransduction cascade for agonist activation is receptor  G protein phospholipase C  inositol trisphosphate  intracellularCa2+ concentration ([Ca2+]i)  calmodulin  myosin light chain kinase  phosphorylated myosin  force development. Light stimulation of isolated sphincters pupillaecan be very precisely controlled, and precise reproducible photomechanical responses (PMRs) result. This precision makes the PMRideal for testing models of regulation of smooth muscle myosinphosphorylation. We measured force and[Ca2+]i concurrently in sphincter pupillaefollowing stimulation by light flashes of varying duration andintensity. We sampled at unusually short (0.01-0.02 s) intervalsto adequately test a PMR model based on the myosin phosphorylationcascade. We found, surprisingly, contrary to the behavior of intestinalmuscle and predictions of the phosphorylation model, that during PMRsforce begins to decay while [Ca2+]i is stillrising. We conclude that control of contraction in the sphincterpupillae probably involves an inhibitory process as well as activationby [Ca2+]i.

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5.
Tumor necrosisfactor (TNF)- has a biphasic effect on heart contractility andstimulates phospholipase A2 (PLA2) incardiomyocytes. Because arachidonic acid (AA) exerts a dual effect onintracellular Ca2+ concentration([Ca2+]i) transients, we investigated thepossible role of AA as a mediator of TNF- on[Ca2+]i transients and contraction withelectrically stimulated adult rat cardiac myocytes. At a lowconcentration (10 ng/ml) TNF- produced a 40% increase in theamplitude of both [Ca2+]i transients andcontraction within 40 min. At a high concentration (50 ng/ml) TNF-evoked a biphasic effect comprising an initial positive effect peakingat 5 min, followed by a sustained negative effect leading to50-40% decreases in [Ca2+]i transientsand contraction after 30 min. Both the positive and negative effects ofTNF- were reproduced by AA and blocked by arachidonyltrifluoromethylketone (AACOCF3), an inhibitor of cytosolic PLA2.Lipoxygenase and cyclooxygenase inhibitors reproduced the high-doseeffects of TNF- and AA. The negative effects of TNF- and AA werealso reproduced by sphingosine and were abrogated by the ceramidaseinhibitor n-oleoylethanolamine. These results point out thekey role of the cytosolic PLA2/AA pathway in mediating thecontractile effects of TNF-.

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6.
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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7.
Regulation of arterial tone by smooth muscle myosin type II   总被引:1,自引:0,他引:1  
Theinitiation of contractile force in arterial smooth muscle (SM) isbelieved to be regulated by the intracellular Ca2+concentration and SM myosin type II phosphorylation. We tested thehypothesis that SM myosin type II operates as a molecular motor proteinin electromechanical, but not in protein kinase C (PKC)-induced,contraction of small resistance-sized cerebral arteries. We utilized aSM type II myosin heavy chain (MHC) knockout mouse model and measuredarterial wall Ca2+ concentration([Ca2+]i) and the diameter of pressurizedcerebral arteries (30-100 µm) by means of digital fluorescencevideo imaging. Intravasal pressure elevation caused a graded[Ca2+]i increase and constricted cerebralarteries of neonatal wild-type mice by 20-30%. In contrast,intravasal pressure elevation caused a graded increase of[Ca2+]i without constriction in (/)MHC-deficient arteries. KCl (60 mM) induced a further[Ca2+]i increase but failed to inducevasoconstriction of (/) MHC-deficient cerebral arteries. Activationof PKC by phorbol ester (phorbol 12-myristate 13-acetate, 100 nM)induced a strong, sustained constriction of (/) MHC-deficientcerebral arteries without changing [Ca2+]i.These results demonstrate a major role for SM type II myosin in thedevelopment of myogenic tone and Ca2+-dependentconstriction of resistance-sized cerebral arteries. In contrast, thesustained contractile response did not depend on myosin andintracellular Ca2+ but instead depended on PKC. We suggestthat SM myosin type II operates as a molecular motor protein in thedevelopment of myogenic tone but not in pharmacomechanical coupling byPKC in cerebral arteries. Thus PKC-dependent phosphorylation ofcytoskeletal proteins may be responsible for sustained contraction invascular SM.

