Regulation of anion transport across the uterine epithelium of Gallus domesticus.

For egg shell production, cations, primarily Ca(2+), and anions, primarily HCO(3)(-), must be secreted across the uterine epithelium. Because alterations of HCO(3)(-) transport influence Ca(2+) secretion, the present study was performed to gain insight into the regulation of anion transport (i.e., chloride and HCO(3)(-) transport) across the egg shell gland of the domestic chicken. To this purpose, unstripped uterus epithelia were mounted in modified Ussing chambers and electrogenic [i.e., short circuit current (I(sc))] and electroneutral anion transport were measured. Stimulating adenylate cyclases by forskolin, thereby enhancing the intracellular cyclic adenosine monophosphate( )(cAMP) concentration, evoked 2 patterns of I(sc) responses. Under HCO(3)(-)-buffered conditions, some of the tissues (3/8) showed an increase of I(sc), whereas in others (5/8) a decrease of I(sc) was observed in the presence of the drug. The I(sc) increase existed in HCO(3)(-) secretion because under HCO(3)(-)-free conditions forskolin stimulated only an I(sc) decrease. In addition, methazolamide, a blocker of carboanhydrases, significantly reduced baseline I(sc). The forskolin-induced decrease of I(sc) presented a Cl(-) absorption. In the absence of HCO(3)(-), forskolin yielded only an I(sc) decrease and the transepithelial flux of (36)Cl(-) was reduced. In the presence of mucosal 5-nitro-2-(3-phenylpropylamino) benzoic acid, a nonselective blocker of chloride channels, forskolin-induced I(sc) decrease was inhibited. In addition to cAMP, the regulatory influence of neurons or prostaglandins on baseline I(sc) and anion transport was investigated. Neither tetrodotoxin, a blocker of neuronal Na(+) conductances, electrical field stimulation, nor indomethacin, a blocker of cyclooxygenases, influenced the baseline I(sc) or anion transport. In summary, these data show effects of forskolin (i.e., cAMP) on anion transport across the egg shell gland of the domestic chicken: HCO(3)(-) secretion (supporting Ca(2+) mineralizing of the egg shell) and Cl(-) absorption. Neurons or prostaglandins did not influence the anion transport.

Sites of action of hydrogen peroxide on ion transport across rat distal colon.

BACKGROUND AND PURPOSE: The aim of this study was the identification of the mechanism of oxidant-induced intestinal secretion. EXPERIMENTAL APPROACH: The action of H2O2 on ion transport across rat distal colon was evaluated in Ussing chambers. Changes in cytosolic Ca2+ concentration were measured using fura-2. KEY RESULTS: H2O2 concentration-dependently induced an increase in short-circuit current (Isc), which was due to a stimulation of Cl(-) secretion. The effect of H2O2 was dependent on the presence of serosal Ca2+. It was inhibited after emptying of intracellular Ca2+ stores by cyclopiazonic acid or blockade of ryanodine receptors by ruthenium red, whereas a blocker of inositol 1,4,5-trisphosphate receptors was less effective. Fura 2-experiments confirmed an increase in the cytosolic Ca2+ concentration in the presence of H2O2. Measurements of Cl- currents across the apical membrane at basolaterally depolarized epithelia revealed the activation of a glibenclamide-sensitive, SITS-resistant Cl- conductance by the oxidant. The activation of this conductance was inhibited after blockade of protein kinases with staurosporine. When the apical membrane was permeabilized with nystatin, two sites of action of H2O2 were identified at the basolateral membrane. The oxidant stimulated a basolateral tetrapentylammonium-sensitive K+ conductance and increased the current generated by the Na+-K+ pump. Pretreatment of the tissues with H2O2 reduced the action of subsequently administered Ca2+-, cAMP- and cGMP-dependent secretagogues demonstrating a long-term downregulation after the initial secretory response evoked by the oxidant. CONCLUSIONS AND IMPLICATIONS: H2O2 affects colonic anion secretion by action sites at both the apical, as well as the basolateral membrane.

Ca2+-induced Cl- efflux at rat distal colonic epithelium.

