4-acetamido-4--isothiocyanatostilbene-2-2--disulfonic-acid and 9-anthroic-acid

During anoxia/reoxygenation (A/R) injury, intracellular chloride ion concentration ([Cl(-)](i)) homeostasis may play a role in maintaining the normal physiological function of cardiomyocytes. Various chloride transport systems could have influenced the concentration of chloride ion, but what kinds of chloride transport systems could play an important role in cardiomyocytes subjected to A/R injury and its mechanism are unknown. The aim of our study was to clarify the contributions of various chloride transport systems to anoxia/reoxygenation in rat neonatal cardiac myocytes and further to investigate the involved mechanisms. Oxidative stress and redox-sensitive transcription factor (NF-kappaB) activation are believed to play an important role in the A/R injury. To assess whether oxidative stress and NF-kappaB involve [Cl(-)](i) changes resulting in cardiomyocytes injury, the anoxia-reoxygenation (A/R) injury model was successfully established and administered with inhibitors of various chloride transport systems. Administration with Cl(-)-substitution and Cl(-)/HCO(3) (-) exchange inhibitor(SITS) has been shown to produce a protective effect against A/R injury by decreasing [Cl(-)](i) concentration, lipid peroxidation (malondialdehyde (MDA)) levels, and NF-kappaB activity, and by increasing antioxidant enzyme (glutathione peroxidase (GSHPx), superoxide dismutase (SOD), and catalase(CAT)) activity. However, inhibitors for the Cl(-)-channel (9-AC) and Na(+)-K(+)-2Cl(-) co-transporter (bumetanide) had no effects. Our results indicate that Cl(-)/HCO(3) (-) exchange system plays an important role in the cardiocyte A/R injury by influencing [Cl(-)](i) concentration. The protective effects of SITS and Cl(-)-substitution on cardiomyocytes may be due to the attenuation of oxidative stress and inhibition of NF-kappaB activation.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Animals; Anthracenes; Apoptosis; Bumetanide; Catalase; Cell Hypoxia; Cell Survival; Chloride Channels; Chloride-Bicarbonate Antiporters; Chlorides; Cultured Milk Products; Enzyme Assays; Glutathione Peroxidase; L-Lactate Dehydrogenase; Lipid Peroxidation; Male; Myocardial Reperfusion Injury; Myocytes, Cardiac; NF-kappa B; Oxidative Stress; Oxygen; Primary Cell Culture; Protein Transport; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Sodium Potassium Chloride Symporter Inhibitors; Sodium-Potassium-Chloride Symporters; Superoxide Dismutase

GABA is known to be the inhibitory neurotransmitter in the majority of brain stem nuclei. The release of GABA has been extensively studied both in vivo and in vitro in higher brain areas, whereas the mechanisms of release in the brain stem have not been systemically characterized. The properties of preloaded [3H]GABA were now investigated in mouse brain stem slices, using a superfusion system. The basal release was enhanced by K+ stimulation (50 mM K+) and under various cell-damaging conditions (ischemia, hypoglycemia, the presence of free radicals and metabolic poisons). No K+-stimulated release was discernible in the absence of Ca2+, indicating that the release was at least partly Ca2+-dependent. Moreover, the release was increased when Na+ or Cl- was omitted from the superfusion medium. GABA and beta-alanine stimulated the release, confirming the involvement of the reversed function of GABA transporters. Incubation of the slices with the anion channel inhibitors diisothiocyanostilbene and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonate and with the Cl- uptake inhibitor 9-anthracenecarboxylic acid also reduced GABA release, demonstrating that a part of it comprises leakage through anion channels. All these mechanisms were involved in the ischemia-induced GABA release, which was over 4-fold greater than the release in normoxia. Contrary to the other brain areas, GABA release in the brain stem was not affected by ionotropic glutamate receptors but may be modulated by metabotropic receptors. This ischemia-induced GABA release might constitute an important mechanism against excitotoxicity, protecting the brain stem under cell-damaging conditions.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Amiloride; Animals; Anthracenes; Brain Ischemia; Brain Stem; Female; gamma-Aminobutyric Acid; Glutamic Acid; Ion Channels; Male; Mice

