The cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel expressed in epithelia and other tissues, is compromised by genetic mutations that cause cystic fibrosis (CF). Although CFTR is reportedly permeant to both Cl¯ and HCO3¯, high throughput screening studies identified drug scaffolds that enhanced Cl¯ conductance of mutated CFTR by 'correcting' processing deficiets and/or 'potentiating' aggregate conductance of channels in epithelial cell membranes while being agnostic to HCO3-. Ongoing work seeks to determine whether mutated forms of CFTR exhibit typical HCO3¯ conductance and to access clinically-used drugs for their effects on HCO3¯ conductance by wild-type and mutated CFTR. Fisher rat thyroid (FRT) cells, which form electrically tight epithelial monolayers, express no functionally detectable CFTR and which traffick exogenously expressed CFTR to both the apical and basolateral membranes, were employed to assess transepithelial anion conductance in modified Ussing-style flux chambers. Initial experiments employing FRT cells expressing human wtCFTR validated that forskolin-stimulated conductance, which was inhibited by selective CFTR blockers, was proportional to extracellular Cl¯ concentration over the range of ~70-140 mEq/l. Further, a clear increment in forskolin-stimulated conductance was observed when HCO3¯ (25 mEq/l) was incorporated into the media. VX770 potentiated forskolin-stimulated conductance, but had no effect in the absence of forskolin. Foskolin-stimulated and VX770-potentiated conductance was compared in base medium (100 mEq/l Cl-) supplemented with 25 mEq/l HCO3- or Cl- and bicarbonate (B) conductance was quantified relative to Cl- (C) conductance (i.e., gB/gC). Following forskolin exposure the gB/gC ratio for wtCFTR was 0.62±0.20, a value that decreased to 0.11±0.10 following exposure to VX770, suggesting that VX770 may negatively impact gB. Forskolin- and VX770-stimulated conductance across cells expressing F508del CFTR was nearly undetectable unless cells were pretreated with CFTR correctors. Following corrector exposure, forskolin stimulated and VX770 potentiated gC, but gB was virtually undetectable, suggesting that this variant may lack gB. Alternatively, forskolin stimulated little conductance across cells expressing G551D CFTR, but a substantial potentiation with VX770 was observed. Notably, the gB/gC ratio approached 1.0 for both foskolin stimulation and VX770 potentiation. Taken together, these results suggest that CFTR HCO3- conductance differs by disease-associated variant and that gB of some variants are sensitive to VX770. Ongoing experiments are designed to determine the optimal forskolin concentration to maximize VX770 potentiation. Initial outcomes suggest that there is interaction between the CFTR variant and forskolin concentration dependence such that VX770-potentiated conductance is maximized at different forskolin concentrations. Taken together, results from this investigation suggest that additional or alternative interventions may be required to optimize bicarbonate secretion and pH regulation in CF patients expressing alternative CFTR variants.

The advent of targeted CFTR therapeutics allows us to assess differences in epithelial Cl− and HCO3− conductances that might be attributed to unique combinations of CFTR variants (mutations), CFTR modulators (corrector(s) and potentiators) as well as secretory agonists. To address these questions, Fisher rat thyroid cells expressing wt or mutant CFTR were cultured on permeable supports for the measurement of transepithelial conductance (Gt) in defined ionic conditions. Initial experiments demonstrated stepwise reduction in the forskolin (Forsk)+ivacaftor (VX770)‐stimulated change in Gt as bath NaCl was partially replaced isosmotically with mannitol. Subsequent experiments were conducted in buffers containing 100 mEq/l Cl− with the addition of either 25 mM alternative anions (Na+ salts) or 50 mM mannitol. Outcomes showed forskolin‐induced Gt changes consistent with reported CFTR permselectivity: Cl− > NO3− ≥ Br− > isethionate ≥ gluconate > mannitol. All subsequent experiments were conducted to assess Gt with 100 mEq/l Cl− plus either 25 mEq/l HCO3−, 50 mM mannitol or 25 mEq/l Cl−, in a 5% CO2 atmosphere. Epithelia expressing wtCFTR respond to forskolin with a substantial increase in Gt. The response in HCO3− was ~75% of that observed with Cl−. Exposure to VX770 increased conductance, but a disproportionately lesser change was observed in HCO3− (vs. Cl−), providing an initial suggestion that VX770 promotes differential ion selectivity.Exposure of cells expressing F508del CFTR to correctors, either C17 or C18, allowed for forskolin‐induced stimulation of Gt with a modest increment of HCO3− conductance. In these conditions, VX770 induced additional Cl− conductance, but no enhancement of HCO3− conductance could be detected. Alternatively, cells exposed to the corrector combination (C17+C18) showed a substantial forskolin‐stimulated increase in anion conductance and a modest increase in CFTR‐associated HCO3− conductance. Thus, the combination of C17 and C18 gives a different outcome regarding F508del‐CFTR HCO3− conductance than either corrector alone.Cells expressing G551D CFTR exhibited little response to forskolin, but substantial responses to VX770. Responses to VX770 in HCO3− approached the magnitude of the responses observed in Cl−.Taken together, the results suggest that CFTR correctors and potentiators can enhance CFTR‐mediated anion conductance, but that significant differences in the conductances of HCO3− relative to Cl− are observed between CFTR variants and that responses can be influenced by the correctors employed. Ultimately, these results suggest that CFTR‐associated pH regulation will be impacted by both the patient’s mutation and the CFTR modulator treatment. Furthermore, additional studies are required to evaluate the CFTR modulators for their effects on Cl− and HCO3− conductances on the many CF causing CFTR variants.Support or Funding InformationCFF BRIDGE18XX0; K‐State CVM SMILE

