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1.
Crypts of Lieberkühn were isolatedfrom human colon, and differential interference contrast microscopydistinguished goblet and columnar cells. Activation with carbachol(CCh, 100 µM) or histamine (10 µM) released contents from gobletgranules. Stimulation with prostaglandinE2(PGE2, 5 µM) or adenosine (10 µM) did not release goblet granules but caused the apical margin ofcolumnar cells to recede. Goblet volume was lost during stimulationwith CCh or histamine (~160 fl/cell), but not withPGE2 or adenosine. Three-quartersof goblet cells were responsive to CCh but released only 30% of gobletvolume. Half-time for goblet volume release was 3.7 min.PGE2 stimulated a prolonged fluidsecretion that attained a rate of ~350 pl/min. Columnar cells lost~50% of apical volume during maximalPGE2 stimulation, with a half-timeof 3.3 min. In crypts from individuals with ulcerative colitis, goblet cells were hypersensitive to CCh for release of goblet volume. Theseresults support separate regulation for mucus secretions from gobletcells and from columnar cells, with control mechanisms restrictingtotal release of mucus stores. 相似文献
2.
Crypts of Lieberkühn were isolated from human colon, and differential interference contrast microscopy distinguished goblet and columnar cells. Activation with carbachol (CCh, 100 microM) or histamine (10 microM) released contents from goblet granules. Stimulation with prostaglandin E(2) (PGE(2), 5 microM) or adenosine (10 microM) did not release goblet granules but caused the apical margin of columnar cells to recede. Goblet volume was lost during stimulation with CCh or histamine ( approximately 160 fl/cell), but not with PGE(2) or adenosine. Three-quarters of goblet cells were responsive to CCh but released only 30% of goblet volume. Half-time for goblet volume release was 3.7 min. PGE(2) stimulated a prolonged fluid secretion that attained a rate of approximately 350 pl/min. Columnar cells lost approximately 50% of apical volume during maximal PGE(2) stimulation, with a half-time of 3.3 min. In crypts from individuals with ulcerative colitis, goblet cells were hypersensitive to CCh for release of goblet volume. These results support separate regulation for mucus secretions from goblet cells and from columnar cells, with control mechanisms restricting total release of mucus stores. 相似文献
3.
p38 mitogen-activated protein kinase inhibits calcium-dependent chloride secretion in T84 colonic epithelial cells 总被引:1,自引:0,他引:1
We havepreviously shown that Ca2+-dependent Clsecretion across intestinal epithelial cells is limited by a signalingpathway involving transactivation of the epidermal growth factorreceptor (EGFR) and activation of ERK mitogen-activated protein kinase (MAPK). Here, we have investigated a possible role for p38 MAPK inregulation of Ca2+-dependent Cl secretion.Western blot analysis of T84 colonic epithelial cells revealed that the muscarinic agonist carbachol (CCh; 100 µM)stimulated phosphorylation and activation of p38 MAPK. The p38inhibitor SB-203580 (10 µM) potentiated and prolonged short-circuitcurrent (Isc) responses to CCh acrossvoltage-clamped T84 cells to 157.4 ± 6.9% of thosein control cells (n = 21; P < 0.001).CCh-induced p38 phosphorylation was attenuated by the EGFR inhibitortyrphostin AG-1478 (0.1 nM-10 µM) and by the Src family kinaseinhibitor PP2 (20 nM-2 µM). The effects of CCh on p38phosphorylation were mimicked by thapsigargin (TG; 2 µM), whichspecifically elevates intracellular Ca2+, and wereabolished by the Ca2+ chelator BAPTA-AM (20 µM), implyinga role for intracellular Ca2+ in mediating p38 activation.SB-203580 (10 µM) potentiated Isc responses toTG to 172.4 ± 18.1% of those in control cells (n = 18; P < 0.001). When cells were pretreated withSB-203580 and PD-98059 to simultaneously inhibit p38 and ERK MAPKs,respectively, Isc responses to TG and CCh weresignificantly greater than those observed with either inhibitor alone.We conclude that Ca2+-dependent agonists stimulate p38 MAPKin T84 cells by a mechanism involving intracellularCa2+, Src family kinases, and the EGFR. CCh-stimulated p38activation constitutes a similar, but distinct and complementary,antisecretory signaling pathway to that of ERK MAPK. 相似文献
4.
