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K Kunzelmann

Publications and source records attributed to K Kunzelmann.

At least 91 records · Page 5Linked to original sources
PubMed1994

Forskolin and PMA pretreatment of HT29 cells alters their chloride conductance induced by cAMP, Ca2+ and hypotonic cell swelling.

HT29 cells were preincubated with forskolin (10(-5) mol/l, FORHT) or phorbol 12-myristate 13-acetate (PMA) (10(-7) mol/l, PMAHT) for 20 h, which has been shown previously and is also shown here, to upregulate and downregulate, respectively, the expression of the cystic fibrosis transmembrane conductance regulator (CFTR). CFPAC-1 cells underwent the same protocols. HT29 cells were examined by slow (SWC) and fast (FWC) whole-cell patch-clamp techniques. The results of SWC and FWC were indistinguishable and were pooled. CFPAC-1 cells were examined with FWC. The membrane voltage (V) of FORHT was -41.8 +/- 1.4 mV (n = 77) and that of PMAHT was -43.6 +/- 2.4 mV (n = 76). The conductance (G) of FORHT (9.4 +/- 0.9 nS, n = 77) was significantly larger than that of PMAHT (3.7 +/- 0.4 nS, n = 76). Acute application of forskolin (10(-5) mol/l, FOR) plus 0.5 mmol/l 8-(4-chlorophenylthio)-cAMP (cAMP) depolarized V by 12 (FORHT) and 8 (PMAHT) mV, respectively. The acute increase of G by FOR plus cAMP was by 7.6 +/- 1.9 nS for FORHT (n = 22) and only 2.2 +/- 1 nS for PMAHT (n = 13). ATP (10(-4) mol/l) depolarized V in both types of cells. It enhanced G by 16.7 +/- 4.1 nS in FORHT (n = 14) and significantly less (by 5.5 +/- 1.2 nS, n = 14) in PMAHT. Also the G increase lasted longer in FORHT. Neurotensin (NT, 10(-8) mol/l) also had a stronger and longer lasting effect in FORHT.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1994

CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells.

A major limitation in the study of vectorial ion transport, secretion, and differentiated function in the human airway epithelium has been the lack of suitable cell culture systems. Progress in this direction has been made through the transformation of primary cultured epithelial cells. However, these transformants tend to lose differentiated properties with increasing serial passage, particularly following crisis. The successful establishment of a postcrisis SV40 large T-antigen transformed epithelial cell line derived from human bronchial epithelium is described. This cell line, 16HBE14o-, retains differentiated epithelial morphology and functions. Cell cultures show the presence of tight junctions and cilia, and monolayers generate transepithelial resistance, as measured in Ussing chambers, and retain beta-adrenergic stimulation of cAMP-dependent chloride ion transport, measured either by 36Cl- efflux or as short-circuit current in Ussing chambers. The cells also increase chloride transport in response to bradykinin or calcium ionophore. In addition, 16HBE14o- cells express levels of both the cystic fibrosis transmembrane conductance regulator (CFTR) mRNA and protein readily detectable by Northern and Western hybridization analysis, respectively. These cells provide a valuable resource for studying the modulation of CFTR and its role in regulation of chloride ion transport in human airway epithelium as well as other aspects of human airway cell biology.

Bronchi↗
PubMed1993

Small and intermediate conductance chloride channels in HT29 cells.

Recently, it has been shown that intermediate conductance outwardly rectifying chloride channels (ICOR) are blocked by cytosolic inhibitor (C. I.) found in the cytosol of human placenta and epithelial cells. C. I. also reduced the baseline current in excised membrane patches of HT29 cells. In the present study, this effect of C. I. was characterized further. Heat treated human placental cytosol was extracted in organic solvents and dissolved in different electrolyte solutions. It is shown that the reduction of baseline conductance (g(o)) is caused by inhibition of small non-resolvable channels, which are impermeable to Na+ and SO4(2-), but permeable to Cl-. The regulation of these small Cl(-)-conducting channels (g(o)) and of ICOR was examined further. First, no activating effects of protein kinase A (PKA) on the open probability (Po) of the ICOR or on the g(o)) were observed. The Po of the ICOR was reduced by 22% in a Ca(2+)-free solution. g(o) was insensitive to changes in the Ca2+ activity. The effects of C. I. from a cystic fibrosis (CF) placenta and the CF pancreatic duct cell line CFPAC-1 were compared with the effects of corresponding control cytosols, and no significant differences between CF and control cytosols were found. We conclude that the excised patches of HT29 cells contain ICOR and small non-resolvable Cl(-)-conducting channels which are similarly inhibited by C. I.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1993

