Stimulation Of Isc Research Articles

CAMP Stimulates Transepithelial Short-Circuit Current and Fluid Transport Across Porcine Ciliary Epithelium.

To investigate the effects of cAMP on transepithelial electrical parameters and fluid transport across porcine ciliary epithelium. Transepithelial electrical parameters were determined by mounting freshly isolated porcine ciliary epithelium in a modified Ussing chamber. Similarly, fluid movement across intact ciliary body was measured with a custom-made fluid flow chamber. Addition of 1, 10, and 100 μM 8-Br-cAMP (cAMP) to the aqueous side (nonpigmented ciliary epithelium, NPE) induced a sustained increase in short-circuit current (Isc). Addition of niflumic acid (NFA) to the aqueous surface effectively blocked the cAMP-induced Isc stimulation. The administration of cAMP to the stromal side (pigmented ciliary epithelium, PE) triggered a significant stimulation of Isc only at 100 μM. No additive effect was observed with bilateral application of cAMP. Likewise, forskolin caused a significant stimulation of Isc when applied to the aqueous side. Concomitantly, cAMP and forskolin increased fluid transport across porcine ciliary epithelium, and this stimulation was effectively inhibited by aqueous NFA. Depleting Cl- in the bathing solution abolished the baseline Isc and inhibited the subsequent stimulation by cAMP. Pretreatment with protein kinase A (PKA) blockers (H89/KT5720) significantly inhibited the cAMP- and forskolin-induced Isc responses. Our results suggest that cAMP triggers a sustained stimulation of Cl- and fluid transport across porcine ciliary epithelium; Cl- channels in the NPE cells are potentially a cellular site for this PKA-sensitive cAMP-mediated response.

Bile acids stimulate chloride secretion through CFTR and calcium-activated Cl- channels in Calu-3 airway epithelial cells.

Bile acids resulting from the aspiration of gastroesophageal refluxate are often present in the lower airways of people with cystic fibrosis and other respiratory distress diseases. Surprisingly, there is little or no information on the modulation of airway epithelial ion transport by bile acids. The secretory effect of a variety of conjugated and unconjugated secondary bile acids was investigated in Calu-3 airway epithelial cells grown under an air-liquid interface and mounted in Ussing chambers. Electrogenic transepithelial ion transport was measured as short-circuit current (Isc). The taurine-conjugated secondary bile acid, taurodeoxycholic acid (TDCA), was found to be the most potent modulator of basal ion transport. Acute treatment (5 min) of Calu-3 cells with TDCA (25 μM) on the basolateral side caused a stimulation of Isc, and removal of extracellular Cl(-) abolished this response. TDCA produced an increase in the cystic fibrosis transmembrane conductance regulator (CFTR)-dependent current that was abolished by pretreatment with the CFTR inhibitor CFTRinh172. TDCA treatment also increased Cl(-) secretion through calcium-activated chloride (CaCC) channels and increased the Na(+)/K(+) pump current. Acute treatment with TDCA resulted in a rapid cellular influx of Ca(2+) and increased cAMP levels in Calu-3 cells. Bile acid receptor-selective activation with INT-777 revealed TGR5 localized at the basolateral membrane as the receptor involved in TDCA-induced Cl(-) secretion. In summary, we demonstrate for the first time that low concentrations of bile acids can modulate Cl(-) secretion in airway epithelial cells, and this effect is dependent on both the duration and sidedness of exposure to the bile acid.

AVT is involved in the regulation of ion transport in the intestine of the sea bream (Sparus aurata)

The intestine of marine fish plays a crucial role in ion homeostasis by selective processing of ingested fluid. Although arginine vasotocin (AVT) is suggested to play a role in ion regulation in fish, its action in the intestine has not been demonstrated. Thus, the present study investigated in vitro the putative role of AVT in intestinal ion transport in the sea bream (Sparus aurata). A cDNA encoding part of an AVT receptor was isolated and phylogenetic analysis revealed it clustered with the V1a2-type receptor clade. V1a2 transcripts were expressed throughout the gastrointestinal tract, from esophagus to rectum, and were most abundant in the rectum regardless of long-term exposure to external salinities of 12, 35 or 55p.p.t. Basolateral addition of AVT (10−6M) to the anterior intestine and rectum of sea bream adapted to 12, 35 or 55p.p.t. mounted in Ussing chambers produced rapid salinity and region dependent responses in short circuit current (Isc), always in the absorptive direction. In addition, AVT stimulation of absorptive Isc conformed to a dose–response curve, with significant effects achieved at 10−8M, which corresponds to physiological values of plasma AVT for this species. The effect of AVT on intestinal Isc was insensitive to the CFTR selective inhibitor NPPB (200μM) applied apically, but was completely abolished in the presence of apical bumetanide (200μM). We propose a role for AVT in the regulation of ion absorption in the intestine of the sea bream mediated by an absorptive bumetanide-sensitive mechanism, likely NKCC2.

