aI-Adrenergic cells involves

signaling in human airway epithelial inositol lipid and phosphate metabolism

CAROLE M. LIEDTKE Cystic Fibrosis Center, Department of Pediatrics at Rainbow Babies and Children Hospital, and Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106 Liedtke, Carole M. cul-Adrenergic signaling in human airway epithelial cells involves inositol lipid and phosphate metabolism. Am. J. Physiol. 262 (Lung Cell. Mol. PhysioZ. 6): L183L191, 1992.-A role for phospholipase C (PLC) hydrolysis of phosphatidylinositol 4,Sbisphosphate (PIP,) as a mechanism of cul-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 &i myo- [“H]inositol/ml for 72 h. Breakdown of polyphosphoinositides was measured using thin-layer chromatography to detect phosphatidylinositol, phosphatidylinositol 4-phosphate (PIP), and PIP,. Inositol phosphates were separated by ion-exchange column chromatography. In normal AEC, the addition of the endogeneous catecholamine Z-epinephrine produced a rapid, transient accumulation of inositol 1,4,5-trisphosphate (IP3) and inositol 1,4bisphosphate (IP2) and breakdown of PIP and PIP,. IP, increased 1.7-fold and IP, 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 Z-epinephrine were not blocked by the ,8-adrenergic antagonist dl-propranolol but were mimicked by the al-adrenergic agonist methoxamine. Prazosin, an al-adrenergic antagonist, and pertussis toxin (PTX) blocked the effects of I-epinephrine and methoxamine. Addition of I-epinephrine and methoxamine to CF nasal epithelial cells also induced prazosin-sensitive polyphosphoinositide breakdown and inositol phosphate accumulation. A 2.2fold accumulation of IP, was observed after 10 s and 2.0-fold increase in IP, after 20 s. Maximal decreases of 32% PIP, and 23% PIP levels were observed after 20-s incubation with Zepinephrine. PTX reduced the effects of Z-epinephrine and significantly blocked the effects of methoxamine. The results suggest that in human AEC aI-adrenergic agents elicit rapid breakdown of polyphosphoinositides that is consistent with PTX-sensitive activation of PLC. a-adrenergic receptor; transmembrane signaling; pertussis toxin; prazosin; epinephrine; nasal polyps; methoxamine; tracheal epithelium LINING THE LARGE airways acts as a protective barrier for the lungs by providing an environment for trapping and removing foreign particles and bacteria. To optimize its functions, the epithelium mediates vectorial transport of the electrolytes sodium and chloride and movement of water in response to environmental and hormonal stimuli. Absorption and secretion of electrolytes balance the movement of fluid across the airway epithelium. Human large airway and nasal epithelia spontaneously absorb sodium but can be induced to secrete chloride through the action of mediators, hormones, and prostaglandins (4). Models of airway epithelial chloride secretion depict the synergistic activity of three electrolyte transporters, in addition to the Na-Kadenosinetriphosphatase (ATPase), that achieve net translocation of the electrolytes sodium and chloride and water into the lumen. During chloride secretion, a basoEPITHELIUM

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lateral NaCl(K) cotransporter mediates coupled uptake of sodium and chloride. Chloride that accumulates in the cells exits at the apical membrane via a channel that is regulated by the second messengers adenosine 3’,5’cyclic monophosphate (CAMP) and Ca2+. For charge compensation, potassium flows from the cell through a basolateral Ca2+-activated potassium channel. Sodium pumped from the cell by a basolateral Na-K-ATPase diffuses to the lumen by a paracellular route. The net flow of electrolytes and water is then toward the airway lumen. Several different intracellular signaling mechanisms regulate chloride secretion in human airway epithelial cells (AEC). Most studies agree that ,&adrenergic agonists stimulate chloride secretion through the intracellular second messenger CAMP with phosphorylation of apical chloride channels or closely associated regulatory proteins as a critical step in signal transduction (21, 28). Indeed, CAMP alone is sufficient to stimulate chloride secretion most likely by activating endogeneous CAMPdependent protein kinase A (33,34). More recent studies implicate a role for Ca2+ in conjunction with the membrane-associated enzyme protein kinase C in the activation of apical chloride channels (4, 14, 20, 37). In contrast to apical chloride channels, basolateral transporters critical for chloride secretion in human AEC are regulated by an aI-adrenergic mechanism (23, 32). Potassium channels and NaCl(K) cotransport display a sensitivity to intracellular Ca2+ that suggests a pivotal role for this divalent cation in transporter activation. The exact mechanism whereby Ca2+ transduces the aladrenergic signal in human AEC remains unclear. In many cells and tissues, al-adrenergic receptors act by stimulating phospholipase C (PLC) hydrolysis of phosphatidylinositol 4,5bisphosphate (PIP2) to Q-diacylglycerol (DAG) and inositol 1,4,5trisphosphate (IP3) (3, 27). Both these metabolites have specific functions as second messengers; IP3 causes mobilization of intracellular Ca2+ and DAG together with Ca2+ activates protein kinase C. In these studies, we explore the possibility that, in human AEC, inositol phospholipid metabolism transduces al-adrenergic activation of basolateral transporters. We compared the kinetics of cellular responses of normal cells and epithelial cells isolated from cystic fibrosis (CF) nasal polyps. In CF, a common genetic disease of Caucasians, airway epithelial chloride secretion is compromised due to defective regulation of apical membrane chloride channels by CAMP and Ca2+ (14, 20, 21, 28, 35). Basolateral NaCl(K) cotransport and potassium channels, however, remain unaffected by the CF defect (23, 35), suggesting normal cu-adrenergic signal transduction. The studies reported here provide evi-

