Postimpact earliest Paleogene warming shown by fish debris oxygen isotopes (El Kef, Tunisia).

Greenhouse warming is a predicted consequence of the Chicxulub impact, but supporting data are sparse. This shortcoming compromises understanding of the impact's effects, and it has persisted due to an absence of sections that both contain suitable material for traditional carbonate- or organic-based paleothermometry and are complete and expanded enough to resolve changes on short time scales. We address the problem by analyzing the oxygen isotopic composition of fish debris, phosphatic microfossils that are relatively resistant to diagenetic alteration, from the Global Stratotype Section and Point for the Cretaceous/Paleogene boundary at El Kef, Tunisia. We report an ~1 per mil decrease in oxygen isotopic values (~5°C warming) beginning at the boundary and spanning ~300 centimeters of section (~100,000 years). The pattern found matches expectations for impact-initiated greenhouse warming.

Surface fluid absorption and secretion in small airways.

Native small airways must remain wet enough to be pliable and support ciliary clearance, but dry enough to remain patent for gas flow. The airway epithelial lining must both absorb and secrete ions to maintain a critical level of fluid on its surface. Despite frequent involvement in lung diseases, the minuscule size has limited studies of peripheral airways. To meet this challenge, we used a capillary to construct an Ussing chamber (area <1 mm(2)) to measure electrolyte transport across small native airways (∼1 mm ø) from pig lung. Transepithelial potentials (V(t)) were recorded in open circuit conditions while applying constant current pulses across the luminal surface of dissected airways to calculate transepithelial electrical conductance (G(t)) and equivalent short circuit current (I(eq)(sc)) in the presence and absence of selected Na(+) and Cl(-) transport inhibitors (amiloride, GlyH-101, Niflumic acid) and agonists (Forskolin + IBMX, UTP). Considered together the responses suggest an organ composed of both secreting and absorbing epithelia that constitutively and concurrently transport fluids into and out of the airway, i.e. in opposite directions. Since the epithelial lining of small airways is arranged in long, accordion-like rows of pleats and folds that run axially down the lumen, we surmise that cells within the pleats are mainly secretory while the cells of the folds are principally absorptive. This structural arrangement could provide local fluid transport from within the pleats toward the luminal folds that may autonomously regulate the local surface fluid volume for homeostasis while permitting acute responses to maintain clearance.

PKA mediates constitutive activation of CFTR in human sweat duct.

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels are constitutively activated in sweat ducts. Since phosphorylation-dependent and -independent mechanisms can activate CFTR, we sought to determine the actual mechanism responsible for constitutive activation of these channels in vivo. We show that the constitutively activated CFTR Cl(-) conductance (gCFTR) in the apical membrane is completely deactivated following alpha-toxin permeabilization of the basolateral membrane. We investigated whether such inhibition of gCFTR following permeabilization is due to the loss of cytoplasmic glutamate or due to dephosphorylation of CFTR by an endogenous phosphatase in the absence of kinase activity (due to the loss of kinase agonist cAMP, cGMP or GTP through alpha-toxin pores). In order to distinguish between these two possibilities, we examined the effect of inhibiting the endogenous phosphatase activity with okadaic acid (10(-8) M) on the permeabilization-induced deactivation of gCFTR. We show that okadaic acid (1) inhibits an endogenous phosphatase responsible for dephosphorylating cAMP but not cGMP or G protein-activated CFTR and (2) prevents deactivation of CFTR following permeabilization of the basolateral membrane. These results indicate that distinctly different phosphatases may be responsible for dephosphorylating different kinase-specific sites on CFTR. We conclude that the phosphorylation by PKA alone appears to be primarily responsible for constitutive activation of gCFTR in vivo.

Sweat gland bioelectrics differ in cystic fibrosis: a new concept for potential diagnosis and assessment of CFTR function in cystic fibrosis.

