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1.
The human bronchial cell line16HBE14o– was used as a model of airway epithelial cells to study the Ca2+-dependent Cl secretion and the identity of KCa channels involved in the generation of a favorable driving force for Cl exit. After ionomycin application, a calcium-activated short-circuit current (I sc) developed, presenting a transient peak followed by a plateau phase. Both phases were inhibited to different degrees by NFA, glybenclamide and NPPB but DIDS was only effective on the peak phase. 86Rb effluxes through both apical and basolateral membranes were stimulated by calcium, blocked by charybdotoxin, clotrimazole and TPA. 1-EBIO, a SK-channel opener, stimulated 86Rb effluxes. Block of basolateral KCa channels resulted in I sc inhibition but, while reduced, I sc was still observed if mucosal Cl was lowered. Among SK family members, only SK4 and SK1 mRNAs were detected by RT-PCR. KCNQ1 mRNAs were also identified, but involvement of KcAMP channels in Cl secretion was unlikely, since cAMP application had no effect on 86Rb effluxes. Moreover, chromanol 293B or clofilium, specific inhibitors of KCNQ1 channels, had no effect on cAMP-dependent I sc. In conclusion, two distinct components of Cl secretion were identified by a pharmacological approach after a Ca i 2+ rise. KCa channels presenting the pharmacology of SK4 channels are present on both apical and basolateral membranes, but it is the basolateral SK4-like channels that play a major role in calcium-dependent chloride secretion in 16HBE14o– cells.  相似文献   

2.
The effect of adenosine regulation on sodium and chloride transport was examined in cultured A6 renal epithelial cells. Adenosine and its analogue N6-cyclopentyladenosine (CPA) had different effects on short-circuit current (I sc) depending on the side of addition. Basolateral CPA addition induced an approximately threefold increase of the I sc that reached a maximum effect 20 min after addition and was completely inhibited by preincubation with either an A2 selective antagonist, CSC, or the sodium channel blocker, amiloride. Apical CPA addition induced a biphasic I sc response characterized by a rapid fourfold transient increase over its baseline followed by a decline and a plateau phase that were amiloride insensitive. The A1 adenosine antagonist, CPX, completely prevented this response. This I sc response to apical CPA was also strongly reduced in Cl-free media and was significantly inhibited either by basolateral bumetanide or apical DPC preincubation. Only basolateral CPA addition was able to induce an increase in cAMP level. CPA, added to cells in suspension, caused a rapid rise in [Ca2+] i that was antagonized by CPX, not affected by CSC and prevented by thapsigargin preincubation. These data suggest that basolateral CPA regulates active sodium transport via A2 adenosine receptors stimulating adenylate cyclase while apical CPA regulates Cl secretion via A1 receptor-mediated changes in [Ca2+] i .  相似文献   

3.
In the epithelium of rat distal colon the acetylcholine analogue carbachol induces a transient increase of short-circuit current (Isc) via stimulation of cellular K+ conductances. Inhibition of the turnover of inositol-1,4,5-trisphosphate (IP3) by LiCl significantly reduced both the amplitude and the duration of this response. When the apical membrane was permeabilized with nystatin, LiCl nearly abolished the carbachol-induced activation of basolateral K+ conductances. In contrast, in epithelia, in which the basolateral membrane was bypassed by a basolateral depolarization, carbachol induced a biphasic increase in the K+ current across the apical membrane consisting of an early component carried by charybdotoxin- and tetraethylammonium-sensitive K+ channels followed by a sustained plateau carried by channels insensitive against these blockers. Only the latter was sensitive against LiCl or inhibition of protein kinases. In contrast, the stimulation of the early apical K+ conductance by carbachol proved to be resistant against inhibition of phospholipase C or protein kinases. However, apical dichlorobenzamil, an inhibitor of Na+/Ca2+ exchangers, or a Ca2+-free mucosal buffer solution significantly reduced the early component of the carbachol-induced apical K+ current. The presence of an apically localized Na+/Ca2+-exchanger was proven immunohistochemically. Taken together these experiments reveal divergent regulatory mechanisms for the stimulation of apical Ca2+-dependent K+ channels in this secretory epithelium, part of them being activated by an inflow of Ca2+ across the apical membrane.
G. SchultheissEmail:
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4.
1. Hen tracheal epithelium can be stimulated by serosal application of acetylcholine (ACh) to secrete Cl equal to ~ 60–90 μA/cm2.2. Radio-ligand-displacement for IP3, cAMP and cGMP and ion channel selective drugs in voltage clamp setups were employed to characterize second messengers and Cl, K+ and Ca2+ channels involved in the ACh response.3. ACh induced a significant rise in IP, in isolated tracheocytes, while ACh did not influence the production of cAMP in whole tissue, isolated tracheocytes or basolateral cell membrane vesicles. Further ACh desensitization did not effect cAMP level in tracheocytes. In addition neither ACh stimulation nor desensitization interfered with cAMP production in presence of 4.5 μM forskolin in tracheocytes, a level of forskolin rising base level cAMP by around five fold.4. Around 35% of ACh Cl secretion depends on Ca2+ mobilization from internal stores and about 65% on Ca2+ influx over basolateral membrane. The activated Ca2+ channel is insensitive to class I, II, III and IV Ca2+ antagonists.5. A 23187 can mimic the ACh effect although 30% is indomethacin-sensitive demonstrating a prostaglandin activated adenylyl cyclase.6. Two K+ channels are involved in ACh secretion, one sensitive to Ba2+ and quinine and both insensitive to 4-aminopyridine, apamin, charybdotoxin and TEA.7. Flufenamate and triaminopyrimidine block a non-selective ion channel likely involved in the ACh response. An ACh activated apical Cl channel is NPPB-sensitive.  相似文献   

