Putative Cl Research Articles

The Organellular Chloride Channel Protein CLIC4/mtCLIC Translocates to the Nucleus in Response to Cellular Stress and Accelerates Apoptosis

CLIC4/mtCLIC, a chloride intracellular channel protein, localizes to the mitochondria and cytoplasm of keratinocytes and participates in the apoptotic response to stress. We now show that multiple stress inducers cause the translocation of cytoplasmic CLIC4 to the nucleus. Immunogold electron microscopy and confocal analyses indicate that nuclear CLIC4 is detected prior to the apoptotic phenotype. CLIC4 associates with the Ran, NTF2, and Importin-alpha nuclear import complexes in immunoprecipitates of lysates from cells treated with apoptotic/stress-inducing agents. Deletion or mutation of the nuclear localization signal in the C terminus of CLIC4 eliminates nuclear translocation, whereas N terminus deletion enhances nuclear localization. Targeting CLIC4 to the nucleus via adenoviral transduction accelerates apoptosis when compared with cytoplasmic CLIC4, and only nuclear-targeted CLIC4 causes apoptosis in Apaf null mouse fibroblasts or in Bcl-2-overexpressing keratinocytes. These results indicate that CLIC4 nuclear translocation is an integral part of the cellular response to stress and may contribute to the initiation of nuclear alterations that are associated with apoptosis.

Specificity of Classical and Putative Cl- Transport Inhibitors on Membrane Transport Pathways in Human Erythrocytes

The majority of anion transport inhibitors tend to be non-specific. This is problematic from a research point of view as caution is required when defining pathways purely based on pharmacology. Here we have tested a range of classical and putative Cl^- transport inhibitors on three Cl^- carrier systems (the KCl cotransporter (KCC), the NaK2Cl cotransporter (NKCC), and the Band 3 anion exchanger (AE)) found in human erythrocytes, using radiolabel tracer experiments. The study confirms the cross-reactivity of many anion transport inhibitors. However, two compounds, H25 and H156, were found to be both potent (IC₅₀ values < 0.1 mM) and specific (at least 1000-fold more effective against one carrier compared to the other two) inhibitors of NKCC and AE, respectively.

Actions of putative chloride channel blocking agents on canine lower esophageal sphincter (LES)

Niflumic acid (NA), a putative Cl–-channel blocker, has provided pharmacological evidence that Cl–-channel closures mediate hyperpolarization caused by NO in gastrointestinal smooth muscle. However, NA caused concentration- dependent relaxation of canine lower esophageal sphincter (LES) and failed to inhibit NO-mediated relaxations. DIDS also did not inhibit NO-mediated relaxations, but did abolish them when present with 20 mM TEA (tetraethyl ammonium ion), which was also ineffective alone. TEA reversed NA-induced relaxations, but with NA it did not inhibit NO-mediated relaxations. We investigated the modes of action of these agents further. Neither nerve-function block nor block of NOS activity affected the inhibition of LES tone by NA. In patch-clamp studies, NA increased outward currents from –30 to + 90 mV when [Ca2+]pipette was 50 nM. This was prevented by 20 mM TEA, but not by prior inhibition of NOS. At 200 nM [Ca2+]pipette, TEA markedly reduced outward currents, but did not prevent the increase from subsequent NA. In contrast, under similar conditions, application of DIDS after 20 mM TEA further reduced outward currents. When the patch pipette contained CsCl and TEA to block K+ currents, NA had no significant effect on currents between –50 and +90 mV. Thus, NA acted by opening K+ channels: some TEA-sensitive and some not. It had no detectable effect on currents when K+ channels were blocked. We conclude that NA is an unreliable pharmacological tool to evaluate Cl–-channel contributions to smooth muscle function. DIDS did not open K+ channels. Decreases in outward currents from DIDS may result from inhibition of K+ currents or currents carried by Cl– at depolarized membrane potentials.Key words: DIDS, niflumic acid, NO actions, smooth muscle.