BACKGROUND: For nearly 50 years the diagnosis of cystic fibrosis (CF) has depended on measurements of sweat chloride concentration. While the validity of this test is universally accepted, increasing diagnostic challenges and the search for adequate biomarker assays to support curative-orientated clinical drug trials have created a new demand for accurate, reliable and more practical CF tests. A novel concept is proposed that may provide a more efficient real-time method for assessing CFTR function in vivo. METHODS: Cholinergic and beta-adrenergic agonists were iontophoresed to stimulate sweating. The bioelectric potential from stimulated sweat glands (SPD) was measured in vivo using a standard ECG electrode applied to the skin surface. SPD and sweat chloride concentrations were compared in cohorts predicted to express a range of CFTR function as presented by healthy controls (HC), heterozygotes (Hz), pancreatic sufficient (CFPS) and pancreatic insufficient patients with CF (CFPI). RESULTS: The median SPD was hyperpolarized in patients with CF compared with control subjects (-47.4 mV vs -14.5 mV, p<0.001). In distinguishing between control and CF subjects, SPD (area under receiver operator curve (AUC) = 0.997) was similar to sweat chloride concentration (AUC = 0.986). Sequential cholinergic/beta-adrenergic sweat stimulation dramatically depolarised the SPD in patients with CF (p<0.001) but had no effect in control subjects (p = 0.6) or on the sweat chloride concentration in either group (p>0.5). Furthermore, the positive SPD response was larger in CFPI than in CFPS subjects (p = 0.04). CONCLUSION: These results support the concept that skin surface voltages arising from stimulated sweat glands can be exploited to assess expressed CFTR function in vivo and may prove to be a useful diagnostic tool.

Effect of cytosolic pH on epithelial Na+ channel in normal and cystic fibrosis sweat ducts.

The activities of cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel and the amiloride-sensitive epithelial Na(+) channel (ENaC) are acutely coordinated in the sweat duct. However, the mechanisms responsible for cross-talk between these ion channels are unknown. Previous studies indicated that luminal pH of sweat ducts varies over 3 pH units and that the cytoplasmic pH affects both CFTR and ENaC. Therefore, using basolaterally alpha-toxin-permeabilized apical membrane preparations of sweat ducts as an experimental system, we tested the hypothesis that the cytosolic pH may mediate the cross-talk between CFTR and ENaC. We showed that while luminal pH had no effect, cytosolic pH acutely affected ENaC activity. That is, acidic pH inhibited, while basic pH activated, ENaC. pH regulation of ENaC appears to be independent of CFTR or endogenous kinase activities because basic pH independently stimulated ENaC (1) in normal ducts even when CFTR was deactivated, (2) in CF ducts that lack CFTR in the plasma membranes and (3) after blocking endogenous kinase activity with staurosporine. Considering the evidence of Na(+)/H(+) exchange (NHE) activity as shown by the expression of mRNA and function of NHE in the basolateral membrane of the sweat duct, we postulate that changes in cytosolic Na(+) ([Na(+)]( i )) may alter cytosolic pH (pH( i )) as salt loads into the cell during electrolyte absorption. These changes may play a role in coordinating the activities of ENaC and CFTR during transepithelial salt transport.

Iontophoretic beta-adrenergic stimulation of human sweat glands: possible assay for cystic fibrosis transmembrane conductance regulator activity in vivo.

