Unidirectional Fluxes Of Chloride Research Articles

Toxoplasma gondii: changes of transepithelial ion transport in infected HT29/B6 cell monolayers.

An in vitro system was established to study the effect of coccidian parasites on ion transport systems in epithelial tissues using HT29/B6, a human colon carcinoma cell line, and Toxoplasma gondii as a model parasite. Ion transport was measured in perfusion chambers 5, 10 and 15 h post-infection using monolayers in which approximately 30% of the cells were parasitized. The infection had rapid effects on the conductance and unidirectional chloride fluxes of infected cell monolayers, which were two to three times higher than those of uninfected HT29/B6 cell monolayers throughout the observation period. However, the chloride net fluxes and short-circuit current were unaffected by the parasites, while the decrease of chloride seromucosal fluxes and conductance after addition of bumetanide were affected by the infection. The unidirectional mannitol fluxes, which correspond with water motion through paracellular pathways, were increased in infected HT29/B6 cell monolayers.

Proton pump activity is required for active uptake of chloride in isolated amphibian skin exposed to freshwater.

Net proton secretion and unidirectional chloride fluxes were measured in isolated skin of toads ( Bufo bufo) and frogs ( Rana esculenta) mounted in an Ussing chamber and exposed to a Ringer's solution on the serosal side and a freshwater-like solution (1-3 mM Cl(-)) on the external side. Active proton secretion was 34.2+/-2.0 pmol.cm(-2).s(-1) ( n=18) in frog skin, and 16.7+/-1.7 pmol.cm(-2).s(-1) ( n=10) in toad skin. Proton secretion by toad skin was dependent on the transepithelial potential ( V(T)), and an amiloride-insensitive short-circuit current was stimulated by exogenous CO(2)/HCO(3)(-), indicating the presence of a rheogenic proton pump. Cl(-) influx was 37.4+/-7.5 pmol.cm(-2).s(-1) ( n=14) in frog skin and 19.5+/-3.5 pmol.cm(-2).s(-1) ( n=11) in toad skin. In toad skin, the mean Cl(-) flux ratio was larger than expected for simple electro-diffusion. In 8 of 11 sets of paired skins, influx was greater than the efflux indicating active uptake of Cl(-). Cl(-) influx in toad skin was unaffected by large perturbations (100-150 mV) of V(T), which was accomplished by adding amiloride to the outer bath under open circuit conditions. A component of the Cl(-) efflux seemed to be dependent on V(T). 4,4'-Diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS; 0.3 mM or 1.3 mM) inhibited Cl(-) influx and, surprisingly, increased Cl(-) efflux in toad skin. Influx and efflux of Cl(-) in toad skin were highly dependent on the external [Cl(-)] in the freshwater range (0.1-4 mM). (36)Cl(-) influx decreased whereas the total Cl(-) efflux increased as a function of external [Cl(-)]. These data indicate the presence of a DIDS-sensitive, electroneutral carrier mechanism with an external binding site for Cl(-). Ethoxzolamide (100 micro M), an inhibitor of carbonic anhydrase, reduced proton secretion and Cl(-) influx in frog skin. Concanamycin A (0.1-10 micro M), a specific vacuolar-type proton pump (V-ATPase) inhibitor, significantly reduced proton secretion in frog skin. In addition, concanamycin A (1 micro M) significantly reduced Cl(-) influx in frog skin. We suggest that the active proton secretion and Cl(-) influx are coupled. We hypothesise that an apical V-ATPase is capable of energising active Cl(-) uptake in fresh water by creating a favourable gradient for an apical HCO(3)(-) exit in exchange for external Cl(-). The data also suggest that a carbonic anhydrase activity provides H(+) and HCO(3)(-) for apically co-expressed proton pumps and Cl(-)/HCO(3)(-) exchangers.

