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1.
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. 相似文献
2.
Na(+)/Ca(2+) exchange and its role in intracellular Ca(2+) regulation in guinea pig detrusor smooth muscle 总被引:3,自引:0,他引:3
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. 相似文献
3.
Intracellular Ca(2+) stores in chemoreceptor cells of the rabbit carotid body: significance for chemoreception 总被引:1,自引:0,他引:1
Vicario I Obeso A Rocher A López-Lopez JR González C 《American journal of physiology. Cell physiology》2000,279(1):C51-C61
Thenotion that intracellular Ca2+ (Cai2+)stores play a significant role in the chemoreception process inchemoreceptor cells of the carotid body (CB) appears in the literaturein a recurrent manner. However, the structural identity of theCa2+ stores and their real significance in the function ofchemoreceptor cells are unknown. To assess the functional significanceof Cai2+ stores in chemoreceptor cells, we havemonitored 1) the release of catecholamines (CA) from thecells using an in vitro preparation of intact rabbit CB and2) the intracellular Ca2+ concentration([Ca2+]i) using isolated chemoreceptor cells;both parameters were measured in the absence or the presence of agentsinterfering with the storage of Ca2+. We found thatthreshold [Ca2+]i for high extracellularK+ (Ke+) to elicit a release response is250 nM. Caffeine (10-40 mM), ryanodine (0.5 µM), thapsigargin(0.05-1 µM), and cyclopiazonic acid (10 µM) did not alter thebasal or the stimulus (hypoxia, high Ke+)-inducedrelease of CA. The same agents produced Cai2+transients of amplitude below secretory threshold; ryanodine (0.5 µM), thapsigargin (1 µM), and cyclopiazonic acid (10 µM) did notalter the magnitude or time course of the Cai2+responses elicited by high Ke+. Several potentialactivators of the phospholipase C system (bethanechol, ATP, andbradykinin), and thereby of inositol 1,4,5-trisphosphate receptors,produced minimal or no changes in [Ca2+]i anddid not affect the basal release of CA. It is concluded that, in therabbit CB chemoreceptor cells, Cai2+ stores do not playa significant role in the instant-to-instant chemoreception process. 相似文献
4.
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. 相似文献
5.
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. 相似文献
6.
Pei Jian-Ming; Yu Xiao-Chun; Bian Jin-Song; Wong Tak-Ming 《American journal of physiology. Cell physiology》1999,277(3):C492
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. 相似文献
7.
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. 相似文献
8.
In fura 2-loaded N1E-115 cells, regulationof intracellular Ca2+ concentration([Ca2+]i) following a Ca2+ loadinduced by 1 µM thapsigargin and 10 µM carbonylcyanidep-trifluoromethyoxyphenylhydrazone (FCCP) wasNa+ dependent and inhibited by 5 mM Ni2+. Incells with normal intracellular Na+ concentration([Na+]i), removal of bath Na+,which should result in reversal of Na+/Ca2+exchange, did not increase [Ca2+]i unlesscell Ca2+ buffer capacity was reduced. When N1E-115 cellswere Na+ loaded using 100 µM veratridine and 4 µg/mlscorpion venom, the rate of the reverse mode of theNa+/Ca2+ exchanger was apparently enhanced,since an ~4- to 6-fold increase in [Ca2+]ioccurred despite normal cell Ca2+ buffering. In SBFI-loadedcells, we were able to demonstrate forward operation of theNa+/Ca2+ exchanger (net efflux ofCa2+) by observing increases (~ 6 mM) in[Na+]i. These Ni2+ (5 mM)-inhibited increases in [Na+]i could onlybe observed when a continuous ionomycin-induced influx ofCa2+ occurred. The voltage-sensitive dyebis-(1,3-diethylthiobarbituric acid) trimethine oxonol was used tomeasure changes in membrane potential. Ionomycin (1 µM) depolarizedN1E-115 cells (~25 mV). This depolarization was Na+dependent and blocked by 5 mM Ni2+ and 250-500 µMbenzamil. These data provide evidence for the presence of anelectrogenic Na+/Ca2+ exchanger that is capableof regulating [Ca2+]i after release ofCa2+ from cell stores. 相似文献
9.