Actions of putative chloride channel blocking agents on canine lower esophageal sphincter (LES)

Niflumic acid (NA), a putative Cl(-)-channel blocker, has provided pharmacological evidence that Cl(-)-channel closures mediate hyperpolarization caused by NO in gastrointestinal smooth muscle. However, NA caused concentration-dependent relaxation of canine lower esophageal sphincter (LES) and failed to inhibit NO-mediated relaxations. DIDS also did not inhibit NO-mediated relaxations, but did abolish them when present with 20 mM TEA (tetraethyl ammonium ion), which was also ineffective alone. TEA reversed NA-induced relaxations, but with NA it did not inhibit NO-mediated relaxations. We investigated the modes of action of these agents further. Neither nerve-function block nor block of NOS activity affected the inhibition of LES tone by NA. In patch-clamp studies, NA increased outward currents from -30 to + 90 mV when [Ca2+]pipette was 50 nM. This was prevented by 20 mM TEA, but not by prior inhibition of NOS. At 200 nM [Ca2+]pipette, TEA markedly reduced outward currents, but did not prevent the increase from subsequent NA. In contrast, under similar conditions, application of DIDS after 20 mM TEA further reduced outward currents. When the patch pipette contained CsCl and TEA to block K+ currents, NA had no significant effect on currents between -50 and +90 mV. Thus, NA acted by opening K+ channels: some TEA-sensitive and some not. It had no detectable effect on currents when K+ channels were blocked. We conclude that NA is an unreliable pharmacological tool to evaluate Cl(-)-channel contributions to smooth muscle function. DIDS did not open K+ channels. Decreases in outward currents from DIDS may result from inhibition of K+ currents or currents carried by Cl- at depolarized membrane potentials.

Permeabilization via the P2X7 Purinoreceptor Reveals the Presence of a Ca2+-activated Cl−Conductance in the Apical Membrane of Murine Tracheal Epithelial Cells

Calcium-activated Cl(-) secretion is an important modulator of regulated ion transport in murine airway epithelium and is mediated by an unidentified Ca(2+)-stimulated Cl(-) channel. We have transfected immortalized murine tracheal epithelial cells with the cDNA encoding the permeabilizing P2X(7) purinoreceptor (P2X(7)-R) to selectively permeabilize the basolateral membrane and thereby isolate the apical membrane Ca(2+)-activated Cl(-) current. In P2X(7)-R-permeabilized cells, we have demonstrated that UTP stimulates a Cl(-) current across the apical membrane of CF and normal murine tracheal epithelial cells. The magnitude of the UTP-stimulated current was significantly greater in CF than in normal cells. Ion substitution studies demonstrated that the current exhibited a permselectivity sequence of Cl(-) > I(-) > Br(-) > gluconate(-). We have also determined a rank order of potency for putative Cl(-) channel blockers: niflumic acid > or = 5-nitro-2-(3-phenylpropylamino)benzoic acid > 4, 4'-diisothiocyanostilbene-2,2'-disulfonate > glybenclamide >> diphenlyamine-2-carboxylate, tamoxifen, and p-tetra-sulfonato-tetra-methoxy-calix[4]arene. Complete characterization of this current and the corresponding single channel properties could lead to the development of a new therapy to correct the defective airway surface liquid in cystic fibrosis patients.

Identification of an acid-activated Cl(-) channel from human skeletal muscles.

ClC-4 gene was isolated as a putative Cl(-) channel. Due to a lack of functional expression of ClC-4, its physiological role remains unknown. We isolated a human ClC-4 clone (hClC-4sk) from human skeletal muscles and stably transfected it to Chinese hamster ovary cells. Whole cell patch-clamp studies showed that the hClC-4sk channel was activated by external acidic pH and inhibited by DIDS. It passed a strong outward Cl(-) current with a permeability sequence of I(-) > Cl(-) > F(-). The hClC-4sk has consensus sites for phosphorylation by protein kinase A (PKA); however, stimulation of PKA had no effect on the currents. hClC-4sk mRNA was expressed in excitable tissues, such as heart, brain, and skeletal muscle. These functional characteristics of hClC-4sk provide a clue to its physiological role in excitable cells.

Chloride conductance in HT29 cells: investigations with apical membrane vesicles and RT-PCR.