CAMP-stimulated Cl- secretion is increased by glucocorticoids and inhibited by bumetanide in semicircular canal duct epithelium

BackgroundThe vestibular system controls the ion composition of its luminal fluid through several epithelial cell transport mechanisms under hormonal regulation. The semicircular canal duct (SCCD) epithelium has been shown to secrete Cl- under β2-adrenergic stimulation. In the current study, we sought to determine the ion transporters involved in Cl- secretion and whether secretion is regulated by PKA and glucocorticoids.ResultsShort circuit current (Isc) from rat SCCD epithelia demonstrated stimulation by forskolin (EC50: 0.8 μM), 8-Br-cAMP (EC50: 180 μM), 8-pCPT-cAMP (100 μM), IBMX (250 μM), and RO-20-1724 (100 μM). The PKA activator N6-BNZ-cAMP (0.1, 0.3 & 1 mM) also stimulated Isc. Partial inhibition of stimulated Isc individually by bumetanide (10 & 50 μM), and [(dihydroindenyl)oxy]alkanoic acid (DIOA, 100 μM) were additive and complete. Stimulated Isc was also partially inhibited by CFTRinh-172 (5 & 30 μM), flufenamic acid (5 μM) and diphenylamine-2,2′-dicarboxylic acid (DPC; 1 mM). Native canals of CFTR+/− mice showed a stimulation of Isc from isoproterenol and forskolin+IBMX but not in the presence of both bumetanide and DIOA, while canals from CFTR−/− mice had no responses. Nonetheless, CFTR−/− mice showed no difference from CFTR+/− mice in their ability to balance (rota-rod). Stimulated Isc was greater after chronic incubation (24 hr) with the glucocorticoids dexamethasone (0.1 & 0.3 μM), prednisolone (0.3, 1 & 3 μM), hydrocortisone (0.01, 0.1 & 1 μM), and corticosterone (0.1 & 1 μM) and mineralocorticoid aldosterone (1 μM). Steroid action was blocked by mifepristone but not by spironolactone, indicating all the steroids activated the glucocorticoid, but not mineralocorticoid, receptor. Expression of transcripts for CFTR; for KCC1, KCC3a, KCC3b and KCC4, but not KCC2; for NKCC1 but not NKCC2 and for WNK1 but only very low WNK4 was determined.ConclusionsThese results are consistent with a model of Cl- secretion whereby Cl- is taken up across the basolateral membrane by a Na+-K+-2Cl- cotransporter (NKCC) and potentially another transporter, is secreted across the apical membrane via a Cl- channel, likely CFTR, and demonstrate the regulation of Cl- secretion by protein kinase A and glucocorticoids.

Methadone but not Morphine Inhibits Lubiprostone-Stimulated Cl− Currents in T84 Intestinal Cells and Recombinant Human ClC-2, but not CFTR Cl− Currents

In clinical trials, methadone, but not morphine, appeared to prevent beneficial effects of lubiprostone, a ClC-2 Cl− channel activator, on opioid-induced constipation. Effects of methadone and morphine on lubiprostone-stimulated Cl− currents were measured by short circuit current (Isc) across T84 cells. Whole cell patch clamp of human ClC-2 (hClC-2) stably expressed in HEK293 cells and in a high expression cell line (HEK293EBNA) as well as human CFTR (hCFTR) stably expressed in HEK293 cells was used to study methadone and morphine effects on recombinant hClC-2 and hCFTR Cl− currents. Methadone but not morphine inhibited lubiprostone-stimulated Isc in T84 cells with half-maximal inhibition at 100 nM. Naloxone did not affect lubiprostone stimulation or methadone inhibition of Isc. Lubiprostone-stimulated Cl− currents in hClC-2/HEK293 cells, but not forskolin/IBMX-stimulated Cl− currents in hCFTR/HEK293 cells, were inhibited by methadone, but not morphine. HEK293EBNA cells expressing hClC-2 showed time-dependent, voltage-activated, CdCl2-inhibited Cl− currents in the absence (control) and the presence of lubiprostone. Methadone, but not morphine, inhibited control and lubiprostone-stimulated hClC-2 Cl− currents with half-maximal inhibition at 100 and 200–230 nM, respectively. Forskolin/IBMX-stimulated hClC-2 Cl− currents were also inhibited by methadone. Myristoylated protein kinase inhibitor (a specific PKA inhibitor) inhibited forskolin/IBMX- but not lubiprostone-stimulated hClC-2 Cl− currents. Methadone caused greater inhibition of lubiprostone-stimulated currents added before patching (66.1 %) compared with after patching (28.7 %). Methadone caused inhibition of lubiprostone-stimulated Cl− currents in T84 cells and control; lubiprostone- and forskolin/IBMX-stimulated recombinant hClC-2 Cl− currents may be the basis for reduced efficacy of lubiprostone in methadone-treated patients.