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8.
We determined the effect of aromatic aminoacid stimulation of the human extracellular Ca2+-sensingreceptor (CaR) on intracellular Ca2+ concentration([Ca2+]i) in single HEK-293 cells. Additionof L-phenylalanine or L-tryptophan (at 5 mM)induced [Ca2+]i oscillations from a restingstate that was quiescent at 1.8 mM extracellular Ca2+concentration ([Ca2+]e). Each[Ca2+]i peak returned to baseline values, andthe average oscillation frequency was ~1 min1 at37°C. Oscillations were not induced or sustained if the[Ca2+]e was reduced to 0.5 mM, even in thecontinued presence of amino acid. Average oscillation frequency inresponse to an increase in [Ca2+]e (from 1.8 to 2.5-5 mM) was much higher (~4 min1) than thatinduced by aromatic amino acids. Oscillations in response to[Ca2+]e were sinusoidal whereas those inducedby amino acids were transient. Thus both amino acids andCa2+, acting through the same CaR, produce oscillatoryincreases in [Ca2+]i, but the resultantoscillation pattern and frequency allow the cell to discriminate whichagonist is bound to the receptor.

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9.
The role of Na+/Ca2+ exchange inregulating intracellular Ca2+ concentration([Ca2+]i) in isolated smooth muscle cellsfrom the guinea pig urinary bladder was investigated. Incrementalreduction of extracellular Na+ concentration resulted in agraded rise of [Ca2+]i; 50-100 µMstrophanthidin also increased [Ca2+]i. Asmall outward current accompanied the rise of[Ca2+]i in low-Na+ solutions(17.1 ± 1.8 pA in 29.4 mM Na+). The quantity ofCa2+ influx through the exchanger was estimated from thecharge carried by the outward current and was ~30 times that which isnecessary to account for the rise of [Ca2+]i,after correction was made for intracellular Ca2+ buffering.Ca2+ influx through the exchanger was able to loadintracellular Ca2+ stores. It is concluded that the levelof resting [Ca2+]i is not determined by theexchanger, and under resting conditions (membrane potential 50 to60 mV), there is little net flux through the exchanger. However, asmall rise of intracellular Na+ concentration would besufficient to generate significant net Ca2+ influx.

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10.
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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11.
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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12.
The subcellular spatial and temporal organization ofagonist-induced Ca2+ signals wasinvestigated in single cultured vascular endothelial cells.Extracellular application of ATP initiated a rapid increase ofintracellular Ca2+ concentration([Ca2+]i)in peripheral cytoplasmic processes from where activation propagated asa[Ca2+]iwave toward the central regions of the cell. The average propagation velocity of the[Ca2+]iwave in the peripheral processes was 20-60 µm/s, whereas in thecentral region the wave propagated at <10 µm/s. The time course ofthe recovery of[Ca2+]idepended on the cell geometry. In the peripheral processes (i.e.,regions with a high surface-to-volume ratio)[Ca2+]ideclined monotonically, whereas in the central region[Ca2+]idecreased in an oscillatory fashion. Propagating[Ca2+]iwaves were preceded by small, highly localized[Ca2+]itransients originating from 1- to 3-µm-wide regions. The average amplitude of these elementary events ofCa2+ release was 23 nM, and theunderlying flux of Ca2+ amountedto ~1-2 × 1018mol/s or ~0.3 pA, consistent with aCa2+ flux through a single orsmall number of endoplasmic reticulum Ca2+-release channels.

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13.
A fluid streamthrough a microtube was applied to cultured human aortic endothelialcells to investigate the endothelial responses of both the ioniccurrents and intracellular Ca2+concentration([Ca2+]i)to mechanical stimulation. The fluid stream induced an increase in[Ca2+]ithat was dependent on both the flow rate and the extracellular Ca2+ concentration.Gd3+ and niflumic acid inhibitedthe fluid stream-induced increase in[Ca2+]i,whereas Ba2+ andtetraethylammonium ion exhibited no effect. The fluid stream-induced [Ca2+]iincrease was accompanied by the activation of an inward current at52.8 mV. The reversal potential of the fluid stream-induced current shifted to positive potentials when the externalCl concentration wasreduced but was not affected by variation of the externalNa+ concentration. During theexposure to the fluid stream,[Ca2+]iwas voltage dependent, i.e., depolarization decreased[Ca2+]i.We therefore conclude that the fluid stream-induced current is largelycarried by Cl and that theCl current may thus play arole in modulating the Ca2+ influxby altering the membrane potential of endothelial cells.

