1. Adrenaline (5 uM) stimulated a K+ secretory current by 2-2 ftequiv h-' cm2 in isolated guinea-pig distal colonic epithelium. This secretory activity was inhibited entirely by addition of the loop diuretic bumetanide to the serosal solution. On-going K+ uptake via the absorptive pathway was unaltered by these changes.2. Prostaglandin E2 (PGE2, 2 /M) stimulated electrogenic K+ secretion and Cl-secretion by 3 0 and 3-6 ,tequiv h-cmM2, respectively. Serosal addition of bumetanide completely inhibited this K+ secretion but blocked only -70% of Cl-secretion. The bumetanideinsensitive Cl-secretory current was dependent on the presence of Cl-and HC03-in the bathing solutions. 3. Stimulation of electrogenic K+ secretion by PGE2 occurred with a half-maximal concentration of 4 nm, an affinity -300 times higher than that for stimulation of Clsecretion by PGE2.4. Forskolin (10 AM) stimulated Cl-secretion by 4-9 tequiv h-' cm2. The apparent K+ secretory rate was increased by only 1-5 ytequiv h-1 cm2. A bumetanide-insensitive shortcircuit current (Isc) was apparent and of the same size as that stimulated by PGE2.5. Addition of the Ca2+ ionophore A23187 (10 fSM), in the presence of indomethacin (1 /SM) to reduce prostaglandin production, inhibited the K+ absorptive pathway by 40% and concurrently stimulated a small rate of electrogenic K+ secretion. 6. Active K+ absorption was inhibited by the addition of ouabain, omeprazole or SCH28080 to the mucosal solution. Both omeprazole and SCH28080 also stimulated a small negative Isc, consistent with electrogenic K+ secretion. 7. Association of K+ absorption, K+ secretion and Cl-secretion is indicated by similarities in transport mechanism and by secretagogue regulation. In particular, maximal rates of K+ secretory current require uptake via apical membrane K+ pumps. Such interrelations support a common cellular locus for these ion transport pathways.Potassium transport across the epithelium lining mammalian distal colon occurs via both active secretory and active absorptive pathways (Halm & Frizzell, 1991). Regulation of these two oppositely directed transport processes permits a range of net flows. Active flow of K+ through the secretory pathway proceeds via a cellular mechanism similar to that for Cl-secretion ; uptake of K+ occurs through Na+-K+ pumps and Na+-K+-2Cl-cotransporters in the basolateral membrane with K+ exit from the cell through channels in the apical and basolateral membranes. The ratio of these K+ conductances together with K+ electrochemical gradients determine the rate of K+ secretion. A cellular mechanism for active K+ absorption has not been resolved as clearly.

Chloride impermeability of epithelial cells can account for many of the experimental and clinical manifestations of cystic fibrosis (CF). Activation of apical-membrane Cl- channels by cyclic AMP-mediated stimuli is defective in CF airway epithelial cells, despite normal agonist-induced increases in cellular cAMP levels. This defect in Cl- channel regulation has been localized to the apical membrane by exposing the cytoplasmic surface of excised membrane patches to the catalytic subunit (C subunit) of cAMP-dependent protein kinase and ATP. In membranes from normal cells, C-subunit activated Cl- channels with properties identical to those stimulated by cAMP-dependent agonists during cell-attached recording. Activation by the C subunit was not observed in CF membranes, but the presence of Cl- channels was verified by voltage-induced activation. The failure of the C subunit to activate the Cl- channels of CF membranes indicates that the block in their cAMP-mediated activation lies distal to induction of cAMP-dependent protein kinase activity and focuses our attention on the Cl- channel and its membrane-associated regulatory proteins as the probable site of the CF defect.

We measured isotopic unidirectional fluxes of K to elucidate the mechanisms of active K transport across the distal colon of the rabbit. Separate pathways for active K absorption and active K secretion were detected using various transport inhibitors and stimulators. The rate and direction of net K transport depend on the activities of these two pathways. K absorption was reduced by orthovanadate (both solutions) or serosal Ba, consistent with ATPase-dependent uptake of K across the apical membrane and exit via a Ba-sensitive basolateral K conductance. K secretion was inhibited by serosal ouabain or mucosal Ba, indicating that K secretion involves basolateral uptake via the Na-K pump and apical exit via a Ba-sensitive K conductance. Active K secretion appears to be electrogenic, since inhibition by ouabain produced equivalent changes in the net K flux and short-circuit current. Addition of bumetanide to the serosal solution or the removal of either Na or Cl from the serosal solution inhibited K secretion; mucosal solution amiloride was without effect. These results indicate that this K secretory process is independent of electrogenic Na absorption but is mechanistically similar to Cl secretory processes. Both epinephrine and prostaglandin E2 (PGE2) stimulate K secretion, but only PGE2 also stimulates Cl secretion. The response to these secretogogues suggests that the mechanisms underlying K and Cl secretion are closely linked but can be regulated independently.

