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HC[O.sup.-.sub.3]-dependent fluid secretion by the corneal endothelium controls corneal hydration and maintains corneal transparency. Recently, it has been shown that mRNA for the cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in the corneal endothelium; however, protein expression, functional localization, and a possible role in HC[O.sub.3] transport have not been reported. Immunoblotting for CFTR showed a single band at ~170 kDa for both freshly isolated and primary cultures of bovine corneal endothelial cells. Indirect immunofluorescence confocal microscopy indicated that CFTR locates to the apical membrane. Relative changes in apical and basolateral chloride permeability were estimated by measuring the rate of fluorescence quenching of the halide-sensitive indicator 6-methoxy-N-ethylquinolinium iodide during [Cl.sup.-] influx in the absence and presence of forskolin (FSK). Apical and basolateral [Cl.sup.-] permeability increased 10- and 3-fold, respectively, in the presence of 50 [micro]M FSK. FSK-activated apical chloride permeability was unaffected by [H.sub.2]DIDs (250 [micro]M); however, 5-nitro-2-(3-phenylpropyl-amino)benzoic acid (NPPB; 50 [micro]M) and glibenclamide (100 [micro]M) inhibited activated [Cl.sup.-] fluxes by 45% and 30%, respectively. FSK-activated basolateral [Cl.sup.-] permeability was insensitive to NPPB, glibenclamide, or furosemide but was inhibited 80% by [H.sub.2]DIDS. HC[O.sup.-.sub.3] permeability was estimated by measuring changes in intracellular pH in response to quickly lowering bath [HC[O.sup.-.sub.3]]. FSK (50 [micro]M) increased apical HC[O.sup.-.sub.3] permeability by twofold, which was inhibited 42% by NPPB and 65% by glibenclamide. Basolateral HC[O.sup.-.sub.3] permeability was unaffected by FSK. Genistein (50 [micro]M) significantly increased apical HC[O.sup.-.sub.3] and [Cl.sup.-] permeability by 1.8- and 16-fold, respectively. When 50 [micro]M genistein was combined with 50 [micro]M FSK, there was no further increase in [Cl.sup.-] permeability; however, HC[O.sup.-.sub.3] permeability was reduced to the control level. In summary, we conclude that CFTR is present in the apical membrane of bovine corneal endothelium and could contribute to transendothelial [Cl.sup.-] and HC[O.sup.-.sub.3] transport. Furthermore, there is a cAMP-activated [Cl.sup.-] pathway on the basolateral membrane that is not CFTR. cornea; endothelium; chloride permeability; MEQ; bicarbonate permeability; intracellular pH; BCECF; forskolin; cAMP; genistein

Resta-Lenert, Silvia, Francis Truong, Kim E. Barrett, and Lars Eckmann. Inhibition of epithelial chloride secretion by butyrate: role of reduced adenylyl cyclase expression and activity. Am J Physiol Cell Physiol 281: C1837-C1849, 2001.--Butyrate and other short-chain fatty acids (SCFAs) are found at high concentrations in the colonic lumen and affect multiple epithelial cell functions. To better understand how SCFAs regulate ion transport, we investigated the effects of SCFAs on [Cl.sup.-] secretion in human colonic epithelial cell line [T.sub.84] Butyrate inhibited [Cl.sup.-] secretory responses to prostaglandin [E.sub.2], forskolin, and cholera toxin. Other SCFAs were less effective or inactive. Reduced secretion was associated with decreased synthesis of the second messenger cAMP rather than increased degradation. Expression and activity of adenylyl cyclase were decreased by butyrate, whereas phosphodiesterase activity was unaffected and phosphodiesterase inhibition did not reverse the effects of butyrate on [Cl.sub.-] secretion. Furthermore, butyrate decreased expression of the basolateral Na-K-2C1 cotrans-porter indicating that it might modulate the secretory capacity of the cells. However, butyrate did not affect secretory responses to the calcium-dependent secretagogue carbachol, cAMP analogs, or uroguanylin, indicating that normal secretory responses to adequate levels of second messengers in butyrate-treated [T.sup.84] cells are possible. These results show that butyrate affects several aspects of epithelial [Cl.sup.-] secretion, including second messenger generation and expression of key ion transporters. However, these effects may not all be equally important in determining [Cl.sup.-] secretion in response to physiologically relevant secretagogues. intestinal epithelial cells; short-chain fatty acids, ion transport Received 21 September 2000; accepted in final form 15 August 2001

A study was conducted to test the hypothesis that endogenously produced nitric oxide (NO) directly reduces NaCl transport by the thick ascending limb of the loop of Henle (TALH). Results indicated a significant role for locally produced NO, which may act through an autocrine mechanism to directly affect TALH sodium chloride transport. Hence, TALH NO synthesis and inhibition of chloride transport may contribute to the diuretic and natriuretic effects of NO seen in vivo.

The reabsorption of isotonic fluid instilled into the alveolar spaces of anesthesized rabbits has been studied. The role of sodium (Na+) and chlorine (Cl-) ions in this process has been examined by measuring normal saline in the presence or absence of amiloride and in chlorine ion-free solutions. The procedure is repeated after the intratracheal introduction of forskolin which increases cAMP in ATII cells. Results show the transcellular movements of Na+ and Cl- ions regulate the magnitude and direction of fluid fluxes across the alveolar epithelium after cAMP simulation.