Liu HJ Wei Y Ferreri NR Nasjletti A Wang WH Fererri NR 《American journal of physiology. Cell physiology》2000,278(5):C905-C913
Vasopressin and prostaglandinE2 (PGE2) are involved in regulating NaClreabsorption in the thick ascending limb (TAL) of the rat kidney. Inthe present study, we used the patch-clamp technique to study theeffects of vasopressin and PGE2 on the apical 70 pSK+ channel in the rat TAL. Addition of vasopressinincreased the channel activity, defined asNPo, from 1.11 to 1.52 (200 pM) and 1.80 (500 pM),respectively. The effect of vasopressin can be mimicked by eitherforskolin (1-5 µM) or 8-bromo-cAMP/dibutyryl-cAMP (8-Br-cAMP/DBcAMP) (200-500 µM). Moreover, the effects of cAMP and vasopressin were not additive and application of 10 µM H-89 abolished the effect of vasopressin. This suggests that the effect ofvasopressin is mediated by a cAMP-dependent pathway. Applying 10 nMPGE2 alone had no significant effect on the channelactivity. However, PGE2 (10 nM) abolished thestimulatory effect of vasopressin. The PGE2-inducedinhibition of the vasopressin effect was the result of decreasing cAMPproduction because addition of 200 µM 8-Br-cAMP/DBcAMPreversed the PGE2-induced inhibition. In addition toantagonizing the vasopressin effect, high concentrations of PGE2 reduced channel activity in the absence of vasopressinby 33% (500 nM) and 51% (1 µM), respectively. The inhibitory effect of high concentrations of PGE2 was not the result ofdecreasing cAMP production because adding the membrane-permeant cAMPanalog failed to restore the channel activity. In contrast, inhibiting protein kinase C (PKC) with calphostin C (100 nM) abolished the effectof 1 µM PGE2. We conclude that PGE2 inhibitsapical K+ channels by two mechanisms: 1) lowconcentrations of PGE2 attenuate the vasopressin-inducedstimulation mainly by reducing cAMP generation, and 2) highconcentrations of PGE2 inhibit the channel activity by aPKC-dependent pathway. 相似文献
5.
Ca2+ and protein kinase C activation of mucin granule exocytosis in permeabilized SPOC1 cells 总被引:2,自引:0,他引:2
Scott C. E.; Abdullah Lubna H.; Davis C. William 《American journal of physiology. Cell physiology》1998,275(1):C285
Mucin secretion by airway goblet cells is under the control ofapical P2Y2, phospholipaseC-coupled purinergic receptors. In SPOC1 cells, the mobilization ofintracellular Ca2+ by ionomycin orthe activation of protein kinase C (PKC) by phorbol 12-myristate13-acetate (PMA) stimulates mucin secretion in a fully additive fashion[L. H. Abdullah, J. D. Conway, J. A. Cohn, and C. W. Davis.Am. J. Physiol. 273 (Lung Cell. Mol. Physiol. 17):L201-L210, 1997]. This apparent independence between PKC andCa2+ in the stimulation of mucinsecretion was tested in streptolysin O-permeabilized SPOC1 cells. Thesecells were fully competent to secrete mucin whenCa2+ was elevated from 100 nM to3.1 µM for 2 min following permeabilization; theCa2+EC50 was 2.29 ± 0.07 µM.Permeabilized SPOC1 cells were exposed to PMA or 4-phorbol atCa2+ activities ranging from 10 nMto 10 µM. PMA, but not 4-phorbol, increased mucin release at allCa2+ activities tested: at 10 nMCa2+ mucin release was 2.1-foldgreater than control and at 4.7 µM Ca2+ mucin release was maximal(3.6-fold increase). PMA stimulated 27% more mucin release at 4.7 µMthan at 10 nM Ca2+. Hence, SPOC1cells possess Ca2+-insensitive,PKC-dependent, and Ca2+-dependentPKC-potentiated pathways for mucin granule exocytosis. 相似文献
6.