While epithelial solute transport predates recorded history, our understanding of epithelial function has risen from the most basic level only recently. This chapter provides an historical perspective of epithelial electrophysiology and an initial foundation for much of the information contained in this three volume 2nd edition. Epithelial cell models are presented in their contemporary contexts to demonstrate the philosophical breakthroughs that they heralded along with the novel techniques that made them possible. The text touches on the roles that unique physiological systems such as eel gill, frog skin, rabbit intestine, and cultured cell lines have contributed to our understanding. Two examples of diseases associated with abnormal Cl− transport, cholera diarrhea and cystic fibrosis, are discussed and underlying mechanisms that contribute to the pathology are identified. A hypothetical cell model with the minimal complement of transport proteins that are required for Cl− secretion (Na+/K+-ATPase, Na+/K+/2Cl− cotransporter, K+ channel, and Cl− channel) along with their required localization to the mucosal (apical) or serosal (basolateral) membrane is presented. Selected examples of these transport mechanisms are presented and discussed in the light of their discoveries, biophysical characteristics, pharmacology, genetic identities, and their molecular partners. A recently published comprehensive cell model is presented as the climax of the chapter that sets the stage for distinct components that are presented in greater detail in various chapters of this volume and the other volumes of this 2nd edition.

The epididymis exhibits a less restrictive physical blood–tissue barrier than the testis and, while numerous immunosuppressive factors have been identified in the latter, no mechanisms for epididymal immunotolerance have been identified to date. Therefore, data are currently insufficient to explain how the immune system tolerates the extremely large load of novel antigens expressed on sperm, which become present in the male body after puberty, i.e., long after central tolerance was established. This study tested the hypothesis that transforming growth factor beta (TGFβ) signaling in dendritic cells (DCs) is required for immunotolerance to sperm located in the epididymis, and that male mice lacking TGFβ signaling in DCs would develop severe epididymal inflammation. To test this, we employed adult Tgfbr2ΔDC males, which exhibit a significant reduction of Tgfbr2 expression and TGFβ signaling in DCs, as reported previously. Results show that Tgfbr2ΔDC males exhibit sperm-specific immune response and severe epididymal leukocytosis. This phenotype is consistent with epididymal loss of immunotolerance to sperm and suggests that TGFβ signaling in DCs is a factor required for a non-inflammatory steady state in the epididymis, and therefore for male tract homeostasis and function.