With the aid of the halide-sensitive dye 6-methoxy-N-ethylquinolinium iodide (MEQ), changes in intracellular Cl(-) concentration were measured to characterize the role of Ca(2+)-dependent Cl(-) channels at the rat distal colon. In order to avoid indirect effects of secretagogues mediated by changes in the driving force for Cl(-) exit (i.e., mediated by opening of Ca(2+)-dependent K(+) channels), all experiments were performed under depolarized conditions, i.e., in the presence of high extracellular K(+) concentrations. The Ca(2+)-dependent secretagogue carbachol induced a stilbene-sensitive Cl(-) efflux, which was mimicked by the Ca(2+) ionophore ionomycin. Surprisingly, the activation of Ca(2+)-dependent Cl(-) efflux was resistant against blockers of classical Ca(2+) signaling pathways such as phospholipase C, protein kinase C and calmodulin. Hence, alternative pathways must be involved in the signaling cascade. One possible signaling molecule seems to be nitric oxide (NO) as the NO donor sodium nitroprusside could induce Cl(- )efflux. Vice versa, the NO synthase inhibitor N-omega-monomethyl-arginine (L: -NMMA) reduced the carbachol-induced Cl(- )efflux. This indicates that NO may be involved in part of the signaling cascade. In order to test the ability of the epithelium to produce NO, the expression of different isoforms of NO synthase was verified by immunohistochemistry. In addition, the cytoskeleton seems to play a role in the activation of Ca(2+)-dependent Cl(-) channels. Inhibitors of microtubule association such as nocodazole and colchicine as well as jasplakinolide, a drug that enhances actin polymerization, inhibited the carbachol-induced Cl(-) efflux. Consequently, the activation of apical Cl(-) channels by muscarinic receptor stimulation differs in signal transduction from the classical phospholipase C/protein kinase C way.

The influence of PEEP and tidal volume on central blood volume.

BACKGROUND AND OBJECTIVE: Measurement of central blood volumes (CBV), such as global end-diastolic volume (GEDV) and right ventricular end-diastolic volume (RVEDV) are considered appropriate estimates of intravascular volume status. However, to apply those parameters for preload assessment in mechanically ventilated patients, the influence of tidal volume (TV) and positive endexpiratory airway pressure (PEEP) on those parameters must be known. METHODS: In 13 mechanically ventilated piglets, the effect of low (10 mL kg(-1)) and high (20 mL kg(-1)) TVs on CBV was investigated in absence and presence of PEEP (0 and 15 cm H(2)O). GEDV, RVEDV, right heart (RHEDV) and left heart end-diastolic volume (LHEDV) were measured by thermodilution. Blood flow on the descending thoracic aorta measured with an ultrasonic flow-probe served to determine stroke volume (SV). Measurements were performed during baseline conditions, after volume loading with previously extracted haemodilution blood (20 mL kg(-1)) and following haemorrhage (30 mL kg(-1)). RESULTS: Application of PEEP decreased GEDV and SV significantly (P < 0.05). Augmenting TV did not reduce GEDV systematically, but significantly reduced SV (P < 0.05). Changes in ventilator settings only influenced RVEDV following volume loading (P < 0.05). RHEDV and LHEDV decreased following application of PEEP, but only RHEDV decreased after augmenting TV at baseline and following volume loading. Correlation of SV with parameters of CBV was r = 0.487 (P < 0.01) for GEDV, r = 0.553 (P < 0.01) for RVEDV, r = 0.596 (P < 0.01) for RHEDV and r = 0.303 (P < 0.01) for LHEDV. CONCLUSION: Application of PEEP decreases CBV and SV. Augmenting TV reduces SV but not CBV. There is a moderate correlation between parameters of CBV and cardiac performance.

Muscarinic receptor stimulation activates a Ca(2+)-dependent Cl(-) conductance in rat distal colon.

