Synergistic activation of non-rectifying small-conductance chloride channels by forskolin and phorbol esters in cell-attached patches of the human colon carcinoma cell line HT-29cl.19A

Cell-attached patch-clamp studies with the human colon carcinoma HT-29cl.19A cells revealed a small chloride channel with a unitary conductance of 6.5 pS at 70 mV and 4.6 pS at –70 mV clamp potential after cAMP was increased by activation of adenylyl cyclase by forskolin. Usually channels inactivated upon patch excision, but in a few excised patches the channels stayed active and displayed a linear I/V relation in symmetrical (150 mmol/l) chloride solutions with a conductance of 7.5 pS. A 16-fold increase in channel incidence was observed when forskolin and phorbol 12,13-dibutyrate (PDB) were present together. The open probability was voltage-independent and was not different in the presence of forskolin plus PDB or with forskolin alone. The conductance sequence of the channel as deduced from outward currents carried by five different anions including chloride was: Cl^–>Br^–>NO₃ ^–>gluconate > I^–. The permeability sequence deduced from the reversal potentials was NO₃ ^–Br^–>Cl^–>I^–>gluconate. With iodide in the pipette the conductance decreased strongly. Moreover, the inward current was reduced by 61%, indicating a strong inhibition of the chloride efflux by iodide. Similarly, the forskolin-induced increase of the short-circuit current (I _sc) in confluent filter-grown monolayers was strongly reduced by iodide in the apical perfusate. Iodide also increased the fractional resistance of the apical membrane and repolarized the membrane potential, indicating an inhibitory action on the forskolin-induced increase of the apical chloride conductance. The PDB-induced I _sc was also reduced by iodide, suggesting that the same chloride conductance is involved in the forskolin and in the PDB response. The results suggest that forskolin via cAMP-dependent protein kinase and PDB via protein kinase C regulate the same non-rectifying small-conductance chloride channels in the HT-29cl.19A cells.     

Characterisation of volume-activated ion transport across epithelial monolayers of human intestinal T84 cells

The effects of hypo-osmolarity upon transepithelial ion transport in human intestinal cell layers have been investigated. Exposure of the basal-lateral surfaces to hypo-osmotic media resulted in a transient stimulation of inward short-circuit current (I _sc). This transient stimulation of inward current by hypo-osmotic media was abolished by 100 mol/l 4,4-diisothiocyanostilbene 2,2-disulphonic acid (DIDS). After prestimulation of inward I _sc by vasoactive intestinal peptide (VIP) or by combinations of carbachol and prostaglandin E₁ hypoosmotic exposure of the basal-lateral surfaces resulted in a further transient stimulation of I _sc. The stimulation of I _sc in these conditions was largely insensitive to DIDS inhibition. Exposure of the basal-lateral surfaces to hypo-osmotic media resulted in a stimulation of loop-diuretic-insensitive ⁸⁶Rb efflux across the basal-lateral surfaces. In addition, hypo-osmotic exposure of T₈₄ cells is also associated with an increase in cytosolic Ca²⁺. It is concluded that the effects of hypo-osmotic exposure of T₈₄ cells on secretory I _sc are consistent with the activation of a DIDS-sensitive apical Cl^– conductance and a basal-lateral K⁺ conductance. With prior activation of inward I _sc by VIP via a cAMP-activated DIDS-insensitive apical Cl^– conductance, augmentation of the secretory current by hypo-osmotic exposure is likely to result primarily from increased basal-lateral K⁺ current and loop-diuretic-sensitive Cl^– uptake.     

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 (P _o) of the ICOR or on the g_o) were observed. The P_o of the ICOR was reduced by 22% in a Ca²⁺-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. Apart from a weak effect of Ca²⁺ on the ICOR, g _o and the ICOR do not seem to be directly controlled by Ca²⁺ or PKA. C. I. of normal and CF epithelia have a similar inhibitory potency on Cl^– channels.     