Role of cyclic ADP-ribose in the regulation of [Ca2+]i in porcine tracheal smooth muscle 总被引:1,自引:0,他引:1
Prakash Y. S.; Kannan Mathur S.; Walseth Timothy F.; Sieck Gary C. 《American journal of physiology. Cell physiology》1998,274(6):C1653
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. 相似文献
10.
Ciglitizone inhibits cell proliferation in human uterine leiomyoma via activation of store-operated Ca2+ channels 总被引:1,自引:0,他引:1
Kim BY Cho CH Song DK Mun KC Suh SI Kim SP Shin DH Jang BC Kwon TK Cha SD Bae I Bae JH 《American journal of physiology. Cell physiology》2005,288(2):C389-C395
This study investigated the acute effects of a peroxisome proliferator-activated receptor (PPAR)- ligand, ciglitizone, on cell proliferation and intracellular Ca2+ signaling in human normal myometrium and uterine leiomyoma. Changes in intracellular Ca2+ concentration ([Ca2+]i) were measured with fura-2 AM, and cellular viabilities were determined by viable cell count and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction assay. Ciglitizone (100 µM) induced greater inhibition of cell proliferation in uterine leiomyoma than in myometrium. Ciglitizone also dose-dependently increased [Ca2+]i in both myometrium and uterine leiomyoma; these [Ca2+]i increases were inhibited by PPAR- antagonists and raloxifene. Ciglitizone-induced [Ca2+]i increase showed only an initial peak in normal myometrial cells, whereas in uterine leiomyoma there was a second sustained [Ca2+]i increase as well. The initial [Ca2+]i increase in both myometrium and uterine leiomyoma resulted from the release of Ca2+ by the sarcoplasmic reticulum via activation of ryanodine receptors. The second [Ca2+]i increase was observed only in uterine leiomyoma because of a Ca2+ influx via an activation of store-operated Ca2+ channels (SOCCs). Cell proliferation was inhibited and secondary [Ca2+]i increase in uterine leiomyoma was attenuated by cotreatment of ciglitizone with a SOCC blocker, lanthanum. The results suggest that ciglitizone inhibits cell proliferation and increases [Ca2+]i through the activation of SOCCs, especially in human uterine leiomyoma. peroxisome proliferator-activated receptor-; intracellular calcium; uterine cells 相似文献
11.
Regulation of arterial tone by smooth muscle myosin type II 总被引:1,自引:0,他引:1
Löhn M Kämpf D Gui-Xuan C Haller H Luft FC Gollasch M 《American journal of physiology. Cell physiology》2002,283(5):C1383-C1389
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. 相似文献
12.
Kaneko Y Ishikawa T Amano S Nakayama K 《American journal of physiology. Cell physiology》2003,284(5):C1215-C1222
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. 相似文献
13.
Various mechanicalstimuli increase the intracellular Ca2+ concentration([Ca2+]i) in vascular smooth muscle cells(VSMC). A part of the increase in [Ca2+]i isdue to the release of Ca2+ from intracellular stores. Wehave investigated the effect of mechanical stimulation produced bycyclical stretch on the release of Ca2+ from theintracellular stores. Permeabilized VSMC loaded with 45Ca2+ were subjected to 7.5% average (15%maximal) cyclical stretch. This resulted in an increase in45Ca2+ rate constant by 0.126 ± 0.0035. Inhibition of inositol 1,4,5-trisphosphate (IP3),ryanodine, and nicotinic acid adenine dinucleotide phosphate channels(NAADP) with 50 µg/ml heparin, 50 µM ruthenium red, and 25 µMthio-NADP, respectively, did not block the increase in45Ca2+ efflux in response to cyclical stretch.However, 10 µM lanthanum, 10 µM gadolinium, and 10 µMcytochalasin D but not 10 µM nocodazole inhibited the increase in45Ca2+ efflux. This supports the existence of anovel stretch-sensitive intracellular Ca2+ store in VSMCthat is distinct from the IP3-, ryanodine-, and NAADP-sensitive stores. 相似文献
14.
