Differential regulation of Cl- transport proteins by PKC in Calu-3 cells.

Cl- transport proteins expressed in a Calu-3 airway epithelial cell line were differentiated by function and regulation by protein kinase C (PKC) isotypes. mRNA expression of Cl- transporters was semiquantitated by RT-PCR after transfection with a sense or antisense oligonucleotide to the PKC isotypes that modulate the activity of the cystic fibrosis transmembrane conductance regulator [CFTR (PKC-epsilon)] or of the Na/K/2Cl (NKCC1) cotransporter (PKC-delta). Expression of NKCC1 and CFTR mRNAs and proteins was independent of antisense oligonucleotide treatment. Transport function was measured in cell monolayers grown on a plastic surface or on filter inserts. With both culture methods, the antisense oligonucleotide to PKC-epsilon decreased the amount of PKC-epsilon and reduced cAMP-dependent activation of CFTR but not alpha(1)-adrenergic activation of NKCC1. The antisense oligonucleotide to PKC-delta did not affect CFTR function but did block alpha(1)-adrenergic activation of NKCC1 and reduce PKC-delta mass. These results provide the first evidence for mRNA and protein expression of NKCC1 in Calu-3 cells and establish the differential regulation of CFTR and NKCC1 function by specific PKC isotypes at a site distal to mRNA expression and translation in airway epithelial cells.

PKC signaling in CF/T43 cell line: regulation of NKCC1 by PKC-delta isotype.

Cystic fibrosis (CF) airway epithelial cells have a reduced mass of ether-linked diacylglycerols which might alter protein kinase C (PKC)-regulated Cl secretion. PKC regulation of basolateral Na-K-2Cl cotransport (NKCC1) was investigated in CF nasal polyp epithelial cells and a CF/T43 cell line to ascertain whether PKC signaling was altered in CF. NKCC1 was detected as bumetanide-sensitive (86)Rb influx. Methoxamine, a alpha(1)-adrenergic agonist, increased PKC activity in cytosol and a particulate fraction for a prolonged time period, as predicted from previous studies on the generation of diglycerides induced with methoxamine. Short-term stimulation of CF/T43 cells for 40 s promoted a shift in PKC-delta and -zeta to a particulate fraction, increased activity of immune complexes of cytosolic PKC-delta and of particulate PKC-zeta and increased activity of NKCC1. Pretreatment with antisense oligonucleotide to PKC-delta blocked methoxamine-stimulated PKC-delta activity, reduced PKC-delta mass by 61.4%, and prevented methoxamine-stimulated activity of NKCC1. Sense and missense oligonucleotide to PKC-delta and antisense oligonucleotide to PKC-zeta did not alter expression of PKC-delta or the effects of methoxamine. These results demonstrate that PKC-delta-dependent activation of NKCC1 is preserved in CF cells and suggest that regulation of NKCC1 is independent of low ether-linked diglyceride mass.

Antisense oligonucleotide to PKC-epsilon alters cAMP-dependent stimulation of CFTR in Calu-3 cells.

Protein kinase C (PKC) regulates cystic fibrosis transmembrane conductance regulator (CFTR) channel activity but the PKC signaling mechanism is not yet known. The goal of these studies was to identify PKC isotype(s) required for control of CFTR function. CFTR activity was measured as 36Cl efflux in a Chinese hamster ovary cell line stably expressing wild-type CFTR (CHO-wtCFTR) and in a Calu-3 cell line. Chelerythrine, a PKC inhibitor, delayed increased CFTR activity induced with phorbol 12-myristate 13-acetate or with the cAMP-generating agents (-)-epinephrine or forskolin plus 8-(4-chlorophenylthio)adenosine 3',5'- cyclic monophosphate. Immunoblot analysis of Calu-3 cells revealed that PKC-alpha, -betaII, -delta, -epsilon, and -zeta were expressed in confluent cell cultures. Pretreatment of cell monolayers with Lipofectin plus antisense oligonucleotide to PKC-epsilon for 48 h prevented stimulation of CFTR with (-)-epinephrine, reduced PKC-epsilon activity in unstimulated cells by 52.1%, and decreased PKC-epsilon mass by 76.1% but did not affect hormone-activated protein kinase A activity. Sense oligonucleotide to PKC-epsilon and antisense oligonucleotide to PKC-delta and -zeta did not alter (-)-epinephrine-stimulated CFTR activity. These results demonstrate the selective regulation of CFTR function by constitutively active PKC-epsilon.