5.
Summary The effects of complete substitution of gluconate for mucosal and/or serosal medium Cl on transepithelial Na+ transport have been studied using toad urinary bladder. With mucosal gluconate, transepithelial potential difference (V T) decreased rapidly, transepithelial resistance (R T) increased, and calculated short-circuit current (I sc) decreased. CalculatedE Na was unaffected, indicating that the inhibition of Na+ transport was a consequence of a decreased apical membrane Na+ conductance. This conclusion was supported by the finding that a higher amiloride concentration was required to inhibit the residual transport. With serosal gluconateV T decreased,R T increased andI sc fell to a new steady-state value following an initial and variable transient increase in transport. Epithelial cells were shrunken markedly as judged histologically. CalculatedE Na fell substantially (from 130 to 68 mV on average). Ba2+ (3mm) reduced calculatedE Na in Cl Ringer's but not in gluconate Ringer's. With replacement of serosal Cl by acetate, transepithelial transport was stimulated, the decrease in cellular volume was prevented andE Na did not fall. Replacement of serosal isosmotic Cl medium by a hypo-osmotic gluconate medium (one-half normal) also prevented cell shrinkage and did not result in inhibition of Na+ transport. Thus the inhibition of Na+ transport can be correlated with changes in cell volume rather than with the change in Cl per se. Nystatin virtually abolished the resistance of the apical plasma membrane as judged by measurement of tissue capacitance. With K+ gluconate mucosa, Na+ gluconate serosa, calculated basolateral membrane resistance was much greater, estimated basolateral emf was much lower, and the Na+/K+ basolateral permeability ratio was much higher than with acetate media. It is concluded the decrease in cellular volume associated with substitution of serosal gluconate for Cl results in a loss of highly specific Ba2+-sensitive K+ conductance channels from the basolateral plasma membrane. It is possible that the number of Na+ pump sites in this membrane is also decreased.  相似文献   

