Increase In Transepithelial Conductance Research Articles

Butyrate Protects Porcine Colon Epithelium from Hypoxia-Induced Damage on a Functional Level.

The large intestinal epithelium is confronted with the necessity to adapt quickly to varying levels of oxygenation. In contrast to other tissues, it meets this requirement successfully and remains unharmed during (limited) hypoxic periods. The large intestine is also the site of bacterial fermentation producing short-chain fatty acids (SCFA). Amongst these SCFA, butyrate has been reported to ameliorate many pathological conditions. Thus, we hypothesized that butyrate protects the colonocytes from hypoxic damage. We used isolated porcine colon epithelium mounted in Ussing chambers, incubated it with or without butyrate and simulated hypoxia by changing the gassing regime to test this hypothesis. We found an increase in transepithelial conductance and a decrease in short-circuit current across the epithelia when simulating hypoxia for more than 30 min. Incubation with 50 mM butyrate significantly ameliorated these changes to the epithelial integrity. In order to characterize the protective mechanism, we compared the effects of butyrate to those of iso-butyrate and propionate. These two SCFAs exerted similar effects to butyrate. Therefore, we propose that the protective effect of butyrate on colon epithelium under hypoxia is not (only) based on its nutritive function, but rather on the intracellular signaling effects of SCFA.

Epithelial Barrier Modulation by a Channel Forming Peptide

NC-1059 is a synthetic channel-forming peptide that provides for ion transport across, and transiently reduces the barrier integrity of, cultured epithelial monolayers derived from canine kidney (MDCK cells). Experiments were conducted to determine whether epithelial cells derived from other sources were similarly affected. Epithelial cells derived from human intestine (T-84), airway (Calu-3), porcine intestine (IPEC-J2) and reproductive duct (PVD9902) were grown on permeable supports. Basal short circuit current (Isc) was <3 microA cm(-2) for T-84, IPEC-J2 and PVD9902 cell monolayers and <8 microA cm(-2) for Calu-3 cells. Apical NC-1059 exposure caused, in all cell types, an increase in Isc to >15 microA cm(-2), indicative of net anion secretion or cation absorption, which was followed by an increase in transepithelial conductance (in mS cm(-2): T-84, 1.6 to 62; PVD9902, 0.2 to 51; IPEC-J2, 0.3 to 26; Calu-3, 2.3 to 13). These results are consistent with the peptide affecting transcellular ion movement, with a likely effect also on the paracellular route. NC-1059 exposure increased dextran permeation when compared to basal permeation, which documents an effect on the paracellular pathway. In order to evaluate membrane ion channels, experiments were conducted to study the dose dependence and stability of the NC-1059-induced membrane conductance in Xenopus laevis oocytes. NC-1059 induced a dose-dependent increase in oocyte membrane conductance that remained stable for greater than 2 h. The results demonstrate that NC-1059 increases transcellular conductance and paracellular permeation in a wide range of epithelia. These effects might be exploited to promote drug delivery across barrier epithelia.

ATP and the Response to Aldosterone

It has been known for many years that adenosine 5′-triphosphate (ATP) production is necessary for the stimulation of sodium reabsorption by aldosterone, a hormone that plays a critical role in regulating sodium homeostasis. However, the exact mechanism whereby aldosterone stimulates sodium reabsorption--and the precise role of ATP in mediating its effects--has been unclear. Using scanning ion conductance microscopy to image the apical membranes of A6 renal epithelial cells in monolayer culture, Gorelik et al. observed that aldosterone produced morphological changes consistent with contraction of clusters of cells. This contraction, which resembled that elicited by hypoosmotic stress of the basolateral surface, was associated with loss of the normal architecture of the microvilli. The progressive recruitment of contracted cells paralleled an increase in Na + permeability of the epithelial sodium channel (ENaC, measured as an amiloride-sensitive increase in transepithelial conductance, G t ENaC ). However, aldosterone-mediated contraction was not sensitive to amiloride but was sensitive to phosphatidylinositol 3-kinase (PI3K) inhibition (which also blocked the increase in conductance). Moreover, patch-clamp analysis of contracted and noncontracted cells revealed that only contracted cells showed ENaC activity. Aldosterone also stimulated basolateral release of ATP, and the nonhydrolyzable ATP analog ATPγS stimulated both G t ENaC and cell contraction. In contrast, trapping free ATP by adding glucose and hexokinase to the basolateral solution eliminated the effects of aldosterone on conductance, as did treatment with a P1 receptor antagonist. Thus, the authors propose that aldosterone stimulates the release of ATP, which, through a purinergic mechanism, stimulates autocrine and paracrine contraction, leading to reorganization of the apical membrane and activation of apical ENaC in the early phase of aldosterone action. J. Gorelik, Y. Zhang, D. Sánchez, A. Shevchuk, G. Frolenkov, M. Lab, D. Klenerman, C. Edwards, Y. Korchev, Aldosterone acts via an ATP autocrine/paracrine system: The Edelman ATP hypothesis revisited. Proc. Natl. Acad. Sci. U.S.A. 102 , 15000-15005 (2005). [Abstract] [Full Text]