Antisense oligodeoxynucleotide to PKC-delta blocks alpha 1-adrenergic activation of Na-K-2Cl cotransport.

A role for protein kinase C (PKC)-delta and -zeta isotypes in alpha 1-adrenergic regulation of human tracheal epithelial Na-K-2Cl cotransport was studied with the use of isotype-specific PKC inhibitors and antisense oligodeoxy-nucleotides to PKC-delta or -zeta mRNA. Rottlerin, a PKC-delta inhibitor, blocked 72% of basolateral-to-apical, bumetanide-sensitive 36Cl flux in nystatin-permeabilized cell monolayers stimulated with methoxamine, an alpha 1-adrenergic agonist, with a 50% inhibitory concentration of 2.3 microM. Methoxamine increased PKC activity in cytosol and a particulate fraction; the response was insensitive to PKC-alpha and -beta II isotype-specific inhibitors, but was blocked by general PKC inhibitors and rottlerin. Rottlerin also inhibited methoxamine-induced PKC activity in immune complexes of PKC-delta, but not PKC-zeta. At the subcellular level, methoxamine selectively elevated cytosolic PKC-delta activity and particulate PKC-zeta activity. Pretreatment of cell monolayers with antisense oligodeoxynucleotide to PKC-delta for 48 h reduced the amount of whole cell and cytosolic PKC-delta, diminished whole cell and cytosolic PKC-delta activity, and blocked methoxamine-stimulated Na-K-2Cl cotransport. Sense oligodeoxynucleotide to PKC-delta and antisense oligodeoxynucleotide to PKC-zeta did not alter methoxamine-induced cotransport activity. These results demonstrate the selective activation of Na-K-2Cl cotransport by cytosolic PKC-delta.

Differential activation of PKC-delta and -zeta by alpha 1-adrenergic stimulation in human airway epithelial cells.

Phorbol ester and alpha 1-adrenergic stimulation of Na-Cl-K cotransport in human tracheal epithelial cells was investigated by determining the expression of protein kinase C (PKC) isotypes and their activation by phorbol 12-myristate 13-acetate (PMA) and methoxamine, an alpha 1-adrenergic agonist. PKC-alpha, -beta II, -delta, -epsilon, and -zeta were expressed in confluent cell cultures. PKC-beta I, -gamma, and -eta were not detected. PKC-alpha and -zeta were localized to the cytosol, and PKC-beta II and -delta were distributed approximately evenly between cytosolic and particulate fractions. Treatment with PMA for 30 min increased PKC activity in subcellular fractions and induced a redistribution of PKC-beta II and -delta to a particulate fraction. PMA treatment for 18 h reduced PKC activity to levels found in untreated cells and reduced, but did not deplete, PKC isotype mass. Methoxamine transiently increased PKC activity, with maximal levels at 40 s, and caused a shift in PKC-delta and -zeta mass to a particulate fraction. Methoxamine selectively induced a sustained increase in PKC-zeta activity but only a transient increase in PKC-delta. These results suggest that PKC-delta and -zeta mediate hormonal activation of Na-Cl-K cotransport.

Alpha 1-adrenergic stimulation differentially regulates ether-linked diacylglycerols in airway epithelial cells from normal and cystic fibrosis patients.

Alpha 1-adrenergic stimulation of human airway epithelial cells induces a transient increase in polyphosphoinositide turnover coincident with augmented Na+Cl-(K+) cotransport activity. This activation of airway epithelial cells also results in a biphasic elevation of diacylglycerols. To better understand the significance of these distinct diacylglycerol pools, we now characterize the mass of ether- and ester-linked diacylglycerol species. We demonstrate that the relative mass of ether-linked diacylglycerols is reduced in airway epithelium from cystic fibrosis patients in the presence or absence of alpha 1-adrenergic stimulation. This reduction in ether-linked diacylglycerol mass may represent a compensatory mechanism to help maintain normal chloride influx in cystic fibrosis patients.

Phorbol ester and okadaic acid regulation of Na-2Cl-K cotransport in rabbit tracheal epithelial cells.