Certain angina and coronary artery disease forms do not respond to Ca2+ channel blockers, and a role for vasoactive eicosanoids such as PGF2 in Ca2+ antagonist-insensitive coronary vasospasm is suggested; however, the signaling mechanisms are unclear. We investigated whether PGF2-induced coronary smooth muscle contraction is Ca2+ antagonist insensitive and involves activation of a PKC-dependent pathway. We measured contraction in single porcine coronary artery smooth muscle cells and intracellular free Ca2+ concentration ([Ca2+]i) in fura 2-loaded cells and examined cytosolic and particulate fractions for PKC activity and reactivity with isoform-specific PKC antibodies. In Hanks' solution (1 mM Ca2+), PGF2 (10-5 M) caused transient [Ca2+]i increase followed by maintained [Ca2+]i increase and 34% cell contraction. Ca2+ channel blockers verapamil and diltiazem (10-6 M) abolished maintained PGF2-induced [Ca2+]i increase but only partially inhibited PGF2-induced cell contraction to 17%. Verapamil-insensitive PGF2 contraction was inhibited by PKC inhibitors GF-109203X, calphostin C, and -PKC V1-2. PGF2 caused Ca2+-dependent -PKC and Ca2+-independent -PKC translocation from cytosolic to particulate fractions that was inhibited by calphostin C. Verapamil abolished PGF2-induced -but not -PKC translocation. PMA (10-6 M), a direct activator of PKC, caused 21% contraction with no significant [Ca2+]i increase and -PKC translocation that were inhibited by calphostin C but not verapamil. Membrane depolarization by 51 mM KCl, which stimulates Ca2+ influx, caused 36% cell contraction and [Ca2+]i increase that were inhibited by verapamil but not GF-109203X or calphostin C and did not cause - or -PKC translocation. Thus a significant component of PGF2-induced contraction of coronary smooth muscle is Ca2+ antagonist insensitive, involves Ca2+-independent -PKC activation and translocation, and may represent a signaling mechanism of Ca2+ antagonist-resistant coronary vasospasm. eicosanoids; calcium; vascular smooth muscle 相似文献
15.
Ca2+-dependent inhibition of NHE3 requires PKC alpha which binds to E3KARP to decrease surface NHE3 containing plasma membrane complexes 总被引:1,自引:0,他引:1
Lee-Kwon W Kim JH Choi JW Kawano K Cha B Dartt DA Zoukhri D Donowitz M 《American journal of physiology. Cell physiology》2003,285(6):C1527-C1536
The intestinal brush border (BB) Na+/H+ exchanger isoform 3 (NHE3) is acutely inhibited by elevation in the concentration of free intracellular Ca2+ ([Ca2+]i) by the cholinergic agonist carbachol and Ca2+ ionophores in a protein kinase C (PKC)-dependent manner. We previously showed that elevating [Ca2+]i with ionomycin rapidly inhibited NHE3 activity and decreased the amount of NHE3 on the plasma membrane in a manner that depended on the presence of the PDZ domain-containing protein E3KARP (NHE3 kinase A regulatory protein, also called NHERF2). The current studies were performed in PS120 fibroblasts (NHE-null cell line) stably transfected with NHE3 and E3KARP to probe the mechanism of PKC involvement in Ca2+ regulation of NHE3. Pretreatment with the general PKC inhibitor, GF109203X prevented ionomycin inhibition of NHE3 without altering basal NHE3 activity. Similarly, the Ca2+-mediated inhibition of NHE3 activity was blocked after pretreatment with the conventional PKC inhibitor Gö-6976 and a specific PKC pseudosubstrate-derived inhibitor peptide. [Ca2+]i elevation caused translocation of PKC from cytosol to membrane. PKC bound to the PDZ1 domain of GST-E3KARP in vitro in a Ca2+-dependent manner. PKC and E3KARP coimmunoprecipitated from cell lysates; this occurred to a lesser extent at basal [Ca2+]i and was increased with ionomycin exposure. Biotinylation studies demonstrated that [Ca2+]i elevation induced oligomerization of NHE3 in total lysates and decreased the amount of plasma membrane NHE3. Treatment with PKC inhibitors did not affect the oligomerization of NHE3 but did prevent the decrease in surface amount of NHE3. These results suggest that PKC is not necessary for the Ca2+-dependent formation of the NHE3 plasma membrane complex, although it is necessary for decreasing the membrane amounts of NHE3, probably by stimulating NHE3 endocytosis. Na absorption; PDZ domains; signal complex 相似文献
16.