Distinct K+ conductive pathways are required for Cl- and K+ secretion across distal colonic epithelium 总被引:1,自引:0,他引:1
Secretion of Cl and K+ in the colonic epithelium operates through a cellular mechanism requiring K+ channels in the basolateral and apical membranes. Transepithelial current [short-circuit current (Isc)] and conductance (Gt) were measured for isolated distal colonic mucosa during secretory activation by epinephrine (Epi) or PGE2 and synergistically by PGE2 and carbachol (PGE2 + CCh). TRAM-34 at 0.5 µM, an inhibitor of KCa3.1 (IK, Kcnn4) K+ channels (H. Wulff, M. J. Miller, W. Hänsel, S. Grissmer, M. D. Cahalan, and K. G. Chandy. Proc Natl Acad Sci USA 97: 81518156, 2000), did not alter secretory Isc or Gt in guinea pig or rat colon. The presence of KCa3.1 in the mucosa was confirmed by immunoblot and immunofluorescence detection. At 100 µM, TRAM-34 inhibited Isc and Gt activated by Epi (4%), PGE2 (30%) and PGE2 + CCh (60%). The IC50 of 4.0 µM implicated involvement of K+ channels other than KCa3.1. The secretory responses augmented by the K+ channel opener 1-EBIO were inhibited only at a high concentration of TRAM-34, suggesting further that KCa3.1 was not involved. Sensitivity of the synergistic response (PGE2 + CCh) to a high concentration TRAM-34 supported a requirement for multiple K+ conductive pathways in secretion. Clofilium (100 µM), a quaternary ammonium, inhibited Cl secretory Isc and Gt activated by PGE2 (20%) but not K+ secretion activated by Epi. Thus Cl secretion activated by physiological secretagogues occurred without apparent activity of KCa3.1 channels but was dependent on other types of K+ channels sensitive to high concentrations of TRAM-34 and/or clofilium. epinephrine; prostaglandin E2; cholinergic; Kcnn4; TRAM-34; clofilium 相似文献
7.
Protease-activated receptor regulation of Cl- secretion in Calu-3 cells requires prostaglandin release and CFTR activation 总被引:1,自引:0,他引:1
Palmer ML Lee SY Maniak PJ Carlson D Fahrenkrug SC O'Grady SM 《American journal of physiology. Cell physiology》2006,290(4):C1189-C1198
Human lung epithelial (Calu-3) cells were used to investigate the effects of protease-activated receptor (PAR) stimulation on Cl secretion. Quantitative RT-PCR (QRT-PCR) showed that Calu-3 cells express PAR-1, -2, and -3 receptor mRNAs, with PAR-2 mRNA in greatest abundance. Addition of either thrombin or the PAR-2 agonist peptide SLIGRL to the basolateral solution of monolayers mounted in Ussing chambers produced a rapid increase in short-circuit current (Isc: thrombin, 21 ± 2 µA; SLIGRL, 83 ± 22 µA), which returned to baseline within 5 min after stimulation. Pretreatment of monolayers with the cell-permeant Ca2+-chelating agent BAPTA-AM (50 µM) abolished the increase in Isc produced by SLIGRL. When monolayers were treated with the cyclooxygenase inhibitor indomethacin (10 µM), nearly complete inhibition of both the thrombin- and SLIGRL-stimulated Isc was observed. In addition, basolateral treatment with the PGE2 receptor antagonist AH-6809 (25 µM) significantly inhibited the effects of SLIGRL on Isc. QRT-PCR revealed that Calu-3 cells express mRNAs for CFTR, the Ca2+-activated KCNN4 K+ channel, and the KCNQ1 K+ channel subunit, which, in association with KCNE3, is known to be regulated by cAMP. Stimulation with SLIGRL produced an increase in apical Cl conductance that was blocked in cells expressing short hairpin RNAs designed to target CFTR. These results support the conclusion that PAR stimulation of Cl secretion occurs by an indirect mechanism involving the synthesis and release of prostaglandins. In addition, PAR-stimulated Cl secretion requires activation of CFTR and at least two distinct K+ channels located in the basolateral membrane. cystic fibrosis transmembrane conductance regulator; KCNQ1; calcium-activated potassium channels; KCNN4; cAMP 相似文献
8.
