Transepithelial Solute Research Articles

Human KCC2a Protein Expression and Phosphorylation is Altered by Elimination of the Second Cryo-EM Identified Chloride Binding Site

Background: Potassium-chloride cotransporters (KCCs) are responsible for the symport of Cl- with K+ in an electroneutral manner. They play important roles in numerous crucial physiological processes, from cell volume regulation to transepithelial solute transport, intracellular ion homeostasis and regulation of GABA neurotransmission. Under isotonicity, except for KCC2, KCCs remain inhibited due to phosphorylation of residues located in cytoplasmic domains, whereas in response to hypotonic cell swelling, dephosphorylation of these residues stimulates transport activity. After several decades of studies, new breakthroughs with the cryo-EM structural determination of KCCs have deepened our understanding of their molecular basis and transport function, including architecture, ion binding sites, and coupled ion transport. In the present study, we mutated and analyzed the two chloride-binding sites in human KCC2a recently reported by cryo-EM to determine whether the phosphorylation status is modified upon their removal. Methods: Xenopus laevis oocytes and HEK-293 cells were either microinjected or transfected with human KCC2a wild type (WT) or chloride-binding site mutants, G136/V137/I138A (S1-Cl), and G436/I437/M438/Y592A (S2-Cl). KCC2a phosphorylation status in response to isotonic and hypotonic conditions was evaluated by immunoblot analysis of regulatory phosphorylation sites pThr926 and pThr1030. In parallel, phosphorylation status of SPAK/OSR1 activating regulatory site pSer373/pSer325 was analyzed under the same conditions. Results: Elimination of either chloride-binding site induced changes in total expression of KCC2a. Removal of S1-Cl slightly increased total KCC2a expression, whereas lack of S2-Cl showed a significant reduction of total protein in Xenopus laevis oocytes and HEK-293 cells. Phosphorylation of regulatory Thr1030 in S2-Cl mutant is significatively decreased on both biological models, even under isotonic inhibitory (phosphorylated) conditions. pThr1030 on S1-Cl mutant behaves similarly to WT KCC2a. Phosphorylation of regulatory SPAK kinase is not affected on both mutants. Conclusions: Our results suggest that while both sites affect KCC2a total expression, only the S2-Cl appears to be affecting KCC2a activating response. It appears S2-Cl mutation dysregulates SPAK signaling pathway, since the kinase osmotic response remains unchanged on presence of KCC2a chloride binding mutants. AM INCar 22-1345 and PdlH UNAM Papiit IN222320 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

E-cigarette constituents propylene glycol and vegetable glycerin decrease glucose uptake and its metabolism in airway epithelial cells in vitro.

Electronic nicotine delivery systems, or e-cigarettes, utilize a liquid solution that normally contains propylene glycol (PG) and vegetable glycerin (VG) to generate vapor and act as a carrier for nicotine and flavorings. Evidence indicated these “carriers” reduced growth and survival of epithelial cells including those of the airway. We hypothesized that 3% PG or PG mixed with VG (3% PG/VG, 55:45) inhibited glucose uptake in human airway epithelial cells as a first step to reducing airway cell survival. Exposure of H441 or human bronchiolar epithelial cells (HBECs) to PG and PG/VG (30–60 min) inhibited glucose uptake and mitochondrial ATP synthesis. PG/VG inhibited glycolysis. PG/VG and mannitol reduced cell volume and height of air-liquid interface cultures. Mannitol, but not PG/VG, increased phosphorylation of p38 MAPK. PG/VG reduced transepithelial electrical resistance, which was associated with increased transepithelial solute permeability. PG/VG decreased fluorescence recovery after photobleaching of green fluorescent protein-linked glucose transporters GLUT1 and GLUT10, indicating that glucose transport function was compromised. Puffing PG/VG vapor onto the apical surface of primary HBECs for 10 min to mimic the effect of e-cigarette smoking also reduced glucose transport. In conclusion, short-term exposure to PG/VG, key components of e-cigarettes, decreased glucose transport and metabolism in airway cells. We propose that this was a result of PG/VG reduced cell volume and membrane fluidity, with further consequences on epithelial barrier function. Taking these results together, we suggest these factors contribute to reduced defensive properties of the epithelium. We propose that repeated/chronic exposure to these agents are likely to contribute to airway damage in e-cigarette users.