Interleukin 10 modulates ion transport in rat small intestine

BACKGROUND & AIMS: It has been proposed that the anti-inflammatory cytokine interleukin (IL)-10 might be an effective therapeutic agent in the treatment of inflammatory bowel disease. This study examined the effects of human recombinant IL-10 on ileal sodium and chloride transport in Sprague-Dawley rats. METHODS: Unidirectional fluxes of sodium and chloride and tissue electrical parameters were measured under voltage-clamped conditions in ussing chambers. Intracellular levels of adenosine 3',5'-cyclic monophosphate (cAMP) were measured in isolated enterocytes. RESULTS: Jejunal and ileal tissue responded to serosal addition of IL-10 with a transient decrease in short-circuit current reflecting an IL-10-induced increase in net sodium and chloride absorption because of an increase in mucosal to serosal ion movement. The IL-10-induced absorption was not prevented by tetrodotoxin but did show tachyphylaxis. IL-10 reversed, or markedly attenuated, forskolin- and carbachol-induced net chloride secretion. The effects of IL-10 on net secretion were accompanied by a reduction in forskolin-stimulated cAMP levels and a decrease in basal cAMP levels. An additional effect of IL-10 was its induction of bicarbonate secretion only in the presence of secretagogues. CONCLUSIONS: This study shows that IL-10 enhances intestinal electroneutral sodium and chloride absorption, inhibits stimulated chloride secretion, and under some secretory conditions stimulates bicarbonate secretion. (Gastroenterology 1996 Oct;111(4):936-44)

Effect of extracellular ATP and UTP on fluid transport by human nasal epithelial cells in culture.

We have investigated the effect of both adenosine triphosphate (ATP) and uridine triphosphate (UTP) on the fluid transport, transepithelial electric potential difference (PD), and unidirectional chloride flux when applied apically to cultured human surface respiratory epithelial (HSRE) cells in a double-compartment chamber. The effects of ATP and UTP (both 100 microM) were examined in cells either untreated or pretreated with 100 microM amiloride in lactated Ringer's solution. ATP or UTP was added to the apical solution in a 100 microliters final volume. After a 2-h incubation period, the change in fluid transport was measured by weighing the apical fluid. Compared with control, amiloride blocked the fluid absorption by HSRE cells. The addition of ATP or UTP, either alone or after pretreatment with amiloride, induced a similar and significant increase in the apical fluid and chloride flux (P < 0.001 and P < 0.005, respectively). The changes in both fluid transport and chloride flux were accompanied by changes in PD. A blocker for chloride transport, 4,4'-diisothiocyano-2,2'-stilbene disulfonate, at 500 microM significantly blocked the ATP-stimulated fluid transport (P < 0.05) and chloride flux (P < 0.01). These results support the hypothesis that extracellular ATP and UTP increase the fluid transport by respiratory epithelial cells and may be useful in the hydration of mucus and respiratory mucosa.

Characteristics of the transport of oxalate and other ions across rabbit proximal colon

In order to characterize oxalate handling by the P2 segment of the rabbit proximal colon, the fluxes of [14C]oxalate, 22Na+, and 36Cl- were measured in vitro using conventional short-circuiting techniques. In standard buffer the proximal colon exhibited net secretion of Na+ (-2.31 +/- 0.64 mu equiv cm-2 h-1), negligible net Cl- transport, and net secretion of oxalate (-12.7 +/- 1.6 pmol cm-2 h-1). Replacement of buffer Na+ or Cl- abolished net oxalate secretion, while HCO(3-)-free media revealed a net absorption of oxalate (19.3 +/- 4.2 pmol cm-2 h-1) and stimulated NaCl absorption. Mucosal amiloride and dimethylamiloride (1 mM) significantly reduced the unidirectional fluxes of oxalate and enhanced sodium secretion by decreasing JNams. The anion exchange inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS; 0.1 mM, both sides) reduced the unidirectional fluxes of oxalate and chloride. Serosal epinephrine (50 microM) stimulated oxalate absorption (21.3 +/- 6.3 pmol cm-2 h-1) and sodium absorption (5.71 +/- 1.20 mu equiv cm-2 h-1), whereas dibutyryl-cAMP enhanced oxalate secretion (-43.4 +/- 6.9 pmol cm-2 h-1) and stimulated chloride secretion (-7.27 +/- 0.64 mu equiv cm-2 h-1). These results indicate that the P2 segment of the proximal colon possesses (a) secretory as well as absorptive capacities, (b) oxalate fluxes that are mediated by pathways involving Na+, Cl-, HCO3- transport and (c) a net oxalate flux that is sensitive to absorptive and secretory stimuli.