Colistin interactions with the mammalian urothelium.

Here we describe the effect of colistin on the barrier function of the mammalian urinary bladder epithelium. Addition of colistin to the mucosal solution of the rabbit urinary bladder epithelium (urothelium) resulted in an increase in the transepithelial conductance. The magnitude of the increase in transepithelial conductance was dependent on the membrane voltage, concentration of colistin, and presence of divalent cations in the bath solution. The initial site of action of colistin was at the apical membrane. Colistin increased the membrane conductance only when the apical membrane potential was cell interior negative. The more negative the membrane potential, the larger the conductance increase. The concentration dependence of the conductance increase saturated, suggesting a membrane binding site. Divalent cations decreased the magnitude of the conductance increase. This divalent cation action occurred at two sites: one in competition with colistin for a membrane binding site, and the other by rapidly blocking the induced conductance. At short exposure times, the increase in conductance was reversed by either removing colistin from the bath or changing the voltage so that the apical membrane was cell interior positive. At long exposure times, the increase was only partially reversible by voltage or removal from the bath. This finding suggests that at long exposure times, there is a toxic effect of colistin on the urothelium.

Long-term effects of urea on urothelial barrier function

Under physiologic conditions, the bladder epithelium (urothelium) is bathed on the urine side (mucosal solution) by a solution that has a variable composition but typically is high in potassium, low in sodium, and has a urea concentration that varies between 100 mmol/L to 500 mmol/L. In contrast, the interstitial side (serosal solution) is bathed in a solution whose composition is similar to that of blood. Previous studies have determined the effect of short-term exposure of the urothelium to serosal urea. It was demonstrated that short-term exposure (<10 minutes) of the serosal side of the urothelium to urea (200 mmol/L or 500 mmol/L) resulted in an increase in the apical (urine facing) membrane ion permeability. On removal of the urea the apical membrane, conductance decreased to control values; thus, the urea effect is reversible. Of interest is that the increase in apical membrane permeability by serosal urea can be completely blocked by the simultaneous presence of mucosal urea. The present study investigates the long-term effects of both mucosal and serosal urea on the mammalian urothelium using electrophysiologic techniques. Addition of urea to the serosal solution (after a short delay) resulted in an exponential increase in the transepithelial conductance followed by a plateau phase and ending in a rapid and irreversible increase in conductance. The exponential increase in transepithelial conductance was due to an increase in cell membrane conductance, whereas the irreversible increase in conductance was due to an increase in the paracellular conductance. The duration of the initial delay and plateau phase was inversely related to the concentration of serosal urea. Addition of urea to both the mucosa and serosal sides of the urothelium resulted in an increase in the duration of the delay, a decrease in the magnitude of the exponential increase, and an increase in the duration of the plateau phase. Thus symmetric urea delays the loss of urothelial barrrier function caused by serosal urea. The ability of mucosal urea to delay the onset of the irreversible increase of urothelial conductance allows the urothelium to perform its primary function, which is to act as a barrier to the movement of water, electrolytes, and nonelectrolytes between the urine and plasma.