Axial compartmentation of descending and ascending thin limbs of Henle's loops

In the inner medulla, radial organization of nephrons and blood vessels around collecting duct (CD) clusters leads to two lateral interstitial regions and preferential intersegmental fluid and solute flows. As the descending (DTLs) and ascending thin limbs (ATLs) pass through these regions, their transepithelial fluid and solute flows are influenced by variable transepithelial solute gradients and structure-to-structure interactions. The goal of this study was to quantify structure-to-structure interactions, so as to better understand compartmentation and flows of transepithelial water, NaCl, and urea and generation of the axial osmotic gradient. To accomplish this, we determined lateral distances of AQP1-positive and AQP1-negative DTLs and ATLs from their nearest CDs, so as to gauge interactions with intercluster and intracluster lateral regions and interactions with interstitial nodal spaces (INSs). DTLs express reduced AQP1 and low transepithelial water permeability along their deepest segments. Deep AQP1-null segments, prebend segments, and ATLs lie equally near to CDs. Prebend segments and ATLs abut CDs and INSs throughout much of their descent and ascent, respectively; however, the distal 30% of ATLs of the longest loops lie distant from CDs as they approach the outer medullary boundary and have minimal interaction with INSs. These relationships occur regardless of loop length. Finally, we show that ascending vasa recta separate intercluster AQP1-positive DTLs from descending vasa recta, thereby minimizing dilution of gradients that drive solute secretion. We hypothesize that DTLs and ATLs enter and exit CD clusters in an orchestrated fashion that is important for generation of the corticopapillary solute gradient by minimizing NaCl and urea loss.

Molecular characterization of Slc26a3 and Slc26a6 anion transporters in guinea pig pancreatic duct

HCO3 in pancreatic juice arises from the pancreatic duct cells. Secretin stimulates HCO3 secretion via a mechanism that involves activation of the adenylate cyclase pathway, activation of both basolateral K channels and the apical CFTR Cl channel, and stimulation of an apical Slc26mediated Cl/HCO3 exchanger (1). The Slc26 anion exchangers, Slc26a3 and Slc26a6, have both been localized to the apical membrane of human pancreatic duct and mouse pancreatic duct although their functional roles in pancreatic HCO3 secretion remain uncertain. The high rates of pancreatic bicarbonate secretion shared by guinea pig (gp) and human, in contrast to the low rates in mouse, prompted us to study the functional characteristics of gp Slc26 anion transporters. Furthermore, recent data from isolated guinea pig pancreatic duct experiments demonstrated that during stimulated HCO3 secretion an apical Slc26a6-like Cl/HCO3 exchanger is functionally coupled with CFTR (2). This study prompted us to clone cDNAs encoding Slc26a3 and Slc26a6 polypeptides from the gp pancreas and to compare their functional characteristics in Xenopus oocytes with those of their mouse and human orthologs (3-5), and with those of luminal Cl/HCO3 exchange measured in isolated gp pancreatic interlobular ducts (2). Whereas gp Slc26a6 mediated Cl/Cl exchange and bidirectional exchange of Cl for oxalate and HCO3, gp Slc26a6 mediated only sulfate influx but not sulfate efflux in preliminary experiments. Gp Slc26a3 mediated robust Cl/Cl exchange and minimal oxalate efflux in exchange for Cl, but did not mediate detectable influx of oxalate or sulfate. Measurement of intracellular pH by BCECF ratio fluorimetry in HEK cells transfected with Slc26a6 or in Slc26a3-expressing Xenopus oocytes during removal and restoration of Clo in the presence of HCO3 indicated that both gp Slc26a6 and Slc26a3 function as Cl/HCO3 exchangers. These substrate specificity experiments reveal that whereas Slc26a3 functions predominantly as a Cl/HCO3 exchanger, Slc26a6 can exchange a wider range of substrates, functioning as a Cl/HCO3 exchanger or a Cl/anion exchanger. This finding allows for the possibility that Slc26a6 may have alternative roles in transepithelial solute transport in the pancreatic duct. The apparent affinity of gp Slc26a6 for extracellular Cl (K1/2) was 2 mM during Cli/Clo exchange and 30 mM during C-oxalatei/Clo exchange. These experiments reveal that binding and/ or translocation rates of extracellular Cl with respect to gp slc26a6 is/are dependent on the internal transanion substrate and show species-specificity (5). The high affinity for extracellular Clo suggests that even when duct luminal Cl is low, such as might occur with passage of pancreatic juice along the duct lumen, Slc26a6-mediated Cl/HCO3 exchange would still be active. Whereas gp Slc26a6-mediated isotopic flux was moderately sensitive to DNDS (500 μM), niflumic acid (100 μM) and DIDS (500 μM), gp Slc26a3 was minimally sensitive to inhibition by DIDS and DNDS but strongly sensitive to niflumic acid. These pharmacological data, combined with EXPANDED ABSTRACT