beta-Adrenergic stimulation reduces albumin permeation across pulmonary artery endothelial monolayers and induces changes in cell morphology that are mediated by Cl- flux. We tested the hypothesis that anion-mediated changes in endothelial cells result in changes in endothelial permeability. We measured permeation of radiolabeled albumin across bovine pulmonary arterial endothelial monolayers when the extracellular anion was Cl-, Br-, I-, F-, acetate (Ac-), gluconate (G-), and propionate (Pr-). Permeability to albumin (Palbumin) was calculated before and after addition of 0.2 mM of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), which reduces permeability. In Cl-, the Palbumin was 3.05 +/- 0.86 x 10(-6) cm/s and fell by 70% with the addition of IBMX. The initial Palbumin was lowest for Pr- and Ac-. Initial Palbumin was higher in Br-, I-, G-, and F- than in Cl-. A permeability ratio was calculated to examine the IBMX effect. The greatest IBMX effect was seen when Cl- was the extracellular anion, and the order among halide anions was Cl- > Br- > I- > F-. Although the level of extracellular Ca2+ concentration ([Ca2+]o) varied over a wide range in the anion solutions, [Ca2+]o did not systematically affect endothelial permeability in this system. When Cl- was the extracellular anion, varying [Ca2+]o from 0.2 to 2.8 mM caused a change in initial Palbumin but no change in the IBMX effect. The anion channel blockers 4-acetamido-4'-isothiocyanotostilbene-2, 2'-disulfonic acid (0.25 mM) and anthracene-9-carboxylic acid (0.5 mM) significantly altered initial Palbumin and the IBMX effect. The anion transport blockers bumetanide (0.2 mM) and furosemide (1 mM) had no such effects. We conclude that extracellular anions influence bovine pulmonary arterial endothelial permeability and that the pharmacological profile fits better with the activity of anion channels than with other anion transport processes.

Topics: 1-Methyl-3-isobutylxanthine; 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Animals; Anions; Anthracenes; Calcium; Capillary Permeability; Cattle; Cells, Cultured; Endothelium, Vascular; Extracellular Space; Phosphodiesterase Inhibitors; Pulmonary Artery

The Cai(2+)-insensitive transient outward current, ilo was studied at 20-24 degrees C in rat ventricular myocytes with the whole cell recording patch-clamp technique. The current was recorded before and after replacement of chloride by methanesulfonate or aspartate or in the absence and the presence of chloride channel blockers, SITS or 9-anthracene carboxylic acid. In control conditions (in the presence of external divalent cations, Ca2+ and Cd2+, Cd2+ being used to suppress Ca2+ current), ilo inactivation was composed of a fast and a slow component. When methanesulfonate was substituted for external Cl-, the peak current decreased to a variable extent, but the inactivation of the remaining current was still composed of a fast and a slow component. In contrast, the inactivation of the difference current was well fitted by a single exponential. The time to peak of the difference current was shorter than that of the current recorded either in the absence or the presence of methanesulfonate. Both activation- and steady-state inactivation-voltage curves were either unchanged (n = 4) or shifted by a few mV (5.5 mV, n = 14) towards positive potentials when methanesulfonate was substituted for Cl-. The current remaining in methanesulfonate reversed at potentials closed to EK. The difference current was composed of a peak and a steady-state component. The peak was suppressed by 4-aminopyridine whereas the steady-state component was not. The peak was also suppressed when pipette solution contained Cs+ instead of K+ but was still present when the Hepes concentration in both external and pipette media was increased 5-fold (50 mM vs. 10 mM). When aspartate was substituted for Cl- or when 2 mM SITS was added to the external solution (in the absence of Ca2+ and Cd2+ because aspartate is known to chelate Ca2+ ions and possibly other divalent cations), ilo was reduced to a similar extent in the two cases and the difference current was composed of a peak (inactivation fitted by a single exponential) and a steady-state component. The SITS-sensitive transient current reversed at a potential close to ECl. When 5 mM 9-anthracene carboxylic acid was added to external solution (in the presence of Ca2+ and Cd2+), the peak of the difference current was similar to that observed when Cl- was substituted by methanesulfonate. The difference current resulting from the substitution of methanesulfonate for chloride was not changed when the pipette solution contained either 50 mM EGTA (

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Animals; Anthracenes; Aspartic Acid; Cells, Cultured; Chloride Channels; Chlorides; Heart Ventricles; Membrane Potentials; Mesylates; Myocardium; Patch-Clamp Techniques; Rats