We characterized the anion channel responsible for the increase in apical membrane Cl secretion using a model salt-secreting epithelium, the T84 colonic cell line. The adenosine 3',5'-cyclic monophosphate (cAMP)-mediated secretagogues, prostaglandin E2, forskolin, and 8-bromo-cAMP, evoked activity of an outwardly rectifying Cl channel in previously quiet cell-attached membrane patches. The channel remained active in excised, inside-out membranes, where its single-channel conductance was 40-45 pS at 0 mV with 160 mM NaCl in pipette and bath. Selectivities were PCl/PNa = 50 and for halides I(1.8)/Br(1.4)/Cl(1.0)/F(0.4). This halide sequence illustrates that the ability of various anions to undergo transepithelial secretion is determined by the selectivity of the basolateral membrane Cl entry step rather than by the apical Cl channel. Open-channel probability increased with depolarization, an effect that would adjust the rate of Cl exit across secretory cell apical membranes with agonist-induced changes in apical membrane potential. Comparison with the properties of Cl channels detected in other cell types suggests that this cAMP-stimulated Cl channel is uniquely present in the apical membranes of salt-secreting epithelial cells.

Crypts of Lieberkühn were isolated from human colon, and differential interference contrast microscopy distinguished goblet and columnar cells. Activation with carbachol (CCh, 100 μM) or histamine (10 μM) released contents from goblet granules. Stimulation with prostaglandin E2(PGE2, 5 μM) or adenosine (10 μM) did not release goblet granules but caused the apical margin of columnar cells to recede. Goblet volume was lost during stimulation with CCh or histamine (∼160 fl/cell), but not with PGE2 or adenosine. Three-quarters of goblet cells were responsive to CCh but released only 30% of goblet volume. Half-time for goblet volume release was 3.7 min. PGE2 stimulated a prolonged fluid secretion that attained a rate of ∼350 pl/min. Columnar cells lost ∼50% of apical volume during maximal PGE2 stimulation, with a half-time of 3.3 min. In crypts from individuals with ulcerative colitis, goblet cells were hypersensitive to CCh for release of goblet volume. These results support separate regulation for mucus secretions from goblet cells and from columnar cells, with control mechanisms restricting total release of mucus stores.

No abstract

Single channel currents though apical membrane CI channels of the secretory epithelial cell line T84 were measured to determine the anionic selectivity and concentration dependence of permeation. The current-voltage relation was rectified with single channel conductance increasing at positive potentials. At 0 mV the single channel conductance was 41 -+ 2 pS. Permeability, determined from reversal potentials, was optimal for anions with diameters between 0.4 and 0.5 nm. Anions of larger diameter had low permeability, consistent with a minimum pore diameter of 0.55 nm. Permeability for anions of similar size was largest for those ions with a more symmetrical charge distribution. Both HCOs and H2PO~ had lower permeability than the similar-sized symmetrical anions, NOs and CIO4. The permeability sequence was SCN > I ---NO~ --C104 > Br > CI > PF6 > HCO~ --F >> H2PO4. Highly permeant anions had lower relative single channel conductance, consistent with longer times of residence in the channel for these ions. The conductance sequence for anion efflux was NO3 > SCN ---CIO4 > CI = I = Br > PF6 > F = HCO3 ~> H~PO4. At high internal concentrations, anions with low permeability and conductance reduced C1 influx consistent with block of the pore. The dependence of current on C1 concentration indicated that CI can also occupy the channel long enough to limit current flow. Interaction of CI and SCN within the conduction pathway is supported by the presence of a minimum in the conductance vs. mole fraction relation. These results indicate that this 40-pS CI channel behaves as a multi-ion pathway in which other permeant anions could alter CI flow across the apical membrane.

Short-circuit current (I(sc)) and transepithelial conductance (Gt) were measured in guinea pig distal colonic mucosa isolated from submucosa and underlying muscle layers. Indomethacin (2 microM) and NS-398 (2 microM) were added to suppress endogenous production of prostanoids. Serosal addition of PGE2 (10 nM) stimulated negative I(sc) consistent with K secretion, and concentrations >30 nM stimulated positive I(sc) consistent with Cl secretion. PGE2 also stimulated Gt at low and high concentrations. Dose responses to prostanoids specific for EP prostanoid receptors were consistent with stimulating K secretion through EP2 receptors, based on a rank order potency (from EC50 values) of PGE2 (1.9 nM) > 11-deoxy-PGE1 (8.3 nM) > 19(R)-hydroxy-PGE2 (13.9 nM) > butaprost (67 nM) > 17-phenyl-trinor-PGE2 (307 nM) >> sulprostone (>10 microM). An isoprostane, 8-iso-PGE2, stimulated K secretion with an EC50 of 33 nM. Cl secretory response was stimulated by PGD2 and BW-245C, a DP prostanoid receptor-specific agonist: BW-245C (15 nM) > PGD2 (30 nM) > PGE2 (203 nM). Agonists specific for FP, IP, and TP prostanoid receptors were ineffective in stimulating I(sc) and Gt at concentrations <1 microM. These results indicate that PGE2 stimulated electrogenic K secretion through activation of EP2 receptors and electrogenic KCl secretion through activation of DP receptors. Thus stimulation of Cl secretion in vivo would occur either via physiological concentrations of PGD2 (<100 nM) or pathophysiological concentrations of PGE2 (>100 nM) that could occur during inflammatory conditions.