This study utilizes morphological and mechanistic endpoints to characterize the onset of bilateral atresia of the vas deferens in a recently derived cystic fibrosis (CF) rat model. Embryonic reproductive structures, including Wolffian (mesonephric) duct, Mullerian (paramesonephric) duct, mesonephric tubules, and gonad, were shown to mature normally through late embryogenesis, with involution of the vas deferens and/or epididymis typically occurring between birth and postnatal day 4 (P4), although timing and degree of atresia varied. No evidence of mucus obstruction, which is associated with pathology in other CF-affected tissues, was observed at any embryological or postnatal time point. Reduced epididymal coiling was noted post-partum and appeared to coincide with, or predate, loss of more distal vas deferens structure. Remarkably, α smooth muscle actin expression in cells surrounding duct epithelia was markedly diminished in CF animals by P2.5 when compared to wild type counterparts, indicating reduced muscle development. RNA-seq and immunohistochemical analysis of affected tissues showed disruption of developmental signaling by Wnt and related pathways. The findings have relevance to vas deferens loss in humans with CF, where timing of ductular damage is not well characterized and underlying mechanisms are not understood. If vas deferens atresia in humans begins in late gestation and continues through early postnatal life, emerging modulator therapies given perinatally might preserve and enhance integrity of the reproductive tract, which is otherwise absent or deficient in 97% of males with cystic fibrosis.

The design, synthesis, modeling and in vitro testing of channel-forming peptides derived from the cys-loop superfamily of ligand-gated ion channels are part of an ongoing research focus. Over 300 different sequences have been prepared based on the M2 transmembrane segment of the spinal cord glycine receptor α-subunit. A number of these sequences are water-soluble monomers that readily insert into biological membranes where they undergo supramolecular assembly, yielding channels with a range of selectivities and conductances. Selection of a sequence for further modifications to yield an optimal lead compound came down to a few key biophysical properties: low solution concentrations that yield channel activity, greater ensemble conductance, and enhanced ion selectivity. The sequence NK4-M2GlyR T19R, S22W (KKKKPARVGLGITTVLTMRTQW) addressed these criteria. The structure of this peptide has been analyzed by solution NMR as a monomer in detergent micelles, simulated as five-helix bundles in a membrane environment, modified by cysteine-scanning and studied for insertion efficiency in liposomes of selected lipid compositions. Taken together, these results define the structural and key biophysical properties of this sequence in a membrane. This model provides an initial scaffold from which rational substitutions can be proposed and tested to modulate anion selectivity. This article is part of a Special Issue entitled: Protein Folding in Membranes.

Mammary epithelial cells express a diversity of membrane transporters including members of organic cation and organic anion (OAT) transporter subfamilies. Four mammal OAT isoforms have been identified: OAT-1, OAT-2, OAT-3, and OAT-4. The pharmacological significance of OAT isoforms has been emphasized because of their role in the movement of a wide variety of substrates across epithelial barriers. The present study identified (molecularly and functionally) bovine OAT isoforms in bovine mammary epithelial (BME-UV) cells. mRNA expression levels of all tested transporters in BME-UV cells were less than expression levels of the corresponding transporters in bovine kidney. Directionality in the flux of P-aminohippuric acid and acetylsalicylate, compounds known to interact with OAT-1 and OAT-2, respectively, across BME-UV monolayers was not observed at the concentrations used in this study. Directionality was, however, observed in the flux of estrone sulfate (EsS). Adding probenecid, penicillin G or nonradiolabeled EsS to the apical donor compartment significantly increased the apical-to-basolateral flux of EsS across the BME-UV monolayer. These results suggest that BME-UV cells express an organic anion transport system, making it a potentially useful model to study the role of this transport system in the mammary epithelial barrier.