1. The effects of some chloride channel antagonists were studied on the calcium-activated chloride current (ICl(Ca)) in smooth muscle cells from the rabbit portal vein with the perforated patch technique. 2. 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS) and 4,4'-diisothiocyanato-stilbene-2,2'-disulphonic acid (DIDS) reduced the amplitude of spontaneous transient inward currents (STICs, calcium-activated chloride currents) in a concentration-dependent manner. The concentrations required to reduce the amplitude by 50% (IC50) of STICs were 2.1 x 10(-4) M and 6.4 x 10(-4) M for DIDS and SITS, respectively. This effect was not voltage-dependent. 3. The time constant of decay of STICs (tau), which is voltage-dependent, was increased by about 30% by SITS and decreased by about 20% by DIDS. The effect of DIDS and SITS on tau was similar at holding potentials of -50 and +50 mV. 4. These compounds did not modify the characteristics of spontaneous transient outward currents (STOCs, calcium-activated potassium currents). 5. DIDS and SITS decreased the amplitude of ICl(Ca) evoked by noradrenaline and caffeine less potently than STICs with IC50 values of 7.5 x 10(-4) M and 1.8 x 10(-3) M, respectively. 6. DIDS and SITS increased the calcium-activated potassium current (IK(Ca) evoked by noradrenaline and caffeine by 3-6 fold. 7. Anthracene-9-carboxylic acid (A-9-C) inhibited STICs in a voltage-dependent fashion and was about 3 fold more active at +50 mV than at -50 mV. A-9-C increased STIC tau and this effect was enhanced by depolarization. 8. A-9-C also inhibited caffeine-evoked IC1(ca) but less potently than STICs and also increased the evoked IK(ca) without altering spontaneous IK(Ca).9. The results from the present work are compared with the pharmacology of other chloride conductances and the mechanism of action of the chloride channel antagonists in vascular smooth muscle is discussed.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Anthracenes; Calcium; Chloride Channels; Female; Male; Muscle, Smooth, Vascular; Portal Vein; Potassium Channels; Rabbits

Preimplantation mammalian development culminates in the generation of a fluid-filled cavity, the blastocoel, which requires the vectorial transport of ions across the trophectoderm, followed by the movement of water. Experiments were carried out to establish the role of Cl- transport in blastocoel formation in the rat. These included investigations of the effect of Cl- substitution and the Cl- transport inhibitors 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, furosemide, and anthracene-9-carboxylic acid, on the development of morulae into blastocysts in culture, and on the rate at which blastocoel fluid is accumulated. In addition, a novel technique was developed in which the Cl(-)-sensitive fluorescent dye 6-methoxy-N-(3-sulfopropyl) quinolium (SPQ) was loaded into the blastocoel cavity, in order to characterize the pathways of transtrophectodermal Cl- transport. We established that 1) blastocoel formation in the rat requires the external presence of Cl- ions; 2) transport across the trophectoderm appears to be via a transcellular pathway, since rates of blastocyst development, fluid accumulation, and SPQ-monitored Cl- efflux are all reduced in the presence of Cl- transport inhibitors; and 3) Cl- transport occurs via Cl-/HCO3- exchange.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Anthracenes; Blastocyst; Calcium; Chlorides; Cyclic AMP; Embryonic and Fetal Development; Female; Fluorescent Dyes; Furosemide; In Vitro Techniques; Ion Transport; Quinolinium Compounds; Rats; Rats, Wistar

In order to find a more specific blocker of the cardiac Cl- channel, we examined the effects of anthracene-9-carboxylic acid (9AC) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) on the beta-adrenoceptor-mediated decreases in resting potential and intracellular chloride ion activity (aiCl) in guinea-pig papillary muscles by using Cl- ion selective microelectrodes. 9AC (1 mM) significantly inhibited the isoproterenol (1 microM)-induced decreases in resting potential and aiCl in quiescent preparations. However, 1 mM DIDS did not significantly affect the changes in aiCl and resting potential during beta-adrenergic stimulation. Thus, in cardiac cells, 9AC is a more potent blocker of the Cl- channels activated by beta-adrenergic stimulation than DIDS.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Anthracenes; Chloride Channels; Chlorides; Female; Guinea Pigs; Heart; In Vitro Techniques; Isoproterenol; Male; Membrane Potentials; Membrane Proteins; Myocardium; Receptors, Adrenergic, beta