Recently, it was observed that the acetylcholine analogue carbachol induces a transient stimulation of an apical Cl(-) conductance in basolaterally depolarized rat distal colonic epithelium (Schultheiss et al., 2003). The further characterization of this conductance was the aim of the present study. All experiments were performed at basolaterally depolarized tissues (111.5 mmol.l(-1) KCl buffer at the serosal side); in the absence of a K(+) gradient, a Cl(-) current was driven across the apical membrane (107 mmol.l(-1) K gluconate/4.5 mmol.l(-1) KCl buffer on the mucosal side). Under these conditions, carbachol evoked an atropine-sensitive biphasic change in short-circuit current (I(SC)), consisting of a transient increase followed by a long-lasting decrease, suggesting a stimulation of apical Cl(-) conductance followed by an inhibition. This conductance was inhibited by SITS, but was resistant against glibenclamide, a blocker of CFTR. The carbachol-induced I(SC) was dependent on the presence of mucosal Ca(2+). Ionomycin, a Ca(2+) ionophore, mimicked the effect of carbachol. An antibody against bovine Ca(2+)-activated Cl(-) channel ClCa 1 stained rat colonic epithelial cells both at the cell membrane as well as intracellularly, suggesting that the action of Ca(2+) may be caused by a stimulation of a ClC a-type anion channel. The activation of apical Cl(-) conductance by carbachol was resistant against any blockers of the phospholipase C/IP3/protein kinase C pathway tested (e.g., U-73122, 2-ABP, Li(+), staurosporine), but was inhibited by the NO-synthase blocker L: -NNA. Vice versa, NO-donating compounds such as GEA 3162 or sodium nitroprusside evoked a transient increase of I(SC). Consequently, NO seems to be involved in the transient stimulation of apical Ca(2+)-dependent Cl(-) conductance after muscarinic receptor stimulation.

Activation of apical K+ conductances by muscarinic receptor stimulation in rat distal colon: fast and slow components.

In the epithelium of rat distal colon the acetylcholine analogue carbachol induces a transient increase of short-circuit current (Isc) via stimulation of cellular K+ conductances. Inhibition of the turnover of inositol-1,4,5-trisphosphate (IP3) by LiCl significantly reduced both the amplitude and the duration of this response. When the apical membrane was permeabilized with nystatin, LiCl nearly abolished the carbachol-induced activation of basolateral K+ conductances. In contrast, in epithelia, in which the basolateral membrane was bypassed by a basolateral depolarization, carbachol induced a biphasic increase in the K+ current across the apical membrane consisting of an early component carried by charybdotoxin- and tetraethylammonium-sensitive K+ channels followed by a sustained plateau carried by channels insensitive against these blockers. Only the latter was sensitive against LiCl or inhibition of protein kinases. In contrast, the stimulation of the early apical K+ conductance by carbachol proved to be resistant against inhibition of phospholipase C or protein kinases. However, apical dichlorobenzamil, an inhibitor of Na+/Ca2+ exchangers, or a Ca2+-free mucosal buffer solution significantly reduced the early component of the carbachol-induced apical K+ current. The presence of an apically localized Na+/Ca2+-exchanger was proven immunohistochemically. Taken together these experiments reveal divergent regulatory mechanisms for the stimulation of apical Ca2+-dependent K+ channels in this secretory epithelium, part of them being activated by an inflow of Ca2+ across the apical membrane.

Fatty acids inhibit anion secretion in rat colon: apical and basolateral action sites.

Ca2+-dependent secretagogues evoke only a transient Cl- secretion in intestinal epithelia, although they induce a prolonged increase in the intracellular Ca2+ concentration, suggesting that they may exert an additional antisecretory action. In order to study the mechanism of this antisecretory effect, Cl- secretion, measured as the increase in short-circuit current (Isc), was evoked by carbachol in the absence and presence of different inhibitors. Neither a calmodulin antagonist, calmidazolium, nor different inhibitors of the nitric oxide (NO) pathway, i.e. Nomega-nitro-L-arginine (L-NNA) and Nomega-nitro-L-arginine methylester (L-NAME), affected the carbachol-induced Isc. However, inhibition of phospholipases A2 (PLA2) by quinacrine or arachidonyltrifluoromethyl ketone (AACOCF3) enhanced the Isc response evoked by carbachol, suggesting a role of fatty acids in the downregulation of anion secretion. Neither econazole, a cytochrome P450 inhibitor, nor nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenases, mimicked the action of the PLA2 blockers. Conversely, short- or medium-chain fatty acids inhibited the carbachol- and forskolin-induced Isc with caprate (C10:0) being the most efficient water-soluble fatty acid. This fatty acid inhibited a Cl- current, which was driven across the apical membrane by a serosally to mucosally directed Cl- gradient after depolarization of the basolateral membrane. A second action site of fatty acids seems to be the basolateral membrane. After permeabilization of the apical membrane with the ionophore nystatin, a mucosally to serosally directed K+ gradient induced a K+ current, which was also inhibited by caprate. These results indicate that carbachol not only acts as a secretagogue but at the same time initializes downregulation by increasing the intracellular concentration of fatty acids, a mechanism limiting the resulting Cl- secretion.