We evaluated a role for protein kinase C (PKC) in the regulation of rabbit tracheal epithelial Na-Cl(K) cotransport. Short-term treatment with phorbol 12-myristate 13-acetate (PMA) dose dependently increased bumetanide-sensitive Na and Cl efflux and elevated staurosporine- and bumetanide-sensitive Na, Cl, and K uptake. PMA and the alpha 2A-adrenergic agonist guanabenz both induced contransport with a stoichiometry of 2 Cl:1 Na and 2 Cl:1 Rb and elevated staurosporine-sensitive PKC activity in cytosolic and particulate fractions. Prolonged PMA treatment did not sustain bumetanide-sensitive 2 Cl:1 Na and 2 Cl:1 Rb transport but did block stimulation of bumetanide-sensitive transport by PMA or guanabenz and elevation of PKC activity by PMA and guanabenz in a particulate fraction. Cells treated with okadaic acid exhibited a staurosporine- and bumetanide-sensitive 2 Cl:1 Na and 2 Cl:1 Rb uptake. In cultured monolayers, basolateral perfusion with epinephrine, isoproterenol, or PMA increased short-circuit current (Isc). Basolateral application of bumetanide reduced elevated Isc to baseline levels, indicating a role for Cl secretory cells in a reconstituted tracheal epithelium. Pretreatment of transmonolayer cultures with PMA diminished the stimulatory response to epinephrine. These results indicate that, in rabbit tracheal epithelial cells, alpha-adrenergic stimulation activated Na-2Cl-K cotransport and that PKC is a critical effector in this process.

The role of protein kinase C in alpha-adrenergic regulation of NaCl(K) cotransport in human airway epithelial cells.

alpha 1-Adrenergic (alpha 1-AR) agents stimulate NaCl(K) cotransport and phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2]-specific phospholipase C in human trachea and nasal polyp epithelial cells. One second messenger generated by PtdIns(4,5)P2 degradation is inositol trisphosphate. We now show that diglycerides (DG) are also generated during alpha 1-AR stimulation. In cells prelabeled with [3H]arachidonic acid, alpha 1-AR agents produced a biphasic DG generation in normal and cystic fibrosis (CF) cells that is blocked by pertussis toxin. The early DG peak closely paralleled PtdIns(4,5)P2 degradation, stimulation of cotransport by phorbol 12-myristate 13-acetate (PMA), and inhibition of cotransport by the protein kinase C (PKC) inhibitor staurosporine. This suggests that cotransporter activation requires PKC-protein phosphorylation. This possibility was tested using the protein phosphatase inhibitor okadaic acid. Okadaic acid elevated bumetanide-sensitive Cl efflux. Staurosporine also blocked > 63% of okadaic-acid-stimulated Cl transport. The late DG peak did not support hormone-stimulated cotransport. The results demonstrate that DGs are a pivotal link between alpha 1-AR stimulation and NaCl(K) cotransport activation with a role for PKC and protein phosphorylation. alpha 1-AR intracellular signaling mechanisms apparently operate normally in CF cells.

Activation of PtdIns(4,5)P2-sensitive phospholipase C in rabbit tracheal epithelial cells.

A role for phospholipase C hydrolysis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] as a mechanism of alpha 2-adrenergic signal transduction in rabbit tracheal epithelial cells (tracheocytes) was investigated in isolated cells grown in in vitro culture and prelabeled with myo-[3H]inositol (3 microCi/ml) for 72 h. Breakdown of polyphosphoinositides was measured by using thin-layer chromatography to detect phosphatidylinositol, phosphatidylinositol 4-phosphate [PtdIns(4)P], and PtdIns(4,5)P2. Inositol phosphates were separated by ion-exchange column chromatography. The endogenous catecholamine l-epinephrine and alpha 2-adrenergic agonists clonidine and 1-(2,6-dichlorobenzylideneamino)guanidine (guanabenz) produced a rapid transient accumulation of inositol trisphosphate and inositol 4,5-bisphosphate and breakdown of [PtdIns(4)P] and PtdIns(4,5)P2. The alpha 2-adrenergic effects were not blocked by the beta-adrenergic antagonist DL-propranolol or by the alpha 1-adrenergic antagonists prazosin and methylurapidil but were inhibited by pertussis toxin and blocked by yohimbine, an alpha 2-adrenergic antagonist. The 50% effective concentration for guanabenz-stimulated inositol trisphosphate generation was right shifted from 0.3 to 0.9 microM by yohimbine. The results provide the first demonstration of alpha 2A-adrenergic activation of pertussis toxin-sensitive PtdIns(4,5)P2-dependent phospholipase C in mammalian tracheocytes. The findings are consistent with previous observations on alpha 2A-adrenergic-mediated activation of NaCl cotransport in these cells.