Lubiprostone reverses the inhibitory action of morphine on mucosal secretion in human small intestine.

Treatments with morphine or opioid agonists cause constipation. Lubiprostone is approved for treatment of adult idiopathic constipation and constipation-predominant IBS in adult women. We tested whether lubiprostone can reverse morphine-suppression of mucosal secretion in human intestine and explored the mechanism of action. Fresh segments of jejunum discarded during Roux-En-Y gastric bypass surgeries were used. Changes in short-circuit current (ΔIsc) were recorded in Ussing flux chambers as a marker for electrogenic chloride secretion during pharmacological interactions between morphine, prostaglandin receptor antagonists, chloride channel blockers and lubiprostone. Morphine suppressed basal Isc. Lubiprostone reversed morphine suppression of basal Isc. Lubiprostone, applied to the mucosa in concentrations ranging from 3 nM to 30 μM, evoked increases in Isc in concentration-dependent manner when applied to the mucosal side of muscle-stripped preparations. Blockade of enteric nerves did not change stimulation of Isc by lubiprostone. Removal of chloride or application of bumetanide or NPPB suppressed or abolished responses to lubiprostone. Antagonists acting at CFTR channels and prostaglandin EP(4) receptors, but not at E(1), EP(1-3) receptors, partially suppressed stimulation of Isc by lubiprostone. Antisecretory action of morphine results from suppression of excitability of secretomotor neurons in the enteric nervous system. Lubiprostone, which does not affect enteric neurons directly, bypasses the action of morphine by directly opening mucosal chloride channels.

T1841 Differential Effect of NHE3 Kinase a Regulatory Protein (E3karp/NHERF2) Knockout on cAMP-, cGMP-, or [Ca2+]-Induced Inhibition of Na+/H+ Exchanger 3 (NHE3) Activity Along the Murine Intestinal Tract

G A A b st ra ct s of calcitonin on ion transport in IECs. Human colonic T84 cells grown on Transwell inserts were utilized as an in-vitro model of IECs. Chloride secretion was assessed by the measurement of short circuit current (Isc) across T84 monolayers mounted in Ussing chambers. We first examined the expression of CTR in IECs. Real time QRT-PCR and western blot analysis demonstrated the expression of CTR in T84 cells. Exposure of T84 cells to calcitonin from the basolateral but not apical side significantly increased Isc (a change of 287 ± 48 μA/cm2 from the baseline). Stimulation of Isc by calcitonin was dose-dependent (1-100 nM) and was completely blocked by the CTR antagonist, CT8-32. In addition, the increase in Isc was blocked by replacing chloride in the bath solutions with equal amount of gluconate (95 ± 6% reduction, P < 0.01) and was significantly inhibited by 10 μM of the specific CFTR inhibitor, CFTR127inh, (88 ± 9% reduction, P < 0.01) indicating the involvement of CFTR chloride channel in CT-induced effects. To further investigate the signaling pathways involved, we examined calcitonin-induced Isc in the presence of the chelator of [Cai], BAPTA-AM and PKA inhibitor, H89. Preincubation with 20 μMof BAPTAAM or 10μM H89 for 45 minutes significantly reduced calcitonin-induced Isc indicating the involvement of both Ca++and PKA-dependent pathways (58±13% reduction, P < 0.01 and 55±7%, P < 0.01, respectively). Simultaneous inhibition of both Ca++ and PKA pathways had an additive effect on the CT-induced stimulation of Isc (78 ± 8% reduction, P < 0.01). In conclusion, calcitonin-induced short circuit current appears to be mediated by CTR via direct activation of the CFTR channel and is Ca++ and PKA-dependent. This novel study showing the expression of calcitonin receptor in intestinal epithelial cells provides further insights into the molecular mechanisms underlying calcitonin-induced diarrhea. (Supported by NIDDK and Dept of Veteran Affairs).

Modulation of NaCl absorption by [HCO(3)(-)] in the marine teleost intestine is mediated by soluble adenylyl cyclase.

