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The intact human reabsorptive sweat duct (RD) has been a reliable model for investigations of the functional role of "endogenous" CFTR (cystic fibrosis transmembrane conductance regulator) in normal and abnormal electrolyte absorptive function. But to overcome the limitations imposed by the use of fresh, intact tissue, we transformed cultured RD cells using the chimeric virus Ad5/SV40 1613 ori-. The resultant cell line, RD2(NL), has remained differentiated forming a polarized epithelium that expressed two fundamental components of absorption, a cAMP activated Cl- conductance (GCl) and an amiloride-sensitive Na+ conductance (GNa). In the unstimulated state, there was a low level of transport activity; however, addition of forskolin (10(-5) M) significantly increased the Cl- diffusion potential (Vt) generated by a luminally directed Cl- gradient from -15.3 +/- 0.7 mV to -23.9 +/- 1.1 mV, n = 39; and decreased the transepithelial resistance (Rt) from 814.8 +/- 56.3 omega.cm2 to 750.5 +/- 47.5 omega.cm2, n = 39, (n = number of cultures). cAMP activation, anion selectivity (Cl- > I- > gluconate), and a dependence upon metabolic energy (metabolic poisoning inhibited GCl), all indicate that the GCl expressed in RD2(NL) is in fact CFTR-GCl. The presence of an apical amiloride-sensitive GNa was shown by the amiloride (10(-5) M) inhibition of GNa as indicated by a reduction of Vt and equivalent short circuit current by 78.0 +/- 3.1% and 77.9 +/- 2.6%, respectively, and an increase in Rt by 7.2 +/- 0.8%, n = 36. In conclusion, the RD2(NL) cell line presents the first model system in which CFTR-GCl is expressed in a purely absorptive tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

The remarkably high level of cystic fibrosis mutations among Caucasians may be due to a reduction in heterozygotes in the severity of the diarrhea caused by enterotoxic bacteria.

Even though the same Cl channel (CFTR) is common to certain fluid transport functions that are oppositely directed, i.e., secretion and absorption, only fluid secretion has clearly been shown to be acutely regulated. It is now clear that fluid secretion activated by beta-adrenergic stimulation is controlled by cAMP-mediated opening and closing of CFTR-Cl channels. Since the conductance of the human sweat duct is almost wholly due to CFTR-Cl conductance (CFTR-GCl), we sought to determine whether salt absorption via CFTR-Cl channels could also be subject to acute regulation in this purely absorptive epithelium. After alpha-toxin permeabilization, we found that addition of cAMP resulted in a large increase in Cl diffusion potentials across the apical membrane and a more than twofold increase in the average membrane conductance. Since the cAMP effects were dependent on Cl alone, not on Na, and since apical Cl conductance appears to be almost exclusively comprised of CFTR-GCl, we surmise that this form of electrolyte absorption like secretion is also subject to acute control through CFTR-GCl. Acute regulation of absorption involves both activation by phosphorylation (PKA) and inactivation by dephosphorylation (unknown endogenous phosphatase) of CFTR. Phosphorylation of CFTR was shown by the facts that CFTR-GCl could be activated by cAMP and inhibited by the kinase antagonist staurosporine, or by removal of either substrate ATP or Mg2+ cofactor. Inactivation of CFTR-GCl by endogenous phosphatase(s) was indicated by a spontaneous but reversible loss of CFTR-GCl upon removal of cAMP. Such loss of CFTR-GCl activity could be prevented either by application of phosphatase inhibitors or by using phosphatase-resistant ATP-gamma-S as substrate to phosphorylate CFTR. We surmise that absorptive function is subject to rapid regulation which can be switched "on" and "off" acutely by a control system that is common to both absorptive and secretory processes and that this control is crucial to switching between conductive and nonconductive transport mechanisms during salt absorption.

In contrast to fluid secretion which in many tissues is well known to be regulated by phosphorylation, little is known of phosphorylation regulation of electrolyte absorption. It is now well established that CFTR (cystic fibrosis transmembrane regulator) is a Cl- channel which is activated by phosphorylation. The human eccrine sweat duct is an absorptive epithelium which expresses relatively large amounts of CFTR. We find that the Cl- conductance (GCl) expressed in the isolated, microperfused sweat duct is also dependent upon phosphorylation activation which can be mediated via cAMP. The activation is Mg2+ dependent, Ca2+ independent, and inhibited by the kinase inhibitor staurosporine. Deactivation appears to occur via endogenous phosphatases that can be inhibited by okadaic acid. We surmise that phosphorylation regulation of CFTR GCl is an important mechanism for shifting Cl- absorption efficiently between electroconductive and carrier mediated transport.

