Absence of the cystic fibrosis transmembrane regulator (Cftr) from myeloid-derived cells slows resolution of inflammation and infection.

The absence or reduction of CFTR function causes CF and results in a pulmonary milieu characterized by bacterial colonization and unresolved inflammation. The ineffectiveness at controlling infection by species such as Pseudomonas aeruginosa suggests defects in innate immunity. Macrophages, neutrophils, and DCs have all been shown to express CFTR mRNA but at low levels, raising the question of whether CFTR has a functional role in these cells. Bone marrow transplants between CF and non-CF mice suggest that these cells are inherently different; we confirm this observation using conditional inactivation of Cftr in myeloid-derived cells. Mice lacking Cftr in myeloid cells overtly appear indistinguishable from non-CF mice until challenged with bacteria instilled into the lungs and airways, at which point, they display survival and inflammatory profiles intermediate in severity as compared with CF mice. These studies demonstrate that Cftr is involved directly in myeloid cell function and imply that these cells contribute to the pathophysiological phenotype of the CF lung.

Phenotypic analysis of conditionally immortalized cells isolated from the BPK model of ARPKD.

To study the pathophysiology of autosomal recessive polycystic kidney disease (ARPKD), we sought to develop conditionally immortalized control and cystic murine collecting tubule (CT) cell lines. CT cells were isolated from intercross breedings between BPK mice (bpk(+/-)), a murine model of ARPKD, and the Immorto mice (H-2K(b)-ts-A58(+/+)). Second-generation outbred offspring (BPK x Immorto) homozygous for the BPK mutation (bpk(-/-); Im(+/+/-); cystic BPK/H-2K(b)-ts-A58), were phenotypically indistinguishable from inbred cystic BPK animals (bpk(-/-)). Cystic BPK/H-2K(b)-ts-A58 mice developed biliary ductal ectasia and massively enlarged kidneys, leading to renal failure and death by postnatal day 24. Principal cells (PC) were isolated from outbred cystic and noncystic BPK/H-2K(b)-ts-A58 littermates at specific developmental stages. Epithelial monolayers were under nonpermissive conditions for markers of epithelial cell polarity and PC function. Cystic and noncystic cells displayed several properties characteristic of PCs in vivo, including amiloride-sensitive sodium transport and aquaporin 2 expression. Cystic cells exhibited apical epidermal growth factor receptor (EGFR) mislocalization but normal expression of ZO-1 and E-cadherin. Hence, these cell lines retain the requisite characteristics of PCs, and cystic BPK/H-2K(b)-ts-A58 PCs retained the abnormal EGFR membrane expression characteristic of ARPKD. These cell lines represent important new reagents for studying the pathogenesis of ARPKD.

Changes in the exocrine pancreas secondary to altered small intestinal function in the CF mouse.

The exocrine pancreas of the cystic fibrosis (CF) mouse (cftr(m1UNC)) is only mildly affected compared with the human disease, providing a useful model to study alterations in exocrine function. The CF mouse pancreas has approximately 50% of normal amylase levels and approximately 200% normal Muclin levels, the major sulfated glycoprotein of the pancreas. Protein biosynthetic rates and mRNA levels for amylase were not altered in CF compared with normal mice, and increases in Muclin biosynthesis and mRNA paralleled the increased protein content. Stimulated pancreatic amylase secretion in vitro and in vivo tended to be increased in CF mice but was not statistically significant compared with normal mice. We show for the first time that the CF mouse duodenum is abnormally acidic (normal intestinal pH = 6.47 +/- 0.05; CF intestinal pH = 6.15 +/- 0.07) and hypothesize that this may result in increased signaling to the exocrine pancreas. There were significant increases in CF intestinal mRNA levels for secretin (310% of normal, P < 0.001) and vasoactive intestinal peptide (148% of normal, P < 0.05). Furthermore, CF pancreatic cAMP levels were 147% of normal (P < 0.01). These data suggest that the CF pancreas may be chronically stimulated by cAMP-mediated signals, which in turn may exacerbate protein plugging in the acinar/ductal lumen, believed to be the primary cause of destruction of the pancreas in CF.

The stoichiometry of the electrogenic sodium bicarbonate cotransporter NBC1 is cell-type dependent.