The sulfonylurea compounds glisoxepide and glibenclamide inhibit the uptake of bile acids into isolated rat hepatocytes. The Ki values for the inhibition of cholate uptake was 9 microM with glibenclamide and 200 microM with glisoxepide. The inhibition of cholate uptake by both sulfonylureas was noncompetitive. Uptake of the conjugated bile acid taurocholate was inhibited by glibenclamide, Ki = 75 microM. Again the inhibition was noncompetitive. Glisoxepide inhibited taurocholate uptake only in the absence of sodium ions. Under sodium-free conditions glisoxepide also strongly inhibited cholate uptake. The inhibition was competitive, Ki = 42 microM. Both bile acids interfered with the hepatocellular uptake of [3H]glisoxepide, with IC50 values of 375 and 467 microM for cholate and taurocholate, respectively. The uptake of [3H]glibenclamide was inhibited by cholate, IC50 = 328 microM, but not by taurocholate. Glisoxepide uptake was further inhibited by blockers of the hepatocellular monocarboxylate transporter, by the loop diuretic bumetanide, by 4,4'-diisothiocyano-2,2'-stilbenedisulfonate (DIDS) and by sulfate. Glibenclamide uptake was weakly inhibited by DIDS and by anthracene-9-carboxylic acid (A-9-C) but not by bumetanide and sulfate. Neither bromosulfophthalein nor the fatty acid oleate inhibited glisoxepide or glibenclamide uptake. These results are consistent with the transport of glisoxepide via the transport system for the unconjugated bile acid cholate. Glibenclamide uptake is mediated by a still unknown hepatocellular transport system.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Anions; Anthracenes; Bile Acids and Salts; Biological Transport; Chlorides; Fatty Acids; Glyburide; Hypoglycemic Agents; Liver; Male; Rats; Rats, Inbred Strains; Serine; Sulfobromophthalein; Sulfonylurea Compounds

The effects of Cl channel blockers on large-conductance (LC-type) Cl channels of cultured vascular smooth muscle cells (VSMC) were studied in order to characterize the channel pharmacologically. Intracellular disulphonic stilbene derivatives, such as 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) and 4-acetamido-4-isothiocyanostilbene-2,2'-disulphonic acid (SITS) inhibited Cl channel activity in a dose-dependent manner. An obvious inhibitory effect of DIDS in this condition was obtained at concentrations higher than 5 microM, and the complete inhibition was obtained at around 100 microM, which was almost 10 times less than the effective dose of SITS. The inhibitory effect of DIDS was reversible at a drug concentration of lower than 50 microM. Single-channel conductance decreased as the concentration of DIDS increased. This decrease in the conductance was a consequence of unresolved openings of the channel due to fast blocking and unblocking rates of the drug. The Cl channel was also obviously inhibited by extracellular DIDS at a concentration of 1 mM. In addition, in cell-attached patches, 500 microM DIDS applied extracellularly inhibited Cl channel activated by the application of polymyxin B. We also investigated the effect of Zn on Cl channels in VSMC. Intracellular Zn dose dependently and reversibly blocked the channel at the positive intracellular potential range, whereas at the negative intracellular potential range it did not block the channel activity. Results in this study suggest the diversity of Cl channels among various tissues.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Anthracenes; Aorta, Thoracic; Cell Line; Chloride Channels; Dose-Response Relationship, Drug; Electric Conductivity; Furosemide; Membrane Proteins; Muscle, Smooth, Vascular; Polymyxin B; Rats; Zinc

Ca(2+)-activated Cl- currents were studied in isolated cells from rat portal vein smooth muscle in short-term primary culture using the whole-cell patch-clamp technique. Cl- currents can be activated separately by Ca2+ release from intracellular stores (in response to external applications of caffeine or noradrenaline) and by Ca2+ influx through voltage-dependent Ca2+ channels. The effects of several Cl- channel blockers and of spironolactone (a substance known to reduce internal Ca2+ loading) on both Cl- and Ca2+ currents were examined. Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), anthracene-9-carboxylic acid (9-AC) and diphenylamine-2,2'-dicarboxylic acid (DPC) inhibited the Ca(2+)-activated Cl- current (IC50 values between 16.5 and 306 microM) with no effects on the inward Ca2+ current and on internal Ca2+ loading (testing by measuring the Ca(2+)-activated K+ current). These results indicate that the inhibition of Cl- current by these compounds is due to a direct interaction with the Cl- channel. In contrast, spironolactone inhibited both K+ and Cl- currents (IC50 = 7.6 microM) by reducing the amount of Ca2+ located in the internal stores, whereas the Cl- current activated by Ca2+ current through T-type Ca2+ channels was unchanged. This preparation and the protocols developed in this study appears to be appropriate for analysis of substances interfering with Cl- channels or intracellular Ca2+ stores.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Anthracenes; Caffeine; Calcium; Cells, Cultured; Chloride Channels; Chlorides; Membrane Proteins; Muscle, Smooth, Vascular; Rats; Spironolactone