6.
Experiments were conducted to determine whether the Cl secretagogue, 1-ethyl-2-benzimidazolinone (EBIO), stimulates Cl transport in the rabbit conjunctival epithelium. For this study, epithelia were isolated in an Ussing-type chamber under short-circuit conditions. The effects of EBIO on the short-circuit current (Isc) and transepithelial resistance (Rt) were measured under physiological conditions, as well as in experiments with altered electrolyte concentrations. Addition of 0.5 mM EBIO to the apical bath stimulated the control Isc by 64% and reduced Rt by 21% (P < 0.05; paired data). Under Cl-free conditions, Isc stimulation using EBIO was markedly attenuated. In the presence of an apical-to-basolateral K+ gradient and permeabilization of the apical membrane, the majority of the Isc reflected the transcellular movement of K+ via basolateral K+ channels. Under these conditions, EBIO in combination with A23187 elicited nearly instantaneous 60–90% increases in Isc that were sensitive to the calmodulin antagonist calmidazolium and the K+ channel blocker tetraethyl ammonium. In the presence of an apical-to-basolateral Cl gradient and nystatin permeabilization of the basolateral aspect, EBIO increased the Cl-dependent Isc, an effect prevented by the channel blocker glibenclamide (0.3 mM). The latter compound also was used to determine the proportion of EBIO-evoked unidirectional 36Cl fluxes in the presence of the Cl gradient that traversed the epithelium transcellularly. Overall, EBIO activated apical Cl channels and basolateral K+ channels (presumably those that are Ca2+ dependent), thereby suggesting that this compound, or related derivatives, may be suitable as topical agents to stimulate fluid transport across the tissue in individuals with lacrimal gland deficiencies. Ussing chamber; short-circuit current; electrolyte transport; chloride secretagogue; potassium conductance; 1-ethyl-2-benzimidazolinone; 1,10-phenanthroline  相似文献   

7.
Dicyclohexylcarbodiimide (DCCD) is a carboxyl group modifier and it is an inhibitor of various ATPases. Present experiments, using an in vitro preparation, were designed to study whether DCCD affected the transporters of the bullfrog cornea epithelium, specifically, the Na+/K+ ATPase pump located in the basolateral membrane. For this purpose, corneas were impaled with microelectrodes and experiments were done under short-circuit current (I sc ) conditions. Addition of DCCD to a concentration of 10−4 m to the tear solution gave a marked decrease in I sc ; a marked depolarization of the intracellular potential, V o ; and a significant decrease in the apical membrane fractional resistance, fR o . There were small and variable although significant changes in the transepithelial conductance, g t . The effects may be explained by a decrease in the basolateral membrane K+ conductance, in combination with a partial inhibition of the Na+/K+-ATPase pump located in the basolateral membrane. There is also evidence for an increase in the apical membrane Cl conductance. Received: 12 August 1999/Revised: 16 November 1999  相似文献   

8.
Alzamora R  O'Mahony F  Harvey BJ 《Steroids》2011,76(9):867-876
Excessive Cl secretion is the driving force for secretory diarrhea. 17β-Estradiol has been shown to inhibit Cl secretion in rat distal colon through a nongenomic pathway. We examined whether 17β-estradiol inhibits Cl secretion in an animal model of secretory diarrhea and the downstream effectors involved. The effect of 17β-estradiol on cholera toxin and heat-stable enterotoxin induced Cl secretion in rat colonic mucosal sheets was studied by current-voltage clamping. Selective permeabilization of apical or basolateral membranes with amphotericin B or nystatin was used to isolate basolateral K+ channel and apical Cl channel activity, respectively. 17β-Estradiol dose-dependently inhibited secretory responses to both toxins with IC50 values of approximately 1 nM. This effect was female-gender specific, with no inhibition observed in male tissues. 17β-Estradiol responses were insensitive to the pure anti-estrogen ICI 182,720. 17β-Estradiol exerted its effects downstream of enterotoxin-induced production of second messengers (cAMP and cGMP) but was dependent on PKCδ activation. In nystatin-permeabilized tissues, apical Cl currents were unaffected by 17β-estradiol treatment while basolateral K+ current was profoundly inhibited by the hormone. This current was sensitive to the specific KCNQ1 channel inhibitors chromanol 293B and HMR-1556. In conclusion, 17β-estradiol inhibits enterotoxin-induced Cl secretion via a PKCδ-dependent mechanism involving inhibition of basolateral KCNQ1 channels. These data elucidate mechanisms of 17β-estradiol inhibition of Cl secretion induced by enterotoxins in intestinal epithelia, which may be relevant for the treatment of diarrheal diseases.  相似文献   