Ion-selective microelectrode techniques were used in the investigation of the effect of simulated ischemia on the intracellular chloride concentration in isolated ventricular papillary muscles of pigs. Results reveal that ischemia induces the elevation of intracellular chloride concentration. This increase in the concentration of chloride inside the cells is probably brought about by a chloride/bicarbonate exchanger.

The role played by the Ca2+-sensing receptor on the reabsorptive function of the cortical part of the thick ascending limb (CTAL) and the transduction pathways has been studied. The Ca2+-sensing receptor is an extracellular calcium ion that acts as a ligand for certain G protein-coupled receptors. Its mRNA and corresponding protein have been detected in rat kidneys. Results show that Ca2+-sensing receptor decreases Cl reabsorption under basal and ADH-stimulated conditions by suppressing the cAMP pathway. This process does not involve the protein kinase C or the phospholipase A2.

The role of chloride ions in the contraction of vascular smooth muscle cells (VSM) is investigated by modifying the medium or the chloride flux across the cell membrane in rat aortic ring preparations and monitoring the response to various vasoconstrictors. Results reveal the the chloride flux across the cell membrane is an important component of membrane depolarization in preparation for VSM contraction induced by epinephrine.

The influence of the vascular endothelium over the contribution of chloride ions in vascular smooth muscle cell contraction induced by norepinephrine is investigated using endothelium-denuded and -intact rat aortic ring preparations and inhibition studies. Results reveal that the vascular endothelium modulates the vascular smooth muscle sensitivity to norepinephrine through the regulation of the voltage-dependent component of contraction by reduced nitric oxide.

Research was undertaken to assess the physiological effect of metabolic acidosis on the cellular reabsorption of sodium chloride (NaCl) in the murine proximal convoluted tubule. It was established that NaCl reabsorption decreased the capacity of proximal tubule during metabolic acidosis. Results revealed possible processes involved in metabolic acidosis. Metabolic acidosis downregulated NaCl transport resulting from organic anion. The process involved in the NaCl transport was the passive paracellular mechanism.

Monolayers of T84 cells were used in the investigation of the inhibition of the calcium-dependent chloride secretion through nuclide efflux techniques using a variety of channel blockers to determine if the inhibition is secondary to an effect on apical chloride conductance or an effect on the basolateral potassium conductance. Results favor the effect on apical chloride conductance as the mechanism of chloride secretion inhibition.

Isolated mouse ciliated nasal epithelial cells from normal controls and cystic fibrosis null subjects were used in patch clamp studies to determine their basal conductances. Results reveal that unstimulated cells have potassium and chloride ion currents and small sodium current under particular conditions. Three chloride ion currents were identified. One of these is modulated by the cystic fibrosis transmembrane conductance regulator.

A cell line derived from the Xenopus kidney, A6, was used to evaluate the effects of arginine vasotocin (AVT) on sodium and chloride transport in the presence of microtubular disarray. The AVT-dependent secretion of chloride was inhibited with the disruption of tubular structure but the amiloride-sensitive sodium flux was spared. Inhibition studies, reverse transcription polymerase chain reaction and immunocytochemistry revealed that chloride conductance in A6 is modulated by the cystic fibrosis transmembrane conductance regulator.

The stroke-prone spontaneously hypertensive rat (SHRSP) is a genetically determined model of 'salt-sensitive' stroke and hypertension whose full phenotypic expression is said to require a diet high in [Na.sup.+] and low in [K.sup.+]. We tested the hypothesis that dietary [Cl.sup.-] determines the phenotypic expression of the SHRSP. In the SHRSP fed a normal NaCl diet, supplementing dietary [K.sup.+] with KCl exacerbated hypertension, whereas supplementing either KHC[O.sub.3] or potassium citrate (KB/C) attenuated hypertension, when blood pressure (BP) was measured radiotelemetrically, directly and continually. Supplemental KCl, but not KB/C, induced strokes, which occurred in all and only those rats in the highest quartiles of both BP and plasma renin activity (PRA). PRA was higher with KCl than with KB/C. These observations demonstrate that with respect to both severity of hypertension and frequency of stroke the phenotypic expression of the SHRSP is (i) either increased or decreased, depending on whether the anionic component of the potassium salt supplemented is, or is not, [Cl.sup.-]; (ii) increased by supplementing [Cl.sup.-] without supplementing [Na.sup.+], and despite supplementing [K.sup.+]; and hence (iii) both selectively [Cl.sup.-]-sensitive and [Cl.sup.-]-determined. The observations suggest that in the SHRSP selectively supplemented with [Cl.sup.-] the likelihood of stroke depends on the extent to which both BP and PRA increase.

The depolarized dog atrial myocytes were analyzed to determine the effects of swelling-induced chloride currents (ICl,swell) on the transmembrane resting potential. The depolarization of dog atrial myocytes led to the activation of ICl,swell which was inhibited by the administration of niflumic acid. The reversal potential for ICl,swell was also shifted by the substitution of extracellular chloride with SCN- ions. Furthermore, the resting membrane was depolarized by the activation of ICl,swell in canine atrial cells.

The modulatory effects of cytostolic Cl- ([Cl-]i) on the voltage-dependent Ca2+ conductance of isolated rat lactotrophs were analyzed to determine the mechanisms that modulate voltage-dependent Ca2+ channel activity. Analysis of voltage-dependent Ca2+ conductance in rodent lactotrophs via microspectrofluometric techniques indicated the role of [Cl-]i in modulating Ca2+ influx. Furthermore, Ca2+ conductance was reduced when Ba2+ replaced Ca2+ as the charge carrier indicating the role of L-type Ca2+ channels in mediating the modulatory effects of [Cl-]i.