1. The pancreatic variant of the sodium bicarbonate cotransporter, pNBC1, mediates basolateral bicarbonate influx in the exocrine pancreas by coupling the transport of bicarbonate to that of sodium, with a 2 HCO3-:1 Na+ stoichiometry. The kidney variant, kNBC1, mediates basolateral bicarbonate efflux in the proximal tubule by coupling the transport of 3 HCO3- to 1 Na+. The molecular basis underlying the different stoichiometries is not known. 2. pNBC1 and kNBC1 are 93 % identical to each other with 41 N-terminal amino acids of kNBC1 replaced by 85 distinct amino acids in pNBC1. In this study we tested the hypothesis that the differences in stoichiometry are related to the difference between the N-termini of the two proteins. 3. Mouse renal proximal tubule and collecting duct cells, deficient in both pNBC1- and kNBC1-mediated electrogenic sodium bicarbonate cotransport function were transfected with either pNBC1 or kNBC1. Cells were grown on a permeable support to confluence, mounted in an Ussing chamber and permeabilized apically with amphotericin B. Current through the cotransporter was isolated as the difference current due to the reversible inhibitor dinitrostilbene disulfonate. The stoichiometry was calculated from the reversal potential by measuring the current-voltage relationships of the cotransporter at different Na+ concentration gradients. 4. Our data indicate that both kNBC1 and pNBC1 can exhibit either a 2:1 or 3:1 stoichiometry depending on the cell type in which each is expressed. In proximal tubule cells, both pNBC1 and kNBC1 exhibit a 3 HCO3-:1 Na+ stoichiometry, whereas in collecting duct cells, they have a 2:1 stoichiometry. These data argue against the hypothesis that the stoichiometric differences are related to the difference between the N-termini of the two proteins. Moreover, the results suggest that as yet unidentified cellular factor(s) may modify the stoichiometry of these cotransporters.

The stoichiometry of the electrogenic sodium bicarbonate cotransporter pNBC1 in mouse pancreatic duct cells is 2 HCO(3)(-):1 Na(+).

The electrogenic sodium bicarbonate cotransporter pNBC1 is believed to play a major role in the secretion of bicarbonate by pancreatic duct cells, by transporting bicarbonate into the cell across the basolateral membrane. Thermodynamics predict that this function can be achieved only if the reversal potential of the cotransporter is negative to the cell's membrane potential, or equivalently that the HCO3-:Na+ stoichiometry is not larger then 2:However, there are no data available on either the reversal potential or the HCO3-:Na+ stoichiometry of pNBC1 in pancreatic cells. We studied pNBC1 function in mouse pancreatic duct cells. RT-PCR analysis of total RNA revealed that these cells contain the message for pNBC1, but not for kNBC1, NBC2 or NBC3. To measure cotransporter activity, mouse pancreatic duct cells were grown to confluence on a porous substrate, mounted in an Ussing chamber, and the apical plasma membrane permeabilized with amphotericin B. Ion flux through pNBC1 was achieved by applying Na+ concentration gradients across the basolateral plasma membrane. The current through the cotransporter was isolated as the difference current due to the reversible inhibitor dinitrostilbene disulfonate (DNDS). Current-voltage relationships for the cotransporter, measured at three different Na+ concentration gradients, were linear over a range of about 100 mV. The reversal potential data, obtained from these current-voltage relationships, all corresponded to a 2 HCO3-:1 Na+ stoichiometry. The data indicate that pNBC1 is functionally expressed in mouse pancreatic duct cells. The cotransporter operates with a 2 HCO3-:1 Na+ stoichiometry in these cells, and mediates the transport of bicarbonate into the cell across the basolateral membrane.

Generation and phenotype of cell lines derived from CF and non-CF mice that carry the H-2K(b)-tsA58 transgene.

Tracheal, renal, salivary, and pancreatic epithelial cells from cystic fibrosis [CF; cystic fibrosis transmembrane conductance regulator (CFTR) -/-] and non-CF mice that carry a temperature-sensitive SV40 large T antigen oncogene (ImmortoMouse) were isolated and maintained in culture under permissive conditions (33 degrees C with interferon-gamma). The resultant cell lines have been in culture for >1 year and 50 passages. Each of the eight cell lines form polarized epithelial barriers and exhibit regulated, electrogenic ion transport. The four non-CF cell lines (mTEC1, mCT1, mSEC1, and mPEC1) express cAMP-regulated Cl(-) permeability and cAMP-stimulated Cl(-) secretion. In contrast, the four CFTR -/- cell lines (mTEC1-CF, mCT1-CF, mSEC1-CF, and mPEC1-CF) each lack cAMP-stimulated Cl(-) secretory responses. Ca(2+)-activated Cl(-) secretion is retained in both CF and non-CF cell lines. Thus we have generated genetically well-matched epithelial cell lines from several tissues relevant to cystic fibrosis that either completely lack CFTR or express endogenous levels of CFTR. These cell lines should prove useful for studies of regulation of epithelial cell function and the role of CFTR in cell physiology.

