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At least 973 records · Page 54Linked to original sources
PubMed1991

A slow calcium-dependent chloride current in rhythmic hyperpolarization in neurones of the rabbit vesical pelvic ganglia.

1. Voltage-clamp recordings were made from neurones of vesical pelvic ganglia isolated from the rabbit urinary bladder. A rhythmic outward current, ISH, which corresponds to the spontaneous hyperpolarization, occurred at fairly constant intervals in fifty-eight of eighty-four neurones superfused with Krebs solution. The peak amplitude of the ISH was 0.5 +/- 0.2 nA (n = 48; mean +/- S.E.M.). 2. The ISH was eliminated in a Krebs solution containing nominally zero calcium and 12 mM-magnesium. Lowering the temperature of the superfusing solution from 36 to 22 degrees C also inhibited the occurrence of the ISH. 3. Bath application of caffeine increased the frequency of ISH. In contrast, ryanodine and procaine reversibly blocked ISH. 4. In thirty-four of fifty-eight neurones, the ISH was composed of two current components, an initial fast ISH with duration of 1-10 s and a slow ISH lasting 15-60 s. In the remaining twenty-four neurones, ISH showed only the fast component. 5. The fast ISH was associated with an increased membrane conductance and the slow ISH was associated with a decreased membrane conductance. The reversal potentials of the fast and the slow ISH were -88 +/- 7 mV (n = 4) and -30 +/- 6 mV (n = 4), respectively. 6. Tetraethylammonium (5 mM) and barium (1 mM) blocked the fast ISH but not the slow ISH. Intracellular caesium injected by ionophoresis through a Cs(+)-filled microelectrode blocked the fast ISH, without affecting the slow ISH. Apamin and (+)-tubocurarine selectively suppressed the fast component of the ISH. 7. Substitution of isethionate (67 mM) for chloride increased the amplitude of the slow ISH and shifted the reversal potential of the slow ISH to +1 +/- 8 mV (n = 5). A slow ISH with amplitude of 0.1-1 nA and was still observed in a low-sodium (26.2 mM) solution. The stilbene derivative, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS), a chloride channel blocker, suppressed the slow ISH. 8. These results suggest that ISH is composed of two distinct calcium-dependent currents, a fast ISH produced by activation of potassium conductance and a slow ISH produced by inactivation of chloride conductance. 9. The after-hyperpolarization (AHP) following the action potential was also composed of apamin-sensitive and insensitive spontaneous hyperpolarizing oscillations. The apamin-insensitive component of IAHP was increased by lowering external chloride activity, while it was depressed by SITS.

Animals↗
PubMed2000

The therapeutic efficacy of VP-343, a selective vasopressin V2 receptor antagonist, in the experimental SIADH rat model.

The aim of this work is to investigate the therapeutic efficacy of VP-343 ((N-[4-[[(2S,3aR)-2-hydroxy-2,3,3a,4-tetrahydropyrrolo[1,2-a]qunoxalin-5(1H)-yl]phenyl]-4'-methyl[1,1'-biphenyl]-2-carboxamide), a selective vasopressin V2 receptor antagonist, using the experimental SIADH (syndrome of inappropriate secretion of antidiuretic hormone) rat model. In the model, which was accomplished by administering continuously 1-desamino-8-D-arginine vasopressin (DDAVP), serum sodium levels (S(Na)) and serum osmolarity levels (S(Osm)) significantly and remarkably decreased, which was accompanied with hyper-osmolarity of urine and oliguria. VP-343 increased rapidly and dose-dependently S(Na) and S(Osm). VP-343 exhibited marked diuretic action and decreased urine osmolarity dose-dependently. In the SIADH rat model, all serum levels of chloride, calcium, creatinine, total cholesterol, and uric acid decreased when compared with normal levels. VP-343 increased all serum levels of chloride, calcium, and total cholesterol. These results indicate that VP-343 has efficacy to normalize the abnormalities in DDAVP-induced SIADH.

Animals↗
PubMed2005

Calcium and chloride channel activation by angiotensin II-AT1 receptors in preglomerular vascular smooth muscle cells.