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a gene that codes for an anion channel that is regulated by cyclic adenosine monophosphate (cAMP) and its associated kinase, protein kinase A (PKA). Numerous physiological agonists are known to stimulate the activity of adenylyl cyclase, which generates cAMP. Our previous work demonstrated that acute exposure to prostaglandin (PG) D 2 or PGE 2 causes a rapid and sustained increase in short circuit current ( I sc ), an indicator of anion secretion, across cultured pig vas deferens epithelial cells, an outcome that is consistent with PG‐induced increases in adenylyl cyclase activity. Also, it was shown that indomethacin, an agent that blocks prostaglandin synthesis, precluded the effects of the stimulant bradykinin, which suggests that all components of the prostaglandin synthesis and the response pathways are present in these cells. However, the molecular identity of the prostaglandin receptor(s), the location(s) of these receptors and second messengers that mediate the responses remain to be determined. Thus, experiments were carried out to determine to localize receptors of PGD 2 and PGE 2 to either the mucosal or serosal aspect of the cells. These experiments were carried out with cultured primary adult pig vas deferens epithelial (1°PVD) cells using a modified Ussing‐style flux chamber. Serosal, but not mucosal exposure of PGE 2 produced a rapid increase in I sc , suggesting serosal localization of EP receptors. Similarly, PGD 2 and a selective DP receptor agonist, BW 245C, produced an effect following serosal exposure. Experiments were also carried out to determine the effect of PGs on cAMP generation using a cAMP enzyme immunoassay kit with 1°PVD cells. PGE2, PGD2, and BW 245C all showed an increase in stimulation of cAMP generation. This stimulation was inhibited by the presence of DP receptor antagonist, BW A868C, and EP2/EP4 receptor antagonists, PF‐04418948 and L‐161,982. The simplest interpretation of data is that both EP and DP receptors are localized to the serosal membrane of vas deferens epithelial cells and cAMP generation is affected by inhibition of these receptors. Activation of these receptors is tightly linked to anion secretion, which would be expected to increase pH and fluid volume in the male reproductive duct. Support or Funding Information IOS‐1238831

The Cl- secretion via Ca2+-activated Cl- channel (CaCC) is critical for fluid secretion in exocrine glands like the salivary gland. Also in the mammary gland, it has been hypothesized that CaCC plays an important role in the secretion of Cl- and aqueous phase of milk. However, there has been no evidence for the functional expression of CaCC in native mammary secretory (MS) cells of lactating animals. We therefore assessed membrane current in MS cells that were freshly isolated from lactating mice using whole cell patch-clamp techniques. In MS cells, we detected CaCC current that exhibited the following characteristics: 1) Ca2+-dependent activation at the concentrations of submicromolar range; 2) voltage-dependent activation; 3) slow kinetics for activation and deactivation; 4) outward rectification of the steady-state current; 5) anion permeability in the sequence of I- > NO3- > Br- > Cl- >> glutamate; 6) inhibition by Cl- channel blockers (niflumic acid, DIDS, and CaCCinh-A01). These characteristics of native CaCC current were similar to reported characteristics of heterologously expressed TMEM16A. RT-PCR analyses showed the expression of multiple CaCC channels including TMEM16A, Best1, and Best3 in the mammary glands of lactating mice. Immunohistochemical staining revealed the localization of TMEM16A protein at the apical membrane of the MS cells. Collectively, our data strongly suggest that MS cells functionally express CaCC, which is at least partly constituted by TMEM16A. The CaCC such as TMEM16A at the apical membrane of the MS cells may influence the quantity and/or quality of milk.

The goal of this study was to test for expression of HCO3 (-) exchangers SLC26A3 and SLC26A6 in primary cultures of porcine vas deferens epithelial cells (1°PVD) and native porcine vas deferens. Quantitative RT-PCR revealed that mRNA coding for SLC26A6 was six times more abundant than mRNA coding for SLC26A3 in 1°PVD cells. Western blot analyses combined with surface biotinylation of 1°PVD demonstrated SLC26A3 and SLC26A6 immunoreactivities in whole-cell lysates and apical surfaces of monolayers. Laser scanning confocal microscopy (LSCM) of the 1°PVD cell monolayers demonstrated that SLC26A3 immunoreactivity was primarily in the apical region but present throughout the basal-apical cellular axis, whereas SLC26A6 immunoreactivity was present in the apical region and sometimes accumulated in the nuclear region. LSCM also demonstrated SLC26A3 and SLC26A6 immunoreactivities present along the entire apical lining of the native porcine vas deferens epithelium and in basal cells. The patterns and apparent abundance of SLC26A3 and SLC26A6 immunoreactivities in the proximal vas deferens were not different from the corresponding immunoreactivities in the distal region. There is no evidence of preferential expression of SLC26A3 or SLC26A6 in any portion of the vas deferens, as has been proposed for epithelia that secrete HCO3 (-) in other duct systems. Thus, vas deferens epithelia express transporters throughout the duct that can contribute to rapid alkalinization of the luminal contents as it has been demonstrated in vivo.

