A primary culture of guinea pig gallbladder epithelial cells that is responsive to secretagogues.

We have developed a cell culture of guinea pig gallbladder epithelial cells with which to study ion transport. When grown on permeable supports, the cultured epithelia developed a transepithelial resistance (R(t)) of approximately 500 Omega. cm(2). The epithelial cell origin of the cell culture was further confirmed by immunocytochemical localization of cytokeratin. Ionomycin and forskolin increased transepithelial voltage and short-circuit current (I(sc)) and decreased R(t). The response to ionomycin was transient, whereas that to forskolin was sustained. Both were attenuated by replacement of Cl(-) and/or HCO(3)(-). Mucosal addition of the anion transport inhibitors DIDS or diphenylamine-2-carboxylic acid (DPC) blocked the response to ionomycin. The response to forskolin was blocked by DPC but not by DIDS. Ionomycin, but not forskolin, increased intracellular Ca(2+) concentration in fura 2-loaded cells. PGE(2), histamine, vasoactive intestinal polypeptide, and secretin elicited a sustained increase in I(sc). Responses to ATP and CCK were transient. Thus cultured guinea pig gallbladder epithelia display the range of responses observed in the native tissue and are an appropriate model for studies of ion transport in gallbladder and intestinal epithelia.

Growth and function of isolated canine pancreatic ductal cells.

These studies investigated the growth characteristics and functional properties of isolated canine pancreatic ductal epithelial cells. Cells were isolated from the accessory pancreatic duct and cultured by using three conditions: on vitrogen-coated petri dishes with fibroblast conditioned medium (nonpolarized); in vitrogen-coated Transwells above a fibroblast feeder layer (polarized); or as organotypic rafts above a fibroblast-embedded collagen layer (polarized). Growth characteristics, transepithelial resistances, and carbonic anhydrase and cyclic adenosine monophosphate (AMP) responses were evaluated. Under polarized conditions, the cells grew as monolayers with columnar epithelial characteristics. The monolayers developed high transepithelial resistance and became impervious to the passage of horseradish peroxidase. Epithelial growth factor (EGF) (2 ng/ml) stimulated ductal cell growth and accelerated the formation of a high-resistance monolayer. Forskolin (10 microM) rapidly decreased transepithelial resistance. Carbonic anhydrase activity, which was lower in nonpolarized compared with polarized conditions, was stimulated by carbachol (175 microM). Secretin, however, did not stimulate carbonic anhydrase activity in these cells. Although secretin stimulated adenylyl cyclase activity in early-passage cells, this response was lost in later-passage cells. Both vasoactive intestinal polypeptide (VIP; 1 microM) and forskolin (10 microM) consistently increased adenylyl cyclase activity. Isolated canine pancreatic ductal epithelial cells proliferate in vitro, develop high-resistance epithelial monolayers, and respond to stimuli that activate adenylyl cyclase. These cells should provide a useful model for regulatory studies of ductal cell functions.

Gamma radiation affects active electrolyte transport by rabbit ileum. II. Correlation of alanine and theophylline response with morphology.

The response of ileal segments isolated from rabbits to an actively transported amino acid and a secretagogue was evaluated following exposure to 10 Gy whole-body gamma irradiation. The ability of ileal segments to respond to the actively transported amino acid, alanine, was not significantly diminished until 96 h postexposure. Decreased responsiveness to the secretagogue, theophylline, occurred earlier at 72 h. These effects did not appear to be accounted for by decreased food intake of irradiated animals alone. Examination of intestinal morphological changes with respect to these changes in electrolyte transport revealed that decreased amino acid transport coincides with loss of intestinal villi. Although a morphological correlate of decreased secretory response was not as striking as that for absorption, the theophylline response appeared to decline concomitant with the appearance of increased mitotic activity in the intestinal crypts. The results of this study indicate that, following a dose of 10 Gy, the inability of these tissues to respond to amino acids is due to a loss of mature villus absorptive cells subsequent to denudation of the intestinal mucosa. There appeared to be little impairment of cell membrane transport processes for alanine. In contrast, the decreased secretory response could not be correlated with the disappearance of any one cell type and perhaps results from increased proliferation in the crypts at the expense of differentiation.