Chloride secretion induced by phorbol dibutyrate and forskolin in the human colonic carcinoma cell line HT-29Cl.19A is regulated by different mechanisms

The human colonic carcinoma cell line HT29cl.19A responds to the protein kinase C activator PDB (4--phorbol 12,13-dibutyrate), as it does to forskolin (an activator of adenylyl cyclase), with a secretory response when the cells are grown on filters and studied at 36 °C. Previously, we showed that when cells were grown on Petri dishes and studied at about 25 °C with the cell-attached patch-clamp technique, forskolin, but not PDB, could activate 8-pS chloride channels (cystic fibrosis transmembrane conductance regulator, CFTR, channels). The present work was carried out to study this discrepancy. Experiments in Ussing chambers, at different temperatures, showed that the responses to PDB and forskolin differ in their temperature sensitivity. This was also found following conventional microelectrode and Ussing chamber studies with nystatin-permeabilized epithelial layers carried out at 25 °C and at 36 °C. Pre-incubation with the microtubular disruptive agents nocodazole or colcemid did not affect the response to PDB or forskolin, suggesting that chloride secretion induced by these agonists in these cells is independent of the microtubular structure. Pre-incubation with brefeldin A strongly inhibited the response to PDB, but the response to forskolin was hardly affected. The differing effect of temperature and brefeldin A on the responses to forskolin and PDB may be due to the activation of two distinct mechanisms by protein kinases A and C.     

The effect of secretagogues on ion conductances of in vitro perfused,isolated rabbit colonic crypts

Several secretagogues were used in this study, including those which enhance intracellular cyclic adenosine monophosphate (cAMP) production, as well as others which elevate intracellular Ca²⁺ activity and are known to increase Cl^– secretion in the intact colon and in colonic carcinoma cell lines. They were examined with respect to their effects on electrophysiological properties in isolated rabbit distal colonic crypts. Crypts were dissected manually and perfused in vitro. Transepithelial voltage (V _te), transepithelial resistance (R _te), membrane voltage across the basolateral membrane (V _bl), and fractional basolateral membrane resistance (FR _bl), were estimated. Basolateral prostaglandin E₂ (PGE₂, 0.1 mol/l), vasoactive intestinal peptide (VIP, 1 nmol/l) and adenosine (0.1 mmol/l) induced an initial depolarisation and a secondary partial repolarisation of (V _bl). In the case of adenosine, the initial depolarization of (V _bl) was by 31±2 mV (n=47).R _te fell significantly from 16.4±3.6 to 14.2±3.7 ·cm² (n= 6), andFR _blincreased significantly from 0.11±0.02 to 0.51±0.10 (n=6). In the second phase the repolarisation of (V _bl) amounted 11±2 mV (n=47) and a steadystate (V _bl) of –51±2 mV (n=47) was reached.R _te fell further and significantly to a steady-state value of 12.4±3.8 ·cm² (n=6) andFR _bl fell significantly to 0.42±0.13 (n=6). In 30% of the experiments, a transient hyperpolarisation of (V _bl) by 8±2 mV (n=14) was seen during wash out of adenosine. In the presence of adenosine, but not under control conditions, lowering of luminal Cl^– concentration from 120 to 32 mmol/l depolarised (V _bl) significantly by 8±1 mV (n=9). Basolateral ATP and ADP (0.1 mmol/l) led to a short initial depolarisation followed by a sustained and significant hyperpolarisation by 6±2 mV (n=27) and 5±4 mV (n=8), respectively. Carbachol (CCH) hyperpolarised (V _bl) in a concentration-dependent manner. At 100 mol/l (bath) the hyperpolarisation was by 14±2 mV (n=11) andFR _bl fell slightly. Neurotensin (10 nmol/l), isoproterenol (10 mol/l) and uridine 5-triphosphate (UTP, 0.1 mmol/l) had no effect. It is concluded that PGE₂, VIP and adenosine upregulate sequentially a luminal Cl^– conductance and a basolateral K⁺ conductance by increasing intracellular cAMP concentration. Ca²⁺ mobilising hormones such as ATP, ADP, and CCH increase the basolateral K⁺ conductance, while the effect on luminal Cl^– conductance appears to be very limited.     