Vesicles enriched in a marker enzyme for apical membranes were isolated from HT29 cells. These vesicles contain an anion conductance with the selectivity gluconate approximately sulphate<F-<Cl-<Br-<NO3-<I-<SCN-. K+ diffusion potential-driven 36Cl- uptake was inhibited by 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB)>4, 4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS)>glibenclamide. The Cl- conductance was insensitive to Ca2+ and to extravesicular cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP) and inosine 5'-triphosphate (ITP). Using the reverse transcription polymerase chain reaction (RT-PCR) technique and sequencing of the amplified products we detected messenger ribonucleic acid (mRNA) for the cystic fibrosis transmembrane conductance regulator (CFTR), the putative Cl- channel or Cl- channel regulator pICln, and the Cl- channels ClC-2, ClC-3, ClC-5 and ClC-6 in HT29 cells. The properties of the vesicles' Cl- conductance resemble those of the intermediate conductance outwardly rectifying Cl- channel and tentatively exclude contributions of CFTR, pICln and ClC-2. Whether ClC-3, ClC-5, ClC-6 are involved in Cl- conductance remains to be determined.

Characterization of an inwardly rectifying chloride conductance expressed by cultured rat cortical astrocytes.

The biophysical and pharmacological properties of the inwardly rectifying Cl- conductance (IClh), expressed in rat type-1 neocortical cultured astrocytes upon a long-term treatment (1-3 weeks) with dibutyryl-cyclic-AMP (dBcAMP), were investigated with the whole-cell patch-clamp technique. Using intra- and extra-cellular solutions with symmetrical high Cl- content and with the monovalent cations replaced with N-methyl-D-glucamine, time- and voltage-dependent Cl- currents were elicited in response to hyperpolarizing voltage steps from a holding potential of 0 mV. The inward currents activated slowly and did not display any time-dependent inactivation. The rising phase of the current traces was best fitted with two exponential components whose time constants decreased with larger hyperpolarization. The steady-state activation of IClh was well described by a single Boltzmann function with a half-maximal activation potential at - 62 mV and a slope of 19 mV that yields to an apparent gating charge of 1.3. The anion selectivity sequence was Cl- = Br- = I- > F- > cyclamate > or = gluconate. External application of the putative Cl- channel blockers 4,4 diisothiocyanatostilbene-2,2 disulphonic acid or 4-acetamido-4-isothiocyanatostilbene-2,2-disulphonic acid did not affect IClh. By contrast, anthracene-9-carboxylic acid, as well as Cd2+ and Zn2+, inhibited, albeit with different potencies, the Cl- current. Taken together, these results indicate that dBcAMP-treated cultured rat cortical astrocytes express a Cl- inward rectifier, which exhibits similar but not identical features compared with those of the cloned and heterologously expressed hyperpolarization-activated Cl- channel ClC-2.

Expansion of the cellular content of ribonucleoside triphosphates induces cell shrinkage and KCl loss in Ehrlich ascites tumor cells and in Chinese hamster ovary cells

The conversion to corresponding triphosphate derivatives of various ribonucleosides has been studied in Ehrlich ascites tumor cells and in Chinese hamster ovary cells under conditions that are optimal for cellular uptake of orthophosphate. The initial cellular uptake of orthophosphate is followed by a cellular loss of Cl − which might be consistent with a H 2PO 4 −/Cl − exchange mechanism. Subsequent addition of ribonucleosides to the medium leads to cellular accumulation of the corresponding triphosphate and to a concomitant loss of KCl and to sustained cell volume reduction. The latter two events are quite unspecific with regard to the nucleobase moiety of the ribonucleoside triphosphate accumulated (adenine, guanine and purine being almost equally effective) and they depend in a rather simple way on the increase of the cellular content of these compounds. The KCl loss seems to depend on opening of the separate K + and Cl − channels. The pharmacological profile of the putative ion channels could not be identified in spite of experiments with conventional blockers. In the case of purine riboside the accumulation of the corresponding triphosphate and concomitant loss of KCl and cell water may be followed by a regain of cell volume due to a continued purine riboside triphosphate accumulation, which apparently depends on the uptake of orthophosphate by cotransport with Na + and which for osmotic reasons is accompanied by the uptake of water and hence volume increase. The possibility that the nucleoside triphosphate induced opening of a putative Cl − channel may be due to a direct effect of triphosphate on a channel protein is discussed.