9.
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   

10.
Background information. TSPO (translocator protein), known previously as PBR (peripheral‐type benzodiazepine receptor), is a 18 kDa protein expressed in the mitochondrial membrane of a variety of tissues. TSPO has been reported to be over‐expressed in human colorectal tumours and cancer cell lines, but its function is not well characterized. Results. We investigated the expression and function of TSPO in the human colon cancer cells HT‐29. Immunohistochemical studies revealed that TSPO is localized in mitochondria, and its endogenous ligand, the polypeptide diazepam‐binding inhibitor, in the cytosol. Radioligand binding studies using the specific high‐affinity drug ligand [3H]PK 11195 and membrane fraction demonstrated saturable binding, with Kd and Bmax values of 13.5±1.5 nM and 10.1±1.0 pmol/mg respectively. PK 11195 induced a rapid and transient dose‐dependent rise in intracellular [Ca2+], which was unaffected by extracellular Ca2+, but was blocked by the PTP (permeability transition pore) inhibitor, cyclosporin A, and by the TSPO partial agonist, flunitrazepam. Using HT‐29 clone 19A cell line, which forms cell monolayers, we demonstrated that TSPO ligand stimulated a Ca2+‐dependent transepithelial Cl? secretion. This secretion was inhibited: (i) after removal of extracellular Cl?; (ii) by apical addition of the Cl? channel blocker NPPB [5‐nitro‐2‐(3‐phenylpropylamino)‐benzoate]; and (iii) by basolateral addition of the Na+–K+–2Cl? co‐transporter inhibitor bumetanide. Furthermore, the intracellular Ca2+ chelator BAPTA/AM [bis‐(o‐aminophenoxy)ethane‐N,N,N′,N′‐tetra‐acetic acid tetrakis(acetoxymethyl ester)] and cyclosporin A abolished the rise in PK 11195‐induced Cl? secretion. Conclusions. These findings indicate that TSPO is located in mitochondrial membranes of HT‐29 and reveal that its activation induces a rise in cytosolic Ca2+, leading to the stimulation of Cl? secretion.  相似文献   

11.
Cyclic AMP-activated intestinal Cl secretion plays an important role in pathogenesis of cholera. This study aimed to investigate the effect of diclofenac on cAMP-activated Cl secretion, its underlying mechanisms, and possible application in the treatment of cholera. Diclofenac inhibited cAMP-activated Cl secretion in human intestinal epithelial (T84) cells with IC50 of ∼20 µM. The effect required no cytochrome P450 enzyme-mediated metabolic activation. Interestingly, exposures of T84 cell monolayers to diclofenac, either in apical or basolateral solutions, produced similar degree of inhibitions. Analyses of the apical Cl current showed that diclofenac reversibly inhibited CFTR Cl channel activity (IC50∼10 µM) via mechanisms not involving either changes in intracellular cAMP levels or CFTR channel inactivation by AMP-activated protein kinase and protein phosphatase. Of interest, diclofenac had no effect on Na+-K+ ATPases and Na+-K+-Cl cotransporters, but inhibited cAMP-activated basolateral K+ channels with IC50 of ∼3 µM. In addition, diclofenac suppressed Ca2+-activated Cl channels, inwardly rectifying Cl channels, and Ca2+-activated basolateral K+ channels. Furthermore, diclofenac (up to 200 µM; 24 h of treatment) had no effect on cell viability and barrier function in T84 cells. Importantly, cholera toxin (CT)-induced Cl secretion across T84 cell monolayers was effectively suppressed by diclofenac. Intraperitoneal administration of diclofenac (30 mg/kg) reduced both CT and Vibrio cholerae-induced intestinal fluid secretion by ∼70% without affecting intestinal fluid absorption in mice. Collectively, our results indicate that diclofenac inhibits both cAMP-activated and Ca2+-activated Cl secretion by inhibiting both apical Cl channels and basolateral K+ channels in intestinal epithelial cells. Diclofenac may be useful in the treatment of cholera and other types of secretory diarrheas resulting from intestinal hypersecretion of Cl.  相似文献   

12.
We investigated the regulation of Cl secretion by adrenoceptors in polarized 16HBE14o- human bronchial epithelial cells. Treatment with the nonselective β adrenoceptor agonist isoprenaline stimulated an increase in short-circuit current (ISC), which was inhibited by the β adrenoceptor blocker propranolol. Treatment with procaterol, an agonist specific for the β2 adrenoceptor subtype, stimulated a similar increase in ISC, which was inhibited by the β2 adrenoceptor antagonist ICI 118551. Inhibitors of cystic fibrosis transmembrane conductance regulator (CFTR) and calcium-activated Cl channel (CaCC), but not K+ channel blockers, were able to inhibit the increase in ISC. “Trimultaneous” recording of ISC and intracellular cyclic adenosine monophosphate (cAMP) and Ca2+ levels in 16HBE14o- epithelia confirmed that the ISC induced by isoprenaline or procaterol involved both cAMP and Ca2+ signaling. Our results demonstrate that β2 adrenoceptors regulate Cl secretion in the human airway epithelium by activating apical CFTRs and CaCCs via cAMP-dependent and intracellular Ca2+-dependent mechanisms, respectively.  相似文献   