UREA MODIFIES THE PERMEABILITY OF THE MAMMALIAN UROTHELIUM

This study investigated the effects of mucosal (urine side) and serosal (blood side) urea on the permeability properties of the in vitro mammalian urinary bladder epithelium. The permeability properties of the rabbit urinary bladder epithelium were studied in modified Ussing chambers using electrophysiological techniques. Addition of two molar urea to the mucosal solution did not cause a significant change in the short circuit current (Isc, a measure of the ion transport capacity of the epithelium), or the transepithelial conductance (Gt, a measure of the ability of ions to diffuse across the epithelium). In contrast, addition of 0.5 M urea to the serosal solution caused an increase in Gt of approximately 35 microS/cm.2 as well as an increase in Isc over a 5 minute period. The site of the conductance increase by short-term serosal urea was at the apical membrane and not at the tight junctions. The urea-induced conductance completely reversed upon removal of urea, was non-selective, and the magnitude was voltage dependent. Long term serosal urea (greater than 30 minutes) resulted in an irreversible increase in transepithelial conductance. Mucosal urea altered the time course but not the magnitude of the serosal urea-induced conductance. The ion permeability of the mammalian urinary bladder is increased by serosal urea. At short times the increase is at the apical membrane, while at long times the increase is at the tight junctions. The presence of mucosal urea slows the loss of urothelial barrier function caused by serosal urea.

Effects of Higenamine on Regulation of Ion Transport in Guinea Pig Distal Colon

Effects of higenamine on Na+, K+ and Cl- transport were studied on stripped guinea pig distal colonic mucosa in vitro using Ussing chambers. Addition of 10(-5) M higenamine induced a biphasic change in short circuit current (Isc): a transient increase followed by a long-lasting decrease that was accompanied by an increase in transepithelial conductance (Gt). The initial phase with an increase in Isc was partially inhibited by serosal bumetanide and abolished by mucosal diphenylamine-2-carboxylate, a chloride channel blocker, indicating transient induction of Cl- secretion. The second phase with a decrease in Isc was composed of two effects: the inhibition of the amiloride-sensitive electrogenic Na+ absorption and the stimulation of the bumetanide-sensitive K+ secretion. However, the initial transient increase was not observed at the lower concentration of higenamine (10(-8)-10(-6) M). All the changes in Isc and Gt induced by higenamine were suppressed by the non-selective beta-adrenergic receptor antagonist propranolol and by the beta2-adrenergic receptor antagonist ICI-118,551, but not by the beta1-adrenergic-receptor-selective antagonist atenolol or by the alpha-antagonists phentolamine, prazosin and yohimbine. These results suggest that higenamine inhibits electrogenic Na+ absorption and stimulates electrogenic K+ and Cl- secretion through beta2-adrenergic receptors in guinea pig distal colon.

Eosinophil peroxidase increases membrane permeability in mammalian urinary bladder epithelium

Eosinophil peroxidase (EPO), a cationic protein found in eosinophils, has been reported to be cytotoxic independent of its peroxidase activity. This study investigated with electrophysiological methods whether EPO is toxic to mammalian urinary bladder epithelium. Results indicate that EPO, when added to the mucosal solution, increases apical membrane conductance of urinary bladder epithelium only when the apical membrane potential is cell interior negative. The EPO-induced conductance was concentration dependent, with a maximum conductance of 411 microseconds/cm2 and a Michaelis-Menten constant of 113 nM. The EPO-induced conductance was nonselective for K+ and Cl-. The conductance was partially reversed using voltage but not by removal of EPO from the bulk solution. Mucosal Ca2+ reversed the EPO-induced conductance by a mechanism involving reversible block of the conductance. Prolonged exposure (up to 1 h) to EPO was toxic to the urinary bladder epithelium, as indicated by an irreversible increase in transepithelial conductance. These results suggest that EPO is indeed toxic to urinary bladder epithelium via a mechanism that involves an increase in membrane permeability.

Chorda tympani responses under lingual voltage clamp: implications for NH4 salt taste transduction.