Intestinal HCO(3)(-) secretion and NaCl absorption are essential for counteracting dehydration in marine teleost fish. We investigated how these two processes are coordinated in toadfish. HCO(3)(-) stimulated a luminal positive short-circuit current (I(sc)) in intestine mounted in Ussing chamber, bathed with the same saline solution on the external and internal sides of the epithelium. The I(sc) increased proportionally to the [HCO(3)(-)] in the bath up to 80 mM NaHCO(3), and it did not occur when NaHCO(3) was replaced with Na(+)-gluconate or with NaHCO(3) in Cl(-)-free saline. HCO(3)(-) (20 mM) induced a approximately 2.5-fold stimulation of I(sc), and this [HCO(3)(-)] was used in all subsequent experiments. The HCO(3)(-)-stimulated I(sc) was prevented or abolished by apical application of 10 muM bumetanide (a specific inhibitor of NKCC) and by 30 microM 4-catechol estrogen [CE; an inhibitor of soluble adenylyl cyclase (sAC)]. The inhibitory effects of bumetanide and CE were not additive. The HCO(3)(-)-stimulated I(sc) was prevented by apical bafilomycin (1 microM) and etoxolamide (1 mM), indicating involvement of V-H(+)-ATPase and carbonic anhydrases, respectively. Immunohistochemistry and Western blot analysis confirmed the presence of an NKCC2-like protein in the apical membrane and subapical area of epithelial intestinal cells, of Na(+)/K(+)-ATPase in basolateral membranes, and of an sAC-like protein in the cytoplasm. We propose that sAC regulates NKCC activity in response to luminal HCO(3)(-), and that V-H(+)-ATPase and intracellular carbonic anhydrase are essential for transducing luminal HCO(3)(-) into the cell by CO(2)/HCO(3)(-) hydration/dehydration. This mechanism putatively coordinates HCO(3)(-) secretion with NaCl and water absorption in toadfish intestine.

Stimulation of mucosal secretion by lubiprostone (SPI-0211) in guinea pig small intestine and colon

Actions of lubiprostone, a selective type-2 chloride channel activator, on mucosal secretion were investigated in guinea pig small intestine and colon. Flat-sheet preparations were mounted in Ussing flux chambers for recording short-circuit current (Isc) as a marker for electrogenic chloride secretion. Lubiprostone, applied to the small intestinal mucosa in eight concentrations ranging from 1-3000 nM, evoked increases in Isc in a concentration-dependent manner with an EC50 of 42.5 nM. Lubiprostone applied to the mucosa of the colon in eight concentrations ranging from 1-3000 nM evoked increases in Isc in a concentration-dependent manner with an EC50 of 31.7 nM. Blockade of enteric nerves by tetrodotoxin did not influence stimulation of Isc by lubiprostone. Antagonists acting at prostaglandin (PG)E2, EP1-3, or EP4 receptors did not suppress stimulation of Isc by lubiprostone but suppressed or abolished PGE2-evoked responses. Substitution of gluconate for chloride abolished all responses to lubiprostone. The selective CFTR channel blocker, CFTR(inh)-172, did not suppress lubiprostone-evoked Isc. The broadly acting blocker, glibenclamide, suppressed (P<0.001) lubiprostone-evoked Isc. Lubiprostone, in the presence of tetrodotoxin, enhanced carbachol-evoked Isc. The cholinergic component, but not the putative vasoactive intestinal peptide component, of neural responses to electrical field stimulation was enhanced by lubiprostone. Application of any of the prostaglandins, E2, F2, or I2, evoked depolarization of the resting membrane potential in enteric neurons. Unlike the prostaglandins, lubiprostone did not alter the electrical behavior of enteric neurons. Exposure to the histamine H2 receptor agonists increased basal Isc followed by persistent cyclical increases in Isc. Lubiprostone increased the peak amplitude of the dimaprit-evoked cycles.

W1374 Excitation of Neurons in the Enteric Nervous System (ENS), Degranulation of Enteric Mast Cells and Direct Action On Enterocytes Underlie Stimulatory Action of Bradykinin (Bk) On Mucosal Secretion in Guinea-Pig Small Intestine