The pathways responsible for the rapid and sustained increases in [Ca(2+)](i) following activation of ANG II receptors (AT(1)) in renal vascular smooth muscle cells were evaluated using fluorescence microscopy. Resting intracellular calcium concentration [Ca(2+)](i) averaged 75 +/- 9 nM. The response to ANG II (100 nM) was characterized by a rapid initial increase of [Ca(2+)](i) by 74 +/- 6 nM (n = 35) followed by a decrease to a sustained level of 12 +/- 2 nM above baseline. The average time from peak to 50% reduction from the peak value (50% time point) was 32 +/- 4 s. AT(1) receptor blockade with 1 microM candesartan (n = 5) prevented the responses to ANG II. In nominally calcium-free conditions (n = 8), the peak increase in [Ca(2+)](i) averaged 42 +/- 7 nM but the sustained phase was absent and the 50% time point was reduced to 11 +/- 4 s. L-type calcium channel blockade with diltiazem reduced the peak [Ca(2+)](i) to 24 +/- 8 nM and the sustained level to 4 +/- 2 nM (n = 10). In cells preincubated in low Cl(-) (3.0 mM), the peak response to ANG II was suppressed as was the sustained response. Blockade of chloride channels with DIDS eliminated both the peak and sustained responses (n = 11); chloride channel blockade with DPC (n = 17) suppressed the peak increase in [Ca(2+)](i) to 18 +/- 5 and also prevented the sustained response. IP3 receptor blockade by 10 microM TMB-8 (n = 6) reduced the peak to 22 +/- 8 and prevented the sustained response. Exposure to 10 microM TMB-8 in the presence of Ca(2+)-free medium prevented the ANG II response (n = 9). In the presence of 100 microM DPC and 10 microM TMB-8 (n = 7), the ANG II response was also prevented. Thus the rapid initial increase in [Ca(2+)](i) is due not only to release from intracellular stores, but also to Ca(2+) influx from the extracellular fluid. Although Ca(2+) entry via L-type calcium channels is responsible for the major portion of the sustained response, other entry pathways participate. The finding that chloride channel blockers markedly attenuate both rapid and sustained responses indicates that chloride channel activation contributes to, rather than being the consequence of, the initial rapid response.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1993

Protein kinase A modulates an endogenous calcium channel, but not the calcium-activated chloride channel, in Xenopus oocytes.

In Xenopus oocytes, Ca2+ influx through an endogenous voltage-gated Ca2+ channel activates a transient outward Cl- current (ICl(Ca)), which is potentiated by cAMP increase. The site of cAMP effect appears to be the Ca2+ channel instead of the Ca(2+)-activated Cl- channel, because cAMP potentiates the Ba2+ current through the Ca2+ channel in a similar way to the ICl(Ca), and cAMP does not potentiate the Ca(2+)-dependent Cl- current in cells treated with Ca2+ ionophore. Using the catalytic subunit of protein kinase A (PKA) and PKA inhibitors, it was shown that PKA is both necessary and sufficient for the cAMP effect on ICl(Ca). Furthermore, the cAMP/PKA-mediated potentiation of ICl(Ca) was inhibited by both type 1 and type 2A protein phosphatases.

Amino Acid Sequence↗
PubMed1974

The sensitivity of Helix aspersa neurones to injected calcium ions.

1. When calcium chloride was injected into Helix aspersa neurones there was a fall in membrane resistance and the membrane potential became hyperpolarized.2. The reversal potential of the response was dependent on the concentration of potassium in the external solution.3. Injection of a calcium-EGTA buffer containing 9 x 10(-7)M free calcium reduced the membrane resistance by 25%. When calcium chloride was injected it was necessary to increase the total intracellular calcium concentration by about 10(-3)M to produce similar change of resistance.4. In sodium-free (Tris) solution there was a slow fall of membrane resistance as if the intracellular calcium concentration had increased. There was a similar resistance change in the presence of 2,4-dinitrophenol and iodoacetate.5. A series of repetitive depolarizing pulses produced a long lasting reduction in membrane resistance which was enhanced by 2,4-dinitrophenol and iodoacetate.6. It is concluded that (a) injection of calcium causes an increase in potassium permeability, (b) the injected calcium is rapidly pumped from the cytoplasm by a sodium-dependent mechanism and by mitochondria, and (c) 1-2 msec depolarizing pulses stimulate an influx of calcium. This influx is rapid enough to trigger potassium activation during an action potential.