Rat chorda tympani (CT) responses to NH4Cl, ammonium acetate (NH4Ac), and ammonium hippurate (NH4Hp) were obtained during simultaneous current and voltage clamping of the lingual field potential. Although functional and developmental similarities for gustation have been reported for NH4+ and K+ salts, we report here that significant differences are discernible in the CT responses to both salts. Unlike neural responses to KCl, those to NH4Cl are voltage sensitive, enhanced by submucosa negative and suppressed by positive voltage clamp. In this regard, NH4Cl responses are qualitatively similar to NaCl responses; however, the magnitude of NH4Cl voltage sensitivity is significantly less than that of NaCl. The concentration dependence of the CT response to NH4Cl manifests a biphasic nonlinear relationship not observed with KCl or NaCl. Below 0.3 M, the CT response increases as if to approach a saturation value. However, beyond 0.3 M an inflection appears in the CT-concentration curve because of an abrupt increase in CT responses. This kinetic profile is Cl-dependent and is correlated with an increase in transepithelial conductance that displays similar NH4Cl concentration dependence. The biphasic relation to salt concentration is not observed when acetate or hippurate is substituted for Cl-. As with Na+ and K+ salts, less mobile anions than Cl- (Ac- and Hp-) lower the CT responses. However, like Na+ salts, but in contrast to K+ salts, the onset kinetics of CT responses to NH4Ac or NH4Hp remained rapid, even under positive voltage-clamp conditions. Amiloride (100 microM) partially suppresses CT responses within the concentration range of 0.05-0.3 M (48-20% suppression). Amiloride also suppresses the voltage sensitivity of NH4Cl CT responses, but does not eliminate the sensitivity as it does for Na+ salts. In conclusion, the data suggest that taste transduction for NH4 salts is mediated over two NH+ conduction pathways in the taste bud. This is especially evident with NH4Cl, where the CT-concentration curves show two distinct kinetic regimes. Below 0.3 M the saturation with increasing concentration, clamp voltage response dependence, and amiloride sensitivity suggest an apical membrane transduction conductance. Above 0.3 M, the high anion dependence of the response and its amiloride insensitivity indicate participation of the paracellular pathway in transduction.

Evidence for a rapid, direct effect on epithelial monolayer integrity and transepithelial transport in response to Salmonella invasion.

In cultured monolayers of high-resistance Madin-Darby Canine Kidney (MDCK) cells, infection with Salmonella typhimurium SL1344 resulted in a dose- and time-dependent increase in transepithelial conductance (Gt) and short-circuit current (Isc). There was a direct linear relationship between the S. typhimurium-induced increments in Isc and Gt suggesting that this early change in epithelial parameters is, in part, the result of a cellular conductance change most probably at the apical membrane. An additional wild-type S. typhimurium strain, SR11, and an invasion-deficient isogenic mutant SB111 carrying a non-polar mutation in invA were used to confirm that the S. typhimurium-induced change in epithelial electrical parameters is directly linked to the invasion process. The S. typhimurium-induced change in epithelial electrical parameters was markedly attenuated in Na+-free choline medium. Addition of piretanide (10(-4) M, basal side) failed to affect the increased epithelial conductance and Isc after a 40-min incubation with S. typhimurium. NPPB (5x10(-4) M) added to the apical medium reduced the S. typhimurium-stimulated Isc by 28%, but Gt was not significantly reduced. It is unlikely that the S. typhimurium-induced Isc is due to Cl- secretion. Staining of S. typhimurium-infected MDCK I monolayers with TRITC-phalloidin revealed marked alterations of F-actin; diffuse intracellular accumulations of F-actin corresponding to the presence of invading bacteria were observed by 15 min. After 60 min, prominent extrusions of the apical membrane corresponding to previously described "membrane ruffles" were noted. Marked accumulations of perijunctional F-actin in infected cells corresponded to contraction of the perijunctional actin ring at the apical pole. In adjacent cells marked distortion and stretch of the apical surface was evident. The invasion-deficient invA mutant SB111 failed to induce these morphological changes. These data demonstrate that S. typhimurium invasion induces increased transcellular conductance which does not result from stimulation of Cl- secretion but instead appears to be predominantly due to increased Na+ permeability. The increased membrane conductance is coincident with increased transepithelial inulin permeability indicating that the increment in Gt has an additional "paracellular" component. The S. typhimurium-induced alterations in epithelial parameters may be related to "membrane ruffling" and/or to the accompanying changes in cell shape.

