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.