With the advent of numerous candidate drugs for therapy in cystic fibrosis (CF), there is an urgent need for easily interpretable assays for testing their therapeutic value. Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) abolished beta-adrenergic but not cholinergic sweating in CF. Therefore, the beta-adrenergic response of the sweat gland may serve both as an in vivo diagnostic tool for CF and as a quantitative assay for testing the efficacy of new drugs designed to restore CFTR function in CF. Hence, with the objective of defining optimal conditions for stimulating beta-adrenergic sweating, we have investigated the components and pharmacology of sweat secretion using cell cultures and intact sweat glands. We studied the electrical responses and ionic mechanisms involved in beta-adrenergic and cholinergic sweating. We also tested the efficacy of different beta-adrenergic agonists. Our results indicated that in normal subjects the cholinergic secretory response is mediated by activation of Ca(2+)-dependent Cl(-) conductance as well as K(+) conductances. In contrast, the beta-adrenergic secretory response is mediated exclusively by activation of a cAMP-dependent CFTR Cl(-) conductance without a concurrent activation of a K(+) conductance. Thus, the electrochemical driving forces generated by beta-adrenergic agonists are significantly smaller compared with those generated by cholinergic agonists, which in turn reflects in smaller beta-adrenergic secretory responses compared with cholinergic secretory responses. Furthermore, the beta-adrenergic agonists, isoproprenaline and salbutamol, induced sweat secretion only when applied in combination with an adenylyl cyclase activator (forskolin) or a phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine, aminophylline or theophylline). We surmise that to obtain consistent beta-adrenergic sweat responses, levels of intracellular cAMP above that achievable with a beta-adrenergic agonist alone are essential. beta-Adrenergic secretion can be stimulated in vivo by concurrent iontophoresis of these drugs in normal, but not in CF, subjects.

Cytosolic potassium controls CFTR deactivation in human sweat duct.

Absorptive epithelial cells must admit large quantities of salt (NaCl) during the transport process. How these cells avoid swelling to protect functional integrity in the face of massive salt influx is a fundamental, unresolved problem. A special preparation of the human sweat duct provides critical insights into this crucial issue. We now show that negative feedback control of apical salt influx by regulating the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel activity is key to this protection. As part of this control process, we report a new physiological role of K(+) in intracellular signaling and provide the first direct evidence of acute in vivo regulation of CFTR dephosphorylation activity. We show that cytosolic K(+) concentration ([K(+)](c)) declines as a function of increasing cellular NaCl content at the onset of absorptive activity. Declining [K(+)](c) cause parallel deactivation of CFTR by dephosphorylation, thereby limiting apical influx of Cl(-) (and its co-ion Na(+)) until [K(+)](c) is stabilized. We surmise that [K(+)](c) stabilizes when Na(+) influx decreases to a level equal to its efflux through the basolateral Na(+)-K(+) pump thereby preventing disruptive changes in cell volume.

Gene delivery to human sweat glands: a model for cystic fibrosis gene therapy.

Gene therapy vectors are mostly studied in cultured cells, rodents, and sometimes in non-human primates, but it is useful to test them in human tissue prior to clinical trials. In this study, we investigated the possibility of using human sweat glands as a model for testing cystic fibrosis (CF) gene therapy vectors. Human sweat glands are relatively easy to obtain from skin biopsy, and can be tested for CFTR function. Using patients' sweat glands could provide a safe model to study the efficacy of CF gene therapy. As the first step to explore using sweat glands as a model for CF gene therapy, we examined various ex vivo gene delivery methods for a helper-dependent adenovirus (HD-Ad) vector. Gene delivery to sweat glands in skin organ culture was studied by topical application, intradermal injection or submerged culture. We found that transduction efficiency can be enhanced by pretreating isolated sweat glands with dispase, which suggests that the basement membrane is a critical barrier to gene delivery by adenoviral vectors. Using this approach, we showed that Cftr could be efficiently delivered to and expressed by the epithelial cells of sweat glands with our helper-dependent adenoviral vector containing cytokeratin 18 regulatory elements. Based on this study we propose that sweat glands might be used as an alternative model to study CF gene therapy in humans.

Normal CFTR Activity and Reversed Skin Potentials in Pseudohypoaldosteronism.