Gating of human ClC‐2 chloride channels and regulation by carboxy‐terminal domains

Eukaryotic ClC channels are dimeric proteins with each subunit forming an individual protopore. Single protopores are gated by a fast gate, whereas the slow gate is assumed to control both protopores through a cooperative movement of the two carboxy-terminal domains. We here study the role of the carboxy-terminal domain in modulating fast and slow gating of human ClC-2 channels, a ubiquitously expressed ClC-type chloride channel involved in transepithelial solute transport and in neuronal chloride homeostasis. Partial truncation of the carboxy-terminus abolishes function of ClC-2 by locking the channel in a closed position. However, unlike other isoforms, its complete removal preserves function of ClC-2. ClC-2 channels without the carboxy-terminus exhibit fast and slow gates that activate and deactivate significantly faster than in WT channels. In contrast to the prevalent view, a single carboxy-terminus suffices for normal slow gating, whereas both domains regulate fast gating of individual protopores. Our findings demonstrate that the carboxy-terminus is not strictly required for slow gating and that the cooperative gating resides in other regions of the channel protein. ClC-2 is expressed in neurons and believed to open at negative potentials and increased internal chloride concentrations after intense synaptic activity. We propose that the function of the ClC-2 carboxy-terminus is to slow down the time course of channel activation in order to stabilize neuronal excitability.

Primary kidney proximal tubule cells.

Primary rabbit kidney epithelial cell cultures can be obtained that express renal proximal tubule functions. Toward these ends, renal proximal tubules are purified from the rabbit kidney by the method of Brendel and Meezan. To summarize, each kidney is perfused with iron oxide, which becomes associated with glomeruli. The renal cortex is sliced and homogenized to liberate nephron segments. Renal proximal tubules and glomeruli are purified by sieving. The glomeruli, covered with iron oxide, are removed using a magnet. After a brief collagenase treatment (to disrupt basement membrane), the tubules are plated in hormonally defined serum-free medium supplemented with 5 micro g/mL bovine insulin, 5 microg/mL human transferrin, and 5 x 10(-8) M hydrocortisone. After 5-6 d of incubation, confluent monolayers are obtained that possess multicellular domes, indicative of their capacity for transepithelial solute transport.

PDZ proteins retain and regulate membrane transporters in polarized epithelial cell membranes

The plasma membrane of epithelial cells is subdivided into two physically separated compartments known as the apical and basolateral membranes. To obtain directional transepithelial solute transport, membrane transporters (i.e., ion channels, cotransporters, exchangers, and ion pumps) need to be targeted selectively to either of these membrane domains. In addition, the transport properties of an epithelial cell will be maintained only if these membrane transporters are retained and properly regulated in their specific membrane compartments. Recent reports have indicated that PDZ domain-containing proteins play a dual role in these processes and, in addition, that different apical and basolateral PDZ proteins perform similar tasks in their respective membrane domains. First, although PDZ-based interactions are dispensable for the biosynthetic targeting to the proper membrane domain, the PDZ network ensures that the membrane proteins are efficiently retained at the cell surface. Second, the close spatial positioning of functionally related proteins (e.g., receptors, kinases, channels) into a signal transduction complex (transducisome) allows fast and efficient control of membrane transport processes.