Vasopressin activates a chloride conductance in cultured cortical collecting duct cells

In rabbit cortical collecting duct (CCD) cells, arginine vasopressin (AVP) causes a transient increase followed by a sustained depression of transepithelial potential difference (PDte). Mechanisms underlying the decrease in PDte are not well understood. In this study, we used primary cultures of rabbit CCD cells to study effects of AVP. Basolateral addition of AVP caused a dose-dependent increase in transepithelial conductance (Gte) and a corresponding decrease in PDte. A significant effect was observed at 1 pM AVP, and half-maximal response occurred at 30 pM AVP; 1 nM AVP increased Gte and decreased PDte. Replacement of apical Na+ with N-methyl-D-glucamine did not prevent the effect of AVP on Gte, suggesting that it is not mediated by an increase in apical Na+ conductance. Similarly, apical Ba2+ (1 mM) or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS, 0.1 mM) failed to prevent the effect of AVP. On the other hand, 5-nitro-2(3-phenylpropylamino)benzoic acid (0.1 mM) caused partial inhibition, whereas substitution of apical Cl- with gluconate or cyclamate almost completely prevented the AVP-induced increase in Gte. In unidirectional ion-flux studies, 1 nM AVP caused only a modest increase in apical-to-basolateral (A-->BL) flux of 22Na and had no effect on transepithelial flux of 86Rb in either direction. On the other hand, AVP caused a pronounced increase in A-->BL flux and BL-->A flux of 36Cl, resulting in an increased net Cl- absorption. The effect of AVP on Gte could be mimicked by 8-bromo-adenosine 3',5'-cyclic monophosphate (8-bromo-cAMP) and isoproterenol, and effects of AVP and isoproterenol were not additive.(ABSTRACT TRUNCATED AT 250 WORDS)

Polycation-induced enhancement of epithelial paracellular permeability is independent of tight junctional characteristics

The nature of polycation-induced change in transepithelial permeability was investigated in strains I ( tight) and II ( leaky) MDCK epithelial monolayers. Apical exposure to poly( l-lysine) ( PLL, mol. wt. ( MW) ≈ 20 000) induced a dose-dependent increase in transepithelial conductance (G T ) in both strains which correlated with increasing transepithelial flux of extracellular markers (thiourea/inulin) indicating that PLL enhanced paracellular permeability in these epithelia. Coincident with the increase in G T , PLL also induced an inward short circuit current (I sc ) which was associated with the early phase of the increase in G T and may be responsible for part of it. Morphological studies showed that immunofluorescent staining of the tight junction protein, ZO-1, was abolished following PLL exposure. In addition, F-actin staining in monolayers challenged with PLL demonstrated breaks in the zonulae occludentes at the apical surface. PLL had similar effects on monolayers of T 84 and HCT-8 human intestinal cells indicating that polycation action may be general for a range of epithelial types. We conclude that epithelial exposure to polycations results in opening of the paracellular route by mechanisms which are independent of tight junction characteristics.

Bradykinin stimulation of electrogenic ion transport in epithelial layers of cultured human endometrium.

Primary cultures of glandular endometrial epithelial cells grown on permeable supports formed monolayers with a high transepithelial electrical resistance [1096 +/- 83 omega.cm2 (n = 34)] and displayed electrogenic ion transport as demonstrated by an inward short circuit current (Isc; 20 +/- 2 microA/cm2). Bradykinin, 10(-8)-10(-6) M, added to either the basolateral or apical solutions enhanced the inward ISC. The concentration-response curves for bradykinin were bell-shaped in nature. The ISC response was more sensitive to apical addition of bradykinin and the maximum response was also greater with apical bradykinin. The increases in ISC were accompanied by two- to three-fold increases in transepithelial conductance. Apical addition of amiloride, 10(-4) M, reduced the unstimulated ISC by 80%. In the presence of amiloride, the response to both apical and basolateral bradykinin was reduced by > 50% in 8 out of 18 layers, and the mean response was reduced by approximately 25%. The data are consistent with a physiological role for bradykinin in the control of the intrauterine electrolyte environment, mediated in part by enhanced Na+ absorption.