Action Potentials↗
PubMed1994

Calcium salts for treating hypocalcemia: carrier effects, acid-base balance, and oral versus rectal administration.

Oral Ca salt supplements are being used to combat hypocalcemia in dairy cows. The absorption of these Ca supplements is dependent on the salt and carrier used. In the studies presented, the magnitude of the rise in plasma Ca concentration following administration of the Ca preparation was used as an index of Ca absorption by the gastrointestinal tract. Calcium chloride preparations in a water-soluble carrier (propylene glycol) were absorbed more readily than Ca propionate in propylene glycol, but Ca propionate effected a more sustained increase in plasma Ca concentration. Vegetable oil preparations of these salts were poorly absorbed. Calcium chloride preparations induced a significant decrease in blood and urine pH. Calcium propionate salts did not alter acid-base balance. Rectal administration of Ca salts could raise plasma Ca concentration rapidly, but caused serious pathological lesions, precluding their use.

Acid-Base Equilibrium↗
PubMed1985

A calcium-activated chloride current generates the after-depolarization of rat sensory neurones in culture.

Neurones from the dorsal root ganglia of 1-day-old rat pups were grown in dissociated culture and voltage clamped using patch electrodes for whole-cell recording. The pipettes were filled with either 140 mM-KCl or CsCl. Depolarizing voltage jumps activated net inward calcium currents in all neurones, which in a subpopulation of 46% were followed by slowly decaying inward tail currents accompanied by large increases in membrane conductance. During voltage jumps to membrane potentials more positive than 0 mV the inward calcium current was contaminated by a slow outward relaxation only in those neurones with slow inward tail currents. The availability curve for the slow inward tail current was U shaped, with a peak at approximately +5 mV in medium containing 2.5 mM-Ca2+; further depolarization reduced the amplitude of the tail current. During perfusion with calcium-free solution, or in the presence of the calcium-channel blockers cadmium or cobalt, or on substitution of barium for calcium, both the slow inward tail currents and outward relaxations were reversibly blocked. The reversal potential of the slow inward tail current, measured using a twin-pulse protocol, was approximately -10 mV. Replacement of sodium by tetraethylammonium (TEA) did not reduce the slow inward tail current, nor change its reversal potential. Reduction of the extracellular chloride activity produced a large increase in the amplitude of the slow inward tail current suggesting an increase in permeability to anions. This conductance, which behaves as though activated by prior or concurrent calcium entry triggered by membrane potential depolarization, is referred to as ICl(Ca). The activation and deactivation kinetics of ICl(Ca) are complex: envelope experiments measuring peak tail current amplitude revealed activation to be described by a single exponential function, of time constant approximately 100 ms at -10 to +8 mV. The integral of the tail currents increased with the duration of depolarizing pre-pulses suggesting accumulation of intracellular calcium. The decay of tail currents activated by short depolarizing voltage jumps was described by a single exponential function of time constant approximately 200 ms at -60 mV; more complex decay kinetics were recorded following activation by voltage jumps of duration greater than 60 ms. Tail current decay was voltage sensitive, becoming faster with hyperpolarization and increasing e-fold per 120 mV change in membrane potential.

Action Potentials↗
PubMed2000

Volume- and calcium-activated chloride channels in human umbilical vein endothelial cells.