The role of depolarizing chloride flux on the activation of voltage-dependent calcium channels was analyzed in rabbit renal afferent arteriolar smooth muscle cells. Analysis of the angiotensin (ANG) II-mediated activation-contraction mechanism in rabbit renal afferent arteriolar smooth muscle cells indicated the role of chloride channel activation in promoting ANG II activity. Furthermore, extracellular chloride was essential for the ability of voltage-dependent Ca2+ channels to induce contractions in afferent arterioles.

The sodium-independent inward Cl pump in rat cardiac ventricular muscle was analyzed by measuring the intracellular Cl concentration with double-barreled microelectrodes. The inward pumping of Cl in rat ventricular muscle was mediated by Na-K-Cl transport and a sodium-independent process labeled as pump III. Pump III contributes to Cl accumulation in ventricular cells by regulating inward and outward Cl currents with Na-K-Cl cotransport in rat cardiac ventricular muscles.

The role of heterologous AE2 anion exchanger on the regulatory volume increase (RVI) of oocytes via functional coupling with endogenous Na+/H+ exchange (NHE) was analyzed in Xenopus oocytes. Analysis of Xenopus oocytes after infusion of N-2-hydroxyethylpiperazine-2-ethanesulfonic acid inhibited hypertonic alkalinization of hydrogen-ion concentration and hypertonic activation of AE2-mediated CL- transport. AE2 anion exchanger also promoted the formation of secondary RVI which is mediated by extracellular Na+ and extracellular Cl-.

Unidirectional maternofetal Cl- transfer in the presence of furonic acids (DIDS) and diphenylamine-2-carboxylate (DPC) were analyzed in isolated dually perfused human placental cotyledons. Quantitative analysis of fetal and maternal Cl- levels in the plasma indicated a unidirectional Cl- transfer from the mother to the fetus. Furthermore, DIDS and DPC affected the small sensitive transcellular components of the maternofetal Cl flux pathway.

The taurine and chloride effluxes of skates were analyzed in isotonic and hyptonic elasmobranch Ringer solutions to determine the pathways of Cl- and taurine transport in the erythrocytes of hypotonically stressed skate. Analysis of skate Cl- and taurine efflux indicated the absence of volume-activated channel activity during erythrocyte-mediated efflux. Furthermore, Cl- efflux from the skate erythrocytes was characterized by a rise in hydrogen-ion concentration indicating an H+-dependent transport pathway.

Mouse inner medullarity collecting duct cells (mIMCD-K2) were analyzed to determine the effects of hyperosmolality on electrogenic Na2+ adsorption (IsupNasubSC) and electrogenic Cl secretion (IsupClsubSC). Acute and chronic hyperosmolality in mIMCD-K2 cell lines reduced basal IsupNasubSc and IsupClsubSC due to elevations in NaCl and urea. Furthermore, the renal transport of urea, water, Na2+ and Cl- across IMCD were dependent on the osmolality of the interstitial fluid.

The role of Cl currents in the membrane capacitance of wild-type cystic fibrosis transmembrane conductance regulators (CFTR) was analyzed by utilizing the Xenopus oocyte expression system. Stimulation of CFTR Cl conductance increased membrane capacitance and Cl current in Xenopus oocytes. CFTR expression in Xenopus oocytes also increased membrane surface area indicating the relationship between CFTR activity and cyclic adenosine monophosphate-regulated membrane trafficking.

The role of short-chain fatty acids (SCFAs) butyrate in Cl- secretion and cyclic adenosine monophosphate (cAMP) levels in rat distal colon was analyzed in mucosal segments from Sprague-Dawley rats and T84 colonic cells. Alterations in hydrogen-ion concentration or SCFA interaction with the basolateral membrane was not affected by the secretory inhibition of SCFA. However, Cl-secretion which is mediated by basal and cAMP in rat distal colon was inhibited by SCFAs in varying levels depending on the degree of cellular metabolism.

Administration of the bradykinin B2 receptor antagonist, HOE-140, increases the absorption of chloride (Cl-) and water in the rat medullary collecting duct (MCD). Though HOE-140 fails to influence the glomerular filtration rate, it reduces the urine flow rate, and the absolute and fractional Cl- excretion. These findings provide evidence for the regulatory role of renal kinins in water and Cl- excretion.

The suppression of the cotransport of Na+ -K+ -Cl- in giant squid axons by the activity of intracellular Cl-, Cl(sub i)-, is not due to the increased activity of the okadaic acid (OKA) sensitive protein phosphatase. When the concentration of intracellular ATP, ATP(sub i), is saturating and Cl(sub i)- concentration is low, OKA, a phosphatase inhibitor, increases the cotransport but has no effect on the cotransport-controlled Cl- influx. However, when the Cl(sub i)- concentration is high, OKA has no effect on the cotransport but increases Cl- influx if the level of ATP(sub i) is low.

Analysis of transepithelial characteristic of monolayers generated by primary cultures of human cystic epithelium reveals that an increase in active chloride anion secretion, controlled by adenosine 3',5'-cyclic monophosphate, causes fluid secretion by cystic epithelial cells. The secretion involves hyperpolarization of the apical-negative V(sub te) and an increase in positive I(sub sc). The experimental procedure of the analysis is discussed

A study of in vitro microperfusion in solutions containing CO2/HCO3 to examine the location of intercalated cell Cl-/base exchange activity and distribution of cortical collecting duct (CCD) intercalated cells confirms that basolateral Cl-/HCO3 exchange activity is present in all CCD intercalated cells. The activity, however, differs in A-type and B-type intercalated cells. Both intercalated cell type and function exhibit axial heterogeneity.