Cellular mechanisms to account for the low Na(+) concentration in human milk are poorly defined. MCF10A cells, which were derived from human mammary epithelium and grown on permeable supports, exhibit amiloride- and benzamil-sensitive short-circuit current (Isc; a sensitive indicator of net ion transport), suggesting activity of the epithelial Na(+) channel ENaC. When cultured in the presence of cholera toxin (Ctx), MCF10A cells exhibit greater amiloride-sensitive Isc at all time points tested (2 h to 7 days), an effect that is not reduced with Ctx washout for 12 h. Amiloride-sensitive Isc remains elevated by Ctx in the presence of inhibitors for PKA (H-89, Rp-cAMP), PI3K (LY294002), and protein trafficking (brefeldin A). Additionally, the Ctx B subunit, alone, does not replicate these effects. RT-PCR and Western blot analyses indicate no significant increase in either the mRNA or protein expression for α-, β-, or, γ-ENaC subunits. Ctx increases the abundance of both β- and γ-ENaC in the apical membrane. Additionally, Ctx increases both phosphorylated and nonphosphorylated Nedd4-2 expression. These results demonstrate that human mammary epithelia express ENaC, which can account for the low Na(+) concentration in milk. Importantly, the results suggest that Ctx increases the expression but reduces the activity of the E3 ubiquitin ligase Nedd4-2, which would tend to reduce the ENaC retrieval and increase steady-state membrane residency. The results reveal a novel mechanism in human mammary gland epithelia by which Ctx regulates ENaC-mediated Na(+) transport, which may have inferences for epithelial ion transport regulation in other tissues throughout the body.

The goal of this study was to determine whether transforming growth factor-β1 (TGF-β1) affects epithelial cells lining the vas deferens, an organ that is universally affected in cystic fibrosis male patients. In PVD9902 cells, which are derived from porcine vas deferens epithelium, TGF-β1 exposure significantly reduced short-circuit current (Isc) stimulated by forskolin or a cell membrane-permeant cAMP analog, 8-pCPT-cAMP, suggesting that TGF-β1 affects targets of the cAMP signaling pathway. Electrophysiological results indicated that TGF-β1 reduces the magnitude of current inhibited by cystic fibrosis transmembrane conductance regulator (CFTR) channel blockers. Real-time RT-PCR revealed that TGF-β1 downregulates the abundance of mRNA coding for CFTR, while biotinylation and Western blot showed that TGF-β1 reduces both total CFTR and apical cell surface CFTR abundance. These results suggest that TGF-β1 causes a reduction in CFTR expression, which limits CFTR-mediated anion secretion. TGF-β1-associated attenuation of anion secretion was abrogated by SB431542, a TGF-β1 receptor I inhibitor. Signaling pathway studies showed that the effect of TGF-β1 on Isc was reduced by SB203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK). TGF-β1 exposure also increased the amount of phospho-p38 MAPK substantially. In addition, anisomycin, a p38 MAPK activator, mimicked the effect of TGF-β1, which further suggests that TGF-β1 affects PVD9902 cells through a p38 MAPK pathway. These observations suggest that TGF-β1, via TGF-β1 receptor I and p38 MAPK signaling, reduces CFTR expression to impair CFTR-mediated anion secretion, which would likely compound the effects associated with mild CFTR mutations and ultimately would compromise male fertility.

Channel replacement therapy, based on synthetic channel-forming peptides (CFPs) with the ability to supersede defective endogenous ion channels, is a novel treatment modality that may augment existing interventions against multiple diseases. Previously, we derived CFPs from the second transmembrane segment of the α-subunit of the glycine receptor, M2GlyR, which forms chloride-selective channels in its native form. The best candidate, NK4-M2GlyR T19R, S22W (p22-T19R, S22W), was water-soluble, incorporated into cell membranes and was nonimmunogenic, but lacked the structural properties for high conductance and anion selectivity when assembled into a pore. Further studies suggested that the threonine residues at positions 13, 17, and 20 line the pore of assembled p22-T19R, S22W, and here we used 2,3-diaminopropionic acid (Dap) substitutions to introduce positive charges to the pore-lining interface of the predicted p22-T19R, S22W channel. Dap-substituted p22-T19R, S22W peptides retained the α-helical secondary structure characteristic of their parent peptide, and induced short-circuit transepithelial currents when exposed to the apical membrane of Madin-Darby canine kidney (MDCK) cells; the sequences containing multiple Dap-substituted residues induced larger currents than the peptides with single or no Dap substitutions. To gain further insights into the effects of Dap residues on the properties of the putative pore, we performed two-electrode voltage clamp electrophysiology on Xenopus oocytes exposed to p22-T19R, S22W or its Dap-modified analogues. We observed that Dap-substituted peptides also induced significantly larger voltage-dependent currents than the parent compound, but there was no apparent change in reversal potential upon replacement of external Na+, Cl- or K+, indicating that these currents remained nonselective. These results suggest that the introduction of positively charged side chains in predicted pore-lining residues does not improve anion-to-cation selectivity, but results in higher conductance, perhaps due to higher oligomerization numbers.