Electrolyte and fluid transport across corneal, conjunctival and lens epithelia

All ocular epithelia examined to date transport fluid as a consequence of a sufficiently high water permeability bestowed by endogenous water channels (aquaporins) and transepithelial solute movement due to active transport mechanisms. This article provides a synopsis of the current understanding of electrolyte and fluid transport across corneal, conjunctival and lens epithelia.

The role of reactive oxygen species in the regulation of tubular function

The phrase reactive oxygen species covers a number of molecules and atoms, including the quintessential member of the group, O2-; singlet oxygen; H2O2; organic peroxides; and OONO-. While nitric oxide (NO) is also technically a member of the reactive oxygen species family, it is generally considered with a different class of compounds and will not be considered here. To our knowledge, there are currently no published data reporting the effects of reactive oxygen species on net transepithelial flux in the proximal nephron. However, there is evidence that OONO- regulates Na+/K+ adenosine triphosphatase (ATPase) activity as well as paracellular permeability. While it is easy to speculate that such an effect on the pump would decrease net transepithelial solute and water reabsorption, one cannot do so without knowing how other transporters are affected. O2- stimulates NaCl absorption by the thick ascending limb by activating protein kinase C and blunting the effects of NO. The effects of O2- on thick ascending limb NaCl absorption may be important for the initiation of salt-sensitive hypertension. To our knowledge, there are no published data concerning the role of reactive oxygen species in the regulation of solute absorption in either the distal convoluted tubule or the collecting duct. However, OONO- inhibits basolateral K+ channels in the cortical collecting duct, although the net effect of such inhibition is unknown. While the regulation of tubular transport by reactive oxygen species is important to overall salt and water balance, we know very little about where and how these regulators act along the nephron.

Identification of a Heteromeric Interaction That Influences the Rectification, Gating, and pH Sensitivity of Kir4.1/Kir5.1 Potassium Channels

Heteromultimerization between different potassium channel subunits can generate channels with novel functional properties and thus contributes to the rich functional diversity of this gene family. The inwardly rectifying potassium channel subunit Kir5.1 exhibits highly selective heteromultimerization with Kir4.1 to generate heteromeric Kir4.1/Kir5.1 channels with unique rectification and kinetic properties. These novel channels are also inhibited by intracellular pH within the physiological range and are thought to play a key role in linking K+ and H+ homeostasis by the kidney. However, the mechanisms that control heteromeric K+ channel assembly and the structural elements that generate their unique functional properties are poorly understood. In this study we identify residues at an intersubunit interface between the cytoplasmic domains of Kir5.1 and Kir4.1 that influence the novel rectification and gating properties of heteromeric Kir4.1/Kir5.1 channels and that also contribute to their pH sensitivity. Furthermore, this interaction presents a structural mechanism for the functional coupling of these properties and explains how specific heteromeric interactions can contribute to the novel functional properties observed in heteromeric Kir channels. The highly conserved nature of this structural association between Kir subunits also has implications for understanding the general mechanisms of Kir channel gating and their regulation by intracellular pH.

Effect of backleak in nephron dynamics

The effect of transepithelial solute, i.e., sodium chloride, backleak is taken into consideration in the spatiotemporal evolution of the chloride concentration along the thick ascending limb of a single nephron. The importance of the mechanism and of its mathematical modeling is argued on physiological grounds. The backleak "strength" is found to significantly modify the threshold for the appearance of temporally oscillatory behavior in the chloride concentration in the thick ascending limb which has previously been experimentally observed in normotensive rats.