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 cellattached 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. For ATP at 10–100 mol/l the depolarization was least with I^–: from –41±1.1 to –36±2.4mV, intermediate for Br^– to –25±1.6 mV, and largest for Cl^– to –20±1.8 mV (n=18). ATP increased G from 3.4±0.3 nS to 12.9±2.8 nS (Cl^–), to 12.9±2.8 nS (Br^–) and to 12.9±2.7 (I^–) (n=18). These data indicate that the ATP-induced anion channel has a permeability sequence of I^–>Br^–>Cl^–. The conductance for all three anions was identical. Hypotonic cell swelling by 160 mosmol/l induced a depolarization that was smallest for I^–, from –42±4 to –32±2.1 mV, intermediate for Br^–: –29±1.8mV, and similar for Cl^–: –28±2 mV (n=20). G was increased from 2.8±0.8 nS to 15±2.5nS in the case of Cl^–, to 15±2.5 nS for Br^– and to 16±2.6 nS for I^– (n=20). Therefore, all three pathways are indistinguishable with respect to their anion selectivity. All three pathways are insensitive towards low concentrations of 4-nitro-2-(3-phenylpropylamino)benzoate, but are all blocked by 4,4-diisothiocyanatostilbene-2,2-disulphonic acid, with a half-maximal inhibition around 0.6 mmol/l. Finally, the possible additivity was examined in three permutations. ATP (0.1 mmol/l) alone (n=14) had a slightly but not significantly larger effect on conductance than the combination of ATP and cAMP (1 mmol/l, n=14) and the combination of ATP and hypotonicity (193 mosmol/l, n=13). Similarly, the effects of hypotonicity and cAMP (n=11) were not additive. These data indicate that all three pathways share common properties. Hence, it is suggested that all three pathways converge on the same small Cl^– channel.Supported by DFG Gr 480/10 and BMFT 01 GA 8816     

Effect of bradykinin and histamine on the membrane voltage,ion conductances and ion channels of human glomerular epithelial cells (hGEC) in culture

The effects of bradykinin (BK) and histamine (Hist) on the membrane voltage (V _m), ion conductances and ion channels of cultured human glomerular epithelial cells (hGEC) were examined with the nystatin patch clamp technique. Cells were studied between passage 3 and 20 in a bath rinsed with Ringer-like solution at 37°C. The mean value of V _m was –41±0.5 mV (n=189). BK (10^–6 mol/l, n=29) and Hist (10^–5 mol/l, n= 55) induced a rapid transient hyperpolarization by 15±1 mV and 18±1 mV, respectively. The hyperpolarization was followed by a long lasting depolarization by 6±1 mV (BK 10^–6 mol/l) and 7±1 mV (Hist 10^–5 mol/l). The ED₅₀ was about 5×10^–8 mol/l for BK and 5×10^–7 mol/l for Hist. In the presence of both agonists, increases of outward and inward currents were observed. A change in the extracellular K⁺ concentration from 3.6 to 30 mmol/l depolarized V _m by 8±1 mV and completely inhibited the hyperpolarizing effect of both agents (n=11). Reduction of extracellular Cl^– concentration from 145 to 30 mmol/l led to a depolarization by 2 ±1 mV (n=25). In 30 mmol/l Cl^– the depolarizations induced by BK (10^–7 mol/l) and Hist (10^–6 mol/l) were augmented to 9±2 mV (n=14) and to 10±2 mV (n=11), respectively. Ba²⁺ (5 mmol/l) depolarized V _m by 19±5 mV (n=6) and completely inhibited the hyperpolarization induced by BK (10^–6 mol/l, n=3) and reduced that of Hist (10^–5 mol/l) markedly (n=3). Preincubation with the K⁺ channel blocker charybdotoxin (1–10 nmol/l) for 3 min had no significant effect on V _m, but reduced markedly the BK(10^–6 mol/l, n=11) and Hist-(10^–5 mol/l, n=6) induced hyperpolarizations. In 10 out of 31 experiments in the cell attached nystatin patch configuration big K⁺ channels with a conductance of 247±17 pS were found. The open probability of these K⁺ channels was increased 3- to 5-fold during the hyperpolarization induced by BK (10^–7 mol/l) or Hist (10^–5 mol/l, both n= 4). In excised inside/out patches this K⁺ channel had a mean conductance of 136±8.5 pS (n=10, clamp voltage 0 mV). The channel was outwardly rectifying and its open probability was increased when Ca²⁺ on the cytosolic side was greater than 0.1 mol/l. The data indicate that BK and Hist activate a and a in hGEC. The hyperpolarization is induced by the activation of a Ca²⁺-dependent maxi K⁺ channel.     