Neurons in the g-pig small intestinal ENS express the BK-B2 receptor. Stimulation of the BK-B2 receptor elevates neuronal excitability (J Pharmacol Exp Ther 2004; 309:310-9; & Br J Pharmacol 2003; 138:1221-32). BK-2 stimulation releases prostaglandins, which feedback in autocoid manner to excite the same neurons that release the prostaglandins. Full thickness and muscle-stripped segments of g-pig ileum were mounted in Ussing flux chambers for recording short-circuit current (Isc), an indicator of Clsecretion. Application of BK in the serosal compartment of the chambers evoked increases in Isc in a concentration-dependent manner with an EC50 8.0±0.1nM. Higher concentrations of 1-5μMwere required for stimulation of Isc with application in the mucosal compartment. Pretreatment with 0.5-μM tetrodotoxin (TTX) partially suppressed the BK-evoked increases in Isc. Transmural electrical field stimulation evoked neurally-mediated increases in Isc. These responses were abolished by TTX in each of 18 preps. Application of the mast cell degranulating agent, compound 48/ 80 (60μg/ml), increased Isc (P 0.05). The BK-B2 antagonist, WIN64388 (1-10μM), partially suppressed BK-evoked increases in Isc. WIN64388 at 10μM suppressed BK-evoked Isc by 62.8% in 12 preps. Pre-incubation for 30 min with the selective cyclooxygenase-1 (COX-1) inhibitor, SC560 (10μM), abolished increases in Isc evoked by 10-nM BK in each of 10 preps. The COX-2 inhibitor, NS398 (2μM) reduced BK-evoked increases in Isc by 28.9 percent in 6 preps. Pre-treatment with a prostanoid receptor antagonist, AH6809 (5μM), significantly suppressed BK-evoked stimulation of Isc (P<0.05) for all of 6 preps. BK-evoked increases in Isc were suppressed by pre-treatment with the mast cell stabilizing agents: cromolyn (10-30μM; P<0.05); doxantrazole (30-50μM; P<0.05) or ketotifen (10μM; P<0.05) with each agent tested on 10 preps. Carbachol acted directly on the epithelium to increase Isc. This well-known action of the muscarinic agonist was suppressed by ketotifen, but not by cromolyn or doxantrazole. The results suggest a role for enteric mast cells in BK-stimulation of mucosal secretion, with a prostaglandin involved as a mediator. Once released from mast cells prostaglandins stimulate secretomotor neuronal activity; as well as, act in direct concert with BK on the enterocytes. All of which might underlie the involvement of BK in inflammation-related secretory diarrhea. Supported by: NIH RO1DK57075 & KO8 DK060468

Atrazine increases the sodium absorption in frog (Rana esculenta) skin

The presence of atrazine in agricultural sites has been linked to the decline in amphibian populations. The efforts of the scientific community generally are directed toward investigating the long-term effect of atrazine on complex functions (reproduction or respiration), but in the present study, we investigated the short-term effect on the short-circuit current (I(sc)), a quantitative measure of the ion transport operated by frog (Rana esculenta) skin. Treatment with 5 microM atrazine (1.08 mg/L) does not affect the transepithelial outfluxes of [14C]mannitol or [14C]urea; therefore, atrazine does not damage the barrier properties of frog skin. Atrazine causes a dose-dependent increase in the short-circuit current, with a minimum of 4.64 +/- 0.76 microA/cm2 (11.05% +/- 1.22%) and a maximum of 12.7 +/- 0.7 microA/cm2 (35% +/- 2.4%) measured at 10 nM and 5 microM, respectively. An increase in Isc also is caused by 5 microM ametryne, prometryn, simazine, terbuthylazine, or terbutryn (other atrazine derivatives). In particular, atrazine increases the transepithelial 22Na+ influx without affecting the outflux. Finally, stimulation of Isc by atrazine is suppressed by SQ 22536, H89, U73122, 2-aminoethoxydiphenyl borate, and W7 (blockers of adenylate cyclase, protein kinase A, phospholipase C, intracellular Ca2+ increase, and calmodulin, respectively), whereas indomethacin and calphostin C (inhibitors of cyclooxygenase and protein kinase C, respectively) have no effect.

Cortisol receptor blockade and seawater adaptation in the euryhaline teleost Fundulus heteroclitus

To examine the role of cortisol in seawater osmoregulation in a euryhaline teleost, adult killifish were acclimated to brackish water (10 per thousand) and RU486 or vehicle was administered orally in peanut oil daily for five days at low (40 mg.kg(-1)) or high dose (200 mg.kg(-1)). Fish were transferred to 1.5 x seawater (45 per thousand) or to brackish water (control) and sampled at 24 h and 48 h after transfer, when Cl- secretion is upregulated. At 24 h, opercular membrane Cl- secretion rate, as Isc, was increased only in the high dose RU486 group. Stimulation of membranes by 3-isobutyl-1-methylxanthine and cAMP increased Isc in vehicle treated controls but those from RU486-treated animals were unchanged and membranes from brackish water animals showed a decrease in Isc. At 48 h, Isc increased and transepithelial resistance decreased in vehicle and RU486 groups, compared to brackish water controls. Plasma cortisol increased in all groups transferred to high salinity, compared to brackish water controls. RU486 treated animals had higher cortisol levels compared to vehicle controls. Vehicle treated controls had lower cortisol levels than untreated or RU486 treated animals, higher stimulation of Isc, and lower hematocrit at 24 h, beneficial effects attributed to increased caloric intake from the peanut oil vehicle. Chloride cell density was significantly increased in the high dose RU486 group at 48 hours, yet Isc was unchanged, suggesting a decrease in Cl- secretion per cell. Thus cortisol enhances NaCl secretion capacity in chloride cells, likely via glucocorticoid type receptors.