Total crypt volume has been estimated by analysis of photographic images of intact viable crypts isolated from guinea-pig small intestine. Exposing these crypts to a hypotonic medium, led to transient swelling followed by regulatory volume decrease (RVD) in 12-20 min. RVD was blocked by inhibitors of K+ and Cl- conductance, suggesting that it occurs by activation of K+ and Cl- permeability pathways and loss of these ions.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Alkaline Phosphatase; Animals; Anthracenes; Barium; Barium Compounds; Chloride Channels; Chlorides; Guinea Pigs; Intestinal Mucosa; Intestine, Small; Ion Channels; Membrane Proteins; Microvilli; Potassium; Potassium Channels; Thymidine Kinase

1. Double-barrelled Cl(-)-sensitive microelectrodes have been used to measure the intracellular Cl- activity (aCli) and membrane potential (Em) in rat lumbrical muscles. The mean Cl- equilibrium potential (ECl), calculated from the measured aCli in sixty fibres, was 2.9 +/- 2.5 mV (S.D. of an observation) less negative than Em. The value of aCli was higher than would be expected for a passive distribution, by a mean 1.4 +/- 1.2 mM. The mean Em was -59.5 +/- 8.2 mV. 2. Removal of external Cl- (Cl-(o)) resulted in a rapid fall in aCli and a transient depolarization. aCli stabilized at an apparent level of 1.7 +/- 1.0 mM (n = 24) while Em became substantially more negative than in normal Krebs solution (mean, -80.1 +/- 12.4 mV). Readdition of Cl-(o) caused a rapid rise in aCli and transient hyperpolarization. ECl quickly became less negative than Em and both then fell in parallel towards the levels previously recorded in normal Krebs solution. 3. If lack of selectivity of the Cl(-)-sensitive ion exchanger and the intracellular presence of interfering anions, assumed to be responsible for the apparent aCli recorded in Cl(-)-depleted fibres, were also responsible for the apparently non-passive Cl- distribution recorded under normal conditions, the difference between the calculated ECl and Em would increase at more negative potentials. This was not observed over a range of Em values between -46 and -84 mV. 4. Inhibition of the Cl- permeability by application of 9-anthracene carboxylic acid (9-AC) resulted in an immediate rise in aCli and hyperpolarization. An aCli up to 40 mM higher, or eleven times higher, than that predicted by a passive distribution was recorded. Application of 9-AC after depletion of intracellular Cl- in Cl(-)-free solution had no effect on either the apparent aCli or Em. 5. It is concluded that Cl- ions are actively accumulated by the skeletal muscle fibre and that the Cl- distribution therefore normally exerts a depolarizing influence. 6. In the presence of 9-AC and nominal absence of CO2 and HCO3-, readdition of Cl-(o) to Cl(-)-depleted fibres resulted in a substantial rise in aCli and a small, maintained depolarization. This clear demonstration of active accumulation was used to investigate the mechanism responsible for inward transport of Cl- ions. 7. Neither application of CO2 and HCO3- nor application of DIDS (4,4'-diisothiocyanostilbene-2,2'-disulphonic acid) had any effect on the accumulation of Cl- ions. This suggests that Cl(-)-H

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Anthracenes; Bicarbonates; Biological Transport, Active; Carbon Dioxide; Cell Membrane Permeability; Chlorides; Furosemide; In Vitro Techniques; Muscles; Potassium; Rats; Rats, Inbred Strains; Sodium

Functional expression of receptors for GnRH was studied using Xenopus laevis oocytes injected with poly(A)+ mRNA extracted from rat anterior pituitary glands. Whole-cell currents were monitored using two-electrode voltage-clamp techniques. In oocytes which responded to both GnRH and TRH, the GnRH response showed a longer latency and time-to-peak than the TRH response. The response to GnRH or an agonist of GnRH receptors, buserelin (1 nM-1 microM) consisted of current fluctuations and occurred in a dose-dependent manner. This GnRH response was blocked by the Cl- channel blockers 9-AC (9-anthracene carboxylic acid; 1 mM), 4,4'-diisothiocyanastilbene-2,2'-disulfonic acid (0.1 mM), and diphenylamine-2-carboxylic acid (0.1 mM). The reversal potential for the GnRH-induced current fluctuations was -25 mV, comparable with the reported Cl- equilibrium potential in Xenopus oocytes, and its shift, when the external concentration of Cl- was changed, was reasonably described by the Nernst equation. These results indicate that the GnRH-induced response was dependent on the activity of Cl- channels. Ca2+ also plays a role, as the GnRH-induced response was reversibly suppressed by a calmodulin inhibitor, chlordiazepoxide (0.2 microM), and by a blocker of intracellular Ca2+ release, TMB-8 (8-(N.N-diethylamino) octyl-3,4,5-trimethoxybenzoate; 0.1-0.2 mM). It is concluded that GnRH (and TRH) receptors, expressed in Xenopus oocytes by injecting exogenous mRNA from rat anterior pituitary glands, operate via activation of Ca2+-dependent Cl- channels.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Anthracenes; Calcium; Chloride Channels; Chlorides; Membrane Proteins; Microinjections; Oocytes; ortho-Aminobenzoates; Pituitary Gland, Anterior; Pituitary Hormone-Releasing Hormones; Receptors, Gonadotropin; RNA, Messenger; Xenopus laevis