An immortalized cystic fibrosis tracheal epithelial cell line homozygous for the delta F508 CFTR mutation.

The development of transformed human airway epithelial cell lines has been important in advancing the understanding of the biochemical and genetic mechanisms underlying the cystic fibrosis (CF) defect. Since the most common mutation associated with CF is a phenylalanine deletion at position 508 (delta F508) in the CF transmembrane conductance regulator (CFTR) gene, a transformed airway epithelial cell line homozygous for this mutation will be important for determining the biologic significance of this mutation in the airways. We report the genotypic and phenotypic characterization of a delta F508 homozygote cell line derived from luminal epithelium in the trachea. The cells were transformed with a plasmid containing an origin of replication defective SV40 genome and have progressed through crisis. Immunocytochemical characterization of the cells shows that they express keratin, indicating epithelial cell origin, and that a calcium-dependent cell adhesion molecule, cellCAM 120/80, is present at plasma membrane junctions between cells. Electrophysiologically, the cells show no cAMP-dependent Cl transport. However, after treatment with the calcium ionophore, ionomycin, cells secrete Cl, albeit at a lower level than that observed in normal cells. Genetically, the cells express CFTR mRNA as determined by polymerase chain reaction amplification and CFTR protein as determined by Western hybridization analysis. Karyotypic analysis shows that 70% of the cells contain two copies of chromosome 7.

Adult↗
PubMed1992

Small-conductance chloride channels induced by cAMP, Ca2+, and hypotonicity in HT29 cells: ion selectivity, additivity and stilbene sensitivity.

Previous studies in HT29 cells utilizing the cell-attached nystatin (CAN) method [Greger R, Kunzelmann K (1991) Pflügers Arch 419:209-211] have revealed that the Cl- channels induced by cAMP or by increasing cytosolic Ca2+, e.g. by addition of ATP, and by hypotonic cell swelling share in common their conductance, which was so small in our studies [Kunzelmann et al. (1992) Pflügers Arch (in press)] that we could not resolve it at the single-channel level. This prompted the question whether these Cl- conductances can be distinguished in terms of their ion selectivity and sensitivity towards inhibitors. Whether these pathways are additive or not was also examined. The present study utilized the whole-cell patch-clamp and the CAN methods. A total of 160 patches were studied. In whole-cell patches 8-(4-chlorophenylthio)-cAMP (cAMP, 0.1 +/- 1 mmol/l) induced a significant depolarization by 5 mV and a twofold increase in conductance (G) from 6.2 +/- 1.5 nS to 11.7 +/- 3.2 nS (n = 15). Total replacement of Cl- by Br- and I- in cAMP-treated cells hyperpolarized the membrane voltage (V) significantly from -35 +/- 2.8 to -39 +/- 3.4 and -45 +/- 3.3 mV respectively, but had no detectable effect on G, which was 11.9 +/- 3.3 nS in the case of Br- and 11.8 +/- 3.3 nS in the case of I-. Hence, the permselectivity of the cAMP pathway was I- > Br- > Cl-, but the conductances for these anions were all indistinguishable.(ABSTRACT TRUNCATED AT 250 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1992

cAMP-dependent activation of small-conductance Cl- channels in HT29 colon carcinoma cells.