Lamb GD El-Hayek R Ikemoto N Stephenson DG 《American journal of physiology. Cell physiology》2000,279(4):C891-C905
Inskeletal muscle fibers, the intracellular loop between domains II andIII of the 1-subunit of the dihydropyridine receptor (DHPR) may directly activate the adjacent Ca2+ releasechannel in the sarcoplasmic reticulum. We examined the effects ofsynthetic peptide segments of this loop on Ca2+ release inmechanically skinned skeletal muscle fibers with functional excitation-contraction coupling. In rat fibers at physiological Mg2+ concentration ([Mg2+]; 1 mM), a20-residue skeletal muscle DHPR peptide[AS(20);Thr671-Leu690; 30 µM], shown previously toinduce Ca2+ release in a triad preparation, caused onlysmall spontaneous force responses in ~40% of fibers, although itpotentiated responses to depolarization and caffeine in all fibers. TheCOOH-terminal half of AS(20)[AS(10)] induced much larger spontaneousresponses but also caused substantial inhibition of Ca2+release to both depolarization and caffeine. Both peptides induced orpotentiated Ca2+ release even when the voltage sensors wereinactivated, indicating direct action on the Ca2+ releasechannels. The corresponding 20-residue cardiac DHPR peptide [AC(20);Thr793-Ala812] was ineffective, but itsCOOH-terminal half [AC(10)] had effects similar to AS(20). In the presence of lower[Mg2+] (0.2 mM), exposure to eitherAS(20) or AC(10) (30 µM) induced substantial Ca2+ release. PeptideCS (100 µM), a loop segment reported to inhibit Ca2+ release in triads, caused partial inhibition ofdepolarization-induced Ca2+ release. In toad fibers, eachof the A peptides had effects similar to or greater than those in ratfibers. These findings suggest that the A and C regions of the skeletalDHPR II-III loop may have important roles in vivo. 相似文献
17.
L-type voltage-dependent Ca2+ channels in cerebral microvascular endothelial cells and ET-1 biosynthesis 总被引:2,自引:0,他引:2
We investigatedthe role of intracellular calcium concentration([Ca2+]i) in endothelin-1 (ET-1) production,the effects of potential vasospastic agents on[Ca2+]i, and the presence of L-typevoltage-dependent Ca2+ channels in cerebral microvascularendothelial cells. Primary cultures of endothelial cells isolated frompiglet cerebral microvessels were used. Confluent cells were exposed toeither the thromboxane receptor agonist U-46619 (1 µM),5-hydroxytryptamine (5-HT; 0.1 mM), or lysophosphatidic acid (LPA; 1 µM) alone or after pretreatment with the Ca2+-chelatingagent EDTA (100 mM), the L-type Ca2+ channel blockerverapamil (10 µM), or the antagonist of receptor-operated Ca2+ channel SKF-96365 HCl (10 µM) for 15 min. ET-1production increased from 1.2 (control) to 8.2 (U-46619), 4.9 (5-HT),or 3.9 (LPA) fmol/µg protein, respectively. Such elevated ET-1biosynthesis was attenuated by verapamil, EDTA, or SKF-96365 HCl. Toinvestigate the presence of L-type voltage-dependent Ca2+channels in endothelial cells, the [Ca2+]isignal was determined fluorometrically by using fura 2-AM. Superfusionof confluent endothelial cells with U-46619, 5-HT, or LPA significantlyincreased [Ca2+]i. Pretreatment ofendothelial cells with high K+ (60 mM) or nifedipine (4 µM) diminished increases in [Ca2+]i inducedby the vasoactive agents. These results indicate that 1)elevated [Ca2+]i signals are involved in ET-1biosynthesis induced by specific spasmogenic agents, 2) theincreases in [Ca2+]i induced by thevasoactive agents tested involve receptor as well as L-typevoltage-dependent Ca2+ channels, and 3) primarycultures of cerebral microvascular endothelial cells express L-typevoltage-dependent Ca2+ channels. 相似文献
18.