CAMP stimulates the in vitro proliferation of renal cyst epithelial cells by activating the extracellular signal-regulated kinase pathway

: Cellular proliferation is a key factor in the enlargement of renal cysts in autosomal dominant polycystic kidney disease (ADPKD). We determined the extent to which adenosine 3':5'-cyclic monophosphate (cAMP) may regulate the in vitro proliferation of cyst epithelial cells derived from human ADPKD cysts. : Epithelial cells from cysts of individuals with ADPKD and from normal human kidney cortex (HKC) of individuals without ADPKD were cultured. The effects of agonists and inhibitors on the rate of cellular proliferation and the activation of extracellular signal-regulated kinase (ERK1/2) were determined. : 8-Br-cAMP (100 micromol/L) stimulated the proliferation of cells from eight different ADPKD subjects to 99.0% above baseline; proliferation was inhibited by protein kinase A (PKA) antagonists H-89 (97%) and Rp-cAMP (90%). Forskolin (10 micromol/L), which activates adenylyl cyclase, increased proliferation 124%, and receptor-mediated agonists arginine vasopressin, desmopressin, secretin, vasoactive intestinal polypeptide, and prostaglandin E2 stimulated proliferation 54.2, 56.3, 46.7, 37.1, and 48.3%, respectively. The mitogen extracellular kinase (MEK) inhibitor PD98059 completely inhibited ADPKD cell proliferation in response to cAMP agonists, but genistein, a receptor tyrosine kinase inhibitor, did not block cAMP-dependent proliferation. cAMP agonists increased the activity of ERK above control levels within five minutes. In contrast to ADPKD, proliferation and ERK activity of cells derived from normal HKC were not stimulated by cAMP agonists, although electrogenic Cl- secretion was increased by these agonists in both ADPKD and HKC cell monolayers. : We conclude that cAMP agonists stimulate the proliferation of ADPKD but not HKC epithelial cells through PKA activation of the ERK pathway at a locus distal to receptor tyrosine kinase. We suggest that the adenylyl cyclase signaling pathway may have a unique role in determining the rate of cyst enlargement in ADPKD through its actions to stimulate cellular proliferation and transepithelial solute and fluid secretion.

Different size limitations for increased transepithelial paracellular solute flux across phorbol ester and tumor necrosis factor‐treated epithelial cell sheets

Different size limitations for increased transepithelial paracellular solute flux across phorbol ester and tumor necrosis factor‐treated epithelial cell sheets

Interaction of human spermatozoa with the zona pellucida of oocyte: development of the acrosome reaction.

Mammalian spermatozoa are not able to fertilize an oocyte upon ejaculation. To gain fertilizing ability, spermatozoa must, either in vivo or in vitro, undergo a process termed capacitation. Since a reliable marker for capacitation does not exist, it is considered that this process is completed when the spermatozoa are able to undergo acrosomal x intestinal mucosa which might require tandem signaling events for this process is the presence of a vascular countercurrent arrangement in the subepithelial compartment. As happens for absorbed solutes, this countercurrent phenomenon may distort transepithelial solute gradient. For example, perfusion of mammalian intestinal loops in vivo with solutions containing fMLP was previously found to induce neutrophil attachment to endothelial cells and structurally defined endothelial activation, but failed to elicit directed migration across the lamina propria (Madara, unpublished observations), suggesting that directed migration may require a more stable gradient than that afforded by the usual soluble signals. For example, once present in inflamed tissue, IL-8 is likely to retain its biological activity for several hours, as shown by local intradermal administration in animals and humans. In contrast to Il-8, chemokines such as fMLP or LTB4 are degraded rapidly by oxidation or hydrolysis. Fertilization is a very complex phenomenon, involving sequential interactions between the fertilizing spermatozoon and cumulus oophorus, ZP, and oolemma. The AR may be playing a key role in penetration of spermatozoa through these egg vestments.

Regulation of the intracellular calcium concentration in epithelial cells.