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
9.
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
10.
The functional coupling of the ciliaryepithelium was studied in isolated pairs (couplets) of pigmentedciliary epithelial (PCE) and nonpigmented ciliary epithelial (NPCE)cells using the whole cell patch clamp and the fluorescent dye luciferyellow. One cell of the pair (usually the NPCE cell of a NPCE-PCE cell couplet) was accessed with a 2-5 M electrode, containing1-2 mM lucifer yellow, in the whole cell configuration of thepatch clamp. After voltage-clamp experiments were completed, cells were viewed under a fluorescent microscope to confirm that the cells werecoupled. The electrical coupling of the cells was also studied bycalculating the capacitance (using the time-domain technique), assuminga "supercell" model for coupled cells. The mean capacitance ofcoupled pairs was 79.8 ± 4.3 (SE) pF(n = 47) compared with single cellcapacitances of 36.8 ± 3.4 pF (n = 10) for PCE cells and 38.1 ± 3.1 pF(n = 15) for NPCE cells. Octanol,carbachol (CCh), and raised extracellularCa2+ concentration([Ca2+]o)all caused uncoupling in pairs (couplets) of coupled NPCE and PCEcells. At room temperature (22-24°C), the capacitance of thecouplets decreased from 70.5 ± 8.0 to 48.0 ± 5.2 pF(n = 5) when exposed to octanol (1 mM), from 73.8 ± 9.2 to 43.2 ± 9.5 pF(n = 4) when exposed to CCh (100 µM), and from 80.5 ± 6.7 to 49.9 ± 7.8 pF(n = 4) when exposed to 10 mM[Ca2+]o.The response to CCh was dose dependent; at higher temperatures of34-37°C, 10 µM CCh caused a 38% reduction in capacitance,from 53.7 ± 9.7 to 33.5 ± 3.3 pF(n = 7) with a half-time of 249 s, and100 µM CCh caused a 49% reduction in capacitance, from 51.3 ± 5.6 to 26.0 ± 2.4 pF (n = 7) witha half-time of 124 s. After pairs uncoupled and the uncoupling agentwas washed out, the cell pairs often exhibited an increase incapacitance that we interpreted as "recoupling" or a reopening ofthe gap junctional communication pathway; the half-time for thisprocess was 729 s after uncoupling with 100 µM CCh and 211 s afteruncoupling with 10 µM CCh. This interpretation was confirmedoptically by the spread of lucifer yellow into both cells of anuncoupled pair with a time course corresponding to the increase inelectrical coupling. The controllable coupling of ciliary epithelialcells extends the idea of a functional syncytium involved in activetransport. PCE cells take up solute and water from the blood, whichthen cross to NPCE cells via gap junctions and from there are secretedinto the posterior chamber of the eye. Modulation of the couplingbetween NPCE and PCE cells may provide a mechanism to controlsecretion. 相似文献
11.
