4-acetamido-4--isothiocyanatostilbene-2-2--disulfonic-acid and 3--5-dichlorodiphenylamine-2-carboxylic-acid

Eel intestinal epithelium when bathed symmetrically with normal Ringer solution develops a net Cl(-) current (short circuit current, Isc) giving rise to a negative transepithelial potential (Vt) at the basolateral side of the epithelium, lower in fresh-water (FW)-acclimated animals with respect to sea-water (SW). The aim of the present work was to study the cell response to hypertonic stress of FW eel intestinal epithelium in relation to Cl(-) absorption. The hypertonicity of the external bathing solutions produced first a transient increase of Vt and Isc, then followed (after 10-15 min) by a gradual and sustained increase which reached the maximum value after 40-60 min. The morphometric analysis of the intestine revealed the shrinkage of the cells after 5 min hypertonicity exposure, and then a regulatory volume increase (RVI) response, which parallels the gradual and sustained increase in the electrophysiological parameters. This last phase is inhibited by drugs known to block Cl(-) absorption in eel intestine, such as luminal bumetanide (10 microM), specific inhibitor of Na(+)-K(+)-2Cl(-) cotransport, or basolateral NPPB (0.5 mM), dichloro-DPC (0.5 mM), inhibitors of basolateral Cl(-) conductance. Serosal dimethyl-amiloride (100 microM), specific inhibitor of the Na(+)/H(+) antiport, was ineffective on the hyperosmotic response. Bicarbonate revealed a crucial role as a modulator of hypertonicity response, since in bicarbonate-free conditions or in the presence of serosal 0.25 mM SITS, blocker of HCO(3)(-) transport systems, the Isc response to hypertonicity was lost. In nominally Ca(2+)-free conditions the Isc response to hypertonicity was abolished. The same results were obtained by bilateral addition of 100 microM verapamil or 50 microM nifedipine or 1 mM lanthanum, known Ca(2+) channel blockers, indicating that extracellular Ca(2+) plays a key role for the activation of Cl(-) current in the response to hypertonic stress. The data show that in the eel intestinal epithelium the hypertonicity of the external medium affects cell volume which in turn might represent the signal to increase the rate of Cl(-) transport. This response is sustained by the activation of the luminal Na(+)-K(+)-2Cl(-) cotransporter and the functionality of basolateral Cl(-) channels.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Adaptation, Physiological; Amiloride; Anguilla; Animals; Bumetanide; Calcium Channel Blockers; Carrier Proteins; Cell Polarity; Cell Size; Chlorides; Diphenylamine; Hydrogen-Ion Concentration; In Vitro Techniques; Intestinal Mucosa; Mannitol; Membrane Potentials; Nitrobenzoates; Osmolar Concentration; Potassium; Sodium; Sodium-Potassium-Chloride Symporters; Water; Water-Electrolyte Balance

The K+ secretory epithelium of the vestibular labyrinth (dark cells) was impaled with glass microelectrodes in order to test the hypothesis that it contains a large Cl- conductance. In the first series of experiments, the short-circuited epithelium was perfused on both sides by a solution containing 150 mmol/l Cl-. The membrane voltage (PD) was -18 +/- 1 mV (N = 101), showed a Gaussian distribution, and the estimated input resistance of the cell (R 'cell') was 17 +/- 3 M omega. The PD responded to 10(-4) mol/l ouabain with a depolarization, suggesting the presence of a (Na(+) + K+)-ATPase. The PD responses to Cl- steps yielded an apparent transference number tCl = 0.34 +/- 0.03 (N = 65) and those to K+ steps yielded a tK = 0.16 +/- 0.01 (N = 48). In the second series of experiments, cells presumed to be Cl(-)-depleted were impaled in Cl(-)-free solutions. The distribution of the PD was not Gaussian; PDs as negative as -90 mV were observed. Cells with a highly negative PD also had a high R 'cell'. With the addition of Cl- the PD collapsed to -19 +/- 1 mV and R collapsed to 16 +/- 3 M omega (N = 145) which are not significantly different from values obtained in the first series of experiments when cells were impaled in a solution containing 150 mmol/l Cl-. Alternating the bath perfusate between Cl(-)-free and Cl(-)-containing solutions led to large PD transients.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Animals; Chloride Channels; Chlorides; Diphenylamine; Diuretics; Electrodes, Implanted; Epithelium; Gerbillinae; Membrane Potentials; Membrane Proteins; Nitrobenzoates; Ouabain; Potassium; Sodium-Potassium-Exchanging ATPase; Sulfonamides; Vestibule, Labyrinth