13.
The response of confluent monolayers of HT29-Cl.16E cells to stimulation by extracellular ATP and ATP analogues was investigated in terms of mucin and electrolyte secretion. Mucin secretion was measured as release of glucosamine-labeled macromolecules trapped at the stacking/running gel interface of polyacrylamide gels and electrolyte secretion as shortcircuit current (Isc). Luminal ATP stimulated a transient increase in the release of mucins and of I sc corresponding to a secretory Cl current. Both secretions peaked at 3 to 5 min after addition of ATP. Maximal ATP-stimulated mucin secretion over 15 min was up to 18-fold above control with an apparent ED50 of approximately 40 m. Maximal peak I sc after stimulation with ATP was approximately 35 A/cm2 with an apparent ED50 of about 0.4 mm. ATP-dependent I sc was at least in part due to Cl secretion since removal of Cl from the medium reduced the peak I sc by 40% and the I sc integrated over 40 min by 80%. The secretory responses were not associated with cell damage as assessed by failure of ethidium bromide to enter into the cells, absence of release of lactate dehydrogenase, maintenance of monolayer conductance, viability, and responses to repeated applications of ATP. The order of efficacy of nucleotide agonists was similar for both processes with ATP>ADP>AMPadenosine. Luminal ATP was much more effective than basolateral addition of this compound. These results suggest involvement of a luminal P2-type receptor which can initiate signaling pathways for granule fusion and mucin release as well as for activation of Cl channels. P2-receptor-stimulated mucin and I sc release was strongly inhibited by a 30 min preincubation with the classical K+ channel blockers quinine (1 mm), quinidine (1 mm), and Ba2+ (3 mm). Experiments with amphotericin B to measure separately the conductance changes of either luminal or basolateral plasma membrane revealed that quinidine did not directly block the ATP-induced basolateral K+ or the luminal anion channels. The quinidine inhibition after preincubation is therefore most easily explained by interference with granule fusion and location of anion channels in granule membranes. Luminal P2 receptors may play a role in intestinal defense mechanisms with both fluid and mucin secretion aiding in the removal of noxious agents from the mucosal surface.Supported by grants from the National Institutes of Health (DK 39658) to U.H., the Philippe Foundation to D.M., the French Cystic Fibrosis Foundation (AFLM) and L'Association Pour La Recherche Sur Le Cancer to C.L. The authors thank Mr. J. Polack for his efforts and skill with electron microscopy and Dr. George Dubyak for helpful discussions. We also acknowledge the Cystic Fibrosis Center Core grant (DK-27651) for its support of electron and light microscopy.  相似文献   

14.
Hypersecretion of chloride can cause diarrhea, a disease frequently occurring in young pigs, particularly around weaning. We investigated the contribution of different channels to intestinal Cl secretion as influenced by age and weaning. Jejunal and colonic epithelia from 4-month-old pigs and 4-week-old piglets were incubated in Ussing chambers and stimulated by carbachol and forskolin. Changes in short-circuit currents were taken as measure of electrogenic net Cl secretion. DIDS or NPPB served to inhibit Ca-activated Cl-channels and outwardly rectifying Cl-channels (ORCC) or cystic fibrosis transmembrane regulator (CFTR), respectively. Depolarizing the basolateral membrane allowed to examine the influence of K+-channels on Cl secretion. Forskolin-stimulated Cl secretion was mediated by CFTR. ORCC were not involved. Carbachol-induced Cl secretion could be ascribed to an enhanced driving force due to the opening of K+-channels, whereas Ca-dependent Cl channels seemed not to be involved. In jejunum, piglets showed higher Cl secretion than pigs. Two days after weaning forskolin induced an I sc overshoot and a faster increase in G t. In colon, Cl secretion was neither influenced by age nor by weaning. The data suggest a disposition of porcine jejunum for a higher Cl secretion in young and freshly weaned piglets, which might be a natural defense mechanism as well as a predisposing factor for diarrhea.  相似文献   