Coupling strength between localized Ca(2+) transients and K(+) channels is regulated by protein kinase C 总被引:1,自引:0,他引:1
Bayguinov O Hagen B Kenyon JL Sanders KM 《American journal of physiology. Cell physiology》2001,281(5):C1512-C1523
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. 相似文献
19.
Huang L Bhattacharjee A Taylor JT Zhang M Keyser BM Marrero L Li M 《American journal of physiology. Cell physiology》2004,286(2):C213-C221
Chronic exposure of pancreatic -cells to high concentrations of glucose impairs the insulin secretory response to further glucose stimulation. This phenomenon is referred to as glucose desensitization. It has been shown that glucose desensitization is associated with abnormal elevation of -cell basal intracellular free Ca2+ concentration ([Ca2+]i). We have investigated the relationship between the basal intracellular free Ca2+ and the L-type (Cav1.3) Ca2+ channel translocation in insulin-secreting cells. Glucose stimulation or membrane depolarization induced a nifedipine-sensitive Ca2+ influx, which was attenuated when the basal [Ca2+]i was elevated. Using voltage-clamp techniques, we found that changing [Ca2+]i could regulate the amplitude of the Ca2+ current. This effect was attenuated by drugs that interfere with the cytoskeleton. Immunofluorescent labeling of Cav1.3 showed an increase in the cytoplasmic distribution of the channels under high [Ca2+]i conditions by deconvolution microscopy. The [Ca2+]i-dependent translocation of Cav1.3 channel was also demonstrated by Western blot analysis of biotinylation/NeutrAvidin-bead-eluted surface proteins in cells preincubated at various [Ca2+]i. These results suggest that Cav1.3 channel trafficking is involved in glucose desensitization of pancreatic -cells. internalization; intracellular free calcium; glucose desensitization 相似文献
20.
Channel phosphorylation and modulation of L-type Ca2+ currents by cytosolic Mg2+ concentration 总被引:1,自引:0,他引:1
Previous studies have shown that inhibition of L-type Ca2+ current (ICa) by cytosolic free Mg2+ concentration ([Mg2+]i) is profoundly affected by activation of cAMP-dependent protein kinase pathways. To investigate the mechanism underlying this counterregulation of ICa, rat cardiac myocytes and tsA201 cells expressing L-type Ca2+ channels were whole cell voltage-clamped with patch pipettes in which [Mg2+] ([Mg2+]p) was buffered by citrate and ATP. In tsA201 cells expressing wild-type Ca2+ channels (1C/2A/2), increasing [Mg2+]p from 0.2 mM to 1.8 mM decreased peak ICa by 76 ± 4.5% (n = 7). Mg2+-dependent modulation of ICa was also observed in cells loaded with ATP--S. With 0.2 mM [Mg2+]p, manipulating phosphorylation conditions by pipette application of protein kinase A (PKA) or phosphatase 2A (PP2A) produced large changes in ICa amplitude; however, with 1.8 mM [Mg2+]p, these same manipulations had no significant effect on ICa. With mutant channels lacking principal PKA phosphorylation sites (1C/S1928A/2A/S478A/S479A/2), increasing [Mg2+]p had only small effects on ICa. However, when channel open probability was increased by 1C-subunit truncation (1C1905/2A/S478A/S479A/2), increasing [Mg2+]p greatly reduced peak ICa. Correspondingly, in myocytes voltage-clamped with pipette PP2A to minimize channel phosphorylation, increasing [Mg2+]p produced a much larger reduction in ICa when channel opening was promoted with BAY K8644. These data suggest that, around its physiological concentration range, cytosolic Mg2+ modulates the extent to which channel phosphorylation regulates ICa. This modulation does not necessarily involve changes in channel phosphorylation per se, but more generally appears to depend on the kinetics of gating induced by channel phosphorylation. voltage-gated Ca2+ channel; cardiac myocytes; human embryonic kidney cells; protein kinase A; protein phosphatase 2A 相似文献