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±1.2nS, 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. Exposure to hypotonic bath solution (160 mosmol/l) depolarized V in both types of cells. The increase in G was by 15±2.2 nS in FORHT (n=18) and by 11±1.3 nS in PMAHT (n=23). After being returned to the normotonic media, the decline of G to the control value was delayed in FORHT when compared to PMAHT. Ionomycin (10^–7 mol/l) increased G significantly more (to 47±8.5 nS, n=13) in FORHT when compared to PMAHT (to 28±4 nS, n=13). The present data indicate that a 20-h exposure of HT₂₉ cells to FOR versus PMA alters markedly the CFTR concentration. The cells with high CFTR (FORHT) when compared to those with low CFTR (PMAHT) differ not only in their acute G response to cAMP, but also in that to ATP, NT, hypotonic cell swelling, and ionomycin. In contrast, the same pretreatment of CFPAC-1 cells did not alter the G changes induced by ionomycin or hypotonic cell swelling. These results indicate that changes in CFTR expression correlate with the Cl^– conductances induced by cAMP, Ca²⁺ and hypotonic cell swelling.     

Differential effects of ADH on sodium,chloride, potassium,calcium and magnesium transport in cortical and medullary thick ascending limbs of mouse nephron

The effect of antidiuretic hormone (arginine vasopressin, AVP) on transepithelial Na⁺, Cl^–, K⁺, Ca²⁺ and Mg²⁺ net transports was investigated in medullary (mTAL) and cortical (cTAL) segments of the thick ascending limb (TAL) of mouse nephron, perfused in vitro. Transepithelial net fluxes (J _Na ⁺,J _Cl ^–,J _K ⁺,J _Ca ²⁺,J _Mg ²⁺) were determined by electron probe analysis of the collected tubular fluid. Transepithelial potential difference (PD_te) and transepithelial resistance (R_te) were measured simultaneously. cTAL segments were bathed and perfused with isoosmolal, HCO ₃ ^– containing Ringer solutions, mTAL segments were bathed and perfused with isoosmolal HCO ₃ ^– free Ringer solutions. In cTAL segments, AVP (10^–10 mol·l^–1) significantly increasedJ _Mg ²⁺ andJ _Ca ²⁺ from 0.39±0.08 to 0.58±0.10 and from 0.86±0.13 to 1.19±0.15 pmol·min^–1 mm^–1 respectively. NeitherJ _Na ⁺ norJ _Cl ^–, (J _Na ⁺: 213±30 versus 221±28 pmol·min^–1 mm^–1,J _Cl ^–: 206±30 versus 220±23 pmol·min^–1 mm^–1) nor PD_te (13.4±1.3 mV versus 14.1±1.9 mV) or R_te (24.6±6.5 cm² versus 22.6±6.4 cm²) were significantly changed by AVP. No significant effect of AVP on net K⁺ transport was observed. In mTAL segments, Mg²⁺ and Ca²⁺ net transports were close to zero and AVP (10^–10 mol·l^–1) elicited no effect. However NaCl net reabsorption was significantly stimulated by the hormone,J _Na ⁺ increased from 107±33 to 148±30 andJ _Cl ^– from 121±33 to 165±32 pmol·min^–1 mm^–1. The rise inJ _NaCl was accompanied by an increase in PD_te from 9.0±0.7 to 13.5±0.9 mV and a decrease in R_te from 14.4±2.0 to 11.2±1.7 cm². No K⁺ net transport was detected, either under control conditions or in the presence of AVP.To test for a possible effect of HCO ₃ ^– on transepithelial ion fluxes, mTAL segments were bathed and perfused with HCO ₃ ^– containing Ringer solutions. With the exception ofJ _Ca ²⁺ which was significantly different from zero (J _Ca ²⁺: 0.26±0.06 pmol·min^–1 mm^–1), net transepithelial fluxes of Na⁺, Cl^–, K⁺ and Mg²⁺ were unaffected by HCO ₃ ^– . In the presence of AVP,J _Mg ²⁺ andJ _Ca ²⁺ were unaltered whereasJ _NaCl was stimulated to the same extent as observed in the absence of HCO ₃ ^– . In conclusion our results indicate heterogeneity of response to AVP in cortical and medullary segments of the TAL segment, since AVP stimulates Ca²⁺ and Mg²⁺ reabsorption in the cortical part and Na⁺ and Cl^– reabsorption in the medullary part of this nephron segment.This study was supported by the Commission des communautés européennes, grant no. ST2J 00951 F(CD), and by Wissenschafts-ausschuß der Nato über den DAAD