Interaction between store-operated non-selective cation channels and the Na(+)-Ca(2+) exchanger during secretion in the rat colon.

The properties of capacitative Ca(2+) influx were studied using the whole-cell patch-clamp technique in crypts isolated from rat distal colon. Store-operated cation influx was evoked by increasing the intracellular buffering capacity for Ca(2+) in the pipette solution; contamination by Cl(-) currents was reduced by the use of NMDG gluconate as the main electrolyte in the pipette solution. The permeability of the non-selective cation conductance stimulated by store depletion had the following sequence for monovalent cations: Cs(+) > Na(+) > or = Li(+). The store-operated conductance is permeable to Na(+) and Ca(2+), but in contrast to Na(+), Ca(2+) also exerts a (feedback) inhibition on its own influx. Other divalent cations shared this inhibitory action with the sequence: Ca(2+) > or = Mg(2+) > or = Ba(2+) > or = Sr(2+). Fura-2 experiments revealed that replacement of extracellular Na(+) by NMDG(+) induced an increase in the intracellular Ca(2+) concentration, which was suppressed by the Na(+)-Ca(2+) exchange inhibitor, dichlorobenzamil, indicating the presence of a Na(+)-Ca(2+) exchanger within the colonic crypt cells. In Ussing chamber experiments dichlorobenzamil induced an increase in short-circuit current (I(sc)) in the majority of tissues tested indicating that this exchanger acts as a Ca(2+)-extruding transporter under physiological conditions. When Ca(2+)-dependent anion secretion was stimulated by the acetylcholine analogue carbachol, dichlorobenzamil no longer evoked an increase in I(sc), indicating that after stimulation of the store-operated cation conductance the Na(+)-Ca(2+) exchanger is turned off. Therefore, it is concluded that the influx of Na(+) across the non-selective store-operated cation conductance serves to reduce the driving force for Ca(2+) extrusion via the Na(+)-Ca(2+) exchanger and thereby maintains the increase in the intracellular Ca(2+) concentration during induction of secretion. Experimental Physiology (2001) 86.4, 461-468.

Adrenoceptor-mediated secretion across the rat colonic epithelium.

Norepinephrine evoked a biphasic change in short-circuit current (Isc) across the proximal and distal colon of the rat. The (1) phase of the current response consisted of a transient increase, which was followed by a long-lasting decrease during the (2) phase. The (1) phase, which is assumed to represent Cl(-) secretion, was resistant against classical adrenoceptor antagonists, but was inhibited by the beta(3)-adrenoceptor antagonist 3-(2-ethylpenoxy)-1-[(1S-1,2,3, 4-tetrahydronaphth-1-ylaminol-(2S)-propranol oxalate (SR 59230A) in the proximal colon and by the non-selective beta-adrenoceptor antagonist bupranolol in both colonic segments. Vice versa, the increase in Isc was mimicked by the beta(3)-adrenoceptor agonist, (R*, R*)-(+/-)-4-[2-[(2-(3-chlorophenyl)-2-hydroxyethyl)amino]propyl]pheno xyacetic acid (BRL 37344). The (2) phase of the norepinephrine-induced Isc, which is assumed to represent K(+) secretion, was inhibited by yohimbine in the proximal colon, suggesting the mediation by alpha(2)-adrenoceptors, whereas in the distal colon, both alpha- and beta-adrenoceptors are involved, as shown by the sensitivity against, e.g. phentolamine and propranolol. These adrenoceptors seem to be located - at least in part - at extraepithelial sites because the (1) phase of the norepinephrine response was sensitive to indomethacin, and the (2) phase, both to indomethacin and tetrodotoxin.

Multiple action sites of flufenamate on ion transport across the rat distal colon.