The efflux of 36C1 from perifused rat lenses consisted of two components: a fast (extracellular) component and a slow (cellular) component. The 36C1 efflux rate constant of the cellular component was 5.7 X 10(-3) min-1. The 36C1- efflux was sensitive to changes in lens potential induced by treatment with high-K+ solutions. The decrease in the 36C1- efflux rate constant caused by high-K+ solutions was consistent with the Goldman model, indicating that, under normal conditions, the majority of the 36C1 efflux is by diffusion. The 36C1- efflux rate constant corresponds to a C1 permeability of 1.3 X 10(8) ms 1. The C1- channel inhibitor anthracene-9-carboxylate (A-9-C), however, caused a relatively small reduction in the efflux rate constant. The anion-exchange inhibitor 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonate (SITS) has little effect on the 36C1 efflux under control conditions. Intracellular acidification, induced by pre-treatment with NH1+, leads to a rapid stimulation of 36C1- efflux. This increased 36C1- efflux is blocked by SITS. Thus, it appears that at low intracellular pH (pH), a normally quiescent, SITS-sensitive, anion-exchange mechanism is activated. The possible role of this exchange mechanism in regulating pH, is discussed.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Animals; Anthracenes; Biological Transport, Active; Chlorides; Hydrogen-Ion Concentration; In Vitro Techniques; Intracellular Fluid; Ion Channels; Ion Exchange; Lens, Crystalline; Membrane Potentials; Perfusion; Rats; Rats, Inbred Strains

The in vitro and in vivo regulation of [3H]Ro 5-4864 binding to peripheral benzodiazepine receptors (PBR) by ion transport/exchange inhibitors was studied in the kidney. The potencies of 9-anthroic acid, furosemide, bumetanide, hydrochlorothiazide and SITS as inhibitors of [3H]Ro 5-4864 binding to renal membranes were consistent with their actions as anion transport inhibitors (Ki approximately equal to 30 - 130 microM). In contrast, spironolactone, amiloride, acetazolamide, and ouabain were less potent (Ki = 100-1000 microM). Administration of furosemide to rats for five days resulted in a profound diuresis (approximately equal to 350% increase in urine volume) accompanied by a significant increase in PBR density (43%) that was apparent by the fifth day of treatment. Administration of hydrochlorothiazide or Ro 5-4864 for five days also caused diuresis and increased renal PBR density. Both the diuresis and increased density of PBR produced by Ro 5-4864 were blocked by coadministration of PK 11195, which alone had no effect on either PBR density or urine volume. The equilibrium binding constants of [3H]Ro 5-4864 to cardiac membranes were unaffected by administration of any of these drugs. These findings suggest that renal PBR may be selectively modulated in vivo and in vitro by administration of ion transport/exchange inhibitors.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Acetazolamide; Amiloride; Animals; Anions; Anthracenes; Biological Transport; Bumetanide; Furosemide; Hydrochlorothiazide; Kidney; Kinetics; Male; Myocardium; Ouabain; Rats; Rats, Inbred Strains; Receptors, GABA-A; Spironolactone