Alpha 2-adrenergic, but not imidazole, agonists activate NaCl cotransport in rabbit tracheal epithelial cells.

The adrenergic agonist clonidine activates NaCl cotransport in rabbit tracheocytes. With the use of the high-affinity analogue p-[125I]iodoclonidine, binding of clonidine to cells was determined to fit a two-site model, with one site of high specificity for alpha 2-adrenergic (alpha 2-AR) and the other with a high affinity for I1-imidazol(in)e (I1) receptors. Total density of binding sites for both receptors was similar at 18 fmol/mg protein. Moxonidine displayed a 166-fold greater specificity for I1 receptors compared with cimetidine. Bumetanide-sensitive Na or Cl transport was stimulated by the alpha 2-AR agonists clonidine or guanabenz but not by the I1 agents cimetidine or moxonidine. I1 agonists-stimulated Na transport was detected only in the presence of bumetanide. Prazosin did not block clonidine-stimulated NaCl uptake or efflux, indicating the presence of an alpha 2A-AR subtype. Addition of clonidine either before or after incubation with l-isoproterenol or forskolin did not attenuate the time- and dose-dependent increase in adenosine 3',5'-cyclic monophosphate (cAMP) levels. Thus clonidine stimulates NaCl cotransport in rabbit tracheocytes through an alpha 2A-AR mechanism that does not require cAMP for signal transduction. In addition, I1-imidazol(in)e receptors stimulate Na transport in rabbit tracheocytes through an unidentified pathway.

Electrolyte transport in the epithelium of pulmonary segments of normal and cystic fibrosis lung.

The epithelium of pulmonary segments from trachea to aveoli actively transports electrolytes and allows osmotic movement of water to maintain the ionic environment in the airway lumen. Models of airway absorption and secretion depict the operation of transporters localized to apical or basolateral membrane. In many epithelia, a variety of electrolyte transporters operate in different combinations to produce absorption or secretion. This also applies to pulmonary epithelium of the large airways (trachea, main-stem bronchi), bronchioles, and alveoli. Na+ absorption occurs in all three pulmonary segments but by different transporters: apical Na+ channels in large airways and bronchioles; Na+/H+ exchange and Na+ channels in adult alveoli. The Na+ channels in each pulmonary segment share a sensitivity to amiloride, a potent inhibitory of epithelial Na+ channels. Fetal alveoli display spontaneous Cl- secretion, as do the large airways of some mammals, such as dog and bovine trachea. Cl- channels differ in conductance properties and in regulation by intracellular second messengers, osmolarity, and voltage mediate stimulated Cl- secretion. Electroneutral carriers, such as NaCl(K) cotransport, Cl-/HCO3- exchange, and Na+/HCO3- exchange, operate in large airways and alveoli during absorption and secretion. Abnormal ion transport in airways of cystic fibrosis (CF) patients is manifest as a reduced Cl- conductance and increased Na+ conductance. Isolation of the CF gene and identification of its product CFTR now allow investigations into the basic defect. Intrinsic to these investigations is the development of systems to study the function of CFTR and its relation to electrolyte transporters and their regulation.

Bumetanide-sensitive NaCl uptake in rabbit tracheal epithelial cells is stimulated by neurohormones and hypertonicity.