A novel fluorescent indicator, N-(ethoxycarbonylmethyl)-6-methoxy-quinolinium bromide (MQAE) is suitable for the determination of intracellular chloride concentration ((Cl-)i) in primary aortic smooth muscle cell cultures in rat. Analysis of the fluorescent and hydrolytic properties reveals that furosemide reduces both initial Cl- influx rate and (Cl-)i while 4,4'-diisothiocyanato-dihydrostilbene-2,2'-disulfonic acid (H2DIDS) reduces only initial Cl- influx rate. The intracellular Stern-Volmer constant is 25 per M while the average Cl- efflux rate is 0.023 plus or minus 0.004 mM/s.

Minute reductions in sympathetic nerve metabolism arterial pressure and heart rate are caused when cobalt chloride is infused into the rostral ventrolateral medulla (RVLM), in small quantities. Somatosympathetic reflexes are totally obstructed by the infusion revealing that the genesis of considerable vasomotor tone does not require synaptic input to RVLM. Pontine reticular formation (PRF) causes tonic stimulation of spinal sympathetic preganglionic neurons which employ RVLM as a relay station. Input from PRF to RVLM is probably controlled by glutamate.

A study was conducted on the signal transduction process by which K-Cl transporter activity is modulated by cell volume changes using measurements of the time course of K-Cl cotransport activation in red cells. A 10-minute delay was observed in swollen cells with normal volume and Mg prior to the achievement of a final steady-state flux. A delay was also observed following cotransport shrinkage inactivation. In low-Mg cells, a delay in cotransport shrinkage inactivation was also detected. These results are discussed in terms of a three-state model.

A study was conducted on the kinetics of chloride transport through the red cell membrane in 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-bound cells (DIDS-C) using 36Cl- efflux. Results revealed that DIDS-C cell chloride transport kinetics differed from that of control cells in terms of outside apparent affinity to chloride and 4,4'-dinitrostilbene-2,2'-disulfonate, to pH and to dependence on temperature. After maximum DIDS binding, residual transporter number is also much lower than that of control cells. Maximum DIDS binding, however, did not fully inhibit mediated chloride transport.

Regional transport characteristics of porcine gallbladder epithelium bathed in Ringer solution in Ussing chamber were studied. Norepinephrine, salbutamol, isoproterenol and dobutamine were administered and their effects on the Na and Cl transport activity observed. Results of the studies confirm regional differences in basal Cl and Na transport. The results also verified alpha2- and beta2-adrenoceptor mediation across the isolated gallbladder epithelium were also noted

The effects of dexamethasone (Dex) on bioelectric characteristics of tracheal epithelium and sodium efflux were studied in feline and canine tracheal epithelium. Dex treatment and subsequent stimulation with isoproterenol (ISP) and metacholine (MCh) had no significant effects on potential difference (PD), short-circuit current (SCC) measurements and rate constant of sodium efflux. However, a dose-related decrease in MCh-induced glandular RC and ISP-induced PD and SCC. The results suggest that the decrease in fluid secretion during stimulation may be attributed to glucocorticoids.

The role of ATP binding in Cl- conductance in cystic fibrosis was examined using the epithelial cell line T84. Results indicated that addition of cAMP to ATP-depleted cells produced insignificant changes in transepithelial potential and conductance which was reversed by introduction of ATP. The activation of Cl- conductance required phosphorylation which implied that high ATP levels were needed for Cl- conductance. The result suggested an interaction between ATP and the cystic fibrosis transmembrane conductance regulator.

The Cl- conductance of rat epididymal cells was studied by observing its response to adenosine 3'-5'-cyclic monophosphate (cAMP) and ionomycin. Whole-cell patch-clamp analysis revealed varied Cl- conductance properties in response to the substances. cAMP-stimulated Cl- conductance displaayed a direct current-voltage relationship and was independent of time and voltage. The converse was observed for ionomycin-stimulated Cl- conductance which also showed a different anion permselectivity. The findings revealed that epididymal cells are related to cystic fibrosis affected epithelium.

Rabbit proximal convoluted tubules (PCT) were utilized in the identification of the transporters responsible for the chloride efflux across the basolateral cell membrane. Results suggest that the net chloride reabsorption is determined by the sodium-sensitive chloride-bicarbonate antiporter whilethe influence of the the other chloride transporters is negligible. Further, the basolateral chloride efflux is sufficient to account for the trancellular transport of chloride ions in the PCT.

A stilbene derivative, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), momentarily increases the short circuit current in T84 monolayer cell cultures. This represent the extrusion of chloride anion induced by intracellular calcium release. This short circuit current increase is transient and inhibition immediately follows. The effect of DIDS mimics that of thapsigargin, the only difference being the time course within which the response is elicited.