Cells of the rabbit renal cortical collecting tubule possess significant regulatory volume decrease (RVD) capabilities. After a 100-mosmol/kg reduction in peritubular osmolality, principal and intercalated cells swell 40-45 and 30-35%, respectively, and immediately activate RVD mechanisms. Both cell types downregulate their volume to within 5-6% of control volume at initial rates of 3-6%/min. Return to isotonic saline causes both cell types to shrink (isotonic shrinkage) 25-35% below control volume due to the loss of osmotically active intracellular solutes during RVD. In most mammalian cells studied to date, RVD is mediated largely by passive KCl efflux via KCl cotransport, parallel K+ and Cl- channels, or parallel K+-H+ and Cl- -HCO3- exchange mechanisms. Peritubular application of 0.1 mM ouabain (0 Na+ lumen), bilateral CO2-HCO3- removal, or bilateral application of 0.02 mM bumetanide, 2.0 mM Ba2+, 2.0 mM anthracene-9-carboxylic acid, or 0.5 mM SITS had no significant effect on rates or magnitudes of RVD and isotonic shrinkage in either cell type. Bilateral elevation of K+ from 5 to 52.5 mM reverses or reduces the electrochemical gradient for K+ movement, causing accumulation of this ion in the cytoplasm, but had no effect on the rates or magnitude of principal and intercalated cell RVD. Principal and intercalated cells from K+- or Cl- -depleted tubules (1 h bilateral perfusion with K+- or Cl- -free saline at 37 degrees C) showed normal rates and magnitudes of RVD in K+- or Cl- -free hypotonic saline. Taken together, these results argue against a significant role of passive KCl efflux pathways in mediating principal and intercalated cell RVD.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Animals; Anthracenes; Bumetanide; Female; In Vitro Techniques; Kidney Cortex; Kidney Tubules; Kidney Tubules, Collecting; Osmolar Concentration; Ouabain; Potassium Chloride; Rabbits

The mechanisms of transmembrane K and anion movements were investigated by measurement of the changes in cell volume, apical membrane potential difference, and intracellular K activity resulting from exposure of Necturus gallbladder to solutions with increased K concentration. Cell swelling occurred when the tissue was exposed bilaterally to 25 mmol/l K. This swelling was both Cl and HCO3 dependent, but was not blocked by DIDS or bumetanide. Unilateral tenfold increases in extracellular K concentration did not cause cell swelling; addition of 5 mmol/l Ba to the contralateral cell surface resulted in cell volume increases comparable to those seen with bilateral K increase. Complete blockage of K channels by Ba could be demonstrated electrophysiologically at normal extracellular K concentrations but not in the presence of increased K. Our results were consistent with the passive movement of K through Ba-sensitive channels in both cell membranes. We were unable to detect other mechanisms for transmembrane K movement. The cell swelling caused by exposure to 25 mmol/l K was not due to intracellular K accumulation and may be related to the effects of membrane depolarization on voltage sensitive anion transport processes.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Anthracenes; Barium; Biological Transport; Bumetanide; Chlorides; Gallbladder; In Vitro Techniques; Ion Channels; Membrane Potentials; Necturus maculosus; Potassium

This report describes a Cl- transport pathway in confluent monolayer cultures of the T84 human colonic carcinoma cell line which is: 1) activated by vasoactive intestinal polypeptide, or other agents which induce or mimic cAMP; 2) independent of extracellular Na+ or K+; 3) refractory to inhibition by 0.1 mM bumetanide and 1 mM 4-acetamido-4'-isothiocyanostilbene-2,-2'-disulfonic acid; 4) competitively inhibited by NO3-, I-, SCN-, and Br-; 5) inhibited in a noncompetitive-complex manner by the putative Cl- channel-blocking agent, N-phenylanthranilic acid; and 6) localized to the apical membrane of confluent monolayers. This Cl- transport system is, therefore, distinct from the bumetanide-sensitive, basolateral membrane-localized, Na+, K+, Cl- cotransport system previously described in these cells (Dharmsathaphorn, K., Mandel, K., Masui, H., and McRoberts, J.A. (1985) J. Clin. Invest. 75, 462-471). Kinetic studies revealed that Cl- transport by this pathway fit simple Michaelis-Menten kinetics with an apparent Km for Cl- of about 6 mM. Activation by vasoactive intestinal polypeptide increased the Vmax but did not alter the apparent Km. We discuss the possibility that this transport system is a Cl- channel which is intimately involved in hormonally mediated, electrogenic Cl- secretion across T84 cell monolayers.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Anthracenes; Biological Transport, Active; Bromides; Bucladesine; Bumetanide; Calcimycin; Cell Line; Cell Membrane; Chlorides; Colon; Cyclic AMP; Dose-Response Relationship, Drug; Epithelium; Humans; Hydrogen-Ion Concentration; Kinetics; ortho-Aminobenzoates; Sodium; Valinomycin; Vasoactive Intestinal Peptide