AIM: To characterize the properties of chloride currents and its modulation in human umbilical vein endothelial cells (HUVEC). METHODS: Using whole-cell patch-clamp recording techniques. RESULTS: Exposure of HUVEC to 13.5% and 27% hypotonic solution (HTS) induced a current ICl, vol. This current was correlated with the changes in cell volume and showed a modest outward rectification. It was slowly inactivated at positive potential (> 50 mV), and it was time- and voltage-independent in kinetics. The current densities (pA/pF) were 20 +/- 3 (13.5% HTS) and 58 +/- 4 (27% HTS, n = 7), respectively at +100 mV test potential. Applying GTP gamma s (300 mumol.L-1) elicited a current similar to ICl, vol, while cAMP (0.5 mmol.L-1) had no effect on the current. Increase in [Ca2+]i, either by directly loading cells with high concentration of Ca2+ (CaCl2), or by perfusing vasoactive agonist ATP (10 mumol.L-1), activated ICl, Ca. The current density was only (23 +/- 5) pA/pF (n = 8 cells). Such current was slowly activated at positive potential, inactivated quickly at negative potential, and showed strong outward rectification. Both currents were inhibited by DIDS and verapamil. Challenging a cell with elevated [Ca2+]i and HTS activated ICl, vol on the top of ICl, Ca in the same cell, suggested co-existence of these two currents and that they were modulated by different ways. cAMP-regulated chloride channel and ClC (chloride channel family) channel were absent. CONCLUSION: HUVEC express two kinds of chloride channels, ICl, vol activated by change in cell volume and ICl, Ca by elevation of [Ca2+]i, respectively.

Adenosine Triphosphate↗
PubMed2002

Presence of a calcium-activated chloride current in mouse ventricular myocytes.

The properties of several components of outward K(+) currents, including the pharmacological and kinetics profiles as well as the respective molecular correlates, have been identified in mouse cardiac myocytes. Surprisingly little is known with regard to the Ca(2+)-activated ionic currents. We studied the Ca(2+)-activated transient outward currents in mouse ventricular myocytes. We have identified a 4-aminopyridine (4-AP)- and tetraethyl ammonium-resistant transient outward current that is Ca(2+) dependent. The current is carried by Cl(-) and is critically dependent on Ca(2+) influx via voltage-gated Ca(2+) channels and the sarcoplasmic reticulum Ca(2+) store. The current can be blocked by the anion transport blockers niflumic acid and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. Single channel recordings reveal small conductance channels (approximately 1 pS in 140 mM Cl(-)) that can be blocked by anion transport blockers. Ensemble-averaged current faithfully mirrors the transient kinetics observed at the whole level. Niflumic acid (in the presence of 4-AP) leads to prolongation of the early repolarization. Thus this current may contribute to early repolarization of action potentials in mouse ventricular myocytes.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2001

Potentiation by stannous chloride of calcium entry into osteoblastic MC3T3-E1 cells through voltage-dependent L-type calcium channels.

The present study was undertaken to confirm that L-type Ca(2+) channels are involved in Ca(2+) entry into osteoblastic MC3T3-E1 cells and to examine the effect of SnCl2, a Ca(2+)]-channel activator, on the intracellular Ca(2+)concentration ([Ca(2+)]i). High K(+)concentration-dependently raised the [Ca(2+)]i. All of the L-type Ca(2+)channel blockers used here, such as nifedipine, nicardipine, verapamil, and diltiazem, and CdCl2 (a non-selective blocker) inhibited the high K(+)-induced [Ca(2+)]i rise, but v-conotoxin GVIA (an N-type blocker) and NiCl2(a T-type blocker) had no effect. Application of SnCl2 alone did not change the [Ca(2+)]i. However, in the presence of high K(+), SnCl2 enhanced the high K(+)-induced [Ca(2+)]i rise, which was inhibited by Ca(2+)]-free medium or nifedipine. In the case where high K(+)was applied prior to SnCl2, SnCl2 alone raised the [Ca(2+)]i by itself. In conclusion, MC3T3-E1 cells possess the voltage-dependent L-type Ca(2+)] channels and SnCl2 facilitates the Ca(2+) entry through the L-type ones under the condition of the membrane depolarization. There is the possibility that Ca(2+) release from intracellular Ca(2+) stores is involved in the action of SnCl2.

3T3 Cells↗
PubMed1993

Lovastatin inhibits cAMP- and calcium-stimulated chloride secretion by T84 cells.

