Relaxation of endothelin-1-induced pulmonary arterial constriction by niflumic acid and NPPB: mechanism(s) independent of chloride channel block. (1/2847)

We investigated the effects of the Cl- channel blockers niflumic acid, 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and 4, 4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) on endothelin-1 (ET-1)-induced constriction of rat small pulmonary arteries (diameter 100-400 microm) in vitro, following endothelium removal. ET-1 (30 nM) induced a sustained constriction of rat pulmonary arteries in physiological salt solution. Arteries preconstricted with ET-1 were relaxed by niflumic acid (IC50: 35.8 microM) and NPPB (IC50: 21.1 microM) in a reversible and concentration-dependent manner. However, at concentrations known to block Ca++-activated Cl- channels, DIDS (+info)

Chloride dependence of hyperpolarization-activated chloride channel gates. (2/2847)

1. ClC proteins are a class of voltage-dependent Cl- channels with several members mutated in human diseases. The prototype ClC-0 Torpedo channel is a dimeric protein; each subunit forms a pore that can gate independently from the other one. A common slower gating mechanism acts on both pores simultaneously; slow gating activates ClC-0 at hyperpolarized voltages. The ClC-2 Cl- channel is also activated by hyperpolarization, as are some ClC-1 mutants (e.g. D136G) and wild-type (WT) ClC-1 at certain pH values. 2. We studied the dependence on internal Cl- ([Cl-]i) of the hyperpolarization-activated gates of several ClC channels (WT ClC-0, ClC-0 mutant P522G, ClC-1 mutant D136G and an N-terminal deletion mutant of ClC-2), by patch clamping channels expressed in Xenopus oocytes. 3. With all these channels, reducing [Cl-]i shifted activation to more negative voltages and reduced the maximal activation at most negative voltages. 4. We also investigated the external halide dependence of WT ClC-2 using two-electrode voltage-clamp recording. Reducing external Cl- ([Cl-]o) activated ClC-2 currents. Replacing [Cl-]o by the less permeant Br- reduced channel activity and accelerated deactivation. 5. Gating of the ClC-2 mutant K566Q in normal [Cl-]o resembled that of WT ClC-2 in low [Cl-]o, i.e. channels had a considerable open probability (Po) at resting membrane potential. Substituting external Cl- by Br- or I- led to a decrease in Po. 6. The [Cl-]i dependence of the hyperpolarization-activated gates of various ClC channels suggests a similar gating mechanism, and raises the possibility that the gating charge for the hyperpolarization-activated gate is provided by Cl-. 7. The external halide dependence of hyperpolarization-activated gating of ClC-2 suggests that it is mediated or modulated by anions as in other ClC channels. In contrast to the depolarization-activated fast gates of ClC-0 and ClC-1, the absence of Cl- favours channel opening. Lysine 556 may be important for the relevant binding site.  (+info)

Volume regulation following hypotonic shock in isolated crypts of mouse distal colon. (3/2847)

1. A video-imaging technique of morphometry was used to measure the diameter as an index of cell volume in intact mouse distal colon crypts submitted to hypotonic shock. 2. Transition from isotonic (310 mosmol l-1) to hypotonic (240 mosmol l-1) saline caused a pronounced increase in crypt diameter immediately followed by regulatory volume decrease (RVD). 3. Exposure of crypts to Cl--free hyposmotic medium increased the rapidity of both cell swelling and RVD. Exposure of crypts to Na+-free hyposmotic medium reduced the total duration of swelling. Return to initial diameter was followed by further shrinkage of the crypt cells. 4. The chloride channel inhibitor NPPB (50 microM) delayed the swelling phase and prevented the subsequent normal decrease in diameter. 5. The K+ channel blockers barium (10 mM), charybdotoxin (10 nM) and TEA (5 mM) inhibited RVD by 51, 44 and 32 %, respectively. 6. Intracellular [Ca2+] rose from a baseline of 174 +/- 17 nM (n = 8) to 448 +/- 45 nM (n = 8) during the initial swelling phase 7. The Ca2+ channel blockers verapamil (50 microM) and nifedipine (10 microM), the chelator of intracellular Ca2+ BAPTA AM (30 microM), or the inhibitor of Ca2+ release TMB-8 (10 microM), dramatically reduced volume recovery, leading to 51 % (n = 9), 25 % (n = 7), 37 % (n = 6), 32 % (n = 8) inhibition of RVD, respectively. TFP (50 microM), an antagonist of the Ca2+-calmodulin complex, significantly slowed RVD. The Ca2+ ionophore A23187 (2 microM) provoked a dramatic reduction of the duration and amplitude of cell swelling followed by extensive shrinkage. The release of Ca2+ from intracellular stores using bradykinin (1 microM) or blockade of reabsorption with thapsigargin (1 microM) decreased the duration of RVD. 8. Prostaglandin E2 (PGE2, 5 microM) slightly delayed RVD, whereas leukotriene D4 (LTD4, 100 nM) and arachidonic acid (10 microM) reduced the duration of RVD. Blockade of phospholipase A2 by quinacrine (10 microM) inhibited RVD by 53 %. Common inhibition of PGE2 and LTD4 synthesis by ETYA (50 microM) or separate blockade of PGE2 synthesis by 1 microM indomethacin reduced the duration of RVD. Blockade of LTD4 synthesis by nordihydroguaiaretic acid (NDGA) did not produce any significant effect on cell swelling or subsequent RVD. 9. Staurosporine (1 microM), an inhibitor of protein kinases, inhibited RVD by 58 %. Taken together the experiments demonstrate that the RVD process is under the control of conductive pathways, extra- and intracellular Ca2+ ions, protein kinases, prostaglandins and leukotrienes.  (+info)