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performed on AEC cultured for 6 to 9 days. On the day of use, culture medium containing radiolabel was discarded and cells were washed twice with 1 ml HBSS. After the addition of 1 ml HBSS, cell cultures were incubated at 37°C with IO mM LiCl for 10 min before addition of drugs or HBSS. Li+ blocks a terminal monophosphodiesterase, thus allowing accumulation of inositol monophosphate (IP) and detection of the rapidly metabolized intracellular messengers IP3 and inositol I,$-bisphosphate (IP2) (26). Analysis of lipids as described in Analysis of polyphosphatidylinositidesshowed baseline incorporation of 3H into the phosphoinositidesof normal AEC with average activities of 35,053 t 3,844 (n = 23), 2,130 t 263 (n = 22), and METHODS AND MATERIALS 535 t 61 (n = 21) disintegrations per minute per microgram (dpm/pg) DNA for phosphatidylinositol (PI), PIP, and PIP2, Cell preparation and culture. Tissue for cell culture was respectively. CF AEC incorporated 49,146 t 4,806 (n = 16), obtained from 11 nasal polyp specimens from CF patients at 2,945 Ifs 446 (n = 16), and 1,531t 218 (n = 15) dpm/pg DNA time of polypectomy and 16 normal tracheae at the time of in phosphatidylinositol (PI), phosphatidylinositol4-phosphate autopsy through the Cystic Fibrosis Center, Case Western (PIP), and PIP2, respectively. A comparison of the two cell Reserve University. The tissue was washed extensively with higher radiolabel incorporation in sterile Hanks’ balanced salt solution buffered with 10 mM 2v- types showed a significantly PIP2 (P < 0.001) and PI (P < 0.025) in CF AEC. 2hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) Analysis of polyphosphatidylinositides and of inositol phosto pH 7.4 (HPSS) containing (per ml) 100 U penicillin, 100 pg phates was performed simultaneously on prelabeled cell culstreptomycin, 50 I,cgtobramycin, and 200 mg piperacillin, then incubated at 37°C with antibiotics for 60 min. The wash and tures. Agents to be tested were added as I- to lo-p1 aliquots of incubation cycles were carried out three times. Cells were concentrated stock solutions Reactions were terminated at indicated times by the addition of 0.8 ml methanol-HCl (100:6, isolated by previously described methods with the following vol/vol) (1). Cells were quickly scraped from the culture wells modification (24, 32). Whole trachea was cut into 1 X 2 cm pieces, and nasal polyps were minced with a fine forceps. Tissue with a Teflon policemen, and 1.5 ml acidified extract was transferred to a precooled conical tube containing 1.5 ml chlopieces were incubated at 4°C for 18 h in 20 ml Ca’+, Mg2’-free roform. Cultures wells were washed with 0.8 ml methanol. HPSS containing 20 mg protease (type XIV) (39) and 2 mg deoxyibonuclease. After overnight incubation, EDTA was Washings were combined with acidified extracts then sonicated for 2 min to ensure cell lysis. Extracts were centrifuged for 10 added to a final concentration of 12 mM, and the tissue was min at 800 g at 4°C to separate phases. The top aqueous and incubated an additional 15 min at 37OC. Tracheal epithelial lower lipid phases were recovered, and the remaining interface cells were gently scraped from the underlying submucosa with material reextracted with 2.4 ml chloroform-methanol-HCl a stainless steel spatula and collected by centrifugation at 125 (50:50:3, vol/vol/vol) and 1.0 ml deionized water. After centrifg for IO min at 15OC. The suspension with nasal polyps was ugation, the second aqueous and lipid phases were pooled with repeatedly pipetted with a lo-ml sterile, plastic pipette from which the tip was removed. The minced tissue was allowed to the first extracts. settle, and the supernatant containing cells was decanted into Analysis of inositol phosphates. Pooled aqueous extracts were a sterile 50-ml plastic conical tube. The tissue was washed twice taken to a final volume of 15 ml with deionized H20 and 25 pug with culture media, and the supernatants were pooled. Epithephytic acid hydrolysate (38) in 7.5 ~1 Hz0 were added to each lial cells were recovered by centrifugation as described above. diluted extract. Inositol phosphates were separated by column Tracheal and nasal epithelial cells were washed twice then chromatography by two different methods. 