Isoprenylated proteins function in the processes of signal transduction and membrane vesicle trafficking. To investigate the role of isoprenylated proteins in secretagogue-stimulated epithelial ion transport, we studied the effects of lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, on adenosine 3',5'-cyclic monophosphate (cAMP)- and Ca(2+)-stimulated Cl- secretion by monolayers of T84 colonic epithelial cells. Lovastatin reduces protein isoprenylation in many cell types. In T84 cells, lovastatin reversibly inhibits forskolin-stimulated equivalent short-circuit current (I(sc)eq) by 50% after 2 days of treatment. The concentration of lovastatin resulting in half-maximal effects on forskolin-stimulated I(sc)eq is consistent with inhibition of protein isoprenylation, and lovastatin effects on forskolin-stimulated I(sc)eq are not associated with inhibition of cholesterol or glycoprotein biosynthesis. Lovastatin blocks N6,2'-O-dibutyryladenosine 3',5'-cyclic monophosphate- and ionomycin-stimulated Isc, suggesting that it inhibits a process beyond the stimulation of cAMP and Ca2+ second-messenger systems. In monolayers in which the basolateral membrane has been permeabilized with nystatin, lovastatin inhibits cAMP activation of a diphenylamine-2-carboxylate-sensitive, apical membrane Cl- conductance. Our results are consistent with the hypothesis that an isoprenylated protein is involved in the regulation of a secretagogue-activated apical membrane Cl- conductance in T84 cells.

Calcium↗
PubMed1992

Calcium-activated chloride channels in cultured embryonic Xenopus spinal neurons.

1. Single-channel currents were recorded from Xenopus spinal neurons developing in vitro using the patch-clamp technique, to identify the channels underlying the large and small macroscopic Ca(2+)-activated Cl- currents (ICl(Ca)) present in these cells. 2. Channels of large (maxi-channels; 310 pS) and smaller conductance (mini-channels; 50-60 pS) are activated by elevation of cytoplasmic Ca2+ concentration. Channel activity is not altered by subsequent removal of Ca2+ from the bath, arguing against a direct ligand-type Ca2+ dependence. The much higher incidence of channel activation in cell-attached patches from cells permeabilized with the Ca2+ ionophore A23187 than in excised patches also suggests the involvement of some unidentified intracellular factor. 3. The reversal potential of maxi-Cl- channels is not altered by changes in Na+ concentration, but is shifted in the negative direction by the substitution of Cl- by methanesulfonate on the intracellular side of the patch, indicating their anionic selectivity. 4. Maxi-Cl- channels exhibited the presence of multiple probable subconductance states and showed marked voltage-dependent inactivation above and below +/- 20 mV. 5. Examination of maxi-Cl- channels at early times in culture (6-9 h) and 24 h later did not reveal any developmental change in the characteristics described above. However, the mean open duration of the channel was found to increase twofold during this period of time. 6. The simultaneous presence of maxi- and mini-Cl- channels prevented detailed characterization of the latter. The anionic selectivity of mini-Cl- channels is suggested by their reversal potential that lies close to the Cl- equilibrium potential.(ABSTRACT TRUNCATED AT 250 WORDS)

Action Potentials↗
PubMed2006

Bestrophin-2 is a candidate calcium-activated chloride channel involved in olfactory transduction.

Ca-activated Cl channels are an important component of olfactory transduction. Odor binding to olfactory receptors in the cilia of olfactory sensory neurons (OSNs) leads to an increase of intraciliary Ca concentration by Ca entry through cyclic nucleotide-gated (CNG) channels. Ca activates a Cl channel that leads to an efflux of Cl from the cilia, contributing to the amplification of the OSN depolarization. The molecular identity of this Cl channel remains elusive. Recent evidence has indicated that bestrophins are able to form Ca-activated Cl channels in heterologous systems. Here we have analyzed the expression of bestrophins in the mouse olfactory epithelium and demonstrated that only mouse bestrophin-2 (mBest2) was expressed. Single-cell RT-PCR showed that mBest2 was expressed in OSNs but not in supporting cells. Immunohistochemistry revealed that mBest2 was expressed on the cilia of OSNs, the site of olfactory transduction, and colocalized with the main CNGA2 channel subunit. Electrophysiological properties of Ca-activated Cl currents from native channels in dendritic knob/cilia of mouse OSNs were compared with those induced by the expression of mBest2 in HEK-293 cells. We found the same anion permeability sequence, small estimated single-channel conductances, a Ca sensitivity difference of one order of magnitude, and the same side-specific blockage of the two Cl channel blockers commonly used to inhibit the odorant-induced Ca-activated Cl current in OSNs, niflumic acid, and 4-acetamido-4'-isothiocyanato-stilben-2,2'-disulfonate (SITS). Therefore, our data suggest that mBest2 is a good candidate for being a molecular component of the olfactory Ca-activated Cl channel.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed2002

P2 purinoceptors regulate calcium-activated chloride and fluid transport in 31EG4 mammary epithelia.