Acetylcholine-induced membrane potential changes in endothelial cells of rabbit aortic valve. (4/2847)

1. Using a microelectrode technique, acetylcholine (ACh)-induced membrane potential changes were characterized using various types of inhibitors of K+ and Cl- channels in rabbit aortic valve endothelial cells (RAVEC). 2. ACh produced transient then sustained membrane hyperpolarizations. Withdrawal of ACh evoked a transient depolarization. 3. High K+ blocked and low K+ potentiated the two ACh-induced hyperpolarizations. Charybdotoxin (ChTX) attenuated the ACh-induced transient and sustained hyperpolarizations; apamin inhibited only the sustained hyperpolarization. In the combined presence of ChTX and apamin, ACh produced a depolarization. 4. In Ca2+-free solution or in the presence of Co2+ or Ni2+, ACh produced a transient hyperpolarization followed by a depolarization. In BAPTA-AM-treated cells, ACh produced only a depolarization. 5. A low concentration of A23187 attenuated the ACh-induced transient, but not the sustained, hyperpolarization. In the presence of cyclopiazonic acid, the hyperpolarization induced by ACh was maintained after ACh removal; this maintained hyperpolarization was blocked by Co2+. 6. Both NPPB and hypertonic solution inhibited the membrane depolarization seen after ACh washout. Bumetanide also attenuated this depolarization. 7. It is concluded that in RAVEC, ACh produces a two-component hyperpolarization followed by a depolarization. It is suggested that ACh-induced Ca2+ release from the storage sites causes a transient hyperpolarization due to activation of ChTX-sensitive K+ channels and that ACh-activated Ca2+ influx causes a sustained hyperpolarization by activating both ChTX- and apamin-sensitive K+ channels. Both volume-sensitive Cl- channels and the Na+-K+-Cl- cotransporter probably contribute to the ACh-induced depolarization.  (+info)

ATP dependence of the ICl,swell channel varies with rate of cell swelling. Evidence for two modes of channel activation. (5/2847)

Swelling-induced activation of the outwardly rectifying anion current, ICl, swell, is modulated by intracellular ATP. The mechanisms by which ATP controls channel activation, however, are unknown. Whole cell patch clamp was employed to begin addressing this issue. Endogenous ATP production was inhibited by dialyzing N1E115 neuroblastoma cells for 4-5 min with solutions containing (microM): 40 oligomycin, 5 iodoacetate, and 20 rotenone. The effect of ATP on current activation was observed in the absence of intracellular Mg2+, in cells exposed to extracellular metabolic inhibitors for 25-35 min followed by intracellular dialysis with oligomycin, iodoacetate, and rotenone, after substitution of ATP with the nonhydrolyzable analogue AMP-PNP, and in the presence of AMP-PNP and alkaline phosphatase to dephosphorylate intracellular proteins. These results demonstrate that the ATP dependence of the channel requires ATP binding rather than hydrolysis and/or phosphorylation reactions. When cells were swollen at 15-55%/min in the absence of intracellular ATP, current activation was slow (0.3-0.8 pA/pF per min). ATP concentration increased the rate of current activation up to maximal values of 4-6 pA/pF per min, but had no effect on the sensitivity of the channel to cell swelling. Rate of current activation was a saturable, hyperbolic function of ATP concentration. The EC50 for ATP varied inversely with the rate of cell swelling. Activation of current was rapid (4-6 pA/pF per min) in the absence of ATP when cells were swollen at rates >/=65%/min. Intracellular ATP concentration had no effect on current activation induced by high rates of swelling. Current activation was transient when endogenous ATP was dialyzed out of the cytoplasm of cells swollen at 15%/min. Rundown of the current was reversed by increasing the rate of swelling to 65%/min. These results indicate that the channel and/or associated regulatory proteins are capable of sensing the rate of cell volume increase. We suggest that channel activation occurs via ATP-dependent and -independent mechanisms. Increasing the rate of cell swelling appears to increase the proportion of channels activating via the ATP-independent pathway. These findings have important physiological implications for understanding ICl, swell regulation, the mechanisms by which cells sense volume changes, and volume homeostasis under conditions where cell metabolism is compromised.  (+info)