The antisecretory effects of flufenamate in the rat distal colon were investigated with the Ussing-chamber and the patch-clamp method as well as by measurements of the intracellular Ca(2+) concentration using fura-2-loaded isolated crypts. Flufenamate (5.10(-4) mol l(-1)) suppressed the short-circuit current (Isc) induced by carbachol (5.10(-5) mol l(-1)), forskolin (5.10(-6) mol l(-1)) and the Isc induced by the membrane-permeable analogue of cyclic AMP, CPT - cyclic AMP (10(-4) mol l(-1)). Indomethacin (10(-6) - 10(-4) mol l(-1)) did not mimic the effect of flufenamate, indicating that the antisecretory effect of flufenamate is not related to the inhibition of the cyclo-oxygenase. When the basolateral membrane was depolarized by a high K(+) concentration and a Cl(-) current was induced by a mucosally directed Cl(-) gradient, the forskolin-stimulated Cl(-) current was blocked by flufenamate, indicating an inhibition of the cyclic AMP-stimulated apical Cl(-) conductance. When the apical membrane was permeabilized by the ionophore, nystatin, flufenamate decreased the basolateral K(+) conductance and inhibited the Na(+) - K(+)-ATPase. Patch-clamp experiments revealed a variable effect of flufenamate on membrane currents. In seven out of 11 crypt cells the drug induced an increase of the K(+) current, whereas in the remaining four cells an inhibition was observed. Experiments with fura-2-loaded isolated crypts indicated that flufenamate increased the basal as well as the carbachol-stimulated intracellular Ca(2+) concentration. These results demonstrate that flufenamate possesses multiple action sites in the rat colon: The apical Cl(-) conductance, basolateral K(+) conductances and the Na(+) - K(+)-ATPase.

Ca-sensitive Na transport in sheep omasum.

Na transport across a preparation of sheep omasum was studied. All tissues exhibited a serosa-positive short-circuit current (Isc), with a range of 1-4 microeq. h-1. cm-2. A Michaelis-Menten-type kinetic was found between the Na concentration and the Isc (Michaelis-Menten constant for transport of Na = 6.7 mM; maximal transport capacity of Na = 4.16 microeq. h-1. cm-2). Mucosal amiloride (1 mM), phenamil (1 or 10 microM), or serosal aldosterone (1 microM for 6 h) did not change Isc. Removal of divalent cations (Ca and Mg) enhanced Isc considerably from 2.61 +/- 0.24 to a peak value of 11.18 +/- 1.1 microeq. h-1. cm-2. The peak Isc (overshoot) immediately declined to a plateau Isc of approximately 6-7 microeq. h-1. cm-2. Na flux measurements showed a close correlation between changes in Isc and Na transport. Transepithelial studies demonstrated that K, Cs, Rb, and Li are transported, indicating putative nonselective cation channels, which are inhibited by divalent cations (including Ca, Mg, Sr, Ba) and by (trivalent) La. Intracellular microelectrode recordings from the luminal side clearly showed changes of voltage divider ratio when mucosal divalent cations were removed. The obtained data support the assumption of a distinct electrogenic Na transport mechanism in sheep omasum.

Inhibition of spontaneous smooth muscle contractions in rat and rabbit intestine by blockers of the thromboxane A2 pathway.

The effect of inhibitors of the thromboxane A2 pathway on spontaneous contractions of intestinal smooth muscle preparations was studied. The thromboxane A2 antagonists Bay u3405, SK and F 88046 and KW-3635 concentration-dependently inhibited both the amplitude and the frequency of spontaneous contractions of the longitudinal muscle from the rat proximal colon. A concentration-dependent inhibition of the myogenic contractions was also observed with the thromboxane A2 synthase inhibitor U-51605, and with the combined cyclooxygenase/lipoxygenase inhibitor nordihydroguaiaretic acid, whereas indomethacin, a pure cyclooxygenase inhibitor, was ineffective. None of these inhibitors affected the contractile response evoked by the cholinergic agonist carbachol, excluding non-specific actions on intestinal motility. A similar response was observed for the rabbit jejunum, which, in contrast to the rat colon, exhibits more regular, high-frequency spontaneous contractions, which were inhibited by Bay u3405, SK and F 88046 and KW-3635 in a concentration-dependent manner, whereas the response to carbachol remained unaffected. These results suggest a role for thromboxane A2 in the generation and/or facilitation of spontaneous smooth muscle contractions in the gut.

Electrogenic Ca2+ entry in the rat colonic epithelium.