The present study was performed to examine the conductance properties in the colon carcinoma cell line HT29 and the activation of Cl- channels by cAMP. A modified cell-attached nystatin patch-clamp technique was used, allowing for the simultaneous recording of the cell membrane potential (PD) and the conductance properties of the cell-attached membrane. In resting cells, PD was -56 +/- 0.4 mV (n = 294). Changing the respective ion concentrations in the bath indicate that these cells possess a dominating K+ conductance and a smaller Cl- conductance. A significant non-selective cation conductance, which could not be inhibited by amiloride, was only observed in cells examined early after plating. The K+ conductance was reversibly inhibited by 1 - 5 mmol/l Ba2+. Stimulation of the cells by the secretagogues isoproterenol and vasointestinal polypeptide (VIP) depolarized PD and induced a Cl- conductance. Similar results were obtained with compounds increasing cytosolic cAMP: forskolin, 3-isobutyl-1-methylxanthine, cholera toxin and 8-bromoadenosine cyclic 3',5'-monophosphate (8-Br-cAMP). VIP (1 nmol/l, n = 10) and isoproterenol (1 mumol/l, n = 12) depolarized the cells dose-dependently and reversibly by 12 +/- 2 mV and 13 +/- 2 mV. The maximal depolarization was reached after some 20 s. The depolarization was due to increases in the fractional Cl- conductance. Simultaneously the conductance of the cell-attached membrane increased from 155 +/- 31 pS to 253 +/- 40 pS (VIP, n = 4) and from 170 +/- 43 pS to 268 +/- 56 pS (isoproterenol, n = 11), reflecting the gating of Cl- channels in the cell-attached membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic beta-Agonists↗
PubMed1992

Small-conductance Cl- channels in HT29 cells: activation by Ca2+, hypotonic cell swelling and 8-Br-cGMP.

The present study demonstrates the activation of Cl- channels in HT29 cells by agonist (ATP, neurotensin, carbachol) increasing cytosolic Ca2+, by hypotonic cell swelling and by cGMP. Cell-attached nystatin patch-clamp (CAN) as well as slow and fast whole-cell recordings were used. The cell membrane potential was depolarized in a dose-dependent manner with half-maximal effects at 0.4 mumol/l for ATP, 60 pmol/l for neurotensin and 0.8 mumol/l for carbachol. The depolarization, which was caused by Cl- conductances increases, occurred within 1 s and was accompanied by a simultaneous and reversible increase of the input conductance of the cell-attached membrane from 295 +/- 32 pS to 1180 +/- 271 pS (ATP; 10 mumol/l, n = 21) and 192 +/- 37 pS to 443 +/- 128 pS (neurotensin; 1 nmol/l, n = 8). The effects of the agonists could be mimicked by ionomycin (0.2 mumol/l), suggesting that an increase in intracellular Ca2+ was responsible for the activation of Cl- channels. The depolarization was followed by a secondary hyperpolarization. Hypotonic cell swelling also depolarized the cells and induced an increase in the membrane conductance. With 120 mmol/l NaCl the depolarization was 10 +/- 0.8 mV and the cell-attached conductance increased from 228 +/- 29 pS to 410 +/- 65 (n = 26) pS. NaCl at 90 mmol/l and 72.5 mmol/l had even stronger effects. Comparable conductance increases were also obtained when the different agonists or hypotonic cell swelling were examined in whole cell experiments. 5-Nitro-2-(3-phenylpropylamino)-benzoate (1 mumol/l) did not prevent the effects of Ca(2+)-increasing hormones and of hypotonic solutions.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1991

Different types of blockers of the intermediate-conductance outwardly rectifying chloride channel in epithelia.