Characterization of a swelling-activated anion conductance in homozygous typing cell hepatoma cells.

Liver cell volume and intracellular ion concentrations are maintained within a narrow physiologic range by regulated changes in membrane ion permeability. These studies of homozygous HTC hepatoma cells, a model liver cell line, evaluate the relationship between cell volume and membrane ion permeability, and assess the possibility that cell swelling allows the efflux of the intracellular osmolite taurine through the opening of a conductive pathway. Cell swelling induced by exposure to hypotonic solutions (203 mOsm) caused a rapid increase in cell volume, followed by recovery toward basal values. Volume recovery was inhibited by Cl- depletion or by exposure to the putative Cl- channel blocker 5-nitro-2-(3-phenylpropyl-amino) benzoic acid (NPPB) (25 micromol/L). Swelling increased the efflux rates of 36Cl (181% +/- 15%, P < .01) and 125I (310% +/- 21%, P < .01). In whole cell patch clamp recordings, cell swelling induced by 1) exposure to hypotonic solution or 2) intracellular perfusion with hypertonic sucrose-containing solutions activated an anion-selective current which was outwardly rectified and showed time-dependent inactivation at depolarizing potentials. The current density at -80 mV increased proportionally with increases in the transmembrane osmotic gradient from basal values of -1 pA/pF to maximal values of 70 pA/pF with 100 mmol/L sucrose in the pipette. Basal taurine permeability was low, but cell swelling increased the efflux of [1,2-3H]taurine to 1,587% +/- 172% of basal levels (P < .05). Intracellular perfusion with hypertonic solutions activated currents carried by anionic taurine, with an estimated taurine/Cl- permeability ratio of .88 +/- .17 for whole cell currents. These studies demonstrate that the HTC membrane anion permeability is closely coupled to changes in cell volume, and that the recovery from swelling depends upon activation of anion-selective conductance pathways permeable to both Cl- and taurine.

Characterization of a swelling-activated anion conductance in homozygous typing cell hepatoma cells

Liver cell volume and intracellular ion concentrations are maintained within a narrow physiologic range by regulated changes in membrane ion permeability. These studies of homozygous HTC hepatoma cells, a model liver cell line, evaluate the relationship between cell volume and membrane ion permeability, and assess the possibility that cell swelling allows the efflux of the intracellular osmolite taurine through the opening of a conductive pathway. Cell swelling induced by exposure to hypotonic solutions (203 mOsm) caused a rapid increase in cell volume, followed by recovery toward basal values. Volume recovery was inhibited by Cl- depletion or by exposure to the putative Cl- channel blocker 5-nitro-2-(3-phenylpropyl-amino) benzoic acid (NPPB) (25 μmol/L). Swelling increased the efflux rates of 36Cl (181% +/- 15%, P < .01) and 125I (310% +/- 21%, P < .01). In whole cell patch clamp recordings, cell swelling induced by 1) exposure to hypotonic solution or 2) intracellular perfusion with hypertonic sucrose-containing solutions activated an anion-selective current which was outwardly rectified and showed time-dependent inactivation at depolarizing potentials. The current density at -80 mV increased proportionally with increases in the transmembrane osmotic gradient from basal values of -1 pA/pF to maximal values of 70 pA/pF with 100 mmol/L sucrose in the pipette. Basal taurine permeability was low, but cell swelling increased the efflux of [1,2-3H]taurine to 1,587% +/- 172% of basal levels (P < .05). Intracellular perfusion with hypertonic solutions activated currents carried by anionic taurine, with an estimated taurine/Cl- permeability ratio of .88 +/- .17 for whole cell currents. These studies demonstrate that the HTC membrane anion permeability is closely coupled to changes in cell volume, and that the recovery from swelling depends upon activation of anion-selective conductance pathways permeable to both Cl- and taurine.(Hepatology 1997 Feb;25(2):403-10)