This study is designed to identify effects of transforming growth factor β1 (TGF‐β1) on ion transport across vas deferens epithelia. In modified Ussing chambers, PVD9902 cells (derived from porcine vas deferens) that were exposed to TGF‐β1 exhibited attenuated forskolin‐stimulated anion secretion and reduced effects of DASU‐02, a CFTR channel blocker. RT‐PCR and western blots revealed that TGF‐β1 down‐regulates mRNA expression and protein expression of CFTR. Biotinylation assays suggested that TGF‐β1 also reduced the abundance of CFTR in the apical membrane. The effect of TGF‐β1 on anion secretion was abrogated by a TGF‐β1 receptor I inhibitor and was reduced by an inhibitor of p38 MAPK, suggesting that TGF‐β1 interacts with its cognate receptor on vas deferens epithelial cells to activate p38 MAPK signaling. This conclusion was further supported by the observation that TGF‐β1 enhances phospho‐p38 MAPK abundance. Additionally, anisomycin, a p38 MAPK activator, mimicked the effect of TGF‐β1 to reduce forskolin‐stimulated anion secretion. Men harboring profound CFTR mutations exhibit infertility and reproductive duct anomalies, whereas mild mutations have variable presentations. Our observations suggest that TGF‐β1 reduces CFTR‐mediated anion secretion, which would likely compound the effects associated with mild CFTR mutations and would compromise male fertility. [NIH HD058398 and RR017686]