Electrogenic anion absorption in rabbit distal colon.

The mechanisms of anion transport in the rabbit distal colon were investigated in vitro under short-circuit conditions by examining the effects of transport inhibitors (the stilbene derivatives SITS and DIDS) under a variety of conditions. These agents consistently inhibited Jm-sCl: SITS (10(-3) M) reduced both unidirectional chloride fluxes to the same degree and did not alter JnetCl. In contrast, 10(-4) M DIDS had no effect on Js-mCl and had a significant chloride antiabsorptive effect. DIDS had no effect on either tissue cyclic AMP levels or on basal flux of potassium. The effects of SITS and the cyclic AMP-related secretagogue theophylline on Isc were independent. Additionally, there was no significant alteration of intracellular potential difference or apical membrane fractional resistance elicited by SITS during microelectrode impalement of colonic surface epithelial cells. These results suggest a complex mechanism of anion transport in the distal colon, with a component of electrogenic anion absorption inhibited by the stilbenes. The subsequent changes in current, conductance, and chloride fluxes are dependent upon additional, independent anion transport processes. These pharmacologic agents exhibit an antiabsorptive effect, rather than a stimulation of electrogenic chloride secretion.

Cyclic AMP effects on chloride transport and carbonic anhydrase activity in frog skin

Unidirectional (36Cl) chloride fluxes across isolated and short-circuited frog skin were measured, with both sides bathed in low chloride solution. Transepithelial chloride influx was inhibited by exogenous cAMP as well as by substances enhancing its cellular concentration, such as epinephrine, isoproterenol, and 3-isobutyl-1-methylxanthine (IBMX). Epinephrine and isoproterenol addition resulted in an increase of transepithelial chloride outflux, but exogenous cAMP or IBMX had no significant effect on this unidirectional flux. Phenylephrine had no significant effect on influx or outflux. Carbonic anhydrase (CA) activity in extracts obtained from frog skin epithelium was inhibited by pretreatment with IBMX at 4-5 degrees C and prolonged exposure to cAMP at freezing point. cAMP or IBMX alone had no significant effects on CA activity. This catalytic activity was chloride insensitive and was abolished by 0.1 microM acetazolamide. Results suggest a Cl(-)-HCO3- exchange inhibition by cAMP via carbonic anhydrase inactivation. Chloride outflux stimulation by beta-adrenergic agonists does not seem to depend solely on an increase in cAMP concentration.

In vitro studies of theophylline-induced changes in Na, K and Cl transport in hen ( Gallus domesticus) colon suggesting bidirectional, basolateral NnK2Cl cotransport

1. 1. In isolated mucosa from a NaCl-loaded hen theophylline stimulates both unidirectional chloride fluxes (J Cl msand J Cl ms). Conductive and electroneutral exchange processes, besides a bumetanidesensitive, rheogenic process contribute. 2. 2. The bumetanide-sensitive fraction of the theophylline-induced ΔJ Cl ms is sodium-dependent. 3. 3. Incubation in nominally K +-free solutions reduces the bumetanide-sensitive fraction ΔJ Cl ms more than treatment with ouabain. 4. 4. With respect to chloride the bumetanide-sensitive fraction of ΔJ Cl ms has a Hill coefficient of 1.93 ± 0.03, a J max of 12.9 ± 0.2 μ mol/cm 2· hr and a K 1 2 of 73 ± 1 mmol/l . 5. 5. After ouabain treatment ΔJ ms Cl and ΔJ ms Cl are equally sensitive to bumetanide, while ΔJ ms Cl is bumetanide insensitive without ouabain treatment.

Reversed short-circuit current across isolated skin of the toad Bufo arenarum.