The human secretin receptor gene: genomic organization and promoter characterization.

Secretin is the most potent regulator of pancreatic bicarbonate, electrolyte and volume secretion. In this report, the organization of the human secretin receptor (hSR) gene was characterized by overlapping genomic phage clones. The hSR gene consists of 13 exons and 12 introns with all the splice donor and acceptor sites conforming to the canonical GT/AG rule. By transient reporter gene assays, the wild-type promoter, containing 3.0 kb of the hSR gene 5' flanking region, was able to drive 5.8 +/- 0.6 and 6.6 +/- 0.2-fold (P < 0.01) increases in luciferase activities in pancreatic ductule-derived PANC-1 and BPD-1 cells, respectively. By subsequent 5' and 3' deletion analysis, a promoter element was identified within -408 to -158, relative to the ATG codon. This promoter element was found to be cell-specific since it could drive reporter gene expression in PANC-1 and BPD-1 cells but not in Hs 262.St, Hs 746T and alphaT3-1 cells. The study of the transcriptional control of human secretin and its receptor should shed light on the pathological developments of pancreatic cancer and autism in the future.

Morphological and functional characterization of a conditionally immortalized collecting tubule cell line.

A conditionally immortalized collecting tubule cell line, mCT1, was derived from the H-2Kb-ts A58 transgenic mouse (ImmortoMouse), which harbors a temperature-sensitive mutant of the SV40 large T antigen oncogene. Cells maintained under permissive conditions [33 degreesC with interferon-gamma (IFN-gamma)] form epithelial monolayers, express large T antigen, and proliferate (>50 passages). The cells retain properties characteristic of the renal collecting tubule (CT) including: vasopressin (VP)-stimulated cAMP accumulation, aquaporin-2 expression, high transepithelial electrical resistance, VP-stimulated ion transport, and amiloride-sensitive sodium absorption. When the cells are transferred to nonpermissive conditions (39 degreesC without IFN-gamma), the steady-state level of large T antigen protein declines (>95% decrease) and cell proliferation is arrested. This conditionally immortalized, murine renal cell line should prove useful for studies of CT physiology and large T antigen biology.

Ion-transport properties of cultured bovine pancreatic duct epithelial cells.

Primary cultures of bovine pancreatic duct epithelial cells grown on permeable supports exhibit electrogenic transepithelial ion transport. The short-circuit current (I(sc)) generated by unstimulated duct cell monolayers and the increase in I(sc) elicited by increased levels of cyclic adenosine monophosphate (cAMP) were greater in monolayers bathed by bicarbonate-containing solution compared with monolayers bathed by nominally bicarbonate-free solution. An inhibitor of epithelial sodium channels (amiloride, 10 microM) had no effect on I(sc), whereas a Cl- channel blocker, N-phenylanthranilic acid (DPC; 1 mM), reduced the forskolin-stimulated I(sc) by approximately 50% in the absence or presence of bicarbonate. Bumetanide (an inhibitor of Na+,K+,2Cl- cotransport activity; 10 microM) reduced forskolin-stimulated I(sc) by 49 +/- 6% in bicarbonate-free bathing solution and by only 18 +/- 1% in bicarbonate-containing solution. Measurements of unidirectional 36Cl- flux across short-circuited ductal monolayers in a bicarbonate-containing solution revealed that net Cl- secretion accounted for the I(sc) during secretin stimulation. However, the basal I(sc) and the I(sc) measured during exposure to secretin plus bumetanide were significantly greater than net Cl- flux. The permeability coefficient for [14C]acetate (a surrogate tracer for bicarbonate) measured in the secretory direction was approximately 1.5-fold greater than the permeability coefficient measured in the absorptive direction, indicating net secretion. These results suggest that primary cultures of bovine pancreatic duct epithelial cells secrete both Cl- and HCO3-.

Regulation of amiloride-sensitive sodium absorption in murine airway epithelium by C-type natriuretic peptide.