Hyperkalemia, an increase in the amount of potassium ions in the blood, is a common electrolyte imbalance, and may be due to a wide variety of different causes. Among these causes is hypoaldosteronism, which is a reduction in the mineralocorticoid hormone aldosterone, which stimulates the kidney to retain sodium and excrete potassium. Kidney disease may also cause hyperkalemia. In pseudohypoaldosteronism, however, the levels of aldosterone may be normal or elevated, and there is no evidence of kidney disease. In type I pseudohypoaldosteronism, the hyperkalemia results from a defect in the responsiveness of the kidney to aldosterone. A second form of pseudohypoaldosteronism, type II, has been described, and it has been suggested that in this case, the excess of potassium in the blood results from a defect in the transport of chloride ions. Researchers have now reported the cases of three patients, a father and two affected children. In normal individuals, an infusion of sodium ions will result in an increase in potassium in the urine. Sodium ions, however, must always be associated with some counterion, such as bicarbonate, sulfate, or chloride. It is therefore possible to tease apart the physiological effects of sodium ions and chloride ions by comparing, for example, an infusion of sodium bicarbonate with an infusion of sodium chloride. Such a test was performed on the patients in the present study, and it was found that infusion of sodium bicarbonate or sodium sulfate resulted in a normal excretion of potassium, while the infusion of an equivalent amount of sodium chloride did not. The results provide further support to the belief that defective chloride transport may be responsible for type II pseudohypoaldosteronism. Furthermore, the case studies suggest that the defect in chloride transport may be inherited, apparently as an autosomal dominant trait. (Consumer Summary produced by Reliance Medical Information, Inc.)

We investigated the dependence of ANG II ([10.sup.-8] M)-induced constriction of outer medullary descending vasa recta (OMDVR) on membrane potential ([Psi]m) and chloride ion. ANG II depolarized OMDVR, as measured by fully loading them with the voltage-sensitive dye bis[1,3-dibutylbarbituric acid-(5)] trimethineoxonol [Di[BAC.sub.4](3)] or selectively loading their pericytes. ANG II was also observed to depolarize pericytes from a resting value of -55.6 [+ or -] 2.6 to -26.2 [+ or -] 5.4 mV when measured with gramicidin D-perforated patches. When measured with Di[BAC.sub.4](3) in unstimulated vessels, neither changing extracellular [Cl.sup.-] concentration ([[Cl.sup.-]]) nor exposure to the chloride channel blocker indanyloxyacetic acid 94 (IAA-94; 30 [micro]M) affected [Psi]m. In contrast, IAA-94 repolarized OMDVR pretreated with ANG II. Neither IAA-94 (30 [micro]M) nor niflumic acid (30 [micro]M, i mM) affected the vasoactivity of unstimulated OMDVR, whereas both dilated ANG II-preconstricted vessels. Reduction of extra-cellular [[Cl.sup.-]] from 150 to 30 meq/l enhanced ANG II-induced constriction. Finally, we identified a [Cl.sup.-] channel in OMDVR pericytes that is activated by ANG II or by excision into extra-cellular buffer. We conclude that constriction of OMDVR by ANG II involves pericyte depolarization due, in part, to increased activity of chloride channels. medulla; kidney; microcirculation; membrane potential; patch clamp; niflumic acid; indanyloxyacetic acid 94

Nongenomic effect of testosterone on chloride secretion in cultured rat efferent duct epithelia. Am J Physiol Cell Physiol 280: C 1160-C 1167, 2001.--Short-circuit current ([I.sub.sc]) technique was used to investigate the role of testosterone in the regulation of chloride secretion in cultured rat efferent duct epithelia. Among the steroids tested, only testosterone, and to a lesser extent, 5[Alpha]-dihydrotestosterone (5[Alpha]-DHT), reduced the basal and forskolin-induced [I.sub.sc] in cultured rat efferent duct epithelia when added to the apical bathing solution. Indomethacin, a 3[Alpha]-hydroxysteroid dehydrogenase, did not affect the inhibitory effect of 5[Alpha]-DHT. The effect of testosterone occurred within 10-20 s upon application and was dose dependent with apparent [IC.sub.50] value of 1 [micro]M. The effect was abolished by removal of [Cl.sup.-] but not [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] from the normal Krebs-Henseleit solution, suggesting that testosterone mainly inhibited [Cl.sup.-] secretion. The efferent duct was found to be most sensitive to testosterone, while the caput and the cauda epididymidis were only mildly sensitive. Cyproterone acetate, a steroidal antiandrogen, or flutamide, a nonsteroidal antiandrogen, did not block the effect of testosterone on the forskolin-induced [I.sub.sc], nor did protein synthesis inhibitors, cycloheximide, or actinomycin D. However, pertussis toxin, a [G.sub.i] protein inhibitor, attenuated the inhibition of forskolin-induced [I.sub.sc] by testosterone. Testosterone caused a dose-dependent inhibition of forskolin-induced rise in cAMP in efferent duct cells. It is suggested that the rapid effect of testosterone was mediated through a membrane receptor that is negatively coupled to adenylate cyclase via [G.sub.i] protein. The role of nongenomic action of testosterone in the regulation of electrolyte and fluid transport in the efferent duct is discussed. testosterone; chloride secretion; efferent duct

Increased functional cell surface expression of CFTR and [Delta]F508-CFTR by the anthracycline doxorubicin. Am J Physiol Cell Physiol 280: C1031-C1037, 2001.--Cystic fibrosis (CF) is a disease that is caused by mutations within the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most common mutation, [Delta]F508, accounts for 70% of all CF alleles and results in a protein that is defective in folding and trafficking to the cell surface. However, [Delta] F508-CFTR is functional when properly localized. We report that a single, noncytotoxic dose of the anthracycline doxorubicin (Dox, 0.25 [micro]M) significantly increased total cellular CFTR protein expression, cell surface CFTR protein expression, and CFTR-associated chloride secretion in cultured T84 epithelial cells. Dox treatment also increased [Delta]F508-CFTR cell surface expression and [Delta]F508-CFTR-associated chloride secretion in stably transfected Madin-Darby canine kidney cells. These results suggest that anthracycline analogs may be useful for the clinical treatment of CF. trafficking; chloride secretion