Cystic fibrosis (CF) transmembrane conductance regulator (CFTR) Cl(-) channel function is required for activating amiloride-sensitive epithelial Na(+) channels (ENaC) in salt-absorbing human sweat duct. It is unclear whether ENaC channel function is also required for CFTR activation. The dysfunctional ENaC mutations in type-1 pseudohypoaldosteronism (PHA-1) provided a good opportunity to study this phenomenon of ion channel interaction between CFTR and ENaC. The PHA-1 ducts completely lacked spontaneous ENaC conductance (gENaC). In contrast, the normal ducts showed large spontaneous gENaC (46 +/- 10 ms, mean +/- SE: ). After permeabilization of the basolateral membrane with alpha-toxin, cAMP + ATP activation of CFTR Cl(-) conductance (gCFTR) or alkalinization of cytosolic pH (6.8 to 8.5) stimulated gENaC of normal but not PHA-1 ducts. In contrast, both spontaneous gCFTR in intact ducts and (cAMP + ATP)-activated gCFTR of permeabilized ducts appeared to be similar in normal and PHA-1 subjects. Lack of gENaC completely blocked salt absorption and caused dramatic reversal of skin potentials associated with pilocarpine-induced sweat secretion from significantly negative in normal subjects (-13 +/- 7.0 mV) to significantly positive (+22 +/- 11.0 mV) in PHA-1 patients. We conclude that virtual lack of ENaC in PHA-1 ducts had little effect on CFTR activity and that the positive skin potentials could potentially serve as a diagnostic tool to identify type-1 pseudohypoaldosteronism.

ENaC activity requires CFTR channel function independently of phosphorylation in sweat duct.

We previously showed that activation of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) Cl- conductance (gCFTR) supports parallel activation of amiloride-sensitive epithelial Na+ channel (ENaC) in the native human sweat duct. However, it is not clear whether phosphorylated CFTR, phosphorylated ENaC, or only Cl(-) -channel function is required for activation. We used basilaterally alpha-toxin-permeabilized human sweat ducts to test the hypothesis that ENaC activation depends only on Cl(-) -channel function and not on phosphorylation of either CFTR or ENaC. CFTR is classically activated by PKA plus millimolar ATP, but cytosolic glutamate activation of gCFTR is independent of ATP and phosphorylation. We show here that both phosphorylation-dependent (PKA) and phosphorylation-independent (glutamate) activation of CFTR Cl- channel function support gENaC activation. We tested whether cytosolic application of 5 mM ATP alone, phosphorylation by cAMP, cGMP, G-protein dependent kinases (all in the presence of 100 microM ATP), or glutamate could support ENaC activation in the absence of gCFTR. We found that none of these agonists activated gENaC by themselves when Cl- current (I(Cl-)) through CFTR was blocked by: 1) Cl- removal, 2) DIDS inhibition, 3) lowering the ATP concentration to 100 microM (instead of 5 mM required to support CFTR channel function), or 4) mutant CFTR (homozygous DeltaF508 CF ducts). However, Cl- gradients in the direction of absorption supported, while Cl- gradients in the direction of secretion prevented ENaC activation. We conclude that the interaction between CFTR and ENaC is dependent on activated I(Cl-) through CFTR in the direction of absorption (Cl- gradient from lumen to cell). But such activation of ENaC is independent of phosphorylation and ATP. However, reversing I(Cl-) through CFTR in the direction of secretion (Cl- gradient from cell to lumen) prevents ENaC activation even in the presence of I(Cl-) through CFTR.

Salivary secretion assay for drug efficacy for cystic fibrosis in mice.

Computerized assays on cultured cells ex vivo have been used to screen thousands of compounds for their effectiveness in correcting the basic physiological defect in cystic fibrosis (CF). While a number of these compounds appear promising, their effectiveness will almost certainly need to be demonstrated in animals before therapeutic tests in humans will be possible. We show herein that the function of salivary secretion in the mouse model for CF could be used as a simple, easy and rapid in vivo assay for drug effects. We demonstrate that salivary secretory capacity stimulated with a beta-adrenergic agonist closely reflects the genotype of origin. Specifically, the mean maximal secretory rate of saliva in normal wild type (+/+) mice was about 1.5 times higher than that of the mean rate in heterozygote (+/-) mice and more than 50 times greater than in CF (-/-) mice. Total saliva secreted per stimulated period obeyed a similar phenotype-genotype segregation. The data indicate that salivary secretory rates in CF mice could be used to assay potential drugs for their effectiveness in correcting the secretory defect in cystic fibrosis.

