Regulation of Ca(2+)-activated chloride channels by cAMP and CFTR in parotid acinar cells

The effect of intracellular cAMP and cystic fibrosis conductance regulator (CFTR) protein on the calcium-activated chloride current (ICaCl) present in parotid acinar cells was studied using the patch clamp technique. Application of 1 mM of 8-(4-chlorophenylthio)adenosine 3':5'-cyclic monophosphate (CPT-cAMP), a permeable analog of cAMP, inhibited ICaCl only at positive potentials. This inhibition was partially abolished in cells dialyzed with 20 nM PKI 6-22 amide, a potent peptide that specifically inhibits PKA. Because cAMP is an activator of the CFTR Cl- channel, a known regulator of ICaCl, we also investigated if the inhibition of ICaCl was mediated by activation of CFTR. To test this idea, we added 1 mM CPT-cAMP to acinar cells isolated from knockout animals that do not express the CFTR channel. In these cells the cAMP effect was totally abolished. Thus, our data provide evidence that cAMP regulates ICaCl by a dual mechanism involving PKA and CFTR.     

Activation of CFTR Cl(-) channel by tyrphostins via a protein tyrosine kinase-independent pathway in forskolin-stimulated renal epithelial A6 cells

We studied effects of tyrphostin A23 (an inhibitor of protein tyrosine kinase; PTK) and tyrphostin A63 (an inactive analog of tyrphostin A23) on forskolin-activated cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels and Cl(-) secretion in renal epithelial A6 cells. Tyrphostin A23 and A63 had no effects on the basal CFTR Cl(-) channel and Cl(-) secretion. However, under the forskolin-stimulated condition, tyrphostin A23 and A63 stimulated Cl(-) secretion by activating CFTR Cl(-) channels. These observations suggest that: 1) tyrphostin A23 and A63 stimulate the cAMP-activated CFTR Cl(-) channel via a PTK-independent, structure-dependent mechanism, and 2) tyrphostin A23 and A63 do not stimulate the basal CFTR Cl(-) channel. These lead us to an idea that: 1) cAMP might cause a conformational change of CFTR Cl(-) channel which is accessible by tyrphostins, and 2) tyrphostins would stimulate translocation of the cAMP-modified channel to the apical membrane by binding to the channel.     

Volume-activated chloride channels in neuroblastoma cells are blocked by the antiestrogen toremifene

Summary The presence of volume-activated chloride channels has been examined in neuroblastoma C1300 cells using the whole-cell configuration of the patch-clamp technique. Chloride channels could not be detected under isotonic conditions. However, hypotonic challenge induced slowly developed inward and outward anionic currents that exhibited outward rectification and inactivation at the most depolarizing potentials, features that were similar to the currents described in other cell preparations where volume-activated Cl^– channels have been associated with the expression of P-glycoprotein. This hypotonicity-activated Cl^– currents could be reversibly blocked by extracellular exposure to toremifene, a novel synthetic antioestrogen. The fact that toremifene and its analog tamoxifen, have been shown to block P-glycoprotein-associated chloride channels and to reverse P-glycoprotein associated multidrug resistance in a number of cell lines suggest that P-glycoprotein could be involved in the generation of hypotomic-induced chloride conductance in neuroblastoma cells.     

Single chloride channels in colon mucosa and isolated colonic enterocytes of the rat

Summary Chloride channels from rat colonic enterocytes were studied using the patch-clamp technique. After removal of mucus, inside-out patches were excised from the apical membrane of intact epithelium located at the luminal surface. They contained spontaneously switching Cl^– channels with a conductance of 35–40 pS. The channels were blocked reversibly by anthracene-9-carboxylic acid (1mm).In excised patches from single enterocytes, isolated by calcium removal, the Cl^– channels were studied in more detail. TheI–V relation was linear between ±80 mV. The selectivity was I^–>Br^–>Cl^–=NO ₃ ^– >F^–=HCO ₃ ^– .Thirty pS Cl^– channels were also found on the basolateral membrane of crypts isolated by brief calcium removal. TheI–V curve of these Cl^– channels was also linear.The results provide direct evidence for the existence of Cl^– channels in the apical membrane of surface cells in colonic mucosa. The properties of these channels are similar to those previously observed when incorporating membrane vesicles into planar lipid bilayers. Both results support the validity of the theoretical models describing intestinal secretion.     