Physiological localization of an agonist-sensitive pool of Ca2+ in parotid acinar cells.

Muscarinic stimulation of fluid secretion by mammalian salivary acinar cells is associated with a rise in the level of intracellular free calcium ([Ca2+]i) and activation of a calcium-sensitive potassium (K+) conductance in the basolateral membrane. To test in the intact cell whether the rise of [Ca2+]i precedes activation of the K+ conductance (as expected if Ca2+ is the intracellular messenger mediating this response), [Ca2+]i and membrane voltage were measured simultaneously in carbachol-stimulated rat parotid acinar cells by using fura-2 and an intracellular microelectrode. Unexpectedly, the cells hyperpolarize (indicating activation of the K+ conductance) before fura-2 detectable [Ca2+]i begins to rise. This occurs even in Ca2+-depleted medium where intracellular stores are the only source of mobilized Ca2+. Nevertheless, when the increase in [Ca2+]i was eliminated by loading cells with the Ca2+ chelator bis(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetate (Me2BAPTA) and stimulating in Ca2+-depleted medium, membrane hyperpolarization was also eliminated, indicating that a rise of [Ca2+] is required for the agonist-induced voltage response. Stimulation of Me2BAPTA-loaded cells in Ca2+-containing medium dramatically accentuates the temporal dissociation between the activation of the K+ conductance and the rise of [Ca2+]i. The data are consistent with the hypothesis that muscarinic stimulation results in a rapid localized increase in [Ca2+]i at the acinar basolateral membrane followed by a somewhat delayed increase in total [Ca2+]i. The localized increase cannot be detected by fura-2 but is sufficient to open the Ca2+-sensitive K+ channels located in the basolateral membrane. We concluded that a receptor-mobilized intracellular store of Ca2+ is localized at or near the basolateral membrane.

Apical membrane potassium conductance in guinea pig gallbladder epithelial cells.

The fractional resistance of the apical membrane (fRa) of guinea pig gallbladder epithelial cells was observed to vary with changes in apical membrane potential (Va). Depolarizing Va from a base-line potential of -60 to -30 mV decreased fRa from 0.79 +/- 0.03 to 0.59 +/- 0.05. A comparable hyperpolarization had no effect on fRa. The potassium channel blocker tetraethylammonium (TEA) inhibited the changes in fRa induced by voltage when added to the mucosal but not when added to the serosal solution. Mucosal addition of Ba2+ and decreased pH also inhibited changes in fRa, whereas quinidine and 4-amino-pyridine did not. These results indicate that an increase in the K+ conductance of the apical membrane is responsible for changes in fRa with membrane depolarization. The current-voltage relation of this TEA-sensitive pathway was determined from differences in transepithelial current in the presence and absence of maximally effective concentrations of TEA and analyzed with respect to the Goldman constant-field equation. Computer-generated, best-fit analysis to the data indicated that they cannot be easily reconciled with K+ movement through a voltage-independent pathway or channel. Taken together, the results suggest that activation of a voltage-dependent K+ conductance in the apical membrane is responsible for changes in fRa with Va. This conductance also appears to be Ca2+-sensitive as ionomycin caused a shift in the relation between Va and fRa.

Gamma radiation affects active electrolyte transport by rabbit ileum: basal Na and Cl transport.

The effect of whole-body gamma radiation (5-12 Gy) on electrolyte transport by rabbit ileum in vitro was assessed for 1-96 h postirradiation using the short-circuit technique and radioisotopic fluxes. Although there was no effect of radiation on short-circuit current (Isc), transepithelial potential, or resistance 1 h after exposure, by 24 h the basal parameters of ileal segments isolated from irradiated animals were significantly greater than those of sham-irradiated controls. The Isc increased in a dose-dependent fashion and was greatest 24 h postexposure. Isotope flux experiments revealed that the increased Isc following irradiation resulted in part from a stimulation of active serosal-to-mucosal net Cl flux. There was no observable change in net Na transport. The results demonstrate that radiation exposure alters cellular transport processes, which may contribute to the fluid and electrolyte imbalance observed following radiation exposure.