Localisation of the vacuolar proton pump (V-H+ -ATPase) and carbonic anhydrase II in the human eccrine sweat gland.

The localisation of the vacuolar proton pump (V-H+ -ATPase) and the enzyme carbonic anhydrase II (CAII) was investigated in the human eccrine sweat gland employing standard immunohistochemical techniques after antigen retrieval using microwave heat treatment and high pressure. The high-pressure antigen retrieval unmasked the presence of V-H+ -ATPase in the clear cells of the secretory coil, with a distribution similar to that previously observed for CAII. However, the dark cells were unreactive to both antibodies. In addition, heat and high-pressure antigen retrieval demonstrated the presence of CAII in the apical zone of luminal cells of the reabsorptive duct, a location not previously reported. The localisation of V-H+ -ATPase and CAII in the secretory coil clear cells suggests that the formation of HCO3- and H+ by carbonic anhydrase II and the transport of H+ by V-H+ -ATPase may play an role in sweat fluid secretion. Their presence at the apex of the duct cells indicates involvement in ductal ion reabsorption.

Effects of a new cystic fibrosis transmembrane conductance regulator inhibitor on Cl- conductance in human sweat ducts.

Effective and specific inhibition of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel in epithelia has long been needed to better understand the role of anion movements in fluid and electrolyte transport. Until now, available inhibitors have required high concentrations, usually in the millimolar or high micromolar range, to effect even an incomplete block of channel conductance. These inhibitors, including 5-nitro-2(3-phenylpropyl-amino)benzoate (NPPB), bumetamide, glibenclamide and DIDS, are also relatively non-specific. Recently a new anion channel inhibitor, a thiazolidinone derivative, termed CFTRInh-172 has been synthesized and introduced with apparently improved inhibitory properties as shown by effects on anion conductance expressed in cell lines and on secretion in vivo. Here, we assay the effect of this inhibitor on a purely salt absorbing native epithelial tissue, the freshly isolated microperfused human sweat duct, known for its inherently high expression of CFTR. We found that the inhibitor at a maximum dose limited by its aqueous solubility of 5 microm partially blocked CFTR when applied to either surface of the membrane; however, it may be somewhat more effective from the cytosolic side (approximately 70% inhibition). It may also partially inhibit Na+ conductance. The inhibition was relatively slow, with a half time for maximum effect of about 3 min, and showed very slow reversibility. Results also suggest that CFTR Cl- conductance (GCl) was blocked in both apical and basal membranes. The inhibitor appears to exert some effect on Na+ transport as well.

Control of dynamic CFTR selectivity by glutamate and ATP in epithelial cells.

Cystic fibrosis is caused by mutations in cystic fibrosis transmembrane conductance regulator (CFTR), an anion channel. Phosphorylation and ATP hydrolysis are generally believed to be indispensable for activating CFTR. Here we report phosphorylation- and ATP-independent activation of CFTR by cytoplasmic glutamate that exclusively elicits Cl-, but not HCO3-, conductance in the human sweat duct. We also report that the anion selectivity of glutamate-activated CFTR is not intrinsically fixed, but can undergo a dynamic shift to conduct HCO3- by a process involving ATP hydrolysis. Duct cells from patients with DeltaF508 mutant CFTR showed no glutamate/ATP activated Cl- or HCO3- conductance. In contrast, duct cells from heterozygous patients with R117H/DeltaF508 mutant CFTR also lost most of the Cl- conductance, yet retained significant HCO3- conductance. Hence, not only does glutamate control neuronal ion channels, as is well known, but it can also regulate anion conductance and selectivity of CFTR in native epithelial cells. The loss of this uniquely regulated HCO3- conductance is most probably responsible for the more severe forms of cystic fibrosis pathology.