Short-circuit current (SCC) across isolated pelvic skin of the toad Bufo arenarum has been shown to be reflected by the algebraic sum of net sodium and chloride transport. After the animals had been maintained in tap water, amiloride--an apical sodium channel blocker--led to a reversal of potential difference (rPD) across this preparation, to which corresponded a reversed short-circuit current (rSCC). Both rSCC and rPD were abolished by dermal treatment of skins with the metabolic inhibitor dinitrophenol, or by omission of chloride ion from the Ringer solution bathing both sides of the skin. There was a significant positive correlation between rSCC and isotopically determined net chloride transport after amiloride. An inhibitory action of amiloride on unidirectional chloride fluxes was detected, but only early after drug addition. rSCC was absent in skins of toads exposed to 110 mmol/l NaCl in tap water during 10 days. Together, our results suggest that amiloride addition--by inhibiting active sodium movement--can in certain conditions reveal the existence of an inward active chloride transport.

Potential dependence of unidirectional chloride fluxes across isolated frog skin

Isolated frog skins were voltage clamped at transepithelial potentials ( V t) ranging from −60 mV to 60 mV to measure transepithelial 36Cl − fluxes from the apical to the basolateral bathing solution ( J 13) and in the opposite direction ( J 31). The potential dependence of fluxes obtained in Na +-free choline Ringer's indicates the presence of conductive components that probably correspond to fluxes through paracellular and cellular pathways, respectively. Rectification of fluxes with reversal of the potential reflects a structural asymmetry, presumably in surface charge density. The data are consistent with a charge density of one negative charge per 280 Å 2 on the apical side. A new model for passive Cl − transport was developed that includes surface charge asymmetry and specifically accounts for the observed variation of conductance with potential. In normal frog Ringer's, J 13 was larger than J 31 at zero potential (active Cl − transport), J 13 rose exponentially with increasing positive potential to reach a maximum at 40 mV (approximately open-circuit), and the predicted partial Cl − conductance exceeded the measured conductance leading to the conclusion that when J 13 is largely driven by Na + transport, much of the coupling occurs via nonconductive pathways. Theophylline stimulates Cl − transport that also occurs via nonconductive pathways as V t becomes more positive.

Concentration dependence of the chloride selfexchange and homoexchange fluxes in human red cell ghosts.

The concentration dependence of the unidirectional chloride flux in human red cell ghosts was studied under selfexchange and under homoexchange conditions. Under selfexchange conditions the intracellular concentration of chloride [Cl]in is equal to the extracellular concentration [Cl]ex and [Cl]in and [Cl]ex are raised concomitantly. Under homoexchange conditions [Cl]in or [Cl]ex were varied separately at a fixed trans-concentration of chloride. The chloride fluxes were calculated from the rate of the tracer efflux and the intracellular chloride. All experiments were executed in isotonic (330 mosM) KCl/K-citrate/sorbitol solutions containing 0-100 mM KCl, 40 mM K-citrate and different concentrations of sorbitol for isoosmotic substitution. The chloride selfexchange and the chloride homoexchange fluxes exhibit a pure saturation kinetics. The halfsaturation constant for the chloride selfexchange was approximately 20 mM, the maximal flux was approx. 3.5 X 10(-4) mol/(min . g cells). The apparent chloride halfsaturation constants from the homoexchange experiments were in the range of 0.9-4.5 mM for the outer and of 5.5-14.5 mM (0 degree C, pH 7.3) for the inner membrane surface, both halfsaturation constants increase with increasing trans-concentrations. At infinite trans-concentrations of chloride, the halfsaturation constant for the outer and the inner membrane surface amounts to approximately 5 mM and approximately 15 mM, respectively. The slope of the double reciprocal plots of flux versus cis-chloride concentration decreases with increasing trans-concentration of chloride. The kinetics of the chloride transport provides evidence for a carrier mediated transport mechanism with a single reciprocating transport site. The translocation of the loaded carrier appears to be much faster than the translocation of the unloaded carrier.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of magnesium on ion transport in short-circuited rabbit terminal ileum.