Effects of Na-coupled alanine transport on intracellular K activities and the K conductance of the basolateral membranes of Necturus small intestine.

Intracellular electrical potentials and K activity, (K)c, were determined simultaneously in Necturus small intestine before and after the addition of alanine to the mucosal solution. As noted previously (Gunter-Smith, Grasset & Schultz, 1982), the addition of alanine to the mucosal solution resulted in a prompt depolarization of the electrical potential difference across the apical membrane (psi mc) and a decrease in the slope resistance of that barrier (rm). This initial response was followed by a slower repolarization of psi mc associated with a decrease in the slope resistance of the basolateral membrane (rs) so that when the steady state was achieved (rm/rs) did not differ significantly from control values in the absence of alanine. In the absence of alanine, psi mc averaged -32 mV and (K)c averaged 67 mM. When a steady state was achieved in the presence of alanine these values averaged -24 mV and 50 mM, respectively. The steady-state electrochemical potential differences for K across the basolateral membrane in the absence and presence of alanine did not differ significantly. Inasmuch as the rate of transcellular active Na transport or "pump activity" was increased two- to threefold in the presence of alanine, it follows that, if active Na extrusion across the basolateral membrane is coupled to active K uptake across that barrier with a fixed stoichiometry then, the decrease in rs must be due to an increase in the conductance of the basolateral membrane to K that parallels the increase in "pump activity". This "homocellular" regulatory mechanism serves to (i) prevent an increase in (K)c due to an increase in pump activity; and, (ii) repolarize psi mc and thus restore the electrical driving force for the rheogenic Na-coupled entry processes.

Contribution of villus and intervillus epithelium to intestinal transmural potential difference and response to theophylline and sugar.

A chamber design is described which permits isolation of villus or intervillus epithelium from proximal segments of Amphiuma intestine and measurement of the transpithelial potential difference (psi ms) and short-circuit current (Isc) produced by each. In media containing Cl- and 10 mequiv./l HCO3- the villus generated a basal psi ms of 0.8 mV (serosa negative) and Isc of 12 microA/cm2 while the intervillus psi ms and Isc were not different from zero. Acetazolamide altered the villus psi ms by 1.2 mV; the intervillus psi ms by only 0.3 mV. Transepithelial gradients of HCO3- appeared to generate diffusion potentials across the intervillus but not the villus epithelium. The actively transported sugar galactose elevated psi ms by 0.6 +/- 0.1 mV in the intervillus epithelium and by 1.5 +/- 0.2 mV in the villus epithelium for a response ratio (0.6/1.5) = 0.4. The response ratio for valine was 0.3. In contrast, the response ratios for theophylline (0.7) and cyclic AMP (0.7) were significantly higher. These observations indicate that the entire epithelium is responsive to theophylline and cyclic AMP while Na+-dependent solute transport and the basal electrogenic ion transport processes are primarily functions of the cells lining the intestinal villus.

Response of Amphiuma small intestine to theophylline: effect on bicarbonate transport.

The electrolyte transport properties of isolated proximal segments of Amphiuma small intestine and their response to theophylline were observed under various conditions. In the absence of theophylline the intestine generates a transepithelial potential (psi ms) serosa negative to mucosa when Cl- and HCO3- are present in the bath. Replacement of Cl- or HCO3- reduced the magnitude and usually reversed the sign of psi ms. Acetazolamide (10(-4) M) nearly abolished the serosa negative psi ms. Theophylline (10 mM) drove psi ms serosa positive, the magnitude depending on the bath Na+ and HCO3- concentrations. Simultaneously it increased the short-circuit current (Isc) and tissue resistance (Rt). The increase in Isc was not due to increase net Na+ transport in Cl-free buffer and was attributed to a residual ion flux. Acetazolamide reduced the Isc, Rt, and the net residual flux observed in theophylline-treated intestine. The magnitude of the acetazolamine effect on Isc was proportional to the Na+ and HCO3- concentrations of the bath. The results suggest that in the absence of theophylline, HCO3-, and Cl- transport are related. Furthermore, acetazolamide inhibits the movement of an ion, possibly HCO3-, secreted in response to theophylline.