Potassium currents in cultured rabbit retinal pigment epithelial cells

Membrane potential and ionic currents were studied in cultured rabbit retinal pigment epithelial (RPE) cells using whole-cell patch clamp and perforated-patch recording techniques. RPE cells exhibited both outward and inward voltage-dependent currents and had a mean membrane capacitance of 26±12 pF (sd, n=92). The resting membrane potential averaged ?31±15 mV (n=37), but it was as high as ?60 mV in some cells. When K⁺ was the principal cation in the recording electrode, depolarization-activated outward currents were apparent in 91% of cells studied. Tail current analysis revealed that the outward currents were primarily K⁺ selective. The most frequently observed outward K⁺ current was a voltage- and time-dependent outward current (I _K) which resembled the delayed rectifier K⁺ current described in other cells. I _K was blocked by tetraethylammonium ions (TEA) and barium (Ba²⁺) and reduced by 4-aminopyridine (4-AP). In a few cells (3–4%), depolarization to ?50 mV or more negative potentials evoked an outwardly rectifying K⁺ current (I _Kt) which showed more rapid inactivation at depolarized potentials. Inwardly rectifying K⁺ current (I _KI) was also present in 41% of cells. I _KI was blocked by extracellular Ba²⁺ or Cs⁺ and exhibited time-dependent decay, due to Na⁺ blockade, at negative potentials. We conclude that cultured rabbit RPE cells exhibit at least three voltage-dependent K⁺ currents. The K⁺ conductances reported here may provide conductive pathways important in maintaining ion and fluid homeostasis in the subretinal space.     

Action of tetraethylammonium on calcium-activated potassium channels in pig pancreatic acinar cells studied by patch-clamp single-channel and whole-cell current recording

Summary The effects of tetraethylammonium ions on currents through high-conductance voltage- and Ca²⁺-activated K⁺ channels have been studied with the help of patch-clamp single-channel and whole-cell current recording on pig pancreatic acinar cells. In excised outside-out membrane patches TEA (1 to 2 mM) added to the bath solution virtually abolishes unitary current activity except at very positive membrane potentials when unitary currents corresponding to a markedly reduced conductance are observed. TEA in a lower concentration (0.2 mM) markedly reduces the open-state probability and causes some reduction of the single-channel conductance. In inside-out membrane patches bath application of TEA in concentrations up to 2 mM has no effect on single-channel currents. At a higher concentration (10 mM) slight reductions in single-channel conductance occur. In whole-cell current recording experiments TEA (1 to 2 mM) added to the bath solution completely suppresses the outward currents associated with depolarizing voltage jumps to membrane potentials of 0 mV and blocks the major part (70 to 90%) of the outward currents even at very positive membrane potentials (30 to 40 mV). In contrast TEA (2 mM) added to the cell interior (pipette solution) has no effect on the outward K⁺ current. Our results demonstrate that TEA in low concentrations (1 to 2 mM) acts specifically on the outside of the plasma membrane to block current through the high-conductance Ca²⁺- and voltage-activated K⁺ channels     

Mechanism of Cl- sensitivity in internal ion receptors of the leech; an inward current gated off by Cl- in the nephridial nerve cells

Summary The nephridial nerve cells of the leech, Hirudo medicinalis, 34 sensory cells, each associated with one nephridium, are sensitive to changes in extracellular Cl^- concentration, an important factor in ion homeostasis. Using single-electrode current- and voltage clamp and ion substitution techniques, the specificity and mechanism of Cl^- sensitivity of the nephridial nerve cell was studied in isolated preparations. Increase of the normally low external Cl^- concentration leads to immediate and sustained hyperpolarization, decrease of the frequency of bursts and decrease of membrane conductance. The response is halogen specific: Cl^- can be replaced by Br^–, but not by organic mono- or divalent anions or inorganic divalent anions.At physiological Cl^- concentrations (36mM extra-cellular Cl^-), the nephridial nerve cell has a high resting conductance for Cl^- and the membrane potential is governed by Cl^-. In high extracellular Cl^- concentrations (110–130 mM), membrane conductance is low, most likely due to the gating off of Cl^- channels. Under these conditions, membrane potential is dominated by the K⁺ distribution and the nephridial nerve cell hyperpolarizes towards E_K.Abbreviations NNC nephridial nerve cell - V _m membrane potential - E _Cl(k) equilibrium potential for Cl (K) - IV-curve current-voltage relationship     