A major step has been made in determining how cytosolic Ca2+ might contribute to the regulation of membrane potential, cell excitability, ion transport and sensation. In this issue of The Journal of Physiology, Adomaviciene et al. (2013) implicate a portion of a putative pore in Ca2+-activated chloride channels (CaCCs) as a non-canonical Ca2+-sensitive region that enhances channel residency in the cell membrane. Many questions remain regarding the time-dependent interplay between Ca2+ concentration and membrane potential in cells expressing CaCCs. While these observations raise questions regarding the positioning of a putative extracellular loop and the structure of the anion permeation pathway, they provide a new direction for the field in the characterization of CaCCs. Much effort has been expended over more than three decades to define the molecular basis of CaCCs. A first goal is to define the molecular identity and structure that accounts for the physiological functions that have been attributed to CaCCs, including epithelial ion transport, smooth muscle contraction, olfaction, gustation, photoreception, somatic sensation and fertilization. Once defined, CaCCs would constitute potential drugable targets to treat or circumvent a variety of diseases, including cancer and cystic fibrosis. Perhaps unwittingly, a major step in defining CaCCs was made in 2003 with the identification of a chromosome 11 gene that coded for a putative transmembrane protein that was expressed in oesophageal, bladder and breast tumours, which was ultimately named Tmem16a (Katoh, 2003). This original identification included the observation that two splice variants (with/without exon 15) were expressed. In silico analysis revealed that Tmem16a was the founder for a 10-member gene family that is characterized by eight putative transmembrane sequences and cytosolic N and C termini. Five years later, three laboratories employing distinctly different approaches independently reported that Tmem16a was (or contributed to) a CaCC (Caputo et al. 2008; Schroeder et al. 2008; Yang et al. 2008). Importantly, Tmem16a exhibited characteristics of classical CaCCs, including Ca2+ and voltage sensitivity, outward rectification at low Ca2+ concentrations with linear conductance at high concentrations, a selectivity sequence of I− > Cl−, permeability to polyatomic anions including SCN− and NO3−, high anion to cation selectivity, and inhibition by niflumic acid. The second family member to be identified, Tmem16b, was also shown to exhibit CaCC characteristics. Based upon the channel function and the structure, it was proposed (Yang et al. 2008) to rename the gene family ‘anoctamins’[anion channels with eight (oct) membrane-spanning segments], which provides the basis for the accepted HUGO nomenclature, ANO1–10. Ultimately, protein identification is only the starting point to define the physiology. Calcium sensitivity is a hallmark of both ANO1 and ANO2, although the Ca2+ concentration dependency differs. ANO1 is reported to have a 10-fold higher sensitivity to Ca2+ (apparent KD 100–500 nm) when compared with ANO2, although direct comparisons in a single expression system have not been reported. The Ca2+ binding site(s) have not been identified; neither canonical helix-loop-helix domains such as the ‘F-hands’ first described for carp parvalbumin, nor classical calmodulin binding domains (i.e., IQ domains) have been identified. Likewise, the voltage sensor has not been defined. Unlike many voltage-sensitive channels, the anoctamins do not harbour a putative transmembrane segment with multiple charged residues. The first putative intracellular loop of ANO1 contains four consecutive glutamate residues, which have been speculated to confer voltage sensitivity. Adjacent to these is a four-residue segment (coded by exon 13) that is absent in some variants that exhibit reduced Ca2+ sensitivity, which suggests that the first intracellular loop may contain critical components for both voltage and Ca2+ sensitivity. Homology modelling suggested that the third extracellular loop includes a component that partly penetrates the membrane and forms a critical portion of the ion channel. Indeed, mutation and cysteine-modifying studies have supported this notion. It is logical to evaluate this putative pore-loop further to define the channel, which Adomaviciene et al. (2013) set out to do. The authors defined a number of parameters that differentiate the whole-cell currents associated with ANO1 and ANO2, including activation and inactivation rates along with voltage and Ca2+ dependency. Permselectivity, however, was similar for a series of anions. Additionally, the outcomes revealed differences in current magnitude and channel density that suggested differences in membrane residency. A chimeric approach was used, and the data set was extended to show that a small segment of the third putative extracellular loop is a determinant of channel density. These 38 residues do not contain a recognized targeting sequence, yet the simplest interpretation of these data is that this segment from ANO1 either increases the trafficking to or decreases the removal from the cell membrane. Additionally, the putative extracellular loop carried with it effects on both voltage and Ca2+ sensitivity. These results suggest that substantial effort is warranted to dissect the many facets of channel behaviour that are imparted by this putative pore-loop sequence. There are many questions regarding the anoctamins and specifically ANO1 and ANO2 that remain to be addressed. Adomaviciene et al. (2013) show that the scaffold around the putative pore-loop also affects channel behaviour. Voltage dependency seems to associate with the pore sequence, but the Boltzmann constant does not, which suggests that additional portions of the protein contribute to the voltage-sensing apparatus. Likewise, the putative pore-loop contributes to Ca2+ sensitivity, but is not the sole determinant. The concentration dependency is not consistent with a simple bimolecular interaction. The Hill coefficient approaches 3, which is greater than the value reported by other researchers. Nonetheless, the simplest interpretation is that multiple Ca2+ binding sites contribute to channel activation, which could reflect an interaction with calmodulin, as has been postulated. Alternatively, the channel may be a homo- or heterodimer, as has been postulated by others, with a requisite Ca2+ binding site on each monomer. The absence of consensus binding sites makes this a difficult but intriguing question to address. Numerous splice variants of ANO1 have been reported, with alternative forms of exon 1 or 6 and the absence or presence of exons 13 and 15. Likewise, splice variants of ANO2 are expressed. There are likely to be differences in developmental expression or tissue distribution, but these differences remain to be defined. Different forms may be targeted to specific cell regions, such as epithelial apical or basolateral membranes, that can contribute to anion secretion or to K+ secretion, as has been suggested. Calcium sensitivity has been associated with residues in the first intracellular loop and now with the putative pore-loop region. Thus, these distinct and distant segments of the protein may work in concert to direct the protein to the appropriate cellular destination. The paper by Adomaviciene et al. (2013) builds substantially on previous work in the field and clearly changes the frontier for ongoing investigations. These authors provide a new starting point for studies that are designed to determine how this putative pore directs CaCC distribution, regulation and gating.