Loop diuretic-sensitive NaCl(K) cotransport plays a fundamental role in absorption and secretion of electrolytes in epithelial tissues. Cotransport activity was measured as uptake of 22Na, 36Cl, or 86Rb at 27 degrees C in isolated rabbit tracheal epithelial cells. Uptake of radiotracer was linear from 1 to 2 min after initiation of radiotracer transport. Bumetanide at 10 microM final concentration did not affect tracer uptake. The endogenous catecholamine l-epinephrine and alpha 2-adrenergic agent clonidine increased sodium and chloride uptake at least 5.5-fold. Bumetanide blocked sodium uptake by 85% and chloride uptake by 72%. 86Rb uptake was not affected by l-epinephrine, clonidine, or bumetanide. The alpha 2-adrenergic antagonist yohimbine blocked the effects of l-epinephrine and clonidine on 22Na and 36Cl uptake. In Ca(2+)-depleted transport medium, baseline levels of sodium and chloride uptake increased 3.8- and 2.4-fold, respectively, in a bumetanide-independent manner. Nevertheless, l-epinephrine and clonidine induced a net stimulation of sodium and chloride uptake similar to that found in Ca(2+)-replete medium. This response was reduced by bumetanide and yohimbine. The Ca(2+)-elevating agent ionomycin increased bumetanide-sensitive sodium and chloride uptake 7.2- and 6.2-fold, respectively. Replacement of chloride with gluconate or sodium with N-methyl-D-glucamine in the extracellular medium inhibited l-epinephrine and clonidine-stimulated bumetanide-sensitive sodium and chloride uptake, respectively. Osmotic shrinkage in hyperosmotic (500 mM NaCl with all other electrolytes at normal concentration) transport medium markedly increased bumetanide-inhibitable sodium and chloride uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Alpha 1-adrenergic signaling in human airway epithelial cells involves inositol lipid and phosphate metabolism.

A role for phospholipase C (PLC) hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) as a mechanism of alpha 1-adrenergic signal transduction in human airway epithelial cells (AEC) was investigated in isolated normal tracheal and cystic fibrosis (CF) nasal epithelial cells grown in in vitro culture and prelabeled with 3 muCi myo-[3H]inositol/ml for 72 h. Breakdown of polyphosphoinositides was measured using thin-layer chromatography to detect phosphatidylinositol, phosphatidylinositol 4-phosphate (PIP), and PIP2. Inositol phosphates were separated by ion-exchange column chromatography. In normal AEC, the addition of the endogenous catecholamine l-epinephrine produced a rapid, transient accumulation of inositol 1,4,5-trisphosphate (IP3) and inositol 1,4-bisphosphate (IP2) and breakdown of PIP and PIP2. IP3 increased 1.7-fold and IP2 1.6-fold after 20 and 40 s, respectively. A maximal decrease of 35% PIP2 and 30% PIP is observed after 20 and 40 s, respectively. The effects of l-epinephrine were not blocked by the beta-adrenergic antagonist dl-propranolol but were mimicked by the alpha 1-adrenergic agonist methoxamine. Prazosin, an alpha 1-adrenergic antagonist, and pertussis toxin (PTX) blocked the effects of l-epinephrine and methoxamine. Addition of l-epinephrine and methoxamine to CF nasal epithelial cells also induced prazosin-sensitive polyphosphoinositide breakdown and inositol phosphate accumulation. A 2.2-fold accumulation of IP3 was observed after 10 s and 2.0-fold increase in IP2 after 20 s. Maximal decreases of 32% PIP2 and 23% PIP levels were observed after 20-s incubation with l-epinephrine. PTX reduced the effects of l-epinephrine and significantly blocked the effects of methoxamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride antagonizes beta-adrenergic stimulation of cAMP synthesis and Cl- secretion in human tracheal epithelial cells.

Amiloride, a potent blocker of the sodium channel in airway epithelium, has been administered by aerosol as a therapeutic agent for cystic fibrosis. Because amiloride in high concentration has been reported to interfere with cell functions, including adrenergic responses, we tested the ability of amiloride to inhibit beta-adrenergic responses in human tracheal epithelial cells. Amiloride (10(-4) M), applied from the basolateral surface of a cell monolayer, inhibited the changes in transepithelial potential and short circuit current to isoproterenol (10(-6) M). The stimulation of cyclic adenosine monophosphate (cAMP) synthesis by isoproterenol was inhibited in dose-dependent fashion by amiloride (P = 0.007 by multivariate ANOVA with multiple samples correction). Amiloride did not affect baseline transepithelial potential, short circuit current, basal cAMP levels, cAMP response to prostaglandin E2, or basal adenylate cyclase activity measured directly in membrane preparations. Therefore, it is unlikely that amiloride exerts a nonspecific toxic effect on adenylate cyclase, receptor-cyclase coupling, or substrate or cofactor supply. The binding of [125I]iodocyanopindolol (ICYP), a beta-adrenergic receptor antagonist, to membranes from human tracheal epithelial cells could be displaced by amiloride with IC50 = 410 microM; displacement was 70% at 10(-3) M amiloride. These data are most consistent with the hypothesis that amiloride inhibits beta-adrenergic responses in airway epithelial cells by occupying beta-adrenergic receptor sites. Therapeutic administration of amiloride should take into account its affinity for adrenergic receptors.