Maxi K+ channels from the apical membranes of rabbit oviduct epithelial cells

Large conductance (approximately 210 pS), K⁺-selective channels were identified in excised, insideout patches obtained from the apical membranes of both ciliated and nonciliated epithelial cells grown as monolayers from the primary culture of rabbit oviduct. The open probability of channels showing stable gating was increased at positive membrane potentials and was sensitive to the concentration of free calcium ions at the cytosolic surface of the patch ([Ca²⁺] _i ). In these respects, the channel resembled maxi K⁺ channels found in a number of other cell types. The distributions of dwell-times in the open state were most consistently described by two exponential components. Four exponential components were fitted to the distributions of dwelltimes in the closed state. Depolarizations and [Ca²⁺] _i increases had similar effects on the distribution of open dwell-times, causing increases in the two open time constants ( _o1 and _o2) and the fraction of events accounted for by the longer component of the distribution. In contrast, calcium ions and voltage had distinct effects on the distribution of closed dwelltimes. While the three shorter closed time constants ( _c1, _c2 and _c3) were reduced by depolarizing membrane potentials, increases in [Ca²⁺] _i caused decreases in the longer time constants ( _c3 and _c4). It is concluded that oviduct large conductance Ca²⁺-activated K⁺ channels can enter at least two major open states and four closed states.A.F.J. was supported by a research fellowship from the Japan Society for the Promotion of Science and received a grant for laboratory expenses from the Ministry of Education, Science and Culture, Japan. The authors wish to thank Dr. Shigetoshi Oiki for valuable discussion of the analysis of gating kinetics and Dr. Jeman Kim (Kyoto Pharmaceutical University) for making the transmission electron micrographs.     

GTP-binding proteins regulate high conductance anion channels in rat bile duct epithelial cells

Summary Epithelial cells from the intrahepatic bile duct contribute to bile formation, but little is known of the cellular mechanisms responsible. In these studies, we have characterized the endogenous GTP-binding proteins (G proteins) present in these cells and evaluated their role in regulation of high conductance anion channels. G proteins were identified in purified plasma membranes of isolated bile duct epithelial cells using specific antisera on Western blots, and ion channel activity was measured in excised inside-out membrane patches using patch-clamp recording techniques. In patches without spontaneous channel activity, addition of cholera toxin to the cytoplasmic surface had no effect (n=10). Addition of pertussis toxin caused an activation of channels in 13/34 (38%) attempts, as detected by an increase in channel open probability. Activated channels were anion selective (gluconate/Cl^– permeability ratio of 0.17±0.04) and had a unitary conductance of 380 pS. Channel open probability was also increased by the nonhydrolyzable GDP analogue guanosine 5-0-(2-thiodiphosphate) in 8/14 (57%) attempts. In contrast, channel open probability was rapidly and reversibly decreased by the nonhydrolyzable analogue of GTP 5guanylylimidodiphosphate in 7/9 (78%) attempts. Western blotting with specific antisera revealed that both G_i –2 and G_i –3 were present in significant amounts, whereas G_i –1 and G_o were not detected. These studies indicate that in bile duct epithelial cells, high conductance anion channels are inhibited, in a membrane-delimited manner, by PTX-sensitive G proteins.We gratefully acknowledge the assistance of Marwan Farouk, M.D. in the preparation of bile duct epithelial cells, Lucy Seger in the identification of the G proteins, C.F. Starmer in channel analysis, and P.J. Casey for the gift of bacteria expressing the different G-protein -subunits. This work was supported in part by grants from the National Institutes of Health DK43278 (to J.G.F.), DK42486 (to T.W.G.), and DK07568 (to J.M.M.); American Gastroenterological Association/G.D. Searle Research Scholar Award (to J.G.F.) and an American Gastroenterological Association Advanced Research Training Award (J.M.M.).     