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14.
We used theCa2+-sensitive fluorescent dyefura 2, together with measurements of intracellularD-myo-inositol1,4,5-trisphosphate [Ins(1,4,5)P3],to assess the inhibitory effects of caffeine on signal transduction viaG protein-coupled receptor pathways in isolated rat mandibular salivaryacinar cells. ACh, norepinephrine (NE), and substance P (SP) all evokedsubstantial increases in the intracellular freeCa2+ concentration([Ca2+]i).Responses to ACh and NE were markedly inhibited by prior application of20 mM caffeine. The inhibitory effect of caffeine was not reproduced byphosphodiesterase inhibition with IBMX or addition of cell-permeantdibutyryl cAMP. In contrast to the ACh and NE responses, the[Ca2+]iresponse to SP was unaffected by caffeine. Despite this, SP and AChappeared to mobilize Ca2+ from acommon intracellular pool. Measurements of agonist-induced changes inIns(1,4,5)P3levels confirmed that caffeine inhibited the stimulus-response couplingpathway at a point beforeIns(1,4,5)P3 generation. Caffeine did not, however, inhibit[Ca2+]iresponses evoked by direct activation of G proteins with 40 mMF. These data show thatcaffeine inhibits G protein-coupled signal transduction in these cellsat some element that is common to the muscarinic and -adrenergicsignaling pathways but is not shared by the SP signaling pathway. Wesuggest that this element might be a specific structural motif on the Gprotein-coupled muscarinic and -adrenergic receptors.  相似文献   

15.
Inisolated rat pancreatic -cells, the nitric oxide (NO) donor NOC-7 at1 µM reduced the amplitude of the oscillations of cytosolicCa2+ concentration ([Ca2+]c)induced by 11.1 mM glucose, and at 10 µM terminated them. In thepresence of NG-nitro-L-arginine(L-NNA), however, NOC-7 at 0.5 and 1 µM increased theamplitude of the [Ca2+]c oscillations,although the NO donor at 10 µM still suppressed them. Aqueous NOsolution also had a dual effect on the[Ca2+]c oscillations. The soluble guanylatecyclase inhibitor LY-83583 and the cGMP-dependent protein kinaseinhibitor KT5823 inhibited the stimulatory effect of NO, and8-bromo-cGMP increased the amplitude of the[Ca2+]c oscillations. Patch-clamp analyses inthe perforated configuration showed that 8-bromo-cGMP inhibited wholecell ATP-sensitive K+ currents in the isolated ratpancreatic -cells, suggesting that the inhibition by cGMP ofATP-sensitive K+ channels is, at least in part, responsiblefor the stimulatory effect of NO on the[Ca2+]c oscillations. In the presence ofL-NNA, the glucose-induced insulin secretion from isolatedislets was facilitated by 0.5 µM NOC-7, whereas it was suppressed by10 µM NOC-7. These results suggest that NO facilitatesglucose-induced [Ca2+]c oscillations of-cells and insulin secretion at low concentrations, which effectsare mediated by cGMP, whereas NO inhibits them in a cGMP-independentmanner at high concentrations.

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16.
-Opioid receptor (-OR)stimulation with U50,488H, a selective -OR agonist, or activation ofprotein kinase C (PKC) with 4-phorbol 12-myristate 13-acetate (PMA), anactivator of PKC, decreased the electrically induced intracellularCa2+ ([Ca2+]i) transient andincreased the intracellular pH (pHi) in single ventricularmyocytes of rats subjected to 10% oxygen for 4 wk. The effects ofU50,488H were abolished by nor-binaltorphimine, a selective -ORantagonist, and calphostin C, a specific inhibitor of PKC, while theeffects of PMA were abolished by calphostin C andethylisopropylamiloride (EIPA), a potent Na+/H+exchange blocker. In both right hypertrophied and leftnonhypertrophied ventricles of chronically hypoxic rats, the effects ofU50,488H or PMA on [Ca2+]i transient andpHi were significantly attenuated and completely abolished,respectively. Results are first evidence that the[Ca2+]i and pHi responses to-OR stimulation are attenuated in the chronically hypoxic rat heart,which may be due to reduced responses to PKC activation. Responses toall treatments were the same for right and left ventricles, indicatingthat the functional impairment is independent of hypertrophy. -ORmRNA expression was the same in right and left ventricles of bothnormoxic and hypoxic rats, indicating no regional specificity.

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17.
To determine whetherthe phosphoinositol/Ca2+ pathwayinteracts with the adenylate cyclase/adenosine 3',5'-cyclicmonophosphate (cAMP) pathway in the cardiac -receptor, the effectsof U-50488, a specific -receptor agonist, on the intracellularCa2+ concentration([Ca2+]i)and forskolin-induced accumulation of cAMP in rat ventricular myocyteswere determined after interference of thephosphoinositol/Ca2+ pathway.U-50488 suppressed the forskolin-induced accumulation of cAMP andelevated[Ca2+]i,which were blocked by norbinaltorphimine, a specific -receptor antagonist, and pertussis toxin. The effects of U-50488 werequalitatively similar to those of A-23187, aCa2+ ionophore, but opposite tothose of1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-acetoxymethyl ester (AM), a[Ca2+]ichelator. Abolition of U-50488-induced elevation of[Ca2+]iby BAPTA-AM also abolished the effect of U-50488 on forskolin-induced accumulation of cAMP. Inhibition of the phospholipase C by specific inhibitors, U-73122 and neomycin, abolished the effects of U-50488 onboth[Ca2+]iand forskolin-induced accumulation of cAMP. The results showed for thefirst time that -receptor stimulation may suppress cAMP accumulationvia activation of thephosphoinositol/Ca2+ pathway inthe rat heart.