We have previously shown that C-type natriuretic peptide (CNP), a guanylate cyclase agonist, can stimulate cystic fibrosis transmembrane conductance regulator (CFTR)-mediated chloride secretion in murine airway epithelial cells via protein kinase (PK) A activation through the inhibition of cGMP-inhibited phosphodiesterases. In this paper, we show that CNP is also capable of reducing amiloride-sensitive sodium absorption in murine airway epithelium through a cGMP-dependent mechanism that is separate from the CFTR regulatory signaling pathway. Both murine tracheal and nasal tissues exhibit sensitivity to amiloride-sensitive sodium regulation by exogenously added CNP. CNP depolarized the nasal transepithelial potential difference by 6.3 +/- 0.5 mV, whereas the cGMP-inhibited phosphodiesterase inhibitor milrinone actually hyperpolarized the nasal transepithelial potential difference by 2.0 +/- 1.2 mV in mice homozygous for a CFTR stop mutation [CFTR(-/-)]. Inhibition of guanylate cyclase activity and PKG activity in normal mice resulted in an increase in amiloride-sensitive sodium absorption, suggesting that tonic regulation of amiloride-sensitive sodium absorption is in part due to basal cGMP levels and PKG activity.

In vivo activation of CFTR-dependent chloride transport in murine airway epithelium by CNP.

Inhibitors of guanosine 3',5'-cyclic monophosphate (cGMP)-inhibited phosphodiesterases stimulate Cl- transport across the nasal epithelia of cystic fibrosis mice carrying the delta F508 mutation [cystic fibrosis transmembrane conductance regulator (CFTR) (delta F/delta F)], suggesting a role for cGMP in regulation of epithelial ion transport. Here we show that activation of membrane-bound guanylate cyclases by C-type natriuretic peptide (CNP) stimulates hyperpolarization of nasal epithelium in both wild-type and delta F508 CFTR mice in vivo but not in nasal epithelium of mice lacking CFTR [CFTR(-/-)]. With the use of a nasal transepithelial potential difference (TEPD) assay, CNP was found to hyperpolarize lumen negative TEPD by 6.1 +/- 0.6 mV in mice carrying wild-type CFTR. This value is consistent with that obtained with 8-bromoguanosine 3',5'-cyclic monophosphate (6.2 +/- 0.9 mV). A combination of the adenylate cyclase agonist forskolin and CNP demonstrated a synergistic ability to induce Cl- secretion across the nasal epithelium of CFTR(delta F/delta F) mice. No effect on TEPD was seen with this combination when used on CFTR(-/-) mice, implying that the CNP-induced change in TEPD in CFTR(delta F/delta F) mice is CFTR-dependent.

Bovine pancreatic duct cells express cAMP- and Ca(2+)-activated apical membrane Cl- conductances.

Secretion of salt and water by the epithelial cells that line pancreatic ducts depends on activation of apical membrane Cl- conductance. In the present study, we characterized two types of Cl- conductances present in the apical cell membrane of bovine pancreatic duct epithelial cells. Primary cultures of bovine main pancreatic duct epithelium and an immortalized cell line (BPD1) derived from primary cultures were used. Elevation of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) or Ca2+ in intact monolayers of duct epithelium induced sustained anion secretion. Agonist-induced changes in plasma membrane Cl- permeability were accessed by 36 Cl- efflux, whole cell current recording, and measurements of transepithelial Cl- current across permeabilized epithelial monolayers. Elevation of intracellular cAMP elicited a sustained increase in Cl- permeability, whereas elevation of intracellular Ca2+ induced only a transient increase in Cl- permeability. Ca(2+)- but not cAMP-induced increases in Cl- permeability were abolished by preincubation of cells with the Ca2+ buffer 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl) ester (BAPTA-AM). N-phenylanthranilic acid (DPC; 1 mM) and glibenclamide (100 microM), but not 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; 500 microM), inhibited the cAMP-induced increase in Cl- permeability. In contrast, DPC and DIDS, but not glibenclamide, inhibited the Ca(2+)-induced increase in Cl- permeability. We conclude from these experiments that bovine pancreatic duct epithelial cells express at least two types of Cl- channels, cAMP and Ca2+ activated, in the apical cell membrane. Because the Ca(2+)-activated increase in Cl- permeability is transient, the extent to which this pathway contributes to sustained anion secretion by the ductal epithelium remains to be determined.

Isolation and culture of bovine pancreatic duct epithelial cells.