The effects of MgCl2 on the electrical characteristics and sodium and chloride transport in short-circuited rabbit terminal ileum in vitro were studied. Increasing the magnesium concentration from a basal concentration of 1.1 mmol/l to either 2.5 or 10.3 mmol/l (mucosal or serosal addition) resulted in increases in the unidirectional flux of chloride from serosa to mucosa (JCl sm), short-circuit current (SCC) and transmucosal potential difference (p.d.). Increasing the magnesium concentration from a low basal concentration (0.3 mmol/l) to 10.3 mmol/l (mucosal or serosal addition) abolished net sodium absorption and converted net chloride absorption into net secretion, as a result of a decrease in the unidirectional flux of sodium from mucosa to serosa (JNa ms), and increases in the unidirectional fluxes of sodium and chloride from serosa to mucosa (JNa sm and JCl sm). Increases in SCC and p.d. occurred after mucosal, but not serosal, addition of magnesium. Incubation of stripped ileal mucosa with MgCl2 (10.3 mmol/l) did not result in increased mucosal concentrations of cAMP or cGMP. These data suggest that at least part of the cathartic effect of magnesium salts is likely to result from marked changes in ileal ion transport and provide a rational explanation for the watery diarrhoea commonly seen after the oral administration of magnesium salts to hypomagnesaemic patients.

Mucosal function of the perfused ileum in patients with and without diarrhoea and dumping after vagotomy and pyloroplasty.

Perfusion studies of the terminal ileum were performed in patients who had previously had vagotomy and pyloroplasty performed for peptic ulcer disease and who at follow-up study had either diarrhoea or dumping, or no symptoms (the control group). The aim was to elucidate possible pathophysiological factors contributing to the sequelae. Net movements of water, electrolytes, and sugars, unidirectional fluxes of sodium and chloride, and the transmural electrical potential difference (PD) were measured simultaneously with and without the influence of chenodeoxycholic acid (CDC), 1.0 mmol/l, in the intestinal lumen. In patients without symptoms or with dumping, water and sodium chloride were absorbed during the control perfusion. Addition of CDC reduced the absorption or evoked secretion. In patients with diarrhoea the control perfusion disclosed a large spontaneous ileal secretion that was not influenced by CDC. The secretion was mainly due to a reduced mucosa-to-serosa flux of sodium and chloride and, to a lesser extent, to an increased serosa-to-mucosa flux. The diarrhoea patients showed an increased xylose absorption rate, suggesting an increased interepithelial leakiness. No change in PD was demonstrated. The effect of CDC on the unidirectional fluxes was identical in all three groups of patients. In conclusion, our findings suggest that the basic biological mechanisms are identical (and normal) in all patients, but the ileal epithelium of patients with diarrhoea secretes spontaneously and seems to be prestimulated by some at present unidentified agent.

Characteristics of bicarbonate, sodium, and chloride fluxes in the rabbit corneal endothelium

Unidirectional fluxes of bicarbonate, sodium and chloride were measured across the isolated rabbit corneal endothelium. Bicarbonate and sodium fluxes were measured between 13 and 37°C and were found to be temperature-sensitive. Ouabain (10 −6 m), which inhibits endothelial fluid transport, reduced the net bicarbonate flux by 32% but had no significant effect on sodium fluxes. Amiloride (10 −4 m) did not alter sodium transport, but slightly increased the unidirectional bicarbonate fluxes. Sodium fluxes were unchanged in chloride-free and potassium-free Ringer solutions, and net sodium transport was unaffected in the presence of carbonic anhydrase, acetazolamide, or low (5 m m) bicarbonate Ringer, but was reduced by 45% in bicarbonate-free Ringer. A net chloride flux was found in the same direction (stroma to aqueous) as bicarbonate and sodium. This chloride flux (2·18 μEq/cm 2/hr) was of the same magnitude as bicarbonate but was sodium-independent. These findings suggest that: (1) like bicarbonate and fluid transport, sodium transport is bicarbonate-dependent, temperature-sensitive, and chloride-independent; (2) sodium transport is not stoichiometrically coupled to bicarbonate; and (3) neither sodium nor bicarbonate transport are coupled to that of chloride.

Effect of furosemide on the electrical parameters of frog skin.