There is ongoing concern about the potential adverse effects of xenobiotic residues in cows' milk to the human consumer. Although drugs that are intentionally administered to lactating dairy cattle are rigorously regulated to prevent harmful residues, there are numerous other potential sources of exposure that are not as easily controlled. For example, cattle may be exposed to mycotoxins, pesticides and/or persistent organic pollutants through feed, water and inhalation of polluted air. Accurate estimates of the rate and extent of excretion of these compounds into milk is important to assess the risk of exposure through cows' milk. In the present study, the expression of carrier mediated transport processes in cultured monolayers of an immortalized bovine mammary epithelial cell line (BME-UV) was determined using a flow-through diffusion cell system, selective substrates and inhibitors of organic cation transporters (OCT) and organic anion transporters (OAT). The basal-to-apical (BL-to-Ap) flux of tetraethylammonium and estrone sulfate significantly exceeded their flux in the opposite direction. The addition of selective inhibitors to the donor compartment significantly decreased the BL-to-Ap flux of either selective substrate. These results suggest that both OCT and OAT are functionally expressed by BME-UV cells.

The aims of this study were to elucidate the mechanisms by which apical electrolytes and cytokines compromise barrier function of bovine mammary epithelium. Previous work demonstrated that bovine mammary epithelial cell monolayers exposed to high apical electrolytes (HE) as compared to low apical electrolytes (LE) exhibited compromised barrier function as assessed by measurement of transepithelial electrical resistance (Rte). BME-UV cells were grown to confluence on permeable supports with a standard basolateral medium and either HE or LE apical medium for 14 days. Rte was highest in monolayers continuously exposed to apical LE. Time-dependent decline in Rte began by 24 hours of HE exposure. Change from HE to LE demonstrated time-dependent increase in Rte. Indirect immunofluorescence employing antibodies for the tight junction proteins occludin (OC) and zonula occludins 1 (ZO) demonstrated significant alteration in OC distribution concomitant with the changes in Rte, but no change in ZO. In a separate set of experiments, BME-UV cell monolayers, cultured in the presence of LE apical medium, were exposed to cytokines associated with mammary inflammation, including tumor necrosis factor alpha (TNF-á; 0.5 ìg/mL), interleukin-1 (IL-1â,; 0.1 ìg/mL) or interleukin-6 (IL-6; 1 ìg/mL) in the apical medium for 8 or 12 hours prior to assay. TNF-á, but not IL-1â or IL-6, caused significant decrease in Rte. These results indicate that mammary epithelium is a dynamic barrier that is acutely modulated by cytokines and the luminal electrolyte environment at least in part via modulation of occludin. [Supported by USDA 2003-35206-14157 & KS Ag Exp Station]

Epithelial ion transport disorders, including cystic fibrosis, adversely affect male reproductive function by nonobstructive mechanisms and by obstruction of the distal duct. Continuous cell lines that could be used to define ion transport mechanisms in this tissue are not readily available. In the present study, porcine vas deferens epithelial cells were isolated by standard techniques, and the cells spontaneously immortalized to form a porcine vas deferens epithelial cell line that we have titled PVD9902. Cells were maintained in continuous culture for >4 yr and 200 passages in a typical growth medium. Frozen stocks were generated, and thawed cells exhibited growth characteristics indistinguishable from their nonfrozen counterparts. Molecular and immunocytochemical studies confirmed the origin and epithelial nature of these cells. When seeded on permeable supports, PVD9902 cells grew as electrically tight (>6,000 ohms x cm2), confluent monolayers that responded to forskolin with an increase in short-circuit current (I(sc); 8 +/- 1 microA/cm2) that required Cl-, HCO3(-), and Na+, and was partially sensitive to bumetanide. mRNA was expressed for a number of anion transporters, including CFTR, electrogenic Na+-HCO3(-) cotransporter 1b (NBCe1b), downregulated in adenoma, pendrin, and Cl-/formate exchanger. Both forskolin and isoproterenol caused an increase in cellular cAMP levels. In addition, PVD9902 cell monolayers responded to physiological (i.e., adenosine, norepinephrine) and pharmacological [i.e., 5'-(N-ethylcarboxamido)adenosine, isoproterenol] agonists with increases in I(sc). Unlike their freshly isolated counterparts, however, PVD9902 cells did not respond to glucocorticoid exposure with an increase in amiloride-sensitive I(sc). RT-PCR analysis revealed the presence of both glucocorticoid and mineralocorticoid receptor mRNA as well as mRNA for the alpha- and gamma-subunits of the epithelia Na+ channels (alpha- and gamma-ENaC), but not beta-ENaC. Nonetheless, PVD9902 cells recapitulated most observations in freshly isolated cells and thus represent a powerful new tool to characterize mechanisms that contribute to male reproductive function.