We describe a method to isolate and culture epithelial cells from the main duct of the bovine pancreas. In primary cultures, secretin caused a dose-dependent increase in intracellular adenosine 3',5'-cyclic monophosphate (cAMP) and stimulated electrogenic transepithelial ion transport. Elevation of intracellular cAMP increased the rate coefficient for 36Cl- efflux from 0.14 +/- 0.03 to 0.47 +/- 0.12 min-1, and plasma membrane conductance, measured by the whole cell patchclamp technique, was increased from 0.7 +/- 0.1 to 6.9 +/- 0.8 nS. The cAMP-activated anion currents had properties similar to those mediated by the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Cells grown on permeable supports formed confluent monolayers with high transepithelial electrical resistance (1.004 +/- 96 omega. cm2) and generated a lumen negative transepithelial voltage difference (-2.5 +/- 0.6 mV). The short-circuit current (Isc) was increased by forskolin or secretin and was inhibited 87 +/- 4% by addition of ouabain (100 microM) to the basolateral bathing solution. Replacement of bathing solution Cl- by cyclamate reduced the forskolin-induced steady-state increase in Isc from 5.3 +/- 0.5 to 0.2 +/- 0.2 microA/cm2, suggesting that the stimulated current is due to anion secretion. The results of these studies demonstrate that large numbers of pancreatic ductal cells can be isolated and grown in primary cell culture. The monolayers express differentiated functions and will be useful for studies of acute and chronic regulation of ion transport in pancreatic duct epithelial cells.

Activation of endogenous deltaF508 cystic fibrosis transmembrane conductance regulator by phosphodiesterase inhibition.

Many heterologously expressed mutants of the cystic fibrosis transmembrane conductance regulator (CFTR) exhibit residual chloride channel activity that can be stimulated by agonists of the adenylate cyclase/protein kinase A pathway. Because of clinical implications for cystic fibrosis of activating mutants in vivo, we are investigating whether deltaF508, the most common disease-associated CFTR mutation, can be activated in airway epithelial cells. We have found that, 36Cl- efflux can be stimulated 19-61% above baseline by beta-adrenoreceptor agonists and cGI-phosphodiesterase inhibitors in transformed nasal polyp (CF-T43) cells homozygous for the deltaF508 mutation. The increase in 36Cl- permeability is diminished by protein kinase A inhibitors and is not mediated by an increase in intracellular calcium concentrations. Preincubation of CF-T43 cells with CFTR anti-sense oligonucleotides prevented an increase in 36Cl- efflux in response to beta-agonist and phosphodiesterase inhibitor. Primary cells isolated from CF nasal polyps gave similar results. These data indicate that endogenous levels of deltaF508 protein can be stimulated to increase 36Cl- permeability in airway epithelial cells.

Immortalization of bovine pancreatic duct epithelial cells.

Pancreatic duct cell lines have been isolated from a number of animal and human tumors, but none appear to express ion transport properties expected for differentiated pancreatic duct epithelial cells. We sought to generate an immortalized ductal cell line from well-differentiated primary cultures of bovine pancreatic duct epithelium. Epithelial cells from the main duct of the bovine pancreas were isolated and immortalized by transfection with a DNA construct encoding simian virus 40 large T antigen. A single clone (BPD1) survived negative selection and was maintained in culture for > 100 passages over 2 yr. The cells grow readily in culture as monolayers and express several properties characteristic of differentiated pancreatic ductal epithelium. The cells do not appear to form a functional tight junction complex, since the transepithelial resistance of the monolayer cultures grown on a permeable support is < 10 omega.cm2. Northern blot analysis revealed that the cells continue to express simian virus 40 large T antigen and contain significant levels of mRNA for proteins thought to be important in transepithelial bicarbonate secretion [carbonic anhydrase II, Cl-/HCO3- exchanger, Na+/H+ exchanger, and cystic fibrosis transmembrane conductance regulator (CFTR)]. In vivo pancreatic ductal secretion is stimulated by the peptide hormone secretin. The secretin receptor is expressed and functionally coupled to adenylate cyclase in the immortalized cells, since secretin caused a dose-dependent accumulation of adenosine 3'5'-cyclic monophosphate (cAMP; approximately 20-fold increase over basal levels) with a mean effective concentration of 15 nM. Elevation of intracellular cAMP by exposure of the cells to forskolin (10 microM) or secretin (0.1 microM) increase plasma membrane Cl- permeability, most likely mediated by activation of CFTR. The results of these studies demonstrate that the pancreatic duct cell line (BPD1) retains several properties exhibited by the secretory epithelial cells that line the pancreatic ductal tree. This cell line should prove useful for studies of expression, function, and regulation of pancreatic duct cell proteins.