Functional interaction of CFTR and ENaC in sweat glands.

The cystic fibrosis transmembrane conductance regulator (CFTR) plays a significant role in transepithelial salt absorption as well as secretion by a number of epithelial tissues including sweat glands, airways and intestine. Early studies suggested that in absorption significant cross talk occurs between CFTR Cl(-) channels and epithelial Na(+) channels (ENaC). Studies based primarily on cultured cells of the airways and on ex vivo expression systems suggested that activating CFTR inhibits ENaC channels so that activation of CFTR and deactivation of ENaC seem reciprocal. Lack of CFTR Cl(-) conductance (g(CFTR)) in the plasma membranes was seen to enhance ENaC conductance (g(ENaC)) and Na(+) absorption from the airway surface liquid causing airway pathology in cystic fibrosis (CF). To determine if these events hold true for a purely absorptive epithelium, we investigated the role of CFTR in regulating g(ENaC) in native human sweat gland ducts. After permeabilizing the basilateral membrane of the duct with alpha-toxin, the relative activities of ENaC and CFTR in the apical membrane were characterized by correlating the effect of activating CFTR with ENaC function. We found that in contrast to reciprocal activities, activating g(CFTR) by either cAMP, cGMP or the G-proteins plus 5 mM ATP was accompanied by a concomitant activation, not inhibition, of g(ENaC). The activation of g(ENaC) appeared to be critically dependent on CFTR Cl(-) channel function because removal of Cl(-) from the medium, blockage of CFTR with inhibitor DIDS or the absence of CFTR in the DeltaF508 CF ducts prevented activation of g(ENaC) by cAMP, GMP or G-proteins. Most significantly, g(ENaC) was dramatically reduced, not increased, in CF as compared to non-CF sweat ducts. These results showed that lack of CFTR in the plasma membranes is not characteristically coupled to elevated ENaC activity or to increased Na(+) absorption in CF epithelial cells. Not only are CFTR and ENaC activated together in duct salt absorption, but ENaC activation depends on functioning CFTR. NaCl is poorly absorbed in the CF duct because CFTR activity appears to impose a loss of ENaC activity as well.

Effect of anion transport blockers on CFTR in the human sweat duct.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a protein kinase A (PKA) and ATP regulated Cl- channel. Studies using mostly ex vivo systems suggested diphenylamine-2-carboxylate (DPC), 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and glybenclamide inhibit CFTR Cl- conductance (CFTR GCl). However, the properties of inhibition in a native epithelial membrane have not been well defined. The objective of this study was to determine and compare the inhibitory properties of the aforementioned inhibitors as well as the structurally related anion-exchange blockers (stilbenes) including 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) in the microperfused intact and basilaterally permeabilized native sweat duct epithelium. All of these inhibitors blocked CFTR in a dose-dependent manner from the cytoplasmic side of the basilaterally permeabilized ducts, but none of these inhibitors blocked CFTR GCl from the luminal surface. We excluded inhibitor interference with a protein kinase phosphorylation activation process by "irreversibly" thiophosphorylating CFTR prior to inhibitor application. We then activated CFTR GCl by adding 5 mM ATP. At a concentration of 10(-4) M, NPPB, DPC, glybenclamide, and DIDS were equipotent and blocked approximately 50% of irreversibly phosphorylated and ATP-activated CFTR GCl (DIDS = 49 +/- 10% > NPPB = 46 +/- 10% > DPC = 38 +/- 7% > glybenclamide = 34 +/- 5%; values are mean +/- SE expressed as % inhibition from the control). The degree of inhibition may be limited by inhibitor solubility limits, since DIDS, which is soluble to 1 mM concentration, inhibited 85% of CFTR GCl at this concentration. All the inhibitors studied primarily blocked CFTR from the cytoplasmic side and all inhibition appeared to be independent of metabolic and phosphorylation processes.

cAMP-independent phosphorylation activation of CFTR by G proteins in native human sweat duct.