Epithelial chloride channels can be blocked by various inhibitors, which show considerable differences in their molecular structure. In the present patch-clamp study, we compared different blockers of one type of epithelial Cl- channel with respect to their inhibitory potency. We applied the blockers to excised inside-out-or outside-out-oriented membrane patches of cultured HT29 colon carcinoma and respiratory epithelial cells (REC) containing the outwardly rectifying intermediate-conductance (ICOR) chloride channel. Four types of inhibitory compounds were tested: stilbene disulphonate derivatives, indanyloxyacetic acid, amidine, and arylaminobenzoates. The concentrations for half-maximal inhibition (IC50) for the different channel blockers were (mumol/l): 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulphonic acid 100; 4,4'-diisothiocyanato-stilbene-2,2'-disulphonic acid 80; indanyloxyacetic acid 9; 4,4'-dinitrostilbene-2,2'-disulphonic acid 8; amidine 8 and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) 0.9. All compounds, when applied to the cytosolic side of the channel, induced a flicker-type block of the ICOR Cl- channel at lower concentrations and a complete channel inhibition at higher concentrations. The inhibitory potency of NPPB was much higher when it was added to the external surface of the channel in outside-out-oriented membrane patches. At 1 mumol/l the inhibition was complete. All blocker effects were fully reversible. The probe with the highest affinity (NPPB) and a closely related compound 5-nitro-2-(3-phenylethylamino)-benzoate (NPEB) were used to construct macromolecular probes by linking these blockers to aminopolyethyleneglycol (PEG) or amino-ethyl-O-dextran (5 kDa).2+ These macromolecular NPPB and NPEB derivatives inhibited the ICOR Cl- channels only from the outside but had no effect on the cytosolic side.(ABSTRACT TRUNCATED AT 250 WORDS)

Amidines↗
PubMed1991

Bicarbonate permeability of epithelial chloride channels.

Bicarbonate permeability of epithelial chloride channels has been studied using the patch-clamp technique. The experiments were performed in excised inside-out oriented membrane patches from three different cultured cell types: (a) HT29 colon carcinoma cell line, (b) T84 colon carcinoma cell line, and (c) respiratory epithelial cells (REC) in primary culture. In all three preparations we observed outwardly rectifying chloride channels with similar conductances with 145 mmol/l NaCl solution in the pipette and in the bath (Cl- pipette/Cl- bath). When Cl- was replaced by HCO3- in the bath (Cl-/HCO3-) the conductance of the channel at negative clamp voltages was reduced significantly by 40% for HT29 (n = 6), 39% for T84 (n = 7), and 38% for REC (n = 6). Similarly, the zero-current potential (V1 = 0) was shifted from 0 mV (Cl-/Cl-) to negative values (Cl-/HCO3-) revealing permeability ratios PCl/PHCO3 of 2.4 +/- 0.1 for HT29 (n = 6), 2.0 +/- 0.1 for T84 (n = 7), and 1.8 +/- 0.1 for REC (n = 7). With NaHCO3 as the pipette solution and NaCl in the bath, the V1 = 0 was positive and a PCl/PHCO3 value of 2.3 +/- 0.1 was determined for HT29 (n = 5). Replacement of Cl- in the bath by HCO3- reduced V1 = 0 to values close to 0 mV. In another series of experiments, the pipette was filled with 145 mmol/l NaCl and the bath contained 35 mmol/l NaCl to which 35 mmol/l NaHCO3 were added. We found that neither the conductance for the inward current nor V1 = 0 was changed significantly with the addition of NaHCO3 (HT29, n = 6). We conclude that the HCO3- permeability and HCO3- conductance of these channels is about half of that for Cl-.

Bicarbonates↗
PubMed1991

Simultaneous recording of the cell membrane potential and properties of the cell attached membrane of HT29 colon carcinoma and CF-PAC cells.

Reconciliation of the properties of excised single channels and whole cell conductances is one of the major problems in the interpretation of patch clamp data. To combine cell attached and whole cell recordings we have modified the nystatin technique. Low concentrations of nystatin (less than or equal to 3 * 10(-5) mol/l) were added to the filling solution of the patch pipettes. This permeabilized the cell attached membrane partially and made it possible to measure the potential difference (PD) of the cell in current clamp mode. The input resistance (Rl) of the cell attached patch was only slightly decreased by nystatin and stayed in the GO range, allowing for the simultaneous recording of single channel activity and the input conductance of the cell attached membrane. This technique was examined in HT29 colon carcinoma and CF-PAC cells. In both cells it was shown that this method provides reliable PD measurements. The method was used then to test which type of Cl- channel is activated by carbachol. The PD of HT29 cells was depolarized by carbachol. The depolarization was mainly due to an increase in the Cl- conductance of the cell membrane and was followed by a slight and transient hyperpolarization. No detectable Cl- channels (conductance greater than 4-8 pS, 300 Hz) were activated in the cell attached membrane, but the input conductance (Go) increased concomitantly with cell depolarization. These results suggest that carbachol induces the opening of very small conductance or very rapidly opening and closing Cl- channels in these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbachol↗
PubMed1991

Potassium conductance of smooth muscle cells from rabbit aorta in primary culture.