It has been reported that secretory mammary epithelial cells (MEC) release ATP, UTP, and UDP upon mechanical stimulation. Here we examined the physiological changes caused by ATP/UTP in nontransformed, clonal mouse mammary epithelia (31EG4 cells). In control conditions, transepithelial potential (apical side negative) and resistance were -4.4 +/- 1.3 mV (mean +/- SD, n = 12) and 517.7 +/- 39.4 Omega. cm(2), respectively. The apical membrane potential was -43.9 +/- 1.7 mV, and the ratio of apical to basolateral membrane resistance (R(A)/R(B)) was 3.5 +/- 0.2. Addition of ATP or UTP to the apical or basolateral membranes caused large voltage and resistance changes with an EC(50) of approximately 24 microM (apical) and approximately 30 microM (basal). Apical ATP/UTP (100 microM) depolarized apical membrane potential by 17.6 +/- 0.8 mV (n = 7) and decreased R(A)/R(B) by a factor of approximately 3. The addition of adenosine to either side (100 microM) had no effect on any of these parameters. The ATP/UTP responses were partially inhibited by DIDS and suramin and mediated by a transient increase in free intracellular Ca(2+) concentration (427 +/- 206 nM; 15-25 microM ATP, apical; n = 6). This Ca(2+) increase was blocked by cyclopiazonic acid, by BAPTA, or by xestospongin C. 31EG4 MEC monolayers also secreted or absorbed fluid in the resting state, and ATP or UTP increased fluid secretion by 5.6 +/- 3 microl x cm(-2) x h(-1) (n = 10). Pharmacology experiments indicate that 31EG4 epithelia contain P2Y(2) purinoceptors on the apical and basolateral membranes, which upon activation stimulate apical Ca(2+)-dependent Cl channels and cause fluid secretion across the monolayer. This suggests that extracellular nucleotides could play a fundamental role in mammary gland paracrine signaling and the regulation of milk composition in vivo.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1995

Normalization of raised sodium absorption and raised calcium-mediated chloride secretion by adenovirus-mediated expression of cystic fibrosis transmembrane conductance regulator in primary human cystic fibrosis airway epithelial cells.

Cystic fibrosis airway epithelia exhibit a spectrum of ion transport properties that differ from normal, including not only defective cAMP-mediated Cl- secretion, but also increased Na+ absorption and increased Ca(2+)-mediated Cl- secretion. In the present study, we examined whether adenovirus-mediated (Ad5) transduction of CFTR can correct all of these CF ion transport abnormalities. Polarized primary cultures of human CF and normal nasal epithelial cells were infected with Ad5-CBCFTR at an moi (10(4)) which transduced virtually all cells or Ad5-CMV lacZ as a control. Consistent with previous reports, Ad5-CBCFTR, but not Ad5-CMV lacZ, corrected defective CF cAMP-mediated Cl- secretion. Basal Na+ transport rates (basal Ieq) in CF airway epithelial sheets (-78.5 +/- 9.8 microA/cm2) were reduced to levels measured in normal epithelial sheets (-30.0 +/- 2.0 microA/cm2) by Ad5-CBCFTR (-36.9 +/- 4.8 microA/cm2), but not Ad5-CMV lacZ (-65.8 +/- 6.1 microA/cm2). Surprisingly, a significant reduction in delta Ieq in response to ionomycin, a measure of Ca(2+)-mediated Cl- secretion, was observed in CFTR-expressing (corrected) CF epithelial sheets (-6.9 +/- 11.8 microA/cm2) when compared to uninfected CF epithelial sheets (-76.2 +/- 15.1 microA/cm2). Dose response effects of Ad5-CBCFTR on basal Na+ transport rates and Ca(2+)-mediated Cl- secretion suggest that the mechanism of regulation of these two ion transport functions by CFTR may be different. In conclusion, efficient transduction of CFTR corrects hyperabsorption of Na+ in primary CF airway epithelial cells and restores Ca(2+)-mediated Cl- secretion to levels observed in normal airway epithelial cells. Moreover, assessment of these ion transport abnormalities may represent important endpoints for testing the efficacy of gene therapy for cystic fibrosis.