Further evidence for the regulation of the tight junction ion selectivity by cAMP in goldfish intestinal mucosa

It has been reported that cAMP controls the transepithelial Cl− conductance in fish intestine (Bakker, R., Groot, J.A., 1984,Am. J. Physiol.246:G213–G217; Krasny, E.J., Madara, J.L., DiBona, D.L., Frizzell, R.A., 1983,Fed. Proc.42:1100). In both studies, the cAMP effect was interpreted as an increase in tight junction Cl− conductance, because cAMP did not change the membrane potential or membrane resistance ratio. However, the activation of a Cl− conductance in the membranes of a subset of the epithelial cells might be difficult to discern from an increase in tight junction Cl− conductance. Here we report experiments that were designed to distinguish a tight junction Cl− conductance from a membrane Cl− conductance in a subpopulation of the epithelial cells. The effect of hypotonicity on the cAMP-induced increase in transepithelial conductance showed that cAMP-induced conductance is located in series with the lateral intercellular spaces. Transepithelial serosa to mucosa direct current caused an increase in resistance due to so-called transport number effects. Forskolin abolished the transport number effects, indicating that cAMP increases the Cl− conductance of the tight junctions. Increasing cAMP did not change mannitol fluxes, whereas Cl− fluxes more than doubled. Changes in dilution potential and transepithelial resistance demonstrated that the cAMP-induced conductance is specific for Cl− and Br− as opposed to I−, NO 3 − , SO 4 2− and gluconate−. In contranst, cytochalasin D also decreased the transepithelial resistance and dilution potential in Nagluconate Ringer's. This demonstrates that cAMP acts on the tight junctions in a more specific manner than cytochalasin D.

Nasal drug delivery: An in vitro characterization of transepithelial electrical properties and fluxes in the presence or absence of enhancers

The study of the mechanisms of nasal drug absorption and factors affecting nasal drug absorption have been studied in vivo, but to date lack a simple, effective in vitro model system. We describe the use of Ussing chambers to study the transport of substances across, and the effect of chemicals on nasal mucosal tissue. Sheep nasal mucosa mounted in an Ussing chamber maintained viability for up to 8 hours as determined by measuring the electrical properties of the tissue, response to ion transport modifiers and histological assessment. Inulin (0.01 m M), mannitol (0.01 m M). and propranolol (0.008 m M.) were transported from the mucosal (m) to the serosal (s) side of the tissue at rates of 0.079, 0.249 and 1.02 nmol/cm 2/h, respectively. Transport from s-to-m and m-to-s occurred at equivalent rates, consistent with a passive transport mechanism. The rate of transport of propranolol was directly proportional to concentration over a range of 0.008 to 0.8 m M. The bile salt, deoxycholate, added to the mucosal bathing solution at 1 %, caused an increase in transepithelial conductance and irreversible tissue damage (cell necrosis). At 0.1%, deoxycholate reversibly increased transepithelial conductance (absence of cell necrosis) and enhanced the transport of mannitol and inulin 10–20 fold. These results demonstrate that the Ussing chamber provides a useful technique to investigate the nasal transport of drugs and allows evaluation of the effects of absorption enhancers on both absorption and tissue integrity.

Effect of theophylline on the apical sodium and chloride permeabilities of amphibian skin.

1. The effects of theophylline (1 mmol/l) on the sodium transport (short-circuit current, Isc) and transepithelial conductance (Gtotal) through toad (Bufo viridis) and frog (Rana temporaria and Rana esculenta) skin were investigated. 2. In toad skin incubated with nitrate Ringer solution on the apical side, theophylline induced an increase in Isc similar to that in frog skin bathed with chloride Ringer solution. 3. The increase in Isc could be attributed to recruitment of sodium channels, without affecting the single-channel current. 4. Chloride-bathed toad skin responded to theophylline with a large increase in transepithelial conductance, in addition to the increased Isc. 5. Chloride replacement by nitrate eliminated the effect of theophylline on the conductance increase, but the Isc response was even larger. 6. The results are discussed in relation to the localization of the cellular chloride pathway to the mitochondria-rich cells.