Extracellular Ca2+ regulates the stimulation of Na+ transport in A6 renal epithelia.

We investigated the involvement of intracellular and extracellular Ca2+ in the stimulation of Na+ transport during hyposmotic treatment of A6 renal epithelia. A sudden osmotic decrease elicits a biphasic stimulation of Na+ transport, recorded as increase in amiloride-sensitive short-circuit current (Isc) from 3.4 +/- 0.4 to 24.0 +/- 1.3 microA/cm2 (n = 6). Changes in intracellular Ca2+ concentration ([Ca2+]i) were prevented by blocking basolateral Ca2+ entry with Mg2+ and emptying the intracellular Ca2+ stores before the hyposmotic challenge. This treatment did not noticeably affect the hypotonicity-induced stimulation of Isc. However, the absence of extracellular Ca2+ severely attenuated Na+ transport stimulation by the hyposmotic shock, and Isc merely increased from 2.2 +/- 0.3 to 4.8 +/- 0.7 microA/cm2. Interestingly, several agonists of the Ca2+-sensing receptor, Mg2+ (2 mM), Gd3+ (0.1 mM), neomycin (0.1 mM), and spermine (1 mM) were able to substitute for extracellular Ca2+. When added to the basolateral solution, these agents restored the stimulatory effect of the hyposmotic solutions on Isc in the absence of extracellular Ca2+ to levels that were comparable to control conditions. None of the above-mentioned agonists induced a change in [Ca2+]i. Quinacrine, an inhibitor of PLA2, overruled the effect of the agonists on Na+ transport. In conclusion, we suggest that a Ca2+-sensing receptor in A6 epithelia mediates the stimulation of Na+ transport without the interference of changes in [Ca2+]i.

Functional and molecular biological evidence of SGLT-1 in the ruminal epithelium of sheep.

Because of the effective catabolism of D-glucose to short-chain fatty acids by intraruminal microorganisms, the absorption of D-glucose from the rumen was thought to be of minor importance. However, clinical studies suggested that significant quantities of D-glucose are transported from the ruminal contents to the blood. We therefore tested the ruminal epithelium of sheep for the presence of Na(+)-glucose cotransporter 1 (SGLT-1) on both the functional and mRNA levels. In the absence of an electrochemical gradient, 3-O-methylglucose (3-OMG) was net absorbed across isolated ruminal epithelia mounted in Ussing chambers. The net transport of 3-OMG followed Michaelis-Menten kinetics and was sensitive to phlorizin or decreasing Na(+) concentrations. The mucosal addition of 10 mM D-glucose induced an immediate, phlorizin-sensitive increase in short-circuit current (I(sc)). I(sc) could also be increased by serosal addition of D-glucose or D-mannose, but electrogenic uptake of D-glucose or 3-OMG added on the mucosal side was still detectable after serosal stimulation of I(sc). RT-PCR using primers specific for the ovine intestinal SGLT-1 with subsequent TA cloning and sequencing revealed 100% identity between the cloned cDNA and mRNA fragment 187-621 of ovine intestinal SGLT-1. In conclusion, the ruminal epithelium has a high-affinity SGLT-1, which indicates that it maintains the capacity for D-glucose absorption.

Calcium and ATP regulation of ion transport in larval frog skin.

Ion transport measured as short circuit current (Isc) across the skin of larval frogs is activated by amiloride, acetylcholine, and ATP. In many epithelia, ATP stimulation of Isc involves an increase in intracellular calcium. To define the role of changes in intracellular calcium in ATP stimulation of Isc in larval frog skin, epithelial cells were loaded with calcium by adding 5 microM ionomycin to a 2 mM calcium apical Ringer's solution. Calcium loading had no observable effect on baseline Isc or on stimulation by ATP. Minimizing changes in intracellular calcium by loading the cell with the calcium chelator BAPTA also had no measurable effect on ATP stimulation of Isc. When the apical side was bathed with Ca(2+)-free Ringer's solution, ionomycin increased Isc up to 15 microA. This increase was partially blocked by 2 mM Ca2+, 2 mM Mg2+, and 10 microM W-7. Other experiments showed that baseline-stimulated and ATP-stimulated Isc were always larger in 2 mM Mg2+ Ringer's compared to 2 mM Ca2+. In dissociated cells bathed in 2 mM Ca2+ Ringer's, ATP had no effect on intracellular calcium as measured by Fluo-LR fluorescence changes. In conclusion, ATP apparently stimulates Isc without concomitant changes in intracellular calcium. This is consistent with a directly ligand-gated receptor at the apical membrane with P2X-like characteristics.