The electrical response ofAvena coleoptile cortex to auxins

The electrical response to auxin perfusion by cortex tissue of peeledAvena saliva L. coleoptiles was examined using microelectrode measurements of the membrane potential. Applied at 10 μM, the known auxins indole-3-acetic acid (IAA), α-naphthalene acetic acid (αNAA), 2,4-dichlorophenoxyacetic acid, and indole-3-butyric acid, the two analogues β-naphthalene acetic acid and 2,3-dichlorophenoxyacetic acid, and acetic acid transiently (1–30 min) depolarized the membrane potential; but a subsequent hyperpolarization, possibly resulting from activation of the plasma-membrane H+-ATPase, occurred only with active auxins. The magnitude and duration of the depolarizations induced by the different compounds varied. The αNAA-induced depolarization consisted of two depolarization events. The first reached a maximum within 2–3 min and the second between 6 and 10 min. The magnitude of the first αNAA-induced depolarization varied with external concentration of Cl− suggesting that it represents an efflux of Cl−. A known anion-channel blocker, anthracene-9-carboxylic acid, at 1 μM prevented the Cl−-sensitive depolarization induced by αNAA. The same concentration failed to block αNAA-induced elongation of peeled coleoptile segments, though higher concentrations (20 μM and 100 μM) increasingly inhibited growth. Applied at 10 μM, the naturally occurring auxin, IAA, induced a single depolarization (maximal between 6 and 8 min) which was insensitive to external Cl− concentration. The depolarization induced by 10 μM IAA, as well as that induced by acetic acid and the second depolarization induced by 10 μM αNAA, increased in magnitude when the pH of the perfusate was lowered from 6.0 to 4.5. When IAA was applied at a higher concentration (100 μM) the magnitude of the depolarization increased, lengthened, and also contained an early, Cl−-sensitive, depolarization (maximal between 2 and 4 min). The Cl−-sensitive depolarization induced by αNAA also increased when αNAA was increased from 10 to 100 μM, but, while 10 and 100 μM IAA and 10 μM αNAA induced elongation of peeledAvena coleoptile segments, 100 μM αNAA did not. Our results suggest that: (i) the putative Cl− currents induced by 10 μM αNAA and 100 μM IAA are the result of the opening of a population of Cl−-permeable anion channels, (ii) activation of these channels is not required for the auxin-induced growth ofAvena coleoptiles, (iii) the pH-sensitive depolarization induced by αNAA and IAA is not an auxin-specific response and might result from a weak acid protonophore effect, and (iv) the delayed hyperpolarization is a specific response to auxins inAvena coleoptiles.

Ca2+ release and activation of K+ and Cl- currents by extracellular ATP in distal nephron epithelial cells.

We have measured ionic currents and changes in intracellular Ca2+ concentration ([Ca2+]i) induced by extracellular ATP in single epithelial cells of the distal nephron from toad (A6 cells). ATP increased [Ca2+]i and concomitantly activated ionic currents. The ATP concentration for half-maximal increase in [Ca2+]i was approximately 10 microM. Current activation and elevation of [Ca2+]i also occurred in Ca(2+)-free bath solutions but were abolished by loading the cells via the patch pipette with 10 mM 1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid (BAPTA) or by preincubating the cells with 10 microM BAPTA-acetoxymethyl ester for 120 min. ATP-activated currents reversed at -53.9 +/- 1.9 mV (n = 22). Tetraethylammonium (TEA, 25 mM), a K+ channel blocker, partially blocked this current but did not affect the Ca2+ transients. The TEA-insensitive component of the current reversed close to Cl- equilibrium potential. 5-Nitro-2-(3-phenylpropylamino) benzoic acid, a putative Cl- channel blocker (100 microM), abolished nearly completely the ATP-activated current. Suramin (100 microM), a P2-purinergic receptor antagonist, strongly attenuated both Ca2+ transients and currents. In cell-attached patches, single channel currents activated by ATP could be observed, i.e., an inwardly rectifying K+ channel with a slope conductance for inward currents of approximately 32 pS and an ohmic Cl- channel with a conductance of 34 pS. It is concluded that ATP activates both Cl- and K+ channels in distal nephron epithelial cells by a Ca(2+)-dependent mechanism.

Chloride Channels in Brown Adipocyte Plasma Membranes: Candidates for Mediation of α 1-Adrenergic Depolarization?