Vascular smooth muscle cells were obtained from rabbit aorta and were studied in primary culture on days 1-7 after seeding with electrophysiological techniques. In impalement experiments a mean membrane potential difference (PD) of -50 +/- 0.3 mV (n = 387) was obtained with Ringer-type solution in the bath. PD was depolarized by 6 +/- 0.3 mV (n = 45) and 16 +/- 2 mV (n = 5) when the bath K+ concentration was increased from the control value of 3.6 mmol/l to 13.6 and 23.6 mmol/l, respectively. Ba2+ (0.1-1 mmol/l) depolarized PD. Tetraethylammonium (TEA, 10 mmol/l) depolarized PD only slightly but significantly. Verapamil (0.1 mmol/l) and charybdotoxin (10 nmol/l) had no effect on PD. The conductance properties of these cells were further examined with the patch-clamp technique. K+ channels were spontaneously present in cell-attached patches. When the pipette was filled with 145 mmol/l KCl, a mean conductance (gK) of 209.6 +/- 4.6 mV (n = 17) was read from the current/voltage curves at a clamp voltage (Vc) of 0 mV. After excision K+ channels were found in 129 patches with inside-out and in 50 with outside-out configuration. With KCl on one and NaCl on the other side the mean gK at a Vc of 0 mV was 134.6 +/- 3.9 pS (n = 179). The mean permeability was 0.89 +/- 0.03 x 10(-12) cm3/s. With symmetrical KCl solution the mean gK was 227 +/- 6 pS (n = 17). The conductance sequence was gK much greater than gRb = gCs = gNa = 0. TEA blocked dose-dependently only from the outside (1-10 mmol/l). Lidocaine (5 mmol/l) quinidine (0.01-1 mmol/l) and quinine (0.01-1 mmol/l) blocked from both sides. Charybdotoxin (0.5-5 nmol/l) blocked only from the extracellular side. Ba2+ blocked from the cytosolic side and the inhibition was increased by depolarization and reduced by hyperpolarization. At a Vc of 0 mV a half-maximal inhibition (IC50) of 2 mumol/l was obtained. Verapamil and diltiazem blocked from both sides, verapamil with an IC50 of 2 mumol/l and diltiazem with an IC50 of 10 mumol/l. The open probability of this channel was increased by CA2+ on the cytosolic side at activities greater than 0.1 mumol/l. Half-maximal activation occurred at Ca2+ activities exceeding 1 mumol/l. The present data indicate that the vascular smooth muscle cells of rabbit aorta in primary culture possess a K+ conductance. In excised patches only a maxi K+ channel was detected. This channel has properties different from the macroscopic K+ conductance.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Effect of depolarizing and hyperpolarizing agents on the membrane potential difference of primary cultures of rabbit aorta vascular smooth muscle cells.

Vascular smooth muscle cells of rabbit aorta were enzymatically dispersed, kept in primary culture, and studied between days 1 and 7 in a bath rinsed with Ringer-like solution at 37 degrees C. The electrical membrane potential difference (PD) was measured with microelectrodes. The mean value of PD was -50 +/- 0.4 mV (n = 53). Cromakalim (BRL 34915), 1 mumol/l and 10 mumol/l, hyperpolarized the membrane potential by 9 +/- 1 mV (n = 11) and 15 +/- 1 mV (n = 53) respectively. Glibenclamide (10 mumol/l) abolished the hyperpolarizing effect of chromakalim (n = 6). Simultaneous addition of cromakalim and glibenclamide (both 10 mumol/l, n = 11) and glibenclamide itself (10 mumol/l, n = 7) had no effect on PD. In patch-clamp experiments in outside-out-oriented Ca(2+)-sensitive K+ channels, cromakalim increased the open probability (Po) only slightly and only with a cytosolic Ca2+ activity of 1 mumol/l. In all other series cromakalim had no effect on the Po of these channels. Forskolin (10 mumol/l) hyperpolarized PD by 6 +/- 1 mV (n = 13). The nucleotides UTP, ATP and ITP (10 mumol/l) depolarized PD by 12 +/- 1 mV (n = 7), 8 +/- 1 mV (n = 65) and 5 +/- 1 mV (n = 6) respectively. GTP, [alpha, beta-methylene]ATP and adenosine had no significant effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Inhibition of epithelial chloride channels by cytosol.