Epithelia, like other vertebrate cells, are equipped with an extensive network of signaling pathways, involved in the regulation of a large variety of distinct cellular functions. Amongst many the intracellular Ca2+ [Ca2+]i signal is the most common one. On the one hand, [Ca2+]i is involved in regulation functions of general cell physiology, such as the control of cell volume, cell growth or differentiation. On the other hand, [Ca2+]i controls epithelia-specific functions like the regulation of transepithelial solute transport. This short overview addresses the various aspects of Ca2+ signaling in epithelia. It is emphasized that [Ca2+]i is a spatially restricted signal, permitting the distinct regulation of one cellular response. Evidence will be discussed indicating that the functional epithelial polarity is paralleled by polarized membrane receptors and polarized [Ca2+]i signals, regulating the apical and basolateral membranes differentially.

Taurine efflux is a cell volume regulatory process in proximal renal tubules from the teleost Carassius auratus.

The potential role of taurine transport associated with volume regulation in renal tissue and isolated proximal renal tubules was studied in the teleost Carassius auratus (goldfish). The cellular taurine content in renal tissue fragments incubated in isosmotic solution (290 mOsm) (7.8 +/- 0.9 (SD) micromol g wet wt(-1)) decreased by 60% following exposure to hyposmotic medium (100 mOsm). The rate coefficient for [14C]taurine efflux in renal tissue and in isolated proximal renal tubules was strongly stimulated following hyposmotically or urea-activated cellular swelling. The stimulated basolateral taurine efflux pathway exhibited channel-like functional characteristics since (a) [14C]taurine influx was stimulated in parallel with the osmolality-dependent taurine efflux and (b) this efflux could not be stimulated by high medium taurine concentrations (40 mM) applied 10 min following the osmolality reduction (trans-stimulation test). Administration of the 5-lipoxygenase inhibitor ETH 615-139 (20 microM) during hyposmotic stimulation inhibited regulatory volume decreases but had no effect on taurine efflux. In addition, hyposmotically induced taurine efflux was slightly but significantly inhibited by the cyclooxygenase inhibitor indomethacin (10 microM). The taurine efflux was also dependent on both extra- and intracellular Ca2+. It is concluded that taurine is likely to coparticipate with KCl as an osmoeffector during RVD in Carassius proximal renal tubule cells. Cellular swelling seems to activate a basolateral taurine transport pathway with functional properties of a channel. This efflux mechanism appears to be partly regulated by Ca2+. Such a transport pathway could play a role in the cell volume regulatory mechanisms participating during transepithelial solute and water transport.

Dual effect of nitrogen dioxide on barrier properties of guinea pig tracheobronchial epithelial monolayers cultured in an air interface.

Nitrogen dioxide (NO2) is an oxidant gas that may injure the airway epithelial lining, leading to decrements in barrier and active ion transport properties. The present studies examined alterations of bioelectric properties and solute flux by guinea pig tracheobronchial epithelial (GPTE) monolayers exposed in vitro to NO2. Confluent GPTE monolayers were exposed to NO2 levels between 0.5 and 5 ppm, while controls were exposed to air. Following exposure, monolayers were mounted in Ussing chambers for measurement of transepithelial resistance (Rte) and short-circuit current (SCC). A 1-h exposure to 1 ppm NO2 significantly increased SCC to 131.3 +/- 8.7% of air controls, while Rte with a value of 109.3 +/- 13.8% was unchanged. In contrast, a 1-h exposure to 2 or 5 ppm NO2 significantly decreased Rte to 39.0 +/- 1.6 or 35.5 +/- 7.3% of air controls, respectively, while SCC values of 140.3 +/- 10.4 or 153.3 +/- 8.6%, respectively, were also significantly elevated. A 1-h exposure to 2 or 5 ppm NO2 significantly increased sucrose permeability across GPTE monolayers to 446.8 +/- 117 or 313.3 +/- 39.5% of air controls, respectively, while glycerol permeability was unchanged. In contrast, a 1-h exposure to 1 ppm NO2 produced no alterations of sucrose or glycerol flux. The SCC of control GPTE monolayers (1-h air exposure) consisted of 50% bumetanide-sensitive and 40% amiloride-sensitive current; exposure for 1 h to 2 ppm NO2 led to no changes in the corresponding SCC components. Active ion transport (i.e., SCC) across the airway epithelium was significantly increased after exposure to NO2 levels < or = 1 ppm with no change of paracellular pathways for diffusion, suggesting that this reactive gas alters cell membrane function. The increased SCC may lead to impairment of fluid balance and mucociliary clearance. NO2-mediated tissue injury with levels > or = 2 ppm primarily affects passive airway epithelial barrier functions, probably by altering tight junctions, which could result in increased transepithelial solute and fluid leakage in vivo.