Effect of Extracellular Calcium Depletion on Epithelial Structure and Function in Rabbit Ileum: A Model for Selective Crypt or Villus Epithelial Cell Damage and Suggestion of Secretion by Villus Epithelial Cells

The epithelium of mammalian small intestine can be conveniently divided into two compartments: the crypt and the villus. These compartments are not only structurally distinct but also may be functionally distinct with regard to sodium and chloride ion transport. Villus epithelial cells are thought to be responsible for electrolyte absorption and crypt cells for secretion of ions and water. We show that exposure of rabbit ilea] mucosal sheets in vitro to bathing solutions depleted of calcium with 1 mM ethyleneglycol-bis (β-aminoethylether)-N,N'-tetraacetic acid on either the mucosal or the serosal surface produces selective damage of one epithelial compartment while leaving the other structurally intact. Thus these preparations represent a potential in vitro model for studying the relative contribution of crypt and villus epithelium to net transepithelial active ion transport. The effects of selective mucosal or serosal extracellular calcium depletion on epithelial structure were studied in detail using light and electron microscopy and freeze-fracture techniques. The functional significance of the structural abnormalities produced in these models was evaluated by measuring unidirectional sodium and chloride ion fluxes and serosal theophylline-dependent and mucosal glucose-dependent increases in short-circuit current. Serosal depletion of extracellular calcium produced diffuse damage to crypt epithelial cells, which consisted of vacuolization, detachment from the basement membrane, and dissolution of tight junctions, but which produced no detectable structural damage to villus epithelial cells. These structural alterations produced a decrease in basal shortcircuit current, an increase in transepithelial conductance and in net sodium and chloride ion absorption, no effect on the glucose-induced increase in short-circuit current and a significant decrease in the theophylline-induced short-circuit current response. In contrast, mucosal depletion of extracellular calcium resulted in vacuolization of a large portion of villus epithelial cells with concomitant detachment from the basement membrane and tight junction dissolution, but produced no structural defect in crypt epithelial cells. These alterations were associated with an increase in transepithelial conductance, no significant change in basal net sodium and chloride ion transport, and significant decreases in both glucose and theophylline-induced short-circuit current response. Furthermore, the decrease in theophylline-induced short-circuit current responses seen in tissues with selective villus damage but structurally intact crypts was at least as great as that seen with selective crypt damage. These data support the concept that crypt epithelium actively secretes and villus epithelium actively absorbs sodium and chloride ions. In addition, however, they suggest that a population of epithelial cells on the intestinal villi may also have the capacity to secrete electrolytes.

Kinins stimulate net chloride secretion by the rat colon.

1 Short circuit current (SCC), transepithelial conductance and ion fluxes were measured across the isolated descending colon of the rat in response to bradykinin or kallidin. 2 Kinins added to the serosal bath caused immediate increases in SCC but were ineffective when added to the mucosal bath. Increases in SCC were accompanied by significant increases in transepithelial conductance. Threshold kinin concentration was 0.5nM and maximal increases were seen at 50-100 nM. 3 A rat glandular kallikrein (7nM) or mellitin (2 microM) also increased SCC if added to the serosal bath. 4 Responses to kinins were unaffected by mucosal amiloride (100 micron) but attenuated or blocked by serosal frusemide (100 microM), indomethacin (1 microM) or mepacrine (50 microM). 5 Replacement of chloride ion in the serosal bath by gluconate and sulphate ions abolished responses to kinins which reappeared after chloride re-addition. 6 Measurement of 36Cl, 22Na and 86Rb fluxes across the tissue showed that the kinin-induced increase in SCC resulted principally from increased net chloride secretion. Effects upon 22Na or 86Rb flux were minimal and made no contribution to the current responses observed in this tissue. 7 The results prove that kinins stimulate net chloride secretion in the rat colon, most probably via a prostaglandin-dependent pathway.