Ion channels activated by swelling of Madin Darby Canine Kidney (MDCK) cells

Summary According to previous studies hyposmotic swelling of Madin Darby Canine Kidney (MDCK) cells leads to a marked decrease of cell membrane resistance. The present study has been performed to identify the underlying ion channels using the patchclamp technique: reduction of extracellular osmolarity to 230 mmol/liter leads to a transient activation of K⁺ channels and a sustained activation of anion channels. The K⁺ channels are inwardly rectifying with a single-channel slope conductance of 56 ± 3 pS at –50 mV (cell negative) and of 29 ± 2 pS at 0 mV PD across the patch 150 mmol/liter K⁺ in pipette). The same channels are activated by an increase of intracellular calcium activity, as shown previously. The anion channels display a single-channel slope conductance of 41 ± 4 pS at –50 mV (cell negative) and of 25 ± 3 pS at 0 mV PD across the patch (150 mmol/liter Cl^– in pipette). The channel is anion selective and conducts both bicarbonate and chloride with a preference for bicarbonate. Its open probability is not affected by changing intracellular calcium from 0.1–10 mol/liter. The channels observed explain the effects of cell swelling on PD, ion selectivity and resistance of the cell membrane in MDCK cells.The authors gratefully acknowledge the valuable discussion with Drs. P. Deetjen, E. Wöll and F. Friedrich, the skilled technical assistance of G. Siber and S. David, and the excellent mechanic and electronic support by K.-H. Streicher, Ing. M. Hirsch and M. Plank. This study was supported by the Fonds zur Förderung der wissenschaftlichen Forschung, Grant No. P5813 and P6792M.     

Chloride channels in human platelets: Evidence for activation by internal calcium

Summary Whole-cell patch-clamp recordings were made from freshly isolated human platelets. The pipette contained a high concentration of divalent cations, which permitted easy disruption of cell-attached membrane patches by suction. Single-channel currents were measured when the pipette contained isotonic BaCl₂ or MgCl₂ saline; over 30 sec –5 min an increasing number of channels appeared until conductance steps through individual channels could no longer be distinguished. The current-voltage relationship was curvilinear; chord conductance at –35 mV was 25 pS increasing to 45 to 52 pS at +45 mV. Ion substitution experiments showed the current to be primarily carried by Cl^–.E _rev was shifted 30 mV/10-fold change in external Cl^– (replaced by gluconate), was similar with BaCl₂ or MgCl₂ in the pipette and was not significantly shifted by replacing external Na⁺ with K⁺. Addition of 1mm BAPTA to the MgCl₂ pipette saline prevented activation of Cl^– currents; with isotonic CaCl₂ internal saline, current appeared immediately upon patch rupture, suggesting that the Cl^– channels are dependent on internal Ca²⁺, 5-nitro-2-(3-phenylpropylamino)-benzoate, reported to block a Cl^– conductance in studies of rat epithelial cells, caused a potent flickery block and may be a useful tool with which to investigate the physiological role of Cl^– currents in human platelets.     

Diversity of K+ channels in the basolateral membrane of restingnecturus oxyntic cells

Summary Patch-clamp techniques have been applied to characterize the channels in the basolateral membrane of resting (cimetidine-treated, nonacid secreting) oxyntic cells isolated from the gastric mucosa ofNecturus maculosa. In cell-attached patches with pipette solution containing 100mm KCl, four major classes of K⁺ channels can be distinguished on the basis of their kinetic behavior and conductance: (1) 40% of the patches contained either voltage-independent (a) or hyperpolarization-activated (b), inward-rectifying channels with short mean open times (16 msec fora, and 8 msec forb). Some channels showed subconductance levels. The maximal inward conductanceg _max was 31±5 pS (n=13) and the reversal potentialE _rev was atV _p=–34±6 mV (n=9). (2) 10% of the patches contained depolarization-activated and inward-rectifying channels withg _max=40 ±18 pS (n=3) andE _rev was atV _p=–31±5 mV (n=3). With hyperpolarization, the channels open in bursts with rapid flickerings within bursts. Addition of carbachol (1mm) to the bath solution in cell-attached patches increased the open probabilityP _o of these channels. (3) 10% of the patches contained voltage-independent inward-rectifying channels withg _max=21±3 pS (n=4) andE _rev was atV _p=–24±9 mV (n=4). These channels exhibited very high open probability (P _o=0.9) and long mean open time (1.6 sec) at the resting potential. (4) 20% of the patches contained voltage-independent channels with limiting inward conductance of 26±2 pS (n=3) andE _rev atV _p=–33±3 mV (n=3). The channels opened in bursts consisting of sequential activation of multiple channels with very brief mean open times (10 msec). In addition, channels with conductances less than 6 pS were observed in 20% of the patches. In all nine experiments with K⁺ in the pipette solution replaced by Na⁺, unitary currents were outward, and inward currents were observed only for large hyperpolarizing potentials. This indicates that the channels are more selective for K⁺ over Na⁺ and Cl^–. A variety of K⁺ channels contributes to the basolateral K⁺ conductance of resting oxyntic cells.     