Tanimura Akihiko; Nezu Akihiro; Tojyo Yosuke; Matsumoto Yoshito 《American journal of physiology. Cell physiology》1999,276(6):C1282
The effects of the cAMP pathway on theCa2+ response elicited byphospholipase C-coupled receptor stimulations were studied in ratparotid cells. Although 1 µM isoproterenol (Iso) itself had no effect on the cytosolicCa2+ concentration, thepretreatment with Iso potentiatedCa2+ responses evoked byphenylephrine. The potentiating effect of Iso was attributed to ashifting of the concentration-response curves of phenylephrine to theleft and an increase in the maximal response. Half-maximal potentiationoccurred at 3 nM Iso. Iso also potentiated theCa2+ response elicited bycarbachol. The potentiating effect of Iso was mimicked by forskolin (10 µM) and dibutyryl adenosine 3',5'-cyclic monophosphate (2 mM) and was blocked by 10 µM H-89. Iso potentiated thephenylephrine-induced Ca2+response in the absence of extracellularCa2+, but Iso did not increase theinositol trisphosphate (IP3)production induced by phenylephrine. These results suggest that thepotentiation of the Ca2+ responsecan be attributed to a sensitization ofIP3 receptors by cAMP-dependentprotein kinase. 相似文献
12.
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
13.
P2 purinoceptors regulate calcium-activated chloride and fluid transport in 31EG4 mammary epithelia 总被引:2,自引:0,他引:2
Blaug S Rymer J Jalickee S Miller SS 《American journal of physiology. Cell physiology》2003,284(4):C897-C909
It has been reported thatsecretory mammary epithelial cells (MEC) release ATP, UTP, and UDP uponmechanical stimulation. Here we examined the physiological changescaused by ATP/UTP in nontransformed, clonal mouse mammary epithelia(31EG4 cells). In control conditions, transepithelial potential (apicalside negative) and resistance were 4.4 ± 1.3 mV (mean ± SD, n = 12) and 517.7 ± 39.4 · cm2, respectively. The apicalmembrane potential was 43.9 ± 1.7 mV, and the ratio of apicalto basolateral membrane resistance (RA/RB) was 3.5 ± 0.2. Addition of ATP or UTP to the apical or basolateral membranescaused large voltage and resistance changes with an EC50 of~24 µM (apical) and ~30 µM (basal). Apical ATP/UTP (100 µM)depolarized apical membrane potential by 17.6 ± 0.8 mV (n = 7) and decreasedRA/RB by a factor of3. The addition of adenosine to either side (100 µM) hadno effect on any of these parameters. The ATP/UTP responses werepartially inhibited by DIDS and suramin and mediated by a transientincrease in free intracellular Ca2+ concentration (427 ± 206 nM; 15-25 µM ATP, apical; n = 6). This Ca2+ increase was blocked by cyclopiazonic acid, by BAPTA,or by xestospongin C. 31EG4 MEC monolayers also secreted or absorbedfluid in the resting state, and ATP or UTP increased fluid secretion by5.6 ± 3 µl · cm2 · h1(n = 10). Pharmacology experiments indicate that 31EG4epithelia contain P2Y2 purinoceptors on the apical andbasolateral membranes, which upon activation stimulate apicalCa2+-dependent Cl channels and cause fluid secretion acrossthe monolayer. This suggests that extracellular nucleotides could playa fundamental role in mammary gland paracrine signaling and theregulation of milk composition in vivo. 相似文献
14.
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
15.