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18.
Prakash, Y. S., H. F. M. van der Heijden, M. S. Kannan, andG. C. Sieck. Effects of salbutamol on intracellular calcium oscillations in porcine airway smooth muscle. J. Appl.Physiol. 82(6): 1836-1843, 1997.Relaxation ofairway smooth muscle (ASM) by -adrenoceptor agonists involvesreduction of intracellular Ca2+concentration([Ca2+]i).In porcine ASM cells, acetylcholine induces[Ca2+]ioscillations that display frequency modulation by agonist concentration and basal[Ca2+]i.We used real-time confocal microscopy to examine the effect ofsalbutamol (1 nM to 1 µM), a2-adrenoceptor agonist, on[Ca2+]ioscillations in freshly dissociated porcine ASM cells. Salbutamol decreased the frequency of[Ca2+]ioscillations in a concentration-dependent fashion, completely inhibiting the oscillations at 1 µM. These effects were mimicked by acell-permeant analog of adenosine 3,5-cyclicmonophosphate. The inhibitory effect of salbutamol was partiallyreversed by BAY K 8644. Salbutamol reduced[Ca2+]ieven when sarcoplasmic reticulum (SR)Ca2+ reuptake andCa2+ influx were blocked.Lanthanum blockade of Ca2+ effluxattenuated the inhibitory effect of salbutamol on[Ca2+]i.The[Ca2+]iresponse to caffeine was unaffected by salbutamol. On the basis ofthese results, we conclude that2-adrenoceptor agonists have little effect on SR Ca2+ releasein ASM cells but reduce[Ca2+]iby inhibiting Ca2+ influx throughvoltage-gated channels and by enhancingCa2+ efflux.

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19.
The presentstudy used real-time confocal microscopy to examine the effects of the2-adrenoceptor agonistsalbutamol on regulation of intracellularCa2+ concentration([Ca2+]i)in myotubes derived from neonatal mouse limb muscles.Immunocytochemical staining for ryanodine receptors and skeletal musclemyosin confirmed the presence of sarcomeres. The myotubes displayedboth spontaneous and ACh-induced rapid (<2-ms rise time)[Ca2+]itransients. The[Ca2+]itransients were frequency modulated by both low and high concentrations of salbutamol. Exposure to -bungarotoxin and tetrodotoxin inhibited ACh-induced[Ca2+]itransients and the response to low concentrations of salbutamol but notthe response to higher concentrations. Preexposure to caffeineinhibited the subsequent[Ca2+]iresponse to lower concentrations of salbutamol and significantly blunted the response to higher concentrations. Preexposure to salbutamol diminished the[Ca2+]iresponse to caffeine. Inhibition of dihydropyridine-sensitive Ca2+ channels with nifedipine orPN-200-110 did not prevent[Ca2+]ielevations induced by higher concentrations of salbutamol. The effectsof salbutamol were mimicked by the membrane-permeant analog dibutyryladenosine 3',5'-cyclic monophosphate. Thesedata indicate that salbutamol effects in skeletal muscle predominantly involve enhanced sarcoplasmic reticulumCa2+ release.  相似文献   

20.
The purpose ofthe present study was to determine whether cyclic ADP-ribose (cADPR)acts as a second messenger forCa2+ release through ryanodinereceptor (RyR) channels in tracheal smooth muscle (TSM). Freshlydissociated porcine TSM cells were permeabilized with -escin, andreal-time confocal microscopy was used to examine changes inintracellular Ca2+ concentration([Ca2+]i).cADPR (10 nM-10 µM) induced a dose-dependent increase in [Ca2+]i,which was blocked by the cADPR receptor antagonist 8-amino-cADPR (20 µM) and by the RyR blockers ruthenium red (10 µM) and ryanodine (10 µM), but not by the inositol 1,4,5-trisphosphate receptor blockerheparin (0.5 mg/ml). During steady-state[Ca2+]ioscillations induced by acetylcholine (ACh), addition of 100 nM and 1 µM cADPR increased oscillation frequency and decreased peak-to-troughamplitude. ACh-induced[Ca2+]ioscillations were blocked by 8-amino-cADPR; however, 8-amino-cADPR didnot block the[Ca2+]iresponse to a subsequent exposure to caffeine. These results indicatethat cADPR acts as a second messenger forCa2+ release through RyR channelsin TSM cells and may be necessary for initiating ACh-induced[Ca2+]ioscillations.

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