TNF-alpha promotes cell survival through stimulation of K+ channel and NFkappaB activity in corneal epithelial cells

Tumor necrosis factor (TNF-alpha) in various cell types induces either cell death or mitogenesis through different signaling pathways. In the present study, we determined in human corneal epithelial cells how TNF-alpha also promotes cell survival. Human corneal epithelial (HCE) cells were cultured in DMEM/F-12 medium containing 10% FBS. TNF-alpha stimulation induced activation of a voltage-gated K+ channel detected by measuring single channel activity using patch clamp techniques. The effect of TNF-alpha on downstream events included NFkappaB nuclear translocation and increases in DNA binding activities, but did not elicit ERK, JNK, or p38 limb signaling activation. TNF-alpha induced increases in p21 expression resulting in partial cell cycle attenuation in the G1 phase. Cell cycle progression was also mapped by flow cytometer analysis. Blockade of TNF-alpha-induced K+ channel activity effectively prevented NFkappaB nuclear translocation and binding to DNA, diminishing the cell-survival protective effect of TNF-alpha. In conclusion, TNF-alpha promotes survival of HCE cells through sequential stimulation of K+ channel and NFkappaB activities. This response to TNF-alpha is dependent on stimulating K+ channel activity because following suppression of K+ channel activity TNF-alpha failed to activate NFkappaB nuclear translocation and binding to nuclear DNA.     

Open-channel block of Na+ channels by intracellular Mg2+

1. Macroscopic and single-channel currents through several types of cloned rat brain Na⁺ channels, expressed in Xenopus oocytes, were measured using the patch-clamp technique. 2. For all cloned channel types and for endogenous Na⁺ channels in chromaffin cells, intracellular Mg²⁺ blocks outward currents in a voltage-dependent manner similar to that in rat brain type II Na⁺ channel (Pusch et al. 1989). 3. A sodium-channel mutant (cZ-2) with long single-channel open times was used to examine the voltage-dependent reduction of single-channel outward current amplitudes by intracellular Mg²⁺. This reduction could be described by a simple blocking mechanism with half-maximal blockage at 0 mV in 1.8 mM intracellular Mg²⁺ and a voltage-dependence of e-fold per 39 mV (in 125 mM [Na] _i ); this corresponds to a binding-site at an electrical distance of 0.32 from the inside of the membrane. 4. At low Mg²⁺ concentrations and high voltages, the open-channel current variance is significantly elevated with respect to zero [Mg] _i . This indicates that Mg²⁺ acts as a fast blocker rather than gradually decreasing current, e.g. by screening of surface charges. Analysis of the open-channel variance yielded estimates of the block and unblock rate constants, which are of the order of 2 · 10⁸ M^–1 s^–1 and 3.6 · 10⁵ s^–1 at 0 mV for the mutant cZ-2. 5. A quantitative analysis of tail-currents of wild-type 11 channels showed that the apparent affinity for intracellular Mg²⁺ strongly depends on [Na] _i . This effect could be explained in terms of a multi-ion pore model. 6. Simulated action potentials, calculated on the basis of the Hodgkin-Huxley theory, are significantly reduced in their amplitude and delayed in their onset by postulating Mg²⁺ block at physiological levels of [Mg] _i .abbreviations [Na]_i intracellular Na⁺ concentration - [K] _i intracellular K⁺ concentration - [Mg] _i intracellular Mg²⁺ concentration - HEPES N-2-hydroxylethyl piperazine-N-2-ethanesulfonic acid - EGTA ethyleneglycol-bis-[\-amino-ethyl ether] N,N-tetra acetic acid - TEA tetraethylammonium     