15.
Summary Gluconate substitution for serosal Cl reduces the transepithelial short-circuit current (I sc) and depolarizes shortcircuited frog skins. These effects could result either from inhibition of basolateral K+ conductance, or from two actions to inhibit both apical Na+ permeability (P Na ap ) and basolateral pump activity. We have addressed this question by studying whole-and split-thickness frog skins. Intracellular Na+ concentration (C Na c ) andP Na ap have been monitored by measuring the currentvoltage relationship for apical Na+ entry. This analysis was conducted by applying trains of voltage pulses, with pulse durations of 16 to 32 msec. Estimates ofP Na ap ) and CNa/c were not detectably dependent on pulse duration over the range 16 to 80 msec. Serosal Cl replacement uniformly depolarized short-circuited tissues. The depolarization was associated with inhibition ofI sc across each split skin, but only occasionally across the whole-thickness preparations. This difference may reflect the better ionic exchange between the bulk medium and the extracellular fluid in contact with the basolateral membranes, following removal of the underlying dermis in the split-skin preparations.P Na ap was either unchanged or increased, and CNa/c either unchanged or reduced after the anionic replacement. These data are incompatible with the concept that serosal Cl replacement inhibitsP Na ap and Na, K-pump activity. Gluconate substutition likely reduces cell volume, triggering inhibition of the basolateral K+ channels, consistent with the data and conclusions of S.A. Lewis, A.G. Butt, M.J. Bowler, J.P. Leader and A.D.C Macknight (J. Membrane Biol. 83:119–137, 1985) for toad bladder. The resulting depolarization reduces the electrical force favoring apical Na+ entry. The volume-conductance coupling serves to conserve volume by reducing K+ solute loss. Its molecular basis remains to be identified.  相似文献   

16.
The whole-cell patch-clamp technique has been used to study membrane currents in cultured rabbit medullary thick ascending limb (MTAL) epithelial cells. A Ca2+-activated K+ current was characterized by its voltage-dependent and Ca2+-dependent properties. When the extracellular K+ ion concentration was increased from 2 to 140 mm, the rereversal potential (Ek) was shifted from –85 to 0 mV with a slope of 46 mV per e-fold change. The Ca2+-activated K+ current is blocked by charybdotoxin (CTX) in a manner similar to the apical membrane Ca2+-activated K+ channel studied with the single channel patch-clamp technique. The results suggest that the Ca2+-activated K+ current is the predominant, large conductance and Ca2+-dependent K+ pathway in the cultured MTAL cell apical membrane. The biophysical properties and physiological regulation of a Cl current were also investigated. This current was activated by stimulation of intracellular cAMP using forskolin and isobutyl-1-methylxanthine (IBMX). The current-voltage (I–V) relationship of the Cl current showed an outward-rectifying pattern in symmetrical Cl solution. The Cl selectivity of the whole-cell current was confirmed by tail current analysis in different Cl concentration bath solutions. Several Cl channel blockers were found to be effective in blocking the outward-rectifying Cl current in MTAL cells. The cAMP-dependent Cl transport in MTAL cells was further confirmed by measuring changes in the intensity of Cl sensitive dye using fluorescence microscopy. These results suggest that the Cl channel in the apical or basolateral membrane of MTAL cells may be regulated by cAMP-dependent protein-kinase-induced phosphorylation.This study was supported by the National Institutes of Health grants GM46834 to L.L. and DK32753 to W.B.G., and by a Grant-in-Aid from the American Heart Association of Ohio to L.L.  相似文献   

17.
Forskolin concentration-dependently increased the short-circuit current (Isc) across the isolated mucosa of rat colon, which was carried mainly by Cl secretion from the mucosal membrane. The sulfonylureas such as glibenclamide, tolbutamide, glipizide and the ATP-sensitive K+ channel opener cromakalim inhibited the forskolin (1 μM)-induced increase of short-circuit current (ΔIsc) when these drugs were applied to the basolateral side. The rank order of potency for inhibition of ΔIsc was: glibenclamide > cromakalim > tolbutamide > glipizide. Glibenclamide (100 μM) and cromakalim (100 μM) caused transient or small reduction of the A23187-induced ΔIsc when applied to the basolateral side. Glibenclamide, tolbutamide and cromakalim decreased the forskolin-induced ΔIsc when applied to the mucosal side; however, the responses produced by basolateral application were greater and faster than those elicited by mucosal application. None of these four agents affected the basal transepithelial current. The results indicate that the cAMP-dependent Cl secretion in the rat colon could be modulated by ATP-sensitive K+ channel regulators. © 1996 Wiley-Liss, Inc.  相似文献   