To define the transport pathway for HCO-3 secretion (JHCO3) across the apical and basolateral membranes of turtle bladder, we examined the effects of cAMP, isobutylmethylxanthine (IBMX), the Cl- channel blocker 9-anthroic acid (9-AA), and the disulfonic stilbene DIDS (4,4'-diisothiocyanostilbene-2,2'-sulfonic acid) on the electroneutral and electrogenic components of JHCO3. Total JHCO3 was measured by pH stat titration of the mucosal compartment after Na+ absorption and H+ secretion were abolished by ouabain and a delta pH, respectively. Addition of cAMP or IBMX increased total JHCO3 and induced a short-circuit current (ISC), accounting for a large part of JHCO3; net Cl- absorption was reduced. Mucosal 9-AA inhibited the IBMX-induced electrogenic component of JHCO3, whereas mucosal DIDS inhibited the electroneutral component and acetazolamide reduced both. We suggest that HCO-3 is generated within the cell by a Na-independent primary active acid-base transport at the basolateral membrane (H+ extrusion into the serosal compartment). Cellular HCO-3 accumulation drives JHCO3 via a Cl-HCO3 exchanger at the luminal membrane. IBMX and cAMP activate a 9-AA-sensitive anion conductance parallel to the exchanger. The apparent reversal of the transport elements between the two cell membranes (compared with H+-secreting cells) led to an ultrastructural examination of the carbonic anhydrase-rich cells.

Topics: 1-Methyl-3-isobutylxanthine; 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Anthracenes; Bicarbonates; Biological Transport; Biological Transport, Active; Cell Membrane Permeability; Cyclic AMP; Hydrogen-Ion Concentration; Ion Channels; Models, Biological; Turtles; Urinary Bladder

Rectal gland tubule (RGT) segments of the spiny dogfish (Squalus acanthias) were perfused in vitro. The effects of inhibitors of known mode of action on transepithelial PD (PDte resistance (Rte), the PD across the basolateral membrane (PDbl), the fractional resistance of this membrane (FRbl), and intracellular activities of NA+, Cl-, K+ (apha cell) were examined. Furosemide (5 x 10(-4) mol x 1(-1)) reduced PDte from -12 +/- 0.7 to -2.3 +/- 0.2 mV (n = 63), hyperpolarized PDbl from -71 +/- 1.3 to -79 +/- 0.9 mV (n = 59), FRbl decreased from 0.2 +/- 0.03 to 0.13 +/- 0.01 (n = 21), alpha cell cl- fell from 38 +/- 4 to 11 +/- 2 mmol x 1(-1) (n = 21), alpha cell Na+ fell from 37 +/- 4 to 17 +/- 2 mmol x 1(-1) (n = 12) and alpha cell K+ was constant [113 +/- 14 vs. 117 +/- 15 mmol x 1(-1) (n = 6)]. Furosemide exerted its effects within some 20-40s. Its action was completely reversible. Analysis of the time courses revealed that the furosemide induced initial fall in alpha cell cl- was approximately twice as rapid when compared to that of alpha cell Na+. Ba2+ 0.5 mmol x 1(-1) (bath) reduced PDte from -7.1 +/- 1.2 to -4.1 +/- 0.6 mV (n = 24), increased Rte from 18 +/- 2 to 22 +/- 2.5, omega cm2 (n = 14). PDbl depolarized from -75 +/- 2 to -48 +/- 2 mV (n = 42), FRbl increased from 0.2 +/- 0.02 to 0.34 +/- 0.04 (n = 14) and alpha cell K+ increased from 143 +/-28 to 188 +/- mmol x 1(-1) (n = 4). Ouabain (50 x 10(-6) mol x 1(-1), bath) reduced PDte from -12 +/-2 to -3 +/- 0.5 mV (n = 9), Rte increased from 18 +/- 3 to 21 +/- 3 omega cm2 (n = 5). PDbl depolarized from -67 +/- 4 to -26 + 3 mV (n = 14), FRbl increased from 0.23 +/- 0.04 to 0.45 +/- 0.05 (n = 6), alpha cell K+ fell only slightly from 135 +/- 15 to 112 +/- 30 mmol x 1(-1) (n = 4), but alpha cell cl- increased from 35 +/- 12 to 111 +/- 37 mmol x 1(-1) (n = 3). These effects of ouabain were slow when compared to those exerted by furosemide or Ba2+. The ouabain effects on PDte and PDbl were completely prevented if furosemide was applied first.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Amiloride; Animals; Anthracenes; Biomechanical Phenomena; Dogfish; Dose-Response Relationship, Drug; Furosemide; In Vitro Techniques; Ouabain; Salt Gland; Sodium Chloride