Chloride channels that have an intermediate conductance and are outwardly rectifying were studied by the patch-clamp technique in cell-excised membrane patches from respiratory epithelial cells in primary culture (REC) of normal and cystic fibrosis tissue, HT29 and T84 human colon carcinoma cells and placenta trophoblast cells (PTC). Chloride channels were immediately activated by the exposure of the cytosolic side of the patch to a Ringer-type solution, which lacked cytosolic components normally inhibiting chloride channels in the "on" cell configuration. Tentatively, we labelled the cytosolic component (or components) responsible for this inhibition cytosolic inhibitor (CI). The presence of CI in cytosol derived from HT29 cells was shown by assaying crude cytosol extracts from these cells on Cl- channels from HT29 cells (n = 2) and REC from normal subjects and cystic fibrosis patients (n = 4). In order to examine CI further, PTC were used as a source of cytosol. The cytosol of PTC inhibited HT29 Cl- channels in a dose-dependent manner with a half-maximal inhibition observed at a 1:6 dilution (n = 11) of the native cytosol. CI from PTC was heat-stable (10 min at 100 degrees C, n = 8). When cytosol extract was partitioned into a chloroform phase, Cl- channel inhibition was shown for the lipophilic extract (n = 12) as well as for the aqueous phase (n = 10). The inhibitory potency of the lipid extract was slightly larger than that of the aqueous phase. Several separation procedures were used to determine the molecular size of CI. When CI was filtered through 30-kDa filters at 6000 rpm for 45 min, inhibitory potency was observed in the filtrate and the retained fraction (n = 3). The same was observed with 10-kDa filters (n = 6). When CI was dialysed through a 12-kDa membrane, inhibitory capacity was recovered from the dialysate. Similarly, gel filtration indicated that CI was less than 5 kDa (n = 13) and probably less than 1.5 kDa (n = 11), but greater than 700 kDa (n = 9). CI was exposed to bead-coupled hydrolysing enzymes (trypsin, non-specific protease, lipase, alpha-amylase, nucleotidase), but none of the enzymes used destroyed the inhibitory potency of CI. These data indicate that CI is present in HT29 as well as in PTC. It inhibits reversibly intermediate-conductance outwardly rectifying Cl- channels in REC, HT29, and PTC.(ABSTRACT TRUNCATED AT 400 WORDS)

Chloride Channels↗
PubMed1989

Properties and regulation of chloride channels in cystic fibrosis and normal airway cells.