Adenoviridae↗
PubMed1983

A transient calcium-dependent chloride current in the immature Xenopus oocyte.

Ionic currents were studied in immature full-grown Xenopus oocytes using the two-micro-electrode voltage-clamp technique. Recordings of total membrane current showed a transient outward peak during depolarizations from the approximate resting voltage (-70 or -80 mV) to voltages more positive than -20 mV. The current-voltage relation for peak outward current was U-shaped, with a maximum at about 0 mV. Replacement of external Cl with methanesulphonate reversed this transient outward current to a transient inward current. Current relaxations recorded after the membrane potential was stepped to different voltages at the time of the peak showed a component that inverted at about -25 to -30 mV. This value was close to ECl as determined by measurement of the intracellular Cl ion concentration. The reversal potential for these current relaxations changed with the external Cl concentration as predicted by the Nernst relation. Replacement of external Ca with Mg, Sr or Ba, or addition of low concentrations of Ni in the presence of Ca, eliminated the transient outward current. Increasing the external Ca concentration increased the amplitude of the transient outward current without affecting the amplitude of the steady-state current. It was concluded that the outward peak in records of total membrane current represented the contribution of a transient outward current carried by Cl ions which was dependent on the entry of external Ca. It will be noted as ICl(Ca). Decay of ICl(Ca) could be described at the normal Ca concentration by a single exponential function whose time constant showed a shallow U-shaped voltage dependence. ICl(Ca) was maximally activatable by depolarizations from a holding potential of about -100 mV, but could not be activated by depolarizations from -40 mV. The amplitude of ICl(Ca) showed a large temperature dependence as compared to the steady-state current, suggesting complex control of its activation.

Animals↗
PubMed1994

Characterization of the calcium-activated chloride channel in isolated guinea-pig hepatocytes.

Macroscopic and unitary currents through Ca(2+)-activated Cl- channels were examined in enzymatically isolated guinea-pig hepatocytes using whole-cell, excised outside-out and inside-out configurations of the patch-clamp technique. When K+ conductances were blocked and the intracellular Ca2+ concentration ([Ca2+]i) was set at 1 microM (pCa = 6), membrane currents were observed under whole-cell voltage-clamp conditions. The reversal potential of the current shifted by approximately 60 mV per 10-fold change in the external Cl- concentration. In addition, the current did not appear when Cl- was omitted from the internal and external solutions, indicating that the current was Cl- selective. The current was activated by increasing [Ca2+]i and was inactivated in Ca(2+)-free, 5 mM EGTA internal solution (pCa > 9). The current was inhibited by bath application of 9-anthracenecarboxylic acid (9-AC) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) in a voltage-dependent manner. In single channel recordings from outside-out patches, unitary current activity was observed, whose averaged slope conductance was 7.4 +/- 0.5 pS (n = 18). The single channel activity responded to extracellular Cl- changes as expected for a Cl- channel current. The open time distribution was best described by a single exponential function with mean open lifetime of 97.6 +/- 10.4 ms (n = 11), while at least two exponentials were required to fit the closed time distributions with a time constant for the fast component of 21.5 +/- 2.8 ms (n = 11) and that for the slow component of 411.9 +/- 52.0 ms (n = 11). In excised inside-out patch recordings, channel open probability was sensitive to [Ca2+]i. The relationship between [Ca2+]i and channel activity was fitted by the Hill equation with a Hill coefficient of 3.4 and the half-maximal activation was 0.48 microM. These results suggest that guinea-pig hepatocytes possess Ca(2+)-activated Cl- channels.

Algorithms↗