Capacitative Ca2+ entry in isolated rat colonic crypts was induced by dialysing the cells in the whole-cell patch-clamp mode with a pipette solution having a high Ca(2+)-buffering capacity. Under these conditions crypt cell resting potential was lower than normal. Flufe-namate, La3+ and Gd3+, blockers of non-selective cation channels, hyperpolarized the crypt cells and decreased membrane current. This current exhibited a cation selectivity of Na+>Ca2+. In contrast to Na+, Ca(2+) inhibited the current at concentrations exceeding 1 mmol/l. Indirect evidence suggests that the non-selective cation conductance is activated after stimulation of muscarinic receptors. Carbachol, a cholinergic agonist, evoked a transient hyperpolarization and an increase in membrane outwards current. The half-time of the decay of the carbachol response was shortened strongly in the presence of La3+. Fura-2 experiments with isolated crypts confirmed that La3+ inhibited the carbachol-induced increase in intracellular Ca2+. In parallel Ussing chamber experiments, La3+ suppressed the induction of Cl- secretion by carbachol. These results demonstrate that a non-selective cation conductance activated by store depletion may be involved in the regulation of electrolyte transport by agonists of the Ca2+ signalling pathway.

Segment-specific effects of epinephrine on ion transport in the colon of the rat.

The effect of epinephrine on transport of K+, Na+, Cl-, and HCO-3 across the rat colon was studied using the Ussing chamber technique. Epinephrine (5 x 10(-6) mol/l) induced a biphasic change in short-circuit current (Isc) in distal and proximal colon: a transient increase followed by a long-lasting decay. The first phase of the Isc response was abolished in Cl--poor solution or after bumetanide administration, indicating a transient induction of Cl- secretion. The second phase of the response to epinephrine was suppressed by apical administration of the K+ channel blocker, quinine, and was concomitant with an increase in serosal-to-mucosal Rb+ flux, indicating that epinephrine induced K+ secretion, although this response was much smaller than the change in Isc. In addition, the distal colon displayed a decrease in mucosal-to-serosal and serosal-to-mucosal Cl- fluxes when treated with epinephrine. In the distal colon, indomethacin abolished the first phase of the epinephrine effect, whereas the second phase was suppressed by TTX. In the proximal colon, indomethacin and TTX were ineffective. The neuronally mediated response to epinephrine in the distal colon was suppressed by the nonselective beta-receptor blocker, propranolol, and by the beta2-selective blocker, ICI-118551, whereas the epithelial response in the proximal colon was suppressed by the nonselective alpha-blocker, phentolamine, and by the selective alpha2-blocker, yohimbine. These results indicate a segment-specific action of epinephrine on ion transport: a direct stimulatory action on epithelial alpha2-receptors in the proximal colon and an indirect action on secretomotoneurons via beta2-receptors in the distal colon.

The bumetanide-resistant part of forskolin-induced anion secretion in rat colon.

In order to reveal the contribution of the Na(+)-K(+)-2 Cl(-)- cotransporter to forskolin-induced anion secretion, the inhibition of forskolin-stimulated short-circuit current (Isc) by bumetanide, furosemide, azosemide and piretanide was investigated. In the distal colon, all blockers inhibited the forskolin-stimulated Isc with a maximal efficiency of 70%. In contrast, in the proximal colon, bumetanide and furosemide inhibited only about 40% of the forskolin response, whereas piretanide and azosemide were ineffective. A similar result was observed, when Na+ was replaced by impermeant cations suggesting especially in the proximal colon a great part of forskolin-induced anion secretion to be independent of the Na(+)-K(+)-2 Cl(-)-cotransporter. In anion substitution experiments the forskolin-induced increase in Isc was reduced by 70-80%, if either Cl- or HCO3- were omitted from the buffer solution, whereas in the combined absence of both anions the response was nearly suppressed. Measurement of the mucosal alkalinization revealed that forskolin stimulated a HCO3- secretion, which was, however, too weak to explain the bumetanide-insensitive Isc induced by forskolin. Bumetanide inhibited the serosa-to-mucosa flux of Cl- (JsmCl) stimulated by forskolin; an effect, which was strongly enhanced by subsequent administration of the anion exchange inhibitor, SITS. These data suggest that the bumetanide-resistant part of the forskolin-induced Isc is mainly mediated by a basolateral anion exchanger, probably a Cl(-)-HCO3- exchanger, which contributes to forskolin-evoked Cl- secretion, and in addition by a small HCO3- secretion stimulated by the drug.

K+ and Cl- conductances in the distal colon of the rat.