The present study examines the properties of Cl- channels in cultured respiratory cells of cystic fibrosis (CF) patients and normal (N) individuals. In excised membrane patches the conductances for CF and N Cl- channels were larger at positive as compared to negative clamp voltages (Vc): 74 +/- 2.6 (Vc greater than 0) and 47 +/- 2.0 pS (Vc less than 0) for CF (n = 57) and 69 +/- 3.6 (Vc greater than 0) and 45 +/- 2.3 pS (Vc less than 0) for N (n = 35). The open probability (Po) of the channel increased markedly with depolarization. Both the voltage dependence of the conductance and of Po contribute to the outward rectification of the channel. The time histogram analysis reveals two open and two closed time constants. The selectivity of the channel was Cl- = Br- = I- greater than NO-3 much greater than gluconate. The channel was inhibited reversibly by 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) at 10(-7) mol/l to 10(-5) mol/l. While Cl- channels were present in cell attached patches of N cells, they were absent in those of CF cells. The mean conductance for cell attached (N) Cl- channels was 76 +/- 3.2 pS for positive clamp voltages (Vc) and 46 +/- 3.9 pS for negative Vc (n = 8). When the membrane patches were excised from CF cells Cl- currents appeared spontaneously (n = 19). The immediate appearance (within 1 s) of Cl- channels after excision was observed at positive (n = 6) as well as at negative clamp voltage (n = 13). "Excision activation" of CF Cl- channels was observed at low (less than 10(-9) mol/l) or high (10(-3) mol/l) calcium activities on the cytosolic side of the excised patch. Variation of the Ca+ activity (less than 10(-9)-10(-3) mol/l) or pH (6.5-8.5) on the cytosolic side exerted no effects on these Cl- channels. These results suggest that Cl- channels are present in the apical membrane of CF and N respiratory cells but they seem to be inhibited in intact CF cells. Excision of the patch and hence removal of the cytosolic "inhibitor" leads to an activation of Cl- channels. The Cl- channels in excised patches of N and CF cells have identical properties.

Calcium↗
PubMed1989

Characterization of potassium channels in respiratory cells. I. General properties.

Potassium channels present in the basolateral membrane of respiratory epithelial cells play an important role in the process of chloride secretion. Utilizing the patch clamp technique, we examined human cultured respiratory epithelial cells derived from patients with cystic fibrosis (CF) and normals individual (N) for the existence of and for the properties of K+ channels. We obtained qualitatively and quantitatively identical results for both preparations (CF and N). K+ channels were spontaneously present in cell attached patches. The channels showed burst appearance with rapid flickering within the bursts. When the pipette was filled with 145 mmol/l KCl, a mean conductance of 131 +/- 25 pS (n = 15) was read from the I/V-curve at a clamp voltage (Vc) of 0 mV. After excision, the conductance read from the I/V-curve at Vc = 0 mV was 212 +/- 11 pS (Pipette: 145 mmol/l KCl, bath: 145 mmol/l NaCl) (n = 61). With NaCl in the pipette and KCl in the bath, a similar conductance was obtained (g = 210 pS; n = 2). When both, pipette and bath contained KCl, the conductance was increased to 302 +/- 19 (n = 7). The channel was highly selective for potassium over sodium: PK + /PNa + greater than 40. The channel open probability was only slightly voltage dependent i.e. the open probability increased slightly with depolarisation. For most of the channels one open time constant (to = 6.3 +/- 1.6 ms; n = 22) and one closed time constant (tc = 1.8 +/- 0.3 ms; n = 21) was obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Membrane↗
PubMed1989

Characterization of potassium channels in respiratory cells. II. Inhibitors and regulation.

In a previous study [26] we described the properties of potassium channels in cultured respiratory cells derived from cystic fibrosis patients (CF) and normal individuals (N). In the present study we examine the regulatory mechanisms of these channels by the patch clamp technique. Since there were no apparent differences in the properties of CF and N K+ channels the results were pooled. In the excised inside/out configuration the channel was blocked by different K+ channel blockers. Barium (5.10(-3) mol/l), tetraethylammoniumchloride (5.10(-3) mol/l), quinidine (10(-3) mol/l) and lidocaine (5.10(-3) mol/l), when added to the cytosolic side, inhibited K+ channels reversibly. An increase in the calcium concentration from 10(-7) mol/l to 10(-6) mol/l led to a marked increase in the open channel probability (Po). Further increases in Ca2+ concentration increased Po only slightly. No pH effects on the cytosolic side of the channel were observed. The channel open probability was reduced when ATP was present on the cytosolic side at a concentration of 10(-4) mol/l to 10(-3) mol/l. Non hydrolysable adenosine 5'-[beta, gamma-methylene] triphosphate had the same inhibitory effect as ATP. The inhibition by ATP was blunted by the simultaneous addition of 1 mmol/l ADP. The inhibition of K+ channels by cytosolic ATP may represent a channel regulatory mechanism in the intact cell. This would allow for coupling between the activity of the (Na+ + K+)-pump and the basolateral K+ conductance.

Adenosine Triphosphate↗