Beta1-adrenergic receptors but not beta2-adrenergic or vasopressin receptors regulate K+ secretion in vestibular dark cells of the inner ear.

Receptors were identified pharmacologically in functional studies where K+ secretion was monitored as transepithelial current (Isc). Further, receptors were identified as transcripts by cloning and sequencing of reverse-transcriptase polymerase chain reaction (RT-PCR) products. Isc under control conditions was 796 +/- 15 microA/cm2 (n = 329) in gerbilline VDC and 900 +/- 75 microA/cm2 (n = 6) in murine VDC. Forskolin (10(-5) m) but not 1, 9-dideoxy-forskolin increased Isc by a factor of 1.42 +/- 0.05 (n = 7). 10(-9) m Arg8-vasopressin and 10(-9) m desmopressin had no significant effect in gerbilline and murine VDC. Isoproterenol, norepinephrine, epinephrine and prenalterol stimulated Isc maximally by a factor of 1.38 +/- 0.04 (n = 7), 1.59 +/- 0.06 (n = 6), 1.64 +/- 0.03 (n = 8) and 1.37 +/- 0.03 (n = 6), respectively. The EC50 values were (1.4 +/- 0.7) x 10(-8) m (n = 36), (2.5 +/- 1.0) x 10(-8) m (n = 31), (1.7 +/- 0.7) x 10(-7) m (n = 36) and (5 +/- 4) x 10(-7) m (n = 32), respectively. Propanolol inhibited isoproterenol-induced stimulation of Isc. Atenolol, ICI118551 and CGP20712A inhibited isoproterenol-induced stimulation of Isc with a pKDB of 5.0 x 10(-8) m (pKDB = 7.30 +/- 0.07, n = 38), 4.4 x 10(-8) m (pKDB = 7.36 +/- 0.14, n = 37) and 6.8 x 10(-12) m (pKDB = 11.17 +/- 0.12, n = 37), respectively. RT-PCR of total RNA isolated from microdissected vestibular labyrinth tissue using specific primers revealed products of the predicted sizes for beta1- and beta2-adrenergic receptors but not for beta3-adrenergic receptors. Sequence analysis of the amplified cDNA fragments from gerbilline tissues revealed a 96.4%, 91.5% and 89.6% identity compared to rat beta1-, beta2- and beta3-adrenergic receptors, respectively. These results demonstrate that K+ secretion in VDC is under the control of beta1- but not beta2- or beta3-adrenergic receptors or vasopressin-receptors.

Regulation of Cl- secretion in seawater fish (Dicentrarchus labrax) gill respiratory cells in primary culture.

1. Primary cultures of sea bass (Dicentrarchus labrax) gill cells grown on permeable membranes form a highly differentiated tight epithelium composed of respiratory-like cells. This preparation was also found to provide a functional model for investigating the hormonal regulation of Cl- secretion. 2. In control conditions, i.e. in the absence of hormones or other stimuli, the cultured epithelium showed a short-circuit current (Isc) of 8.8 +/- 0.4 microA cm-2, a transepithelial potential (Vt) of 28.6 +/- 0.6 mV (serosal side positive), and a transepithelial resistance (Rt) of 5026 +/- 127 Omega cm2. Addition of 50 nM PGE2 caused a stimulation of Isc, Vt and transepithelial conductance, Gt. The increase in Isc was probably due to the elevation in Cl- secretion, since it could be correlated with the stimulation of serosal to mucosal 36Cl- flux. Application of the neurohypophyseal peptide arginine vasotocin (AVT; 50 nM) or the beta-adrenergic agonist isoproterenol (isoprenaline; 0. 5 microM) evoked a stimulation in Cl- secretion, as was shown by the increases in Isc and Gt. The excitatory effect of isoproterenol followed by the inhibitory action of propranolol, a beta-adrenergic antagonist, suggested the presence of beta-adrenergic receptors. Noradrenaline (0.1 microM) elicited a reduction in Isc, Vt and Gt, which was counterbalanced by the addition of phentolamine, an alpha-adrenergic antagonist. This suggested an activation of alpha-adrenergic receptors. 3. This study provides evidence for hormonal control of the Cl- secretion in sea bass gill respiratory cells in culture, involving AVT, prostaglandin (PGE2), and beta- and alpha-adrenergic receptors.