Alrefai, W. A., S. Tyagi, F. Mansour, S. Saksena, I. Syed, K. Ramaswamy, and P. K. Dudeja. Sulfate and chloride transport in Caco-2 cells: differential regulation by thyroxine and the possible role of DRA gene. Am J Physiol Gastrointest Liver Physiol 280: G603-G613, 2001.--The current studies were undertaken to establish an in vitro cellular model to study the transport of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] and [Cl.sup.-] and hormonal regulation and to define the possible function of the downregulated in adenoma (DRA) gene. Utilizing a postconfiuent Caco-2 cell line, we studied the [OH.sup.-] gradient-driven [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] and [sup.36][Cl.sup.-] uptake. Our findings consistent with the presence of an apical carrier-mediated [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] exchange process in Caco-2 cells include: 1) demonstration of saturation kinetics [Michaelis-Menten constant ([K.sub.m]) of 0.2 [+ or -] 0.08 mM for [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] and maximum velocity of 1.1 [+ or -] 0.2 pmol [multiplied by] mg [protein.sup.-1] [multiplied by] 2 [min.sup.-1]]; 2) sensitivity to inhibition by DIDS ([K.sub.i] = 0.9 [+ or -] 0.3 [micro]M); and 3) competitive inhibition by oxalate and [Cl.sup.-] but not by nitrate and short chain fatty acids, with a higher [K.sub.i] (5.95 [+ or -] 1 mM) for [Cl.sup.-] compared with oxalate ([K.sub.i] = 0.2 [+ or -] 0.03 mM). Our results also suggested that the [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] and [Cl.sup.-]/[OH.sup.-] exchange processes in Caco-2 cells are distinct based on the following: 1) the [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] exchange was highly sensitive to inhibition by DIDS compared with [Cl.sup.-]/ [OH.sup.-] exchange activity ([K.sub.i] for DIDS of 0.3 [+ or -] 0.1 mM); 2) [Cl.sup.-] competitively inhibited the [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] exchange activity with a high [K.sub.i] compared with the [K.sub.m] for [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII], indicating a lower affinity for [Cl.sup.-]; 3) DIDS competitively inhibited the [Cl.sup.-]/[OH.sup.-] exchange process, whereas it inhibited the [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] exchange activity in a mixed-type manner; and 4) utilizing the RNase protection assay, our results showed that 24-h incubation with 100 nM of thyroxine significantly decreased the relative abundance of DRA mRNA along with the exchange activity but without any change in [Cl.sup.-]/[OH.sup.-] exchange process. In summary, these studies demonstrated the feasibility of utilizing Caco-2 cell line as a model to study the apical [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] and [Cl.sup.-]/[OH.sup.-] exchange processes in the human intestine and indicated that the two transporters are distinct and that DRA may be predominantly a [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] transporter with a capacity to transport [Cl.sup.-] as well. human intestine; anion exchangers; [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] exchange; [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] exchange

Papageorgiou, Panos, Boris E. Shmukler, Alan K. Stuart-Tilley, Lianwei Jiang, and Seth L. Alper. AE anion exchangers in atrial tumor cells. Am J Physiol Heart Circ Physiol 280: H937-H945, 2001.--Intracellular pH homeostasis and intracellular [Cl.sup.-] concentration in cardiac myocytes are regulated by anion exchange mechanisms. In physiological extracellular [Cl.sup.-] concentrations, [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] exchange promotes intracellular acidification and [Cl.sup.-] loading sensitive to inhibition by stilbene disulfonates. We investigated the expression of AE anion exchangers in the AT-1 mouse atrial tumor cell line. Cultured AT-1 cells exhibited a substantial basal [Na.sup.+]-independent [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] (but not [Cl.sup.-]/ [OH.sup.-]) exchange activity that was inhibited by DIDS but not by dibenzamidostilbene disulfonic acid (DBDS). AT-1 cell [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] activity was stimulated two- to threefold by extracellular ATP and ANG II. AE mRNAs detected by RT-PCR in AT-1 cells included brain AE3 (bAE3), cardiac AE3 (cAE3), AE2a, AE2b, AE2c1, AE2c2, and erythroid AE1 (eAE1), but not kidney AE1 (kAE1). Cultured AT-1 cells expressed AE2, cAE3, and bAE3 polypeptides, which were detected by immunoblot and immunocytochemistry. An AE1-like epitope was detected by immunocytochemistry but not by immunoblot. Both bAE3 and cAE3 were present in intact AT-1 tumors. Cultured AT-1 cells provide a useful system for the study of mediators and regulators of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] exchange activity in an atrial cell type. AT-1; band 3; AE1; AE3; chloride-bicarbonate exchange; atrium; heart