It is generally believed that cAMP-dependent phosphorylation is the principle mechanism for activating cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels. However, we showed that activating G proteins in the sweat duct stimulated CFTR Cl(-) conductance (G(Cl)) in the presence of ATP alone without cAMP. The objective of this study was to test whether the G protein stimulation of CFTR G(Cl) is independent of protein kinase A. We activated G proteins and monitored CFTR G(Cl) in basolaterally permeabilized sweat duct. Activating G proteins with guanosine 5'-O-(3-thiotriphosphate) (10-100 microM) stimulated CFTR G(Cl) in the presence of 5 mM ATP alone without cAMP. G protein activation of CFTR G(Cl) required Mg(2+) and ATP hydrolysis (5'-adenylylimidodiphosphate could not substitute for ATP). G protein activation of CFTR G(Cl) was 1) sensitive to inhibition by the kinase inhibitor staurosporine (1 microM), indicating that the activation process requires phosphorylation; 2) insensitive to the adenylate cyclase (AC) inhibitors 2',5'-dideoxyadenosine (1 mM) and SQ-22536 (100 microM); and 3) independent of Ca(2+), suggesting that Ca(2+)-dependent protein kinase C and Ca(2+)/calmodulin-dependent kinase(s) are not involved in the activation process. Activating AC with 10(-6) M forskolin plus 10(-6) M IBMX (in the presence of 5 mM ATP) did not activate CFTR, indicating that cAMP cannot accumulate sufficiently to activate CFTR in permeabilized cells. We concluded that heterotrimeric G proteins activate CFTR G(Cl) endogenously via a cAMP-independent pathway in this native absorptive epithelium.

Apical heterotrimeric g-proteins activate CFTR in the native sweat duct.

Other than the fact that the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel can be activated by cAMP dependent kinase (PKA), little is known about the signal transduction pathways regulating CFTR. Since G-proteins play a principal role in signal transduction regulating several ion channels [4, 5, 9], we sought to test whether G-proteins control CFTR Cl- conductance (CFTR G(Cl)) in the native sweat duct (SD). We permeabilized the basolateral membrane with alpha-toxin so as to manipulate cytosolic nucleotides. We activated G-proteins and monitored CFTR G(Cl) activity as described earlier [20, 23, 25]. We now show that activating G-proteins with GTP-gamma-S (100 microm) also activates CFTR G(Cl) in the presence of 5 mm ATP alone (without exogenous cAMP). GTP-gamma-S increased CFTR G(Cl) by 44 +/- 20 mS/cm(2) (mean +/- se; n = 7). GDP (10 mm) inhibited G-protein activation of CFTR G(Cl) even in the presence of GTP-gamma-S. The heterotrimeric G-protein activator (AlF(4-) in the cytoplasmic bath activated CFTR G(Cl) (increased by 51.5 +/- 9.4 mS/cm(2) in the presence of 5 mm ATP without cAMP, n = 6), the magnitude of which was similar to that induced by GTP-gamma-S. Employing immunocytochemical-labeling techniques, we localized Galphas, Galphai, Galphaq, and Gbeta at the apical membranes of the sweat duct. Further, we showed that the mutant CFTR G(Cl) in ducts from cystic fibrosis (CF) subjects could be partially activated by G-proteins. The magnitude of mutant CFTR G(Cl) activation by G-proteins was smaller as compared to non-CF ducts but comparable to that induced by cAMP in CF ducts. We conclude that heterotrimeric G-proteins are present in the apical membrane of the native human sweat duct which may help regulate salt absorption by controlling CFTR G(Cl) activity.