pH Regulation in tissue-cultured bovine lens epithelial cells

Summary The intracellular pH (pH _i ) of tissue-cultured bovine lens epithelial cells was measured in small groups of 6 to 10 cells using the trapped fluorescent dye 2,7-bis-(2-,carboxyethyl)-5 (and 6)carboxyfluorescein (BCECF). When perifused at 35°C with artificial aqueous humour solution (AAH) containing 16 mM HCO ₃ ^- and 5% CO₂, pH 7.25, pH _i was 7.19±0.02 (sem, n = 95). On removing HCO ₃ ^- and CO₂ there was an initial transient alkalinization followed by a fall in pH to a steady value of 6.97±0.03 (sem, n = 54). Addition of 0.25 mM 4,4-diisothiocyanatostilbene2, 2-disulfonic acid (DIDS) to AAH containing HCO ₃ ^- and CO₂ led to a rapid and pronounced fall in pH. Exposure to Na⁺-free AAH again led to a marked fall in pH _i , but in this case the addition of DIDS did not produce a further fall. Substitution of the impermeant anion gluconate for Cl^– in the presence of HCO ₃ ^- led to a rise in pH _i , while substitution in the absence of HCO ₃ ^- led to a fall in pH _i . The above data indicate a significant role for a sodium-dependent Cl^–-HCO ₃ ^- exchange mechanism in the regulation of pH _i . Addition of 1 mM amiloride to control AAH in both the presence and absence of HCO ₃ ^- led to a marked fall in pH _i , indicating that a Na⁺/H⁺ exchange mechanism also has a significant role in the regulation of pH _i . There is evidence for a lactic acid transport mechanism in bovine lens cells, as addition of lactate to the external medium produced a rapid fall in pH _i . Larger changes in pH _i were observed in control compared to HCO ₃ ^- -free AAH and in the latter case a pronounced alkalinizing overshoot was obtained on removing external lactate. Tissue-cultured bovine lens cells thus possess at least three membrane transport mechanisms that are involved in pH regulation. The buffering capacity of the lens cells was measured by perturbing pH _i with either NH ₄ ⁺ or procaine. The values obtained were similar in both cases and the intrinsic buffering capacity measured in the absence of external HCO ₃ ^- was 5 mm/pH unit (procaine). However, in the presence of HCO ₃ ^- and CO₂ the buffer capacity increases approximately fourfold, indicating that HCO ₃ ^- is the principal intracellular buffer.We acknowledge financial support from the Wellcome Trust and the Humane Research Trust for this project. M.R. Williams was in receipt of a Science & Engineering Research Council studentship.     

Regulation of maxi-K+ channels on pancreatic duct cells by cyclic AMP-dependent phosphorylation

Summary Using the patch-clamp technique we have identified a Ca²⁺-sensitive, voltage-dependent, maxi-K⁺ channel on the basolateral surface of rat pancreatic duct cells. The channel had a conductance of 200 pS in excised patches bathed in symmetrical 150mm K⁺, and was blocked by 1mm Ba²⁺. Channel openstate probability (P _o ) on unstimulated cells was very low, but was markedly increased by exposing the cells to secretin, dibutyryl cyclic AMP, forskolin or isobutylmethylxanthine. Stimulation also shifted theP _o /voltage relationship towards hyperpolarizing potentials, but channel conductance was unchanged. If patches were excised from stimulated cells into the inside-out configuration,P _o remained high, and was not markedly reduced by lowering bath (cytoplasmic) Ca²⁺ concentration from 2mm to 0.1 m. However, activated channels were still blocked by 1mm Ba²⁺. ChannelP _o was also increased by exposing the cytoplasmic face of excised patches to the purified catalytic subunit of cyclic AMP-dependent protein kinase., We conclude that cyclic AMP-dependent phosphorylation can activate maxi-K⁺ channels on pancreatic duct cells via a stable modification of the channel protein itself, or a closely associated regulatory subunit, and that phosphorylation alters the responsiveness of the channels to Ca²⁺. Physiologically, these K⁺ channels may contribute to the basolateral K⁺ conductance of the duct cell and, by providing a pathway for current flow across the basolateral membrane, play an important role in pancreatic bicarbonate secretion.     