When added to the mucosal solution bathing isolated frog skin at concentrations ranging from 5 X 10(-4) to 3 X 10(-3) M, the diuretic furosemide increased both the active transport of sodium and the electrical potential difference across the tissue in a dose-dependent way. The same effect was observed in chloride-free solutions. Mucosal furosemide also decreased the passive unidirectional fluxes of chloride. We believe that as far as electrical parameters are concerned mucosal furosemide has a double effect in frog skin: it increases the active conductance to sodium across the mucosal membrane, thus increasing active transport, and decreases the passive permeability to chloride, thus altering the passive conductance of the skin. The relative increase in short-circuit current was, however, invariably greater than the increase of the active conductance, suggesting the influence of yet a third effect. The effect of mucosal furosemide on active sodium transport was blocked by amiloride (5 X 1-(-5) M) and was independent of vasopressin. Qualitatively the effect was similar to the effect produced by triphenylmethylphosphonium ion.

Permeability Characteristics of Human Jejunum, Ileum, Proximal Colon and Distal Colon: Results of Potential Difference Measurements and Unidirectional Fluxes

In order to assess the passive permeability characteristics of the human intestine in vivo, we measured potential difference in the jejunum, ileum, proximal colon, and distal colon during perfusion of various test solutions that were designed to establish chemical gradients for sodium or chloride, or both or neither. In addition, unidirectional fluxes of sodium and chloride were measured in 30-cm segments of the jejunum and ileum and entire colon during perfusion of balanced electrolyte solution. These studies indicate that there are marked differences in the pathways for passive ion movement in the areas of the intestine studied. In the jejunum, this pathway appears to be highly permeable to both sodium and chloride with modest cation selectivity. In the ileum this pathway is much more cation selective, predominantly because of a relative impermeability to chloride. In the colon, on the other hand, these passive pathways appear to be more anion than cation selective. The implication of these results for normal transport physiology are discussed.

Ion fluxes and electrical characteristics of the short-circuited rat colon in vitro.

The unidirectional fluxes of sodium and chloride across stripped rat colon mucosa were measured and compared with the electrical characteristics of the tissue under voltage-clamped conditions. The relationship between the serosal-mucosal fluxes of the two ions and an imposed potential revealed that the serosal-mucosal flux of sodium was entirely paracellular, whereas there was also a transcellular component of the corresponding chloride flux. In the absence of sodium, the short-circuit current and net chloride flux were abolished; in the absence of chloride, the net sodium flux was reduced but not abolished, and the short-circuit current was unchanged. From an analysis of the effects of the inhibitors, amiloride, theophylline, acetazolamide, furosemide and piretanide, a plausible model was developed to explain the characteristics of these transports. It was proposed that both membranes possess Cl-/HCO-3 antiports, though their sensitivities to inhibitors were different. There is also a Na+/Cl- symport and an electrogenic sodium entry mechanism in the brushborder membrane.

Sodium and chloride transport in the isolated intestine of the earthworm,Lumbricus terrestris(L.)

1. Measurements of electrical potential difference (PD), short-circuit current (SCC) and unidirectional fluxes of sodium and chloride were made across portions of the intestine. Based on the results, the intestine can be divided into at least four physiologically distinct regions. 2. These four physiological regions, designated from anterior to posterior as R I-II, R III A, R III B and R IV, do not completely correspond to the four anatomically distinct regions of the intestine. 3. The PD (serosal side positive) in R I-II, R III A, R III B and R IV is 1.08, 12.4, 5.61 and 31.7 mV, respectively. 4. The SCC in these same regions is 9.9, 50.4, 49.7, and 16.4 micro A cm2, respectively. 5. When short-circuited, net sodium and net chloride fluxes in the above regions are -0.36 and -0.27, 1.46*** and -0.92*, 1.74*** and -0.06 and 1.01*** and 0.07 mumol cm-2 h-1, respectively. Positive fluxes indicate net mucosal to serosal movements and asterisks indicate significant net fluxes (* P less than 0.05, *** P less than 0.001). 6. There is good agreement between the SCC and net sodium transport in R III B. In the other regions of the intestine the ionic basis of the SCC has not been completely explained. 7. The properties of the intestine in vitro appear to make the intestine well suited for the task of conserving sodium, a function which the intestine performs in vivo.