Calcium and alpha-adrenergic regulation of Na-Cl(K) cotransport in rabbit tracheal epithelial cells.

The marked sensitivity of Cl and fluid secretion in mammalian airways to basolateral application of loop diuretics has led to postulates that electrically neutral Na-Cl entry plays a critical role during secretion. Electrically neutral Na-Cl(K) cotransport was investigated by determining the initial rate of 22Na and 36Cl efflux in epithelial cells isolated from rabbit trachea and preequilibrated with radioactive tracer at 25 degrees C. Tracer transport was initiated by 10-fold dilution of an aliquot of cells in radioisotope-free medium. The initial rate of radiolabeled ion transport was calculated from the linear portion of efflux curves. Base-line Na and Cl transport rates were not affected by furosemide or bumetanide. l-Epinephrine stimulated Na and Cl transport rates 1.8-fold each in Ca2(+)-replete and 2.6- and 2.3-fold, respectively, in Ca2(+)-deficient transport medium. Loop diuretics and yohimbine, an alpha 2-adrenergic antagonist, blocked the effects of l-epinephrine, and, clonidine, an alpha 2-adrenergic agonist, stimulated yohimbine- and furosemide-sensitive Cl transport. The beta-adrenergic agonist l-isoproterenol alone did not affect tracer transport and, in combination with clonidine, did not affect the response to clonidine. Elevation of intracellular Ca2+ with ionomycin stimulated tracer transport, and buffering of intracellular Ca2+ with 1,2-bis(aminophenoxy)ethane- N,N,N',N'-tetraacetic acid blocked the stimulatory effects of alpha-adrenergic agents. These results indicate an alpha 2-adrenergic stimulation of loop diuretic-sensitive Na and Cl transport that requires elevated intracellular Ca2+ as the second messenger. The transport mechanism is probably a Na-Cl or Na-K-2Cl cotransport located in the basolateral membrane.

Alpha-adrenergic regulation of Na-Cl cotransport in human airway epithelium.

The demonstration of abnormal beta-adrenergic and cAMP-modulated apical Cl- channels in cystic fibrosis (CF) airway epithelial cells suggests that other transporters, which are required for Cl- secretion, may also be abnormally regulated. A basolateral cotransporter was investigated by determining the initial rate of 36Cl efflux from cells isolated from CF nasal polyps or trachea and non-CF trachea. Cells were preequilibrated with radioactive tracer at 25 degrees C, and tracer transport was initiated by 10-fold dilution of an aliquot of cells in radioisotope-free medium. The initial rate of Cl transport was calculated from the linear portion of the efflux curves. In CF and non-CF cells, base-line Cl- transport was not blocked by furosemide but was stimulated twofold by l-epinephrine in Ca2+-deficient and Ca2+-replete transport medium. In both types of cells, furosemide blocked 70 and 77%, respectively, of the stimulated Cl- transport. Prazosin, an alpha 1-adrenergic antagonist, blocked the effects of l-epinephrine and methoxamine, an alpha 1-adrenergic agonist, stimulated prazosin- and furosemide-sensitive Cl transport. Ionomycin mimicked the effects of l-epinephrine. l-Isoproterenol, a beta-adrenergic agonist, did not affect Cl transport. The results of this study indicate an alpha 1-adrenergic stimulation of furosemide-sensitive Cl transport in human airway epithelium that functions normally in CF airway epithelial cells. The transport mechanism is probably a Na-Cl or Na-K-2Cl cotransport located in the basolateral membrane and requires elevated intracellular Ca2+ for activation.

Regulation of chloride channels by protein kinase C in normal and cystic fibrosis airway epithelia.

Apical membrane chloride channels control chloride secretion by airway epithelial cells. Defective regulation of these channels is a prominent characteristic of cystic fibrosis. In normal intact cells, activation of protein kinase C (PKC) by phorbol ester either stimulated or inhibited chloride secretion, depending on the physiological status of the cell. In cell-free membrane patches, PKC also had a dual effect: at a high calcium concentration, PKC inactivated chloride channels; at a low calcium concentration, PKC activated chloride channels. In cystic fibrosis cells, PKC-dependent channel inactivation was normal, but activation was defective. Thus it appears that PKC phosphorylates and regulates two different sites on the channel or on an associated membrane protein, one of which is defective in cystic fibrosis.