Murthy Karnam S.; Makhlouf Gabriel M. 《American journal of physiology. Cell physiology》1998,274(5):C1199
Recent studies on the role of nitric oxide (NO) ingastrointestinal smooth muscle have raised the possibility thatNO-stimulated cGMP could, in the absence of cGMP-dependent proteinkinase (PKG) activity, act as aCa2+-mobilizing messenger[K. S. Murthy, K.-M. Zhang, J.-G. Jin, J. T. Grider, and G. M. Makhlouf. Am. J. Physiol. 265 (Gastrointest. Liver Physiol. 28):G660-G671, 1993]. This notion was examined indispersed gastric smooth muscle cells with 8-bromo-cGMP (8-BrcGMP) andwith NO and vasoactive intestinal peptide (VIP), which stimulate endogenous cGMP. In muscle cells treated with cAMP-dependent protein kinase (PKA) and PKG inhibitors (H-89 and KT-5823), 8-BrcGMP (10 µM),NO (1 µM), and VIP (1 µM) stimulated45Ca2+release (21 ± 3 to 30 ± 1% decrease in45Ca2+cell content); Ca2+ releasestimulated by 8-BrcGMP was concentration dependent with anEC50 of 0.4 ± 0.1 µM and athreshold of 10 nM. 8-BrcGMP and NO increased cytosolic freeCa2+ concentration([Ca2+]i)and induced contraction; both responses were abolished after Ca2+ stores were depleted withthapsigargin. With VIP, which normally increases[Ca2+]iby stimulating Ca2+ influx,treatment with PKA and PKG inhibitors caused a further increase in[Ca2+]ithat reverted to control levels in cells pretreated with thapsigargin. Neither Ca2+ release norcontraction induced by cGMP and NO in permeabilized muscle cells wasaffected by heparin or ruthenium red.Ca2+ release induced by maximallyeffective concentrations of cGMP and inositol 1,4,5-trisphosphate(IP3) was additive, independent of which agent was applied first. We conclude that, in the absence ofPKA and PKG activity, cGMP stimulatesCa2+ release from anIP3-insensitive store and that itseffect is additive to that of IP3. 相似文献
16.
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
17.
Ovine male genital duct epithelial cells differentiate in vitro and express functional CFTR and ENaC
Bertog M Smith DJ Bielfeld-Ackermann A Bassett J Ferguson DJ Korbmacher C Harris A 《American journal of physiology. Cell physiology》2000,278(5):C885-C894
To investigate the biology of the malegenital duct epithelium, we have established cell cultures from theovine vas deferens and epididymis epithelium. These cells develop tightjunctions, high transepithelial electrical resistance, and alumen-negative transepithelial potential difference as a sign of activetransepithelial ion transport. In epididymis cultures the equivalentshort-circuit current (Isc) averaged 20.8 ± 0.7 µA/cm2 (n = 150) and was partially inhibited byapical application of amiloride with an inhibitor concentration of 0.64 µM. In vas deferens cultures, Isc averaged 14.4 ± 1.1 µA/cm2 (n = 18) and was also inhibited byapical application of amiloride with a half-maximal inhibitorconcentration (Ki) of 0.68 µM. The remainingamiloride-insensitive Isc component in epididymisand vas deferens cells was partially inhibited by apical application ofthe Cl channel blocker diphenylamine-2-carboxylicacid (1 mM). It was largely dependent on extracellularCl and, to a lesser extent, on extracellularHCO3. It was further stimulated bybasolateral application of forskolin (105 M), which increasedIsc by 3.1 ± 0.3 µA/cm2 (n=65) in epididymis and 0.9 ± 0.1 µA/cm2 (n =11) in vas deferens. These findings suggest that cultured ovine vasdeferens and epididymis cells absorb Na+ viaamiloride-sensitive epithelial Na+ channels (ENaC) andsecrete Cl and HCO3via apical cystic fibrosis transmembrane conductance regulator (CFTR)Cl channels. This interpretation is supported byRT-PCR data showing that vas deferens and epididymis cells express CFTRand ENaC mRNA. 相似文献
18.