Mucosal acidification and an acid microclimate in the hen colon in vitro

Experiments were performed on isolated, stripped colonic epithelia of low-salt-adapted hens (Gallus domesticus) in order to characterize acid secretion by this tissue. With symmetric, weak buffer solutions, colonic epithelia acidified both mucosal and serosal sides. Titration measurements of the mucosal acidification rate (pH-stat technique) averaged 1.63±0.25 Eq·cm^-2·h^-1. Mucosal acidification was also evident in colons from high-salt-adapted birds and in low-salt-adapted coprodeum, but was completely abolished in the high-salt coprodeum. Mucosal acidification by low-salt-adapted colonic epithelium was unaffected by sodium replacement, mucosal amiloride (10^-3 mol·l^-1), and serosal ouabain (5x10^-4 mol·l^-1), although all three treatments significantly reduced or reversed the short-circuit current. Acetazolamide (10^-3 mol·l^-1, serosal) reduced mucosal acidification by 15% and simultaneously increased short-circuit current by a similar amount. Colonic epithelia incubated in glucose-free solutions had significantly lower acidification rates (0.59±0.13 Eq·cm^-2·h^-1, P<0.002 versus controls) and addition of glucose (15 mmol·l^-1), but not galactose, partially restored acidification to control levels. Anoxia (N₂ gassing) completely inhibited short-circuit current, but reduced acidification by only 30%. A surface microclimate pH, nearly 2 pH units more acidic than the bath pH of 7.1–7.4 was measured in low-salt-adapted colon and coprodeum. The acid microclimate of both tissues was partially attenuated by adaptation to a high-salt diet. Colonic microclimate pH was dependent on the presence of glucose and sensitive to the bath pH. Histochemical staining for carbonic anhydrase localized this enzyme to cytoplasm and lateral margins of one subfraction of colonic cells, and to cytoplasm in a second subpopulation Intense staining was also evident in subepithelial capillaries. These results suggest that a large part of mucosal acidification and maintenance of the acid microclimate in hen colon may be dependent on glycolysis and metabolic acid production, although a smaller, electrogenic and acetazolamidesensitive component also appears to exist. This latter component may become more prominent under conditions of cellular acidification.Abbreviations CA carbonic anhydrase - I _SC short circuit current - NFM N-ethylmaleimide - PD transepithelial potential - SCFA short chain fatty acids     

Low-conductance chloride channel activated by cAMP in the epithelial cell line T84

We have studied the modulation and pharmacological properties of two anion channels in T₈₄ cells by recording single channel and transepithelial currents. One channel had an outwardly rectifying current-voltage I/V curve, was rarely active in cell-attached patches, and was unaffected by cAMP. The other channel had lower conductance (8.7 pS at 37°C) and a more ohmic I/V relationship. Exposure to cAMP increased the probability of observing low-conductance channel activity in cell-attached patches> 6-fold. Extracellular DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid) or IAA-94 (an indanyloxyacetic acid) inhibited the outward rectifier but did not affect the low-conductance channel or cAMP-stimulated transepithelial current. These results suggest the low-conductance Cl channel may contribute to apical membrane conductance during cAMP-stimulated secretion.     

Differences in open state of NBA-modified cardiac Na+ channels

Patch clamp recordings from neonatal cardiac Na⁺ channels treated with N-bromoacetamide (NBA, 5–50 x 10^-mol/l) showed modified Na⁺ channel activity. By chemical removal of inactivation, repetitive openings with an increased life time and burst-like activity occurred. NBA-modified Na⁺ channels differ in life time and may attain either a slightly (mean open time 3.1±0.2 ms) or a strongly (mean open time 15.2±1.4 ms) prolonged open state. This strongly suggests a heterogeneous population of NBA-modified Na⁺ channels in newborn rat cardiocytes.