Modulation of Chloride Secretion Across the Pigmented Rabbit Conjunctiva

The purpose of the present study was to investigate whether active Cl−secretion in the pigmented rabbit conjunctiva was subject to cAMP, Ca2+and protein kinase C (PKC) modulation. The excised pigmented rabbit conjunctivas were mounted in the modified Ussing-type chambers for measurement of unidirectional36Cl fluxes under the open-circuit condition and of the short-circuit current (Isc), potential difference, and transconjunctival electrical resistance. The results indicate that Cl−secretion across the conjunctiva was abolished by mucosal application of 1 mMN-phenylanthranilic acid and was reduced by 40% by serosal application of 10 μMbumetanide. Net Cl−flux was stimulated by 133% by 1 mM8-Br cAMP, 107% by 10 μMA23187, and 87% by 1 μMphorbol 12-myristate-13-acetate (PMA), suggesting that cAMP, Ca2+, and PKC all modulated active Cl−secretion, respectively. There existed a linear correlation between measured changes in net Cl−flux and observed changes in Isc(r2=0.99). The serial treatment of the conjunctiva with (a) 1 mM8-Br cAMP and 10 μMA23187 and (b) 10 μMA23187 and 1 μMPMA resulted in sequence-independent, additive stimulation of Isc. In the case of 1 mM8-Br cAMP and 1 μMPMA, additive stimulation of Iscwas observed only when 1 mM8-Br cAMP was added prior to 1 μMPMA. These results suggest that a given pharmacological agent may affect more than one channel type and that there might be a possible connection among the channels at the signal transduction level. In summary, Cl−appears to enter the pigmented rabbit conjunctiva from the serosal fluid via Na+-(K+)-2Cl−cotransport process and exit to the mucosal fluid via channels, resulting in active Cl−secretion. Active Cl−secretion in the pigmented rabbit conjunctiva appears to be modulated by cAMP, Ca2+, and PKC.

Inhibitory effects of peptide YY on basal and VIP-stimulated short-circuit current in the rat jejunum: influence of technical conditions on observed results

The interaction of PYY and VIP was studied in stripped and intact rat jejunum preparations mounted in Ussing chambers. PYY decreased basal Isc in intact as well as in stripped jejunum. Stripping was necessary to evidence a stimulation of basal Isc by VIP. When PYY and VIP were administered at the same time in the serosal bath, their effects seemed additive; VIP stimulation took over when VIP was present in ten times larger amounts than PYY, while PYY inhibition predominated at isomolar concentrations (10 −7 M) of both peptides. However, when PYY was administered three to six minutes before isomolar amounts of VIP, the VIP stimulation developed without being notably hampered. At this time, however, the amount of radioimmunoassayable PYY in the serosal compartment represented still 60% of the added amount. In conclusion, the experimental conditions can significantly change the results: stripping the longitudinal muscle/myenteric plexus impairs the effect of PYY and VIP in a different fashion, while the timing and order of administration of the peptides may change the apparent interaction between VIP stimulation and PYY inhibition.

Osmotic regulation of Na+ transport across A6 epithelium: interactions with prostaglandin E2 and cyclic AMP.

Previous work from this laboratory has shown that apical membrane sodium channel activity is stimulated by serosal hyposmotic solutions (Wills, Millinoff & Crowe, 1991). In the present study, we determined whether this stimulation of sodium transport is additive with the actions of prostaglandin E2 (PGE2) or cyclic AMP (cAMP). Addition of exogenous PGE2 (100 nM; serosal bath) to isosmotic solutions led to large increases in the amiloride-sensitive short-circuit current (Isc) and transepithelial conductance (Gt), whereas no significant effects of PGE2 were observed in hyposmotic serosal solutions. Subsequent addition of mucosal amiloride reduced Isc by approximately 95% and Gt by approximately 60%. Inhibition of endogenous PGE2 production by blockers of phospholipase A2 activity (quinacrine or 3[4-octadecyl]-benzoylacrylic acid; OBBA), or inhibition of cyclooxygenase activity by indomethacin reduced the stimulation of Isc and Gt by hyposmotic solutions. Addition of forskolin (FSK) or 3-Isobutyl-1-methylxanthine (IBMX) also resulted in approximately twofold increases in the amiloride-sensitive Isc and Gt and abolished the effects of subsequent hyposmotic challenge. The effects of forskolin, PGE2, and hyposmotic challenge were diminished by pretreatment with H89, a protein kinase A (PKA) inhibitor. We conclude that osmotic regulation of sodium channel activity interacts with multiple intracellular signaling pathways, specifically the arachidonic acid metabolic pathway and the cAMP/PKA intracellular messenger cascade.