The Movement of Solutes and Cells across Tight Junctionsa

The TJ is a highly dynamic rate-limiting barrier for passive transepithelial solute flow. It is not only physiologically regulated but is modulated in various disease states as well. Such modulations occur as a result of epithelial cell interactions with immune cells or immune cell products and thus epithelial barrier function appears to be regulated in disease states.

Anion channels in a human pancreatic cancer cell line (Capan-1) of ductal origin

Transepithelial solute transport and bicarbonate secretion are major functions of pancreatic duct cells, and both functions are thought to involve the presence of chloride channels in the apical membrane of the cell. After being isolated from a human pancreatic adenocarcinoma, the Capan-1 cell line conserves most of the properties of ductal cells and thus constitutes a useful system for investigating the role of chloride channels. Using patch-clamp techniques, we identified three different chloride-selective channels in the apical membrane of confluent Capan-1 cells. Two were non-rectifying chloride channels with low (50 pS) and high (350 pS) unitary conductances. Both channels were active in cell-attached recordings, and they were consistently located together in the same patch. Maxi Cl- channels displayed multiple subconductance states, and were reversibly inactivated by either positive or negative voltage changes, which indicates that they were optimally opened at the cell resting potential. The third was an outwardly rectifying chloride channel with a unitary conductance of 38 pS and 70 pS at negative and positive potentials respectively. Rectifying Cl- channels were clustered in discrete loci. They were silent in situ, but became active after patch excision. In inside-out excised patches, the three channels displayed a high selectivity for Cl- over monovalent cations (Na+ and K+) and gluconate. They were blocked by 20-200 microM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and were insensitive to changes in the Ca2+ concentration. Our results show that the apical membrane of Capan-1 cells contains a high density of chloride channels; these channels may provide pathways for transepithelial solute transport as well as for bicarbonate secretion.

Characterization of a mouse cortical collecting duct cell line.

A cortical collecting duct (CCD) cell line has been developed from a mouse transgenic for the early region of simian virus 40, Tg(SV40E)Bri/7. CCDs were microdissected and placed on collagen gels. Monolayers were subsequently subcultured onto permeable collagen membranes and maintained in serum-supplemented medium. One line, designated M-1, retained many characteristics of the CCD, including a typical epithelial appearance and CCD-specific antigens. M-1 cells, when grown in monolayers on permeable supports, exhibited a high transepithelial resistance (885.7 +/- 109.6 ohms/cm2) and developed a lumen negative transepithelial potential difference (PD) of -45.7 +/- 3.5 mV. The associated short-circuit current (SCC) averaged 71.8 +/- 10.3 microA/cm2, and was reduced by 95% by luminal application of amiloride. The cultured cells responded to arginine vasopressin (AVP) with a significant increase in SCC. M-1 cells generated significant transepithelial solute gradients. After 24 hours incubation, the composition of the luminal (L) and basolateral (B) media (in mM) was: [Na+], L = 106.7 +/- 0.9 and B = 127.4 +/- 0.4; [K+], L = 8.6 +/- 0.6 and B = 2.1 +/- 0.3; [Cl], L = 68.6 +/- 5.8 and B = 101.8 +/- 6.6; [HCO3], L = 15.5 +/- 1.5 and B = 8.6 +/- 1.2; while pH was 7.16 +/- 0.03 at the luminal and 6.94 +/- 0.03 at the basolateral side. The formation of these concentration gradients indicates that the CCD cultures absorb Na+ and Cl- and secrete K+.(ABSTRACT TRUNCATED AT 250 WORDS)