Recent studies suggested dual regulation of the Cl- conductance (GCl) affected in cystic fibrosis, one by protein kinase A-dependent phosphorylation and a second by low-affinity ATP binding. We proposed that ATP binding may couple the transport demands to the energy level of the cell. In the present study we examined this hypothesis further in a purely secretory function using the epithelial cell line T84. We used a depletion-permeabilization protocol on cells grown on permeable supports to deplete the cells of endogenous ATP and to provide access to the intracellular compartment for the impermeable nucleotides adenosine 3',5'-cyclic monophosphate (cAMP) and ATP. In contrast to non-depleted permeabilized cells, which responded to 0.1 mM cAMP with an increase in transepithelial potential (delta Vt = 29.8 +/- 3.0 mV, n = 4) and conductance (delta Gt = 1.23 +/- 0.54 mS/cm2, n = 4), addition of cAMP to ATP-depleted cells resulted in insignificant changes in Vt (delta Vt = 0.7 +/- 0.2 mV, n = 26; P < 0.05) and Gt (delta Gt = 0.020 +/- 0.003 mS/cm2, n = 26; P < 0.05). However, the cAMP response was restored by addition of 5 mM ATP (delta Vt = 21.7 +/- 1.5 mV, n = 26; delta Gt = 0.59 +/- 0.06 mS/cm2, n = 26). ATP dose-response experiments, taken together with the effect of cAMP with and without ATP, suggest that phosphorylation is necessary, but not sufficient, for activation. The data provide evidence for a second level of regulation of GCl, which requires high concentrations of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)

Site-specific mutation and membrane reconstitution experiments provide compelling evidence that the product of the gene which is at fault in the disease cystic fibrosis, termed the cystic fibrosis transmembrane conductance regulator (CFTR), is a small-conductance chloride channel activated by phosphorylation. As transport of chloride ions is passive, the predicted presence of two nucleotide-binding domains in CFTR seems as puzzling as a report that ATP hydrolysis is essential to activate the channel. We now find that in the sweat duct, which expresses high levels of CFTR and has a very high Cl- conductance, intracellular concentrations of ATP must be about normal (5 mM) for activation of this conductance, apparently by a non-hydrolytic, perhaps allosteric, mechanism. This passive dependence on ATP should mean that even a modest depletion of cell energy levels will significantly lower the energy demands of electrolyte transport by decreasing chloride conductance. We believe this direct coupling between cellular ATP levels and chloride channel activity is an adaptive mechanism to protect the tissue from damage resulting from excessive energy depletion.

The T84 cell line possesses an adenosine 3',5'-cyclic monophosphate (cAMP)-activated Cl- conductance and expresses high levels of the cystic fibrosis (CF) gene product, implicating it as a good model for CF research. To evaluate whether T84 Cl- conductance properties are consistent with those described in CF target epithelial, we used transepithelial measurements (verified by selective permeabilization of the basal membrane) to determine the apparent anion selectivity properties of the apical and basolateral membranes of stimulated and unstimulated T84 cells. Unstimulated epithelial cells were almost electrically inert, having a low transepithelial voltage (Vt; -6 mV, apical surface negative), a small equivalent short-circuit current (Isc,(eq.) 2.2 microA/cm2), a very high transepithelial resistance (Rt; 2,500 omega.cm2), and poor anion permselectivity properties at both membrane surfaces (0.8 less than PX/PCl- less than 1.1), where X is NO3-, Br-, I-, or gluconate. When stimulated with forskolin (10(-6) M), Vt increased 8-fold, Isc(eq) increased 30-fold, Rt fell to one-third of unstimulated values, and the apical surface became highly anion selective, i.e., NO3- (1.4) greater than Br- (1.2) greater than Cl- (1.0) greater than I- (0.7) greater than gluconate (0.0), where numbers in parentheses are PX/PCl-. I- was less permeable than Cl- and probably directly inhibits the anion conductance, since Rt was substantially greater after I- substitution than after substitution with the impermeable anion gluconate. Bumetanide (10(-4) M) significantly attenuated the response of Vt to anion substitutions at the basal membrane surface, indicating that the effects of substitution were predominantly on the Na(+)-K(+)-2Cl- cotransporter.(ABSTRACT TRUNCATED AT 250 WORDS)

We studied the Cl- permeability properties of apical and basolateral membranes of human reabsorptive sweat duct (RSD) from normal and cystic fibrosis (CF) subjects. In normal ducts, Cl- substitution by impermeant anion gluconate in the lumen increased the voltage divider ratio (VDR) from 4.8 +/- 0.9 to 7.0 +/- 1.1 (n = 8, P less than 0.05), whereas Cl- substitution in the contraluminal bath decreased the VDR from 3.2 +/- 0.7 to 1.9 +/- 0.4 (n = 7, P less than 0.05). These results are consistent with a significant Cl- permeability in both apical and basolateral membranes of normal ducts. Amiloride (10(-4) M) in the lumen of normal ducts resulted in a small increase in VDR from 4.2 +/- 0.6 to 5.0 +/- 0.8 (n = 10, P less than 0.05), whereas the current-induced basolateral membrane voltage deflections (delta Vb) increased from 6.9 +/- 1.3 to 7.7 +/- 1.2 mV, suggesting that inhibition of Na+ permeability decreased basolateral membrane Cl- permeability. In the absence of luminal Cl-, amiloride decreased delta Vb and induced much greater effect on VDR (from 5.2 +/- 1.1 to 10.8 +/- 2.3, n = 9, P less than 0.05) than in the presence of Cl-. Likewise, in the presence of amiloride, Cl- substitution in the lumen had greater effect on VDR (increased from 3.5 +/- 0.5 0.5 to 10.0 +/- 1.5, n = 15, P less than 0.05) than in the absence of amiloride. These results indicate that Na+ conductance in the apical membrane of the normal duct is significantly smaller than Cl- conductance.(ABSTRACT TRUNCATED AT 250 WORDS)