The presence of plasma membrane Cl − channels was investigated in brown-fat cells differentiated in culture. Single channel activity was followed by the patch-clamp technique, with an NMDG + Cl − pipette solution and a NaCl bath solution. Only rarely was putative Cl − channel activity encountered in the cell-attached mode. However, after excision, ion channel activity of two types was observed in inside-out patches: one type represented the earlier observed non-selective cation channel, and one a putative Cl −channel, with the following characteristics: practically non-rectifying current-voltage relationship with a conductance of ≍50 pS and a reversal potential of ≍0 in symmetrical Cl − solutions, voltage dependent channel activity (P o approached 0.5 at positive holding potentials), and rapid flickering activity in the open state. It is discussed whether this Cl − channel may be responsible for the initial depolarization phase observed after α 1-adrenergic stimulation of brown-fat cells.

Two highly homologous members of the ClC chloride channel family in both rat and human kidney.

We have cloned two closely related putative Cl- channels from both rat kidney (designated rClC-K1 and rClC-K2) and human kidney (hClC-Ka and hClC-Kb) by sequence homology to the ClC family of voltage-gated Cl- channels. While rClC-K1 is nearly identical to ClC-K1, a channel recently isolated by a similar strategy, rClC-K2 is 80% identical to rClC-K1 and is encoded by a different gene. hClC-Ka and hClC-Kb show approximately 90% identity, while being approximately 80% identical to the rat proteins. All ClC-K gene products are expressed predominantly in the kidney. While rClC-K1 is expressed strongly in the cortical thick ascending limb and the distal convoluted tubule, with minor expression in the S3 segment of the proximal tubule and the cortical collecting tubule, rClC-K2 is expressed in all segments of the nephron examined, including the glomerulus. Since they are related more closely to each other than to the rat proteins, hClC-Ka and hClC-Kb cannot be regarded as strict homologs of rClC-K1 or rClC-K2. After injection of ClC-K cRNAs into oocytes, corresponding proteins were made and glycosylated, though no additional Cl- currents were detectable. Glycosylation occurs between domains D8 and D9, leading to a revision of the transmembrane topology model for ClC channels.

Evaluation of the discriminative stimulus effects of the novel sedative-hypnotic CL 284,846

CL 284,846, N-[3-(3-cyanopyrazolo[1, 5-a]pyrimidin-7-yl)phenyl)]-N- ethylacetamide, is a novel non-benzodiazepine sedative-hypnotic with benzodiazepine-like sedative effects, but with less apparent liability for accompanying undesired side effects. In an effort to further characterize its pharmacological activity, CL 284,846 (3.0 mg/kg, IP, 30 min pretreatment) was established as a discriminative stimulus (DS) in rats (n = 7). CL 284,846 (0.3-10.0 mg/kg) showed a dose-related increase in drug-appropriate responding up to the training dose and a dose-related decrease in response rate. The benzodiazepine agonist triazolam (0.1-1.0 mg/kg), the benzodiazepine partial agonist Ro 17-1812 (0.3-3.0 mg/kg) and the triazolopyridazine CL 218,872 (1.0-3.0 mg/kg) substituted for CL 284,846 in all rats, whereas the imidazopyridines zolpidem (3.0-10.0 mg/kg) and alpidem (10.0-30.0 mg/kg), the benzodiazepine partial agonist bretazenil (0.03-10.0 mg/kg) and the novel putative anxiolytic CL 273,547 (10.0-56.0 mg/kg) substituted in most, but not all, rats. Ro 17-1812, bretazenil, and CL 218,872 had no effect on response rate while the other drugs showed a concomitant decrease in rate. The 5-HT1A agonist buspirone (1.0-10.0 mg/kg) and the barbiturate pentobarbital (3.0-17.0 mg/kg) failed to substitute for CL 284,846 up to rate-decreasing doses. The benzodiazepine antagonist flumazenil (3.0-10.0 mg/kg) blocked the DS effects of CL 284,846 in most rats with no effect on response rate. Taken together, these results suggest that the DS effects of CL 284,846 are mediated via benzodiazepine receptors; however, the DS profile of CL 284,846 remains distinct from both benzodiazepine and non-benzodiazepine sedative-hypnotic drugs.

Effect of NPPB on chloride (Cl-) transport in distal colon of potassium (K+) adapted rats.