The muscle chloride channel ClC-1 has a double-barreled appearance that is differentially affected in dominant and recessive myotonia. (6/2847)

Single-channel recordings of the currents mediated by the muscle Cl- channel, ClC-1, expressed in Xenopus oocytes, provide the first direct evidence that this channel has two equidistant open conductance levels like the Torpedo ClC-0 prototype. As for the case of ClC-0, the probabilities and dwell times of the closed and conducting states are consistent with the presence of two independently gated pathways with approximately 1.2 pS conductance enabled in parallel via a common gate. However, the voltage dependence of the common gate is different and the kinetics are much faster than for ClC-0. Estimates of single-channel parameters from the analysis of macroscopic current fluctuations agree with those from single-channel recordings. Fluctuation analysis was used to characterize changes in the apparent double-gate behavior of the ClC-1 mutations I290M and I556N causing, respectively, a dominant and a recessive form of myotonia. We find that both mutations reduce about equally the open probability of single protopores and that mutation I290M yields a stronger reduction of the common gate open probability than mutation I556N. Our results suggest that the mammalian ClC-homologues have the same structure and mechanism proposed for the Torpedo channel ClC-0. Differential effects on the two gates that appear to modulate the activation of ClC-1 channels may be important determinants for the different patterns of inheritance of dominant and recessive ClC-1 mutations.  (+info)

A single hydrophobic residue confers barbiturate sensitivity to gamma-aminobutyric acid type C receptor. (7/2847)

Barbiturate sensitivity was imparted to the human rho1 homooligomeric gamma-aminobutyric acid (GABA) receptor channel by mutation of a tryptophan residue at position 328 (Trp328), which is located within the third transmembrane domain. Substitutions of Trp328 with a spectrum of amino acids revealed that nearly all hydrophobic residues produced receptor channels that were both directly activated and modulated by pentobarbital with similar sensitivities. Previous studies with ligand-gated ion channels (including GABA) have demonstrated that even conservative amino acid substitution within the agonist-dependent activation domain (N-terminal extracellular domain) can markedly impair agonist sensitivity. Thus, the lack of significant variation in pentobarbital sensitivity among the Trp328 mutants attests to an intrinsic difference between pentobarbital- and the GABA-dependent activation domain. Compared with the heterooligomeric alphabetagamma receptor channel, the mode of modulation for homooligomeric Trp328 mutants by pentobarbital was more dependent on the GABA concentration, yielding potentiation only at low concentrations of GABA (fractions of their respective EC50 values), yet causing inhibition at higher concentrations. Agonist-related studies have also demonstrated that residue 328 plays an important role in agonist-dependent activation, suggesting a functional interconnection between the GABA and pentobarbital activation domains.  (+info)

Functional and molecular characterization of a volume-sensitive chloride current in rat brain endothelial cells. (8/2847)

1. Volume-activated chloride currents in cultured rat brain endothelial cells were investigated on a functional level using the whole-cell voltage-clamp technique and on a molecular level using the reverse transcriptase-polymerase chain reaction (RT-PCR). 2. Exposure to a hypotonic solution caused the activation of a large, outward rectifying current, which exhibited a slight time-dependent decrease at strong depolarizing potentials. The anion permeability of the induced current was I- (1.7) > Br- (1.2) > Cl- (1.0) > F- (0. 7) > gluconate (0.18). 3. The chloride channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB, 100 microM) rapidly and reversibly inhibited both inward and outward currents. The chloride transport blocker 4,4'-diisothiocyanatostilbene-2, 2'-disulphonic acid (DIDS, 100 microM) also blocked the hypotonicity-induced current in a reversible manner. In this case, the outward current was more effectively suppressed than the inward current. The volume-activated current was also inhibited by the antioestrogen tamoxifen (10 microM). 4. The current was dependent on intracellular ATP and independent of intracellular Ca2+. 5. Activation of protein kinase C by phorbol 12,13-dibutyrate (PDBu, 100 nM) inhibited the increase in current normally observed following hypotonic challenge. 6. Extracellular ATP (10 mM) inhibited the current with a more pronounced effect on the outward than the inward current. 7. Verapamil (100 microM) decreased both the inward and the outward hypotonicity-activated chloride current. 8. RT-PCR analysis was used to determine possible molecular candidates for the volume-sensitive current. Expression of the ClC-2, ClC-3 and ClC-5 chloride channels, as well as pICln, could be shown at the mRNA level. 9. We conclude that rat brain endothelial cells express chloride channels which are activated by osmotic swelling. The biophysical and pharmacological properties of the current show strong similarities to those of ClC-3 channel currents as described in other cell types.  (+info)