Dietary changes improve survival of CFTR S489X homozygous mutant mouse.

Over 90% of untreated CFTR S489X homozygous (CF) mutant mice reportedly die of intestinal obstruction by 40 days of age, significantly limiting their usefulness as a model for the human disease. Because the period of highest mortality is during the week after weaning, we hypothesized that providing a low-residue liquid diet would improve survival and growth. When 99 CF mice that survived to 10 days of age were fed Peptamen (Clintec Nutrition), an elemental liquid diet, and housed on corn-cob bedding, 88% of them survived to maturity (50 days). The diet causes only minor histologic and ion transport changes in the intestines of normal mice and does not reduce growth rate or size. CF mice raised on Peptamen continue to display severe pathological changes in the intestine and completely lack a adenosine 3',5'-cyclic monophosphate-inducible chloride current in the cecum. This combination of dietary and bedding changes provides a reliable method for keeping CF mice alive well into adulthood and will be useful for the evaluation of the effect and duration of potential therapies for CF.

Relationship of a non-cystic fibrosis transmembrane conductance regulator-mediated chloride conductance to organ-level disease in Cftr(-/-) mice.

Although loss of cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl- channel function is common to all epithelia in cystic fibrosis (CF) patients, the severity of disease varies in different organs. We hypothesized that differences in disease severity in CF relate to the expression of an "alternative" plasma membrane Cl- conductance. In CF mice [Cftr(-/-); mice homozygous for Ser-489 to Xaa mutation], which do not express cAMP CFTR-mediated Cl- secretion, we surveyed organs that exhibit a range of disease severity for a Ca(2+)-mediated apical membrane epithelial Cl- conductance. This alternative conductance (Cl-a) was detected in epithelia of organs from CF mice that exhibit a mild disease phenotype (airway, pancreas) but not in epithelia with a severe phenotype (small, large intestine). We conclude that (i) there is an intracellular Ca(2+)-regulated Cl- conductance that is molecularly distinct from CFTR; and (ii) the level of expression of this alternative Cl- conductance in the epithelium is an important determinant of the severity of organ-level disease in CF.

Pseudo-streaming potentials in Necturus gallbladder epithelium. II. The mechanism is a junctional diffusion potential.

The mechanisms of apparent streaming potentials elicited across Necturus gallbladder epithelium by addition or removal of sucrose from the apical bathing solution were studied by assessing the time courses of: (a) the change in transepithelial voltage (Vms). (b) the change in osmolality at the cell surface (estimated with a tetrabutylammonium [TBA+]-selective microelectrode, using TBA+ as a tracer for sucrose), and (c) the change in cell impermeant solute concentration ([TMA+]i, measured with an intracellular double-barrel TMA(+)-selective microelectrode after loading the cells with TMA+ by transient permeabilization with nystatin). For both sucrose addition and removal, the time courses of Vms were the same as the time courses of the voltage signals produced by [TMA+]i, while the time courses of the voltage signals produced by [TBA+]o were much faster. These results suggest that the apparent streaming potentials are caused by changes of [NaCl] in the lateral intercellular spaces, whose time course reflects the changes in cell water volume (and osmolality) elicited by the alterations in apical solution osmolality. Changes in cell osmolality are slow relative to those of the apical solution osmolality, whereas lateral space osmolality follows cell osmolality rapidly, due to the large surface area of lateral membranes and the small volume of the spaces. Analysis of a simple mathematical model of the epithelium yields an apical membrane Lp in good agreement with previous measurements and suggests that elevations of the apical solution osmolality elicit rapid reductions in junctional ionic selectivity, also in good agreement with experimental determinations. Elevations in apical solution [NaCl] cause biphasic transepithelial voltage changes: a rapid negative Vms change of similar time course to that of a Na+/TBA+ bi-ionic potential and a slow positive Vms change of similar time course to that of the sucrose-induced apparent streaming potential. We conclude that the Vms changes elicited by addition of impermeant solute to the apical bathing solution are pseudo-streaming potentials, i.e., junctional diffusion potentials caused by salt concentration changes in the lateral intercellular spaces secondary to osmotic water flow from the cells to the apical bathing solution and from the lateral intercellular spaces to the cells. Our results do not support the notion of junctional solute-solvent coupling during transepithelial osmotic water flow.