Release of dopamine and norepinephrine by hypoxia from PC-12 cells 总被引:10,自引:0,他引:10
Kumar Ganesh K.; Overholt Jeffrey L.; Bright Gary R.; Hui Kwong Y.; Lu Hongwen; Gratzl Miklos; Prabhakar Nanduri R. 《American journal of physiology. Cell physiology》1998,274(6):C1592
We examined the effects of hypoxia on the release of dopamine(DA) and norepinephrine (NE) from rat pheochromocytoma 12 (PC-12) cellsand assessed the involvement ofCa2+ and protein kinases instimulus-secretion coupling. Catecholamine release was monitored bymicrovoltammetry using a carbon fiber electrode as well as by HPLCcoupled with electrochemical detection (ECD). Microvoltammetricanalysis showed that hypoxia-induced catecholamine secretion(PO2 ofmedium ~40 mmHg) occurred within 1 min after the onset of thestimulus and reached a plateau between 10 and 15 min. HPLC-ECD analysisrevealed that, at any level of PO2, therelease of NE was greater than the release of DA. In contrast, inresponse to K+ (80 mM), DA releasewas ~11-fold greater than NE release. The magnitude ofhypoxia-induced NE and DA releases depended on the passage, source, andculture conditions of the PC-12 cells. Omission of extracellularCa2+ or addition of voltage-gatedCa2+ channel blockers attenuatedhypoxia-induced release of both DA and NE to a similarextent. Protein kinase inhibitors, staurosporine (200 nM) andbisindolylmaleimide I (2 µM), on the other hand, attenuatedhypoxia-induced NE release more than DA release. However, proteinkinase inhibitors had no significant effect onK+-induced NE and DA releases.These results demonstrate that hypoxia releases catecholamines fromPC-12 cells and that, for a given change inPO2, NErelease is greater than DA release. It is suggested that proteinkinases are involved in the enhanced release of NE during hypoxia. 相似文献
19.
Mohanty Madhumita Jena; Ye Maian; Li Xingli; Rossi Noreen F. 《American journal of physiology. Cell physiology》2001,281(2):C555
Hypotonicswelling increases the intracellular Ca2+ concentration([Ca2+]i) in vascular smooth muscle cells(VSMC). The source of this Ca2+ is not clear. To study thesource of increase in [Ca2+]i in response tohypotonic swelling, we measured [Ca2+]i infura 2-loaded cultured VSMC (A7r5 cells). Hypotonic swelling produced a40.7-nM increase in [Ca2+]i that was notinhibited by EGTA but was inhibited by 1 µM thapsigargin. Priordepletion of inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores with vasopressin did not inhibit the increasein [Ca2+]i in response to hypotonic swelling.Exposure of 45Ca2+-loaded intracellular storesto hypotonic swelling in permeabilized VSMC produced an increase in45Ca2+ efflux, which was inhibited by 1 µMthapsigargin but not by 50 µg/ml heparin, 50 µM ruthenium red, or25 µM thio-NADP. Thus hypotonic swelling of VSMC causes a release ofCa2+ from the intracellular stores from a novel sitedistinct from the IP3-, ryanodine-, and nicotinic acidadenine dinucleotide phosphate-sensitive stores. 相似文献
20.
Noll T Schäfer M Schavier-Schmitz U Piper HM 《American journal of physiology. Cell physiology》2000,279(3):C717-C723
In cultured porcine aortic endothelial monolayers, theeffect of ATP on myosin light chain (MLC) phosphorylation, whichcontrols the endothelial contractile machinery, was studied. ATP (10 µM) reduced MLC phosphorylation but increased cytosolicCa2+ concentration ([Ca2+]i).Inhibition of the ATP-evoked [Ca2+]i rise byxestospongin C (10 µM), an inhibitor of the inositol trisphosphate-dependent Ca2+ release from endoplasmicreticulum, did not affect the ATP-induced dephosphorylation of MLC. MLCdephosphorylation was prevented in the presence of calyculin A (10 nM),an inhibitor of protein phosphatases PP-1 and PP-2A. Thus ATP activatesMLC dephosphorylation in a Ca2+-independent manner. In thepresence of calyculin A, MLC phosphorylation was incremented afteraddition of ATP, an effect that could be abolished when cellswere loaded with the Ca2+ chelator1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acidacetoxymethyl ester (10 µM). Thus ATP also activates aCa2+-dependent kinase acting on MLC. In summary, ATPsimultaneously stimulates a functional antagonism toward bothphosphorylation and dephosphorylation of MLC in which thedephosphorylation prevails. In endothelial cells, ATP is the firstphysiological mediator identified to activate MLC dephosphorylation bya Ca2+-independent mechanism. 相似文献