Secondary hyperaldosteronism enhances the rate of K secretion in distal colon, at least in part, through the stimulation of Na(+)-K(+)-Cl- cotransport across the basolateral membrane. To maintain a constant intracellular Cl- activity an increase in Cl- transport out of the cell must be assumed. We explored, under amiloride 10(-4) M and short circuited conditions, conductive pathways for Cl- exit in the distal colon of K(+)-adapted rats by means of a putative Cl- channel blocker, NPPB (5-nitro-2(3-phenyl-propylamino-benzoate. Results prior to NPPB showed an increase in JClms after K+ loading from 5.84 +/- 0.66 to 8.33 +/- 0.86 and JClsm from 4.77 +/- 0.55 to 8.16 +/- 0.96 microEq h-1 cm-2 (P < 0.001), when compared with controls. Net fluxes were not different between groups. Luminal NPPB in K+ adaptation resulted in a decrease of JClsm, from 7.85 +/- 1.5 to 6.69 +/- 1.5 microEq h-1 cm-2 (P < 0.05). There were no changes in both unidirectional Cl- fluxes in controls under luminal NPPB and in potential difference (V) and short-circuit current (Isc) under any condition. Finally, K+ adaptation resulted in an increase of luminal cyclic AMP (cAMP) concentration (0.09 +/- 0.02 to 0.20 +/- 0.03 pmol 100 microliters -1, P < 0.005), when compared with control rats. The data may suggest a transcellular recycling of Cl- and an activated NPPB inhibitable serosal to mucosal Cl- pathway on luminal membrane in the K+ adapted state, possibly mediated by an increase in cAMP production.

Mechanisms of oxalate absorption and secretion across the rabbit distal colon.

To further evaluate the mechanisms of oxalate (Ox2-) transport in the intestine the following studies were performed using isolated, short-circuited segments of the rabbit distal colon (DC). In control buffer, the DC absorbed Ox2- (net Ox2- flux, JNetOx = 5.4 +/- 0.7 pmol.cm-1.h-1). Replacement of Na+ with N-methyl-D-glucamine (NMDG+) abolished Ox2- absorption by decreasing mucosal to serosal Ox2- flux (JmsOx), without affecting Cl- transport, while gluconate substitution for Cl- did not affect JNetOx or net Na+ flux (JNetNa). Addition of Na+ to the serosal side of tissues bathed by NMDG+ buffer increased JmsOx 40% without altering mucosal to serosal Cl- flux (JmsCl). Serosal amiloride or dimethyl amiloride (10(-3) M) abolished JNetOx by decreasing JmsOx, it increased serosal to muscosal Cl- flux (JsmCl) and it gradually inhibited short-circuit current (Isc). Mucosal amiloride (10(-4) M) abolished Ise but had no effect on Ox2- or Cl- fluxes. Serosal 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, 10(-6) M) reduced JmsOx by 20% and JNetOx by 43% without affecting JmsCl or JNetCl. Dibutyryl cyclic adenosine monophosphate (dB-cAMP, 5 x 10(-4) M, both sides) stimulated Ox2- secretion (JNetOx = -12.6 +/- 3.3 pmol.cm-2.h-1). The dB-cAMP-induced secretion of Ox2- and Cl- were fully abolished by serosal furosemide (10(-4) M) and partially inhibited (35%) by 5 x 10(-4) M mucosal NPPB [5-nitro-2-(3-phenylpropylamino)-benzoic acid], a putative Cl- channel blocker.(ABSTRACT TRUNCATED AT 250 WORDS)

Ionic flux contributions to neocortical slow waves and nucleus basalis- mediated activation: whole-cell recordings in vivo