18.
To determine if calcium-dependent secretagogues directly act on epithelial cells to elicit CI secretion, their effects on CI transport and intracellular Ca2+ concentrations ([Ca2+]i) were determined in primary cultures of rabbit distal colonic crypt cells. The Cl sensitive fluorescent probe, 6-methoxyquinolyl acetoethyl ester, MQAE and the Ca2+-sensitive fluorescent probe, fura-2AM were used to assess Cl transport and [Ca2+]i, respectively. Basal Cl transport (0.274 ± 0.09 mM/sec) was inhibited significantly by the Cl channel blocker diphenylamine-2-carboxylate (DPC, 50 μM, 0.068 ± 0.02 mM/sec; P < 0.001) and the Na+/K+/2Cl cotransport inhibitor furosemide (1 μM, 0.137 ± 0.04 mM/sec; P < 0.01). Ion substitution studies using different halides revealed the basal influx to be I > F ≥ Cl > Br. DPC inhibited I influx by ∼50%, F influx by 80%, Cl influx by 85%, and Br influx by 90%. Furosemide significantly inhibited influx of Br (84%) and Cl (81%) but not of F and I. The effects of agents known to alter biological response by increasing [Ca2+]i in other epithelial systems were used to stimulate Cl transport. Cl influx in mM/second was stimulated by 1 μM histamine (0.58 ± 0.05), 10 μM neurotensin (2.07 ± 0.32), 1 μM serotonin (1.63 ± 0.28), and 0.1 μM of the Ca2+ ionophore A23187 (2.05 ± 0.40). The Cl permeability stimulated by neurotensin, serotonin, and A23187 was partially blocked by DPC or furosemide added alone or in combination. Histamine-induced Cl influx was significantly inhibited by only furosemide. Indomethacin blocked histamine-stimulated Cl permeability but had no effect on the actions of the other agents. These studies, focusing on isolated colonocytes without the contribution of submucosal elements, reveal that (1) histamine stimulates Cl transport by activating the Na+/K+/2Cl cotransporter via a cyclooxygenase-dependent pathway; (2) neurotensin, serotonin, and A23187 activate both Cl channels and the cotransporter, and their actions are cyclooxygenase-independent. © 1996 Wiley-Liss, Inc.  相似文献   

19.
Summary Proximal, stripped segments of small intestine from the urodeleAmphiuma were short-circuited in media containing Na+, Cl and HCO 3 . Under these conditions there was a large net absorption of Cl, a small net absorption of Na+ and a residual flux (J Net R ) consistent with HCO 3 secretion. Net Cl absorption correlated with the short-circuit current (I sc); net Na+ absorption correlated negatively withJ Net R . Acetazolamide eliminated theI sc, lowered Cl absorption by 50%, and reduced net Na+ absorption without alteringJ Net R . Benzolamide inhibited theI sc without alteringJ Net R . Benzolamide inhibited theI sc more rapidly when applied on the mucosal surface. Replacement of Na+ or HCO 3 (and CO2) in the media eliminated theI sc, net Cl absorption and the residual flux. Likewise, inclusion of the stilbene SITS in the serosal media eliminated theI sc, net Cl absorption and the residual flux. The cytoplasmic activity of Cl (a ci a ) was determined with single and double-barreled microelectrodes. Thea ci a of villus absorptive cells in normal media was 21.0mm and in excess of that expected on the basis of electrochemical equilibrium of Cl at the mucosal membrane. Active Cl accumulation was also observed in the presence of acetazolamide but was eliminated upon replacement of media Na+ with choline. The mucosal membrane potential was depolarized upon replacement of media Na+. It is concluded that Cl is actively absorbed into intestinal cells ofAmphiuma by an electrogenic process located in the mucosal membrane. Depending on the level of intracellular HCO 3 , accumulated Cl may diffuse passively back into the mucosal media or undergo exchange with bath HCO 3 at the serosal membrane.  相似文献   

20.
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