BACKGROUND:Enterotoxigenic Escherichia coli (ETEC) strains are the leading bacterial cause of diarrhea to humans and farm animals. These ETEC strains produce heat-labile toxin (LT) and/or heat-stable toxins that include type I (STa), type II (STb), and enteroaggregative heat-stable toxin 1 (EAST1). LT, STa, and STb (in pigs) are proven the virulence determinants in ETEC diarrhea. However, significance of EAST1 in ETEC-associated diarrheal has not been determined, even though EAST1 is highly prevalent among ETEC strains.METHODOLOGY/PRINCIPAL FINDINGS:In this study, we constructed E. coli strains to express EAST1 toxin as the only toxin and studied them in cell lines and five-day old gnotobiotic piglets to determine significance of EAST1 toxin. Data from in vitro studies indicated that EAST1 did not stimulate an increase of intracellular cyclic AMP or GMP levels in T-84 cells or porcine cell line IPEC-J2, nor did it enhance LT or STa toxin of ETEC strains in stimulation of cAMP or cGMP in T-84 cells. In addition, 5-day old gnotobiotic pigs challenged with E. coli strains expressing EAST1 as the only toxin did not developed diarrhea or signs of clinical disease during 72 h post-inoculation.CONCLUSION/SIGNIFICANCE:Results from this study indicated that EAST1 alone is not sufficient to cause diarrhea in five-day old gnotobiotic pigs, and suggest that EAST1 likely is not a virulence determinant in ETEC-associated diarrhea.

Epithelial cells lining the male excurrent duct contribute to male fertility by employing a number of physiological mechanisms that generate a luminal microenvironment conducive to spermatozoa maturation and storage. Among these mechanisms, male duct epithelia establish intercellular tight junctions that constitute a barrier to paracellular diffusion of water, solutes, large molecules, and cells. Mechanisms regulating the male duct epithelial barrier remain unidentified. Transforming growth factor beta (TGFB) is a regulatory cytokine present in high concentrations in human semen. This study examined whether TGFB has any effects on epithelial function exhibited by primary cultures of porcine vas deferens epithelia. TGFB1 exposure caused a 70%-99% decrease in basal transepithelial electrical resistance (R-TE, a sensitive indicator of barrier integrity), while a significant decrease in anion secretory response to forskolin was detected at the highest levels of TGFB1 exposure employed. SB431542, a selective TGFB receptor I (TGFBR1) inhibitor, prevented decreases in barrier function. Results also demonstrated that TGFB1 exposure modifies the distribution pattern of tight junction proteins occludin and claudin 7. TGFBR1 is localized at the apical border of the native porcine vas deferens epithelium. Pharmacological inhibition of mitogen-activated protein kinase (MAPK) 11 (also known as p38-MAPK) did not alter the effect of TGFB1 on R-TE significantly. These data suggest that epithelia lining the vas deferens are subject to disruptions in the physical barrier if active TGFB becomes bioavailable in the luminal fluid, which might be expected to compromise fertility.

The importance of intracellular and extracellular pH to physiological function is unquestioned. There are few scenarios in which pathological processes do not include aberrant pH. For example, cystic fibrosis, a genetic disease that adversely affects airways and other epithelia throughout the body, is associated with secretions that have abnormally low pH. Although the contributions of CO2 and HCO3− to acid–base balance have been known for well over a century and carbonic anhydrase was described first in The Journal of Physiology (Meldrum & Roughton 2012) some 80 years ago, much remains to be learned regarding the molecular and biophysical mechanisms that account for HCO3− transport and the resulting buffering capacity and pH regulation in most body compartments.

Epithelial monolayers act as barriers to the movement of small solute molecules – including both inorganic ions and drugs – between body compartments. Ions traverse epithelial apical and basolateral membranes via a combination of tightly regulated ion-specific transporters and channels. Compromised function of any component leads to electrolyte and fluid imbalances resulting in morbidity and potentially, mortality. In the case of cystic fibrosis (CF) the defect lies in various genotypes that result in suboptimal synthesis, folding, transport, or gating of the CF transmembrane conductance regulator (CFTR; an anion channel that has other reported cellular functions). Many of the current therapies involve palliative interventions that address infections, inflammation, nutrition and mucus viscosity issues in patients. While these approaches have increased the life span of CF patients by reducing the rate of decline in lung functions or other health issues, none of them addresses the underlying cause of the disease at the cellular or tissue level, namely reduced anion conductance that sets the chemiosmotic driving force for both paracellular and transcellular fluid movement.