Slow, rhythmic membrane potential (Vm) fluctuations occur spontaneously in cortical neurons of urethane-anesthetized rats, and likely underlie EEG activity in the same low-frequency (1-4 Hz or delta) range. Nucleus basalis (NB) stimulation elicits neocortical activation, simultaneously modifying Vm and EEG fluctuations, by way of cortical muscarinic ACh receptors (Metherate et al., 1992). To investigate the nature of spontaneous fluctuations and their modification by NB stimulation, we have obtained intracellular recordings from auditory cortex using the whole-cell recording technique in vivo. Spontaneous Vm fluctuations appeared to contain three components whose polarity and time course resembled the EPSP, putative Cl(-)-mediated IPSP, and putative K(+)-mediated, long-lasting IPSP elicited by thalamic stimulation. The spontaneous, long-lasting hyperpolarization, whose rhythmic occurrence appeared to set the slow-wave rhythm, was associated with an increased conductance that could shunt the thalamocortical EPSP. We hypothesized that spontaneous Vm fluctuations arise from intermixed rapid depolarizations, rapid Cl(-)-mediated hyperpolarizations, and long-lasting, K(+)-mediated hyperpolarizations. NB-mediated cortical activation might then result from muscarinic suppression of K+ permeability, allowing the rapid depolarizations and Cl- fluxes to continue uninterrupted. Tests of this hypothesis showed that (1) intracellular blockade of K+ channels by rapid diffusion of Cs+ from the recording pipette resulted in suppression of spontaneous, long-lasting hyperpolarizations, mimicking the effect of NB stimulation, and reducing shunting of the thalamocortical EPSP; (2) effects of Cs+ and NB stimulation suggested overlapping, or converging, mechanisms of action; however, there were important differential effects on the spontaneous, long-lasting hyperpolarizations and the K(+)-mediated IPSP; and (3) modifying Cl- fluxes with intracellular picrotoxin or high intracellular Cl- concentrations resulted in spontaneous and NB-elicited large-amplitude depolarizations. We conclude that spontaneous, long-lasting hyperpolarizations are K+ fluxes, but are not "spontaneous" K(+)-mediated IPSPs. Since NB-mediated reduction of spontaneous hyperpolarizations implies muscarinic suppression of a K+ conductance, the spontaneous hyperpolarizations more likely result from the calcium-activated K+ current, IK(Ca). Finally, Cl- fluxes form an important component of activated Vm fluctuations that acts to restrain excessive depolarization.

CAMP-activated apical membrane chloride channels in Necturus gallbladder epithelium. Conductance, selectivity, and block.

Elevation of intracellular cAMP levels in Necturus gallbladder epithelium (NGB) induces an apical membrane Cl- conductance (GaCl). Its characteristics (i.e., magnitude, anion selectivity, and block) were studied with intracellular microelectrode techniques. Under control conditions, the apical membrane conductance (Ga) was 0.17 mS.cm-2, primarily ascribable to GaK. With elevation of cell cAMP to maximum levels, Ga increased to 6.7 mS.cm-2 and became anion selective, with the permeability sequence SCN- > NO3- > I- > Br- > Cl- >> SO4(2-) approximately gluconate approximately cyclamate. GaCl was not affected by the putative Cl- channel blockers Cu2+, DIDS, DNDS, DPC, furosemide, IAA-94, MK-196, NPPB, SITS, verapamil, and glibenclamide. To characterize the cAMP-activated Cl- channels, patch-clamp studies were conducted on the apical membrane of enzyme-treated gallbladders or on dissociated cells from tissues exposed to both theophylline and forskolin. Two kinds of Cl- channels were found. With approximately 100 mM Cl- in both bath and pipette, the most frequent channel had a linear current-voltage relationship with a slope conductance of approximately 10 pS. The less frequent channel was outward rectifying with slope conductances of approximately 10 and 20 pS at -40 and 40 mV, respectively. The Cl- channels colocalized with apical maxi-K+ channels in 70% of the patches. The open probability (Po) of both kinds of Cl- channels was variable from patch to patch (0.3 on average) and insensitive to [Ca2+], membrane voltage, and pH. The channel density (approximately 0.3/patch) was one to two orders of magnitude less than that required to account for GaCl. However, addition of 250 U/ml protein kinase A plus 1 mM ATP to the cytosolic side of excised patches increased the density of the linear 10-pS Cl- channels more than 10-fold to four per patch and the mean Po to 0.5, close to expectations from GaCl. The permeability sequence and blocker insensitivity of the PKA-activated channels were identical to those of the apical membrane. These data strongly suggest that 10-pS Cl- channels are responsible for the cAMP-induced increase in apical membrane conductance of NGB epithelium.