Broughman, James R., Kathy E. Mitchell, Roger L. Sedlacek, Takeo Iwamoto, John M. Tomich, and Bruce D. Schultz. [NH.sub.2]-terminal modification of a channel-forming peptide increases capacity for epithelial anion secretion. Am J Physiol Cell Physiol 280: C451-C458, 2001.--A synthetic, channel-forming peptide, derived from the [Alpha]-subunit of the glycine receptor (M2GlyR), has been synthesized and modified by adding four lysine residues to the [NH.sub.2] terminus (N-[K.sub.4]-M2GlyR). In Ussing chamber experiments, apical N-[K.sub.4]-M2GlyR (250 [micro]M) increased transepithelial short-circuit current ([I.sub.sc]) by 7.7 [+ or -] 1.7 and 10.6 [+ or -] 0.9 [micro]A/[cm.sup.2] in Madin-Darby canine kidney and T84 cell monolayers, respectively; these values are significantly greater than those previously reported for the same peptide modified by adding the lysines at the COOH terminus (Wallace DP, Tomich JM, Iwamoto T, Henderson K, Grantham JJ, and Sullivan LP. Am J Physiol Cell Physiol 272: C1672-C1679, 1997). N-[K.sub.4]-M2GlyR caused a concentration-dependent increase in [I.sub.sc] ([k.sub.[1/2]] = 190 [micro]M) that was potentiated two- to threefold by l-ethyl-2-benzimidazolinone. N-[K.sub.4]-M2GlyR-mediated increases [I.sub.sc] were insensitive to changes in apical cation species. Pharmacological inhibitors of endogenous [Cl.sup.-] conductances [glibenclamide, diphenylamine-2-dicarboxylic acid, 5-nitro-2-(3-phenylpropylamino)benzoic acid, 4,4'-dinitrostilben-2,2'-disulfonic acid, indanyloxyacetic acid, and nifiumic acid] had little effect on N-[K.sub.4]-M2GlyR-mediated [I.sub.sc]. Whole cell membrane patch voltage-clamp studies revealed an N-[K.sub.4]-M2GlyR-induced anion conductance that exhibited modest outward rectification and modest time- and voltage-dependent activation. Planar lipid bilayer studies yielded results indicating that N-[K.sub.4]-M2GlyR forms a 50-pS anion conductance with a [k.sub.[1/2]] for [Cl.sup.-] of 290 meq. These results indicate that N-[K.sub.4]-M2GlyR forms an anion-selective channel in epithelial monolayers and shows therapeutic potential for the treatment of hyposecretory disorders such as cystic fibrosis. synthetic peptide; anion conductance; chloride transport; epithelial cells; cystic fibrosis

Mechanisms of chloride transport in thymic lymphocytes. Am J Physiol Renal Physiol 280: F314-F324, 2001.--This study examined mechanisms of [Cl.sup.-] transport in rat lymphocytes under a variety of conditions. Basal intracellular [Cl.sup.-] concentration ([[[Cl.sup.-]].sub.i]) was not different between cells assayed in the presence of [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] or its absence (HEPES). Removal of external [Cl.sup.-] resulted in a fall in [[[Cl.sup.-]].sub.i] and a rapid rise in intracellular pH ([pH.sub.i]). Both [Cl.sup.-] efflux and the rise in [pH.sub.i] were blocked by DIDS or removal of external [Na.sup.+] but were unaffected by furosemide. The mechanisms governing [Cl.sub.-] influx were assessed in cells that had been [Cl.sup.-] depleted for 1 h. Reexposure to [Cl.sup.-] resulted in a rapid rise in [[[Cl.sup.-]].sub.i] that was partially inhibited by pretreatment with DIDS (57%) and partially inhibited by pretreatment with furosemide (45%). Pretreatment with both compounds together completely blocked [Cl.sup.-] influx. [Cl.sup.-] depletion caused a marked increase in [pH.sub.i] that rapidly declined toward normal when the cells were reexposed to [Cl.sup.-]. Preincubation with DIDS completely blocked this decrease in [pH.sub.i]. In contrast, neither removal of [Na.sup.+] nor preincubation with furosemide affected the decline in [pH.sub.i] when the cells were reexposed to [Cl.sup.-]. We conclude that, in thymic lymphocytes, [Cl.sup.-]/[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] (or [Cl.sup.-]/base exchange) regulates both [Cl.sup.-] influx and efflux. [Cl.sup.-] efflux is totally inhibited by DIDS and is mediated by a [Na.sup.+]-dependent [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] exchanger. [Cl.sup.-] influx is partially DIDS sensitive and partially furosemide sensitive and is mediated by both a [Na.sup.+]-independent [Cl.sup.-][MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] exchanger and by a [Na.sup.+]-[K.sup.+]-2[Cl.sup.-] cotransporter. chloride/bicarbonate exchange; sodium-potassium-2 chloride cotransport; stilbenes; furosemide; 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid

Chloride channel function is linked to epithelium-dependent airway relaxation. Am J Physiol Lung Cell Mol Physiol 280: L334-L341, 2001.--We previously reported that substance P (SP) and ATP evoke transient, epithelium-dependent relaxation of mouse tracheal smooth muscle. Since both SP and ATP are known to evoke transepithelial [Cl.sup.-] secretion across epithelial monolayers, we tested the hypothesis that epithelium-dependent relaxation of mouse trachea depends on [Cl.sup.-] channel function. In perfused mouse tracheas, the responses to SP and ATP were both inhibited by the [Cl.sup.-] channel inhibitors diphenylamine-2-carboxylate and 5-nitro-2-(3-phenylpropylamino)benzoate. Relaxation to ATP or SP was unaffected by 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS), and relaxation to SP was unaffected by either DIDS or DNDS. Replacing [Cl.sup.-] in the buffer solutions with the impermeable anion gluconate on both sides of the trachea inhibited relaxation to SP or ATP. In contrast, increasing the gradient for [Cl.sup.-] secretion using [Cl.sup.-]-free medium only in the tracheal lumen enhanced the relaxation to SP or ATP. We conclude that [Cl.sup.-] channel function is linked to receptor-mediated, epithelium-dependent relaxation. The finding that relaxation to SP was not blocked by DIDS suggested the involvement of a DIDS-insensitive [Cl.sup.-] channel, potentially the cystic fibrosis transmembrane conductance regulator (CFTR) [Cl.sup.-] channel. To test this hypothesis, we evaluated tracheas from CFTR-deficient mice and found that the peak relaxation to SP or ATP was not significantly different from those responses in wild-type littermates. This suggests that a DIDS-insensitive [Cl.sup.-] channel other than CFTR is active in the SP response. This work introduces a possible role for [Cl.sup.-] pathways in the modulation of airway smooth muscle function and may have implications for fundamental studies, of airway function as well as therapeutic approaches to pulmonary disease. smooth muscle; substance P; adenosine triphosphate; trachea; mouse

Extracellular UTP stimulates electrogenic bicarbonate secretion across CFTR knockout gallbladder epithelium. Am J Physiol Gastrointest Liver Physiol 279: G132-G138, 2000.--The loss of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated transepithelial HCO[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] secretion contributes to the pathogenesis of pancreatic and biliary disease in cystic fibrosis (CF) patients. Recent studies have investigated [P2Y.sub.2] nucleotide receptor agonists, e.g., UTP, as a means to bypass the CFTR defect by stimulating [Ca.sup.2+]-activated Cl[sup.-] secretion. However, the value of this treatment in facilitating transepithelial [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] secretion is unknown. Gallbladder mucosae from CFTR knockout mice were used to isolate the [Ca.sup.2+]-dependent anion conductance during activation of luminal [P2Y.sub.2] receptors. In Ussing chamber studies, UTP stimulated a transient peak in short-circuit current ([I.sub.sc]) that declined to a stable plateau phase lasting 30-60 min. The plateau [I.sub.sc] after UTP was [Cl.sup.-] independent, HCO[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] dependent, insensitive to bumetanide, and blocked by luminal DIDS. In pH stat studies, luminal UTP increased both [I.sub.sc] and serosal-to-mucosal HCO[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] flux ([J.sup.s[right arrow]m]) during a 30-min period. Substitution of [Cl.sup.-] with gluconate in the luminal bath to inhibit [C1.sup.-]/HCO[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]exchange did not prevent the increase in [J.sup.s[right arrow]m] and [I.sub.sc] during UTP. In contrast, luminal DIDS completely inhibited UTP-stimulated increases in [J.sup.s[right arrow]m and [I.sub.sc]. We conclude that [P2Y.sub.2] receptor activation results in a sustained (30-60 rain) increase in electrogenic HCO3[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] secretion that is mediated via an intracellular [Ca.sup.2+]-dependent anion conductance in CF gallbladder. cystic fibrosis; biliary system; [P2Y.sub.2] receptor; nucleotide receptor; purinoceptor; chloride

CFTR induces the expression of DRA along with [Cl.sup.-]/[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] exchange activity in tracheal epithelial cells. Am J Physiol Cell Physiol 279: C62-C71, 2000.--Thickening of airway mucus and lung dysfunction in cystic fibrosis (CF) results, at least in part, from abnormal secretion of [Cl.sup.-] and [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] across the tracheal epithelium. The mechanism of the defect in [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] secretion is ill defined; however, a lack of apical [Cl.sup.-]/[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] exchange may exist in CF. To test this hypothesis, we examined the expression of [Cl.sup.-]/[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] exchangers in tracheal epithelial cells exhibiting physiological features prototypical of cystic fibrosis [CFT-1 cells, lacking a functional cystic fibrosis transmembrane conductance regulator (CFTR)] or normal trachea (CFT-1 cells transfected with functional wild-type CFTR, termed CFT-WT). Cells were grown on coverslips and were loaded with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, and intracellular pH was monitored. [Cl.sup.-]/[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] exchange activity increased by ~300% in cells transfected with functional CFTR, with activities increasing from 0.034 pH/min in CFT-1 cells to 0.11 in CFT-WT cells (P [is less than] 0.001, n = 8). This activity was significantly inhibited by DIDS. The mRNA expression of the ubiquitous basolateral AE-2 [Cl.sup.-]/ [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] exchanger remained unchanged. However, mRNA encoding DRA, recently shown to be a [Cl.sup.-]/[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] exchanger (Melvin JE, Park K, Richardson L, Schultheis PJ, and Shull GE. J Biol Chem 274: 22855-22861, 1999.) was abundantly expressed in cells expressing functional CFTR but not in cells that lacked CFTR or that expressed mutant CFTR. In conclusion, CFTR induces the mRNA expression of 'downregulated in adenoma' (DRA) and, as a result, upregulates the apical [Cl.sup.-]/[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] exchanger activity in tracheal cells. We propose that the tracheal [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] secretion defect in patients with CF is partly due to the downregulation of the apical [Cl.sup.-]/[MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] exchange activity mediated by DRA. cystic fibrosis transmembrane conductance regulator; cystic fibrosis; trachea; bicarbonate secretion; downregulated in adenoma; chloride ion/bicarbonate exchange