NC-1059 is a synthetic channel-forming peptide that provides for ion transport (Isc) across, and changes in barrier function of, a variety of epithelia. The goal of this study is to identify the mechanisms by which NC-1059 increases epithelial paracellular permeability. NC-1059 exposure causes a rapid decrease in transepithelial resistance (Rte) that is accompanied by an increase in dextran permeation rate which documents an effect on the paracellular pathway and suggests that tight junction proteins might be affected. Immunolabeling of occludin, ZO-1 and actin and confocal microscopy reveal substantial redistribution or loss of these proteins upon exposure to 200 micromolar NC-1059. The immunolabeling of the tight junction proteins done after 5, 15 and 30 minutes of NC-1059 exposure reveal that the changes begin to occur in the first 15 minutes and the tight junctions are substantially altered within 30 minutes matching the pattern of the change in Rte observed with NC-1059 exposure. The modulation of the epithelial barrier has therapeutic potential to increase the efficiency of drug delivery. (Supported by GM 074096-01)

Expression of pSLC4A4 variant NBCe1B (pancreatic form) is over 30 times larger than that of variant NBCe1A (kidney form) in primary cultures of porcine vas deferens epithelial cells or the PVD9902 cell line. PVD9902 cells constitutively expressing shRNA targeted to a specific 21-bp segment of the common coding sequence shared by porcine SLC4A4 NBCe1A and e1B, revealed, by means of absolute quantitative RT-PCR, a 5.65-fold decrease in the levels of NBCe1B and a 2.55-fold decrease of NBCe1A expression when compared to non-transfected PVD9902 cells, as a clear indication of RNA interference – this cell population is here denominated as pSLC4A4-knockdown (pSLC4A4-kd). HCO3−-dependent ion transport at the basolateral membrane of apical-nystatin permeabilized cells revealed that anion transport in non-transfected cells is 50% larger than in the pSLC4A4-kd cells. Reduction of the HCO3−-dependent anion transport in the apical-nystatin permeabilized model by DNDS is 54% in control cells and 27% in the pSLC4A4-kd. These results indicate that primary and immortalized vas deferens epithelia secreted bicarbonate by an SLC4A4-dependent mechanism that likely influences male fertility. (CFF SCHULT99 and NIH RR-17686 support)

The ion transport mechanisms of the epithelia lining pig vas deferens play an important role in the modulation of luminal environment to which sperm are exposed. One or more basolateral K+ conductances are required to maintain membrane potential in presence of other ion transport mechanisms. We sought to determine the identity of such basolateral K+ conductance(s). The effects of various K+ channel inhibitors were tested on basolateral K+ conductance. Confluent primary cultures were used to measure short circuit current, Isc, an indicator of net ion flux. Forskolin-stimulated Isc that is consistent with anion secretion was significantly reduced by basolateral but not apical application of Ba2+ and clofilium. Clotrimazole, a lipophillic compound also inhibited Isc in a concentration dependent manner. RT-PCR demonstrated the presence of transcripts for SK3, Maxi-K, Kv7.1, and KCNE1. Maxi-K and Kv7.1 were localized to basolateral membrane using confocal microscopy confirming their expression at protein level. The above findings suggest that the clotrimazole sensitivity observed may be attributed to SK3 and Maxi-K while clofilium sensitivity due to Kv7.1 and KCNE1. These data suggest that net ion transport in pig vas deferens epithelia requires the support of multiple K+ conductances. Targeted modulation of such potassium conductances could be useful in managing male fertility. Supported by NIH P20-RR17686