1. K+ and Cl- conductances and their putative regulation have been characterized in the rat colonic epithelium by Ussing-chamber experiments, whole-cell and single-channel patch-clamp recordings. 2. The apical Cl- conductance is under the control of intracellular cAMP. An increase in the concentration of this second messenger induces transepithelial Cl- secretion due to the activation of an apical 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB)- and glibenclamide-sensitive Cl- conductance. 3. In addition to the apical Cl- conductance, the basolateral membrane is equipped with Cl- channels. They are stimulated by cell swelling and play a role in cell volume regulation and transepithelial Cl- absorption. 4. The basolateral K+ conductance is under the dominant control of intracellular Ca2+. An increase in the cytosolic Ca2+ concentration leads to the opening of basolateral K+ channels, which causes a hyperpolarization of the cell membrane, indirectly supporting Cl- secretion owing to an increase in the driving force for Cl- exit. The predominant effect of cAMP on the basolateral K+ conductance is an inhibitory one, probably due to a decrease in the intracellular Ca2+ concentration. 5. The apical K+ conductance, which is involved in transepithelial K+ secretion, is stimulated by an increase in the intracellular Ca2+ concentration. 6. The differential regulation of apical and basolateral ion conductances in the epithelium of the rat distal colon provides an interesting example for the mechanisms underlying vectorial transport of ions across polarized cells.

Inhibition of a K+ conductance by the phosphatase inhibitor calyculin A in rat distal colon.

Basal membrane permeability of epithelial cells from the lower third and the middle of rat colonic crypts is dominated by a K+ conductance as shown by ion replacement experiments. Calyculin A, an inhibitor of protein phosphatases, induced a depolarization of these cells. The depolarization was concomitant with an inhibition of membrane current. The current inhibited by calyculin A had a reversal potential identical with the theoretical K+ equilibrium potential indicating that the drug inhibits a basal K+ conductance. The efficiency of calyculin A was comparable with that of other well-known K+ channel blockers such as Ba2+, tetraethylammonium or quinine. In the intact tissue, calyculin A exerted an inhibitory action on forskolin-induced anion secretion, an effect which may be explained by the decrease in the driving force for Cl- exit after inhibition of cellular K+ conductance. Together with previous results, these data suggest an inhibition of epithelial K+ conductance by phosphorylation.

Regulation of apical and basolateral K+ conductances in rat colon.

1. Apical administration of an ionophore, nystatin, and basolateral depolarization by K+ were used to investigate the regulation of apical and basolateral electrogenic transport pathways for K+ in the rat proximal and distal colon. 2. Administration of nystatin (100 micrograms ml-1 at the mucosal side), in the presence of Na+ and in the presence of a serosally directed K+ gradient, stimulate a large increase in short-circuit current (ISC) and tissue conductance in both colonic segments. This response was composed of a pump current generated by the Na(+)-K(+)-ATPase and of a current cross a quinine-sensitive basolateral K+ conductance. 3. The pump current, measured as Na(+)-dependent or scilliroside-sensitive current in the absence of a K+ gradient, was significantly greater in the distal than in the proximal colon. The pump current was unaltered by pretreatment of the tissue with forskolin (5 x 10(-6) mol 1(-1)). 4. The current across the basolateral K+ conductance, measured as current in the presence of a serosally directed K+ gradient either in the absence of Na+ or in the presence of scilliroside, was increased by the cholinoreceptor agonist, carbachol (5 x 10(-5) mol 1(-1)), but inhibited by forskolin (5 x 10(-6) mol 1(-1)). 5. Basolateral K+ depolarization induced a negative ISC in both colonic segments, which was inhibited by the K+ channel blocker quinine (10(-3) mol 1(-1)) at the mucosal side), but was resistant to tetraethylammonium (5 x 10(-3) mol 1(-1) at the mucosal side). This K+ current across an apical K+ conductance was stimulated in both colonic segments by carbachol, whereas forskolin had no effect, although control experiments revealed that forskolin was still able to open an apical Cl- conductance under these conditions. 6. These results demonstrate that an increase in intracellular Ca2+ concentration induced by carbachol causes an increase in the basolateral and the apical K+ conductance, thereby inducing K+ secretion in parallel with an indirect support of Cl- secretion due to the hyperpolarization of the cell membrane. In contrast, the dominating effect of an increase in the intracellular cyclic AMP concentration is inhibition of a basolateral K+ conductance; a mechanism which might contribute to the inhibition of K+ absorption.