Nonselective Ca2+-sensitive cation channels in the basolateral membrane of isolated cells of the rat exocrine pancreas were investigated with the patch clamp technique. With 1.3 mmol/l Ca2+ on the cytosolic side, the mean open-state probability Po of one channel was about 0.5. In inside-out oriented cell-excised membrane patches the substances diphenylamine-2-carboxylic acid (DPC), 5-nitro-2-(3-phenelpropylamino)-benzoic acid (NPPB) and 3',5-dichlorodiphenylamine-2-carboxylic acid (DCDPC) were applied to the cytosolic side. These compounds inhibited the nonselective cation channels by increasing the mean channel closed time (slow block). 100 mumol/l of NPPB or DPC decreased Po from 0.5 (control conditions) to 0.2 and 0.04, respectively, whereas 100 mumol/l of DCDPC blocked the channel completely. All effects were reversible. 1 mmol/l quinine also reduced Po, but in contrast to the above mentioned substances, it induced fast flickering. Ba2+ (70 mmol/l) and tetraethylammonium (TEA+; 20 mmol/l) had no effects. We investigated also the stilbene disulfonates 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and 4,4'-dinitro-2,2'-stilbenedisulfonate (DNDS). 10 mumol/l SITS applied to the cytosolic side increased Po from 0.5 to 0.7 and with 100 mumol/l SITS the channels remained nearly permanently in its open state (Po approximately equal to 1). A similar activation of the channels was also observed with DIDS and DNDS. These effects were poorly reversible. The stilbene disulfonates acted by increasing the channel mean open time. When the channel was inactivated by decreasing bath Ca2+ concentration to 0.1 mumol/l, addition of 100 mumol/l of SITS had no effect. Similarly, reducing bath Ca2+ concentration from 1.3 mmol/l in presence of 100 mumol/l SITS (channels are maximally activated) to 0.1 mumol/l, inactivated the channels completely. These results demonstrate, that SITS can only activate the channels in the presence of Ca2+. SITS had no effects, when applied to the extracellular side in out-side out patches.(ABSTRACT TRUNCATED AT 400 WORDS)

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Aniline Compounds; Animals; Calcium Channels; Cell Membrane; Cytosol; Diphenylamine; Nitrobenzoates; ortho-Aminobenzoates; Pancreas; Quinine; Rats; Stilbenes

Sheets of utricular epithelium from gerbil were mounted in a micro-Ussing chamber in order to identify and localize chloride conductances. The [Cl-] was rapidly reduced (substituted with isethionate) in the apical or basolateral perfusate and the transepithelial potential difference (Vt) and transepithelial resistance (Rt) were monitored continuously. In addition, agents known to inhibit anion transport in other epithelia were applied. The direction of all initial changes in Vt and Rt due to Cl- substitutions were consistent with the presence of ionic conductances for Cl- on both sides of the epithelium. The time-courses and magnitudes of the fall in Vt and increase in Rt during apical [Cl-] steps in the presence and absence of basolateral bumetanide were monophasic and identical in the two cases. The response of Vt to basolateral [Cl-] steps was biphasic and the initial response was greatly attenuated by bumetanide. These findings demonstrate that the largest conductance for Cl- is in the basolateral cell membrane, but that the paracellular and/or apical pathway also possess a finite Cl- conductance. All three agents tested, 3',5-dichlorodiphenylamine-2-carboxylic acid (DCDPC), 5-nitro-2(3-phenylpropylamino)benzoic acid (NPPB) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), caused an increase in Vt. NPPB and DIDS were more effective from the apical side. DCDPC and DIDS administered from the apical side led to a decrease in Rt. These results suggest that these agents act in this tissue by enhancing a conductive pathway on the apical membrane rather than blocking the basolateral Cl- conductance.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Bumetanide; Chloride Channels; Chlorides; Diphenylamine; Electric Conductivity; Epithelium; Gerbillinae; Kinetics; Membrane Potentials; Membrane Proteins; Nitrobenzoates; Saccule and Utricle