Inhibition by prostaglandins of electrogenic chloride transport across toad ciliary epithelium

Effects of PGE 1 , PGE 2 and PGE 2α on the electrical parameters and unidirectional chloride flux across the isolated toad ciliary epithelium and cyclic-AMP content of the tissue have been investigated. Addition of prostaglandins to the bathing solutions caused a sustained decrease in the short-circuit current without appreciable change in the electrical resistance. The effects of prostaglandin added to the epithelial side were much faster and stronger than when added to the stromal side. The order of potency of the inhibitory action was PGE 1 >PGE 2 ≧PGF 2α , The minimum effect was observed at doses greater than 10 −8 g/ml for PGE 1 and 10 −6 g/ml for PGE 2 and PGF 2α . Addition of Dbc-AMP to the epithelial side caused a similar change in the short-circuit current to those elicited by prostaglandins. Tissue content of cyclic-AMP increased immediately and significantly after the addition of PGE 1 . Unidirectional chloride flux from the stromal to epithelial side was decreased by PGE 1 , while the flux in the opposite direction increased very slightly. Consequently, the net chloride flux toward the epithelial side decreased considerably. These results suggest that prostaglandins exert their inhibitory action on the active chloride transport across the toad ciliary epithelium through the binding with a receptor site at the aqueous-facing membrane, stimulation of the adenylate cyclase activity and elevation of the cellular level of cyclic-AMP. Passive permeability of the epithelium to NaCl appeared to be unaffected by prostaglandins.

Effect of vasopressin on electrical potential difference and chloride transport in mouse medullary thick ascending limb of Henle's loop.

Medullary thick ascending limbs of Henle's loop of the Swiss-Webster mouse were perfused in vitro with an isotonic perfusate and a Ringer's bathing medium. In five studies, addition of a supramaximal concentration of synthetic arginine vasopressin (AVP) to the bathing medium resulted in an increase in electrical potential difference (PD) from 5.0 +/- 1.5 mV, lumen positive, to 10.7 +/- 1.4 mV (P < 0.001). When AVP was removed, the PD returned to 2.6 +/- 0.9 mV (P < 0.001), then increased again to 6.9 +/- 1.7 mV (P < 0.01) when AVP was added a second time. A significant, but submaximal, increase in PD of 2.3 +/- 0.6 MV (P < 0.05) was observed in five medullary thick ascending limbs when AVP was added to the bathing medium at a concentration of 10 microunits/ml. This increase was approximately one-third of the response observed at a concentration of 100 microunits/ml in the same tubule. No further increment in PD was observed in five medullary thick ascending limbs when the AVP concentration was increased from 100 to 1,000 microunits/ml. In seven thick ascendcing limbs, the effect of AVP on PD was reproduced by the addition of 8-[p-chlorophenylthio]-cyclic 3',5'-adenosine monophosphate to the bathing medium at a final concentration of 0.1 mM. AVP increased unidirectional chloride flux from lumen to bath from 29.3 +/- 3.2 to 69.8 +/- 6.2 peq/cm per s (P < 0.001) in spite of an increase in the lumen positive PD from 1.6 +/- 0.5 mV to 7.0 +/- 0.6 mV (P < 0.001). Unidirectional chloride flux from bath to lumen was not affected by AVP. In another series of experiments, net chloride flux increased from 15.6 +/- 3.0 to 41.7 +/- 5.3 peq/cm per s (P < 0.05) after addition of AVP. The effect of AVP on hydraulic water permeability (Lp) was examined by adding raffinose to the bathing medium in both the presence and the absence of AVP. The calculated Lp of 16 +/- 2 nm/s per atm in the absence of AVP, although very low, was significantly different from zero (P < 0.01). However, the Lp did not increase significantly when AVP was added to the bathing medium. These results suggest that AVP has a second site of action in the kidney to increase chloride transport by the medullary thick ascending limb in addition to its well-known effect on the water permeability of the collecting tubule. The former effect would contribute to urinary concentrating ability by increasing the axial osmotic gradient in the renal medulla.