1) The diluted suspended in culture medium. extract was used to charge l.O-ml AG l-X8 (Bio-Rad) anion Cells were counted using a hemocytometer, and viability was exchange columns (200-400 mesh, chloride form). After chargassessed by fluorescent staining with acridine orange-ethidium ing, columns were washed with 10 ml 30 mM HCl to elute 3Hbromide (19). labeled inositol, glycerophosphoinositol, and IP (9, 10). FracIn vitro ceZZculture. Cells were suspended in culture medium tions containing [3H]IP2 and [“H]IP3 were collected by succesand seeded onto tissue culture plastic dishes as previously sive elution with 3 ml 90 mM HCl and 3 ml 500 mM HCl, described with the following modifications (22). Tissue culture respectively. After elution of [3H]IP2, the columns were washed dishes were coated with a solution of human placental collagen with an additional 3 ml 90 mM HCl before elution of [“H]IP, that was prepared by dissolving 50 mg collagen in 100 ml 0.2% with 500 mM HCl. Fractions were collected directly into 20-ml glacial acetic acid (8). The solution was filter-sterilized and scintillation vials; 10 ml scintillation fluid were added to each stored at -20°C. The culture medium consisted of a 1:1 mixture vial, and samples were counted in a Beckman 5801 liquid of Ham’s F12 and Dulbecco’s modified Eagle’s medium supplescintillation counter. Radioactive counts were corrected for mented with insulin (5 pg/ml), hydrocortisone (50 PM), epiderbackground and for loss of sample during extractions. 2) Dima1 growth factor (4 rig/ml), transferrin (5 pg/ml), fibronectin luted aqueous extracts were run through an 0.8 ml AG l-X8 (4 pg/ml), trace elements, selenium (10 nM), 5% newborn calf (Bio-Rad) resin in the formate form (200-400 mesh). Inositol serum, penicillin (100 U/ml), streptomycin (100 pg/ml), and phosphates were separated by sequential elution with increastobramycin (50 pg/ml). Cultures were incubated at 37°C in a ing concentrations of ammonium formate in 0.1 M formic acid humidified atmosphere in 95% air-5% C02. Culture medium (31). The column first was eluted with 16 ml 50 mM sodium was changed every 3 days. In some experiments, cell cultures formate, 5 mM sodium tetraborate to remove 3H-labeled inowere incubated with medium that was supplemented with 100 sitol and glycerophosphoinositol. Three fractions containing ng PTX/ml for 2 h at 37°C in a CO2 incubator. phosphate esters of myo-inositol were next collected as follows: Isotopic labeling of ceZZuZar phosphoinositides. Tracheal and IP with 16 ml 200 mM ammonium formate, 100 mM formic nasal epithelial cell phosphoinositides were isotopically labeled acid, IP2 with 20 ml 400 mM ammonium formate, 100 mM by “H-labeling using 3 &i myo-[3H]inositol in l-ml culture formic acid, and IP3/IP4 with 12 ml of 1 M ammonium formate, 100 mM formic acid. Radioactivity in aliquots of these fractions medium for 72 h. Radiolabel was added to cell cultures that were at or near confluence. Experiments reported here were was determined by scintillation counting. Radioactive counts

dence, for the first time, of pertussis toxin (PTX)sensitive aI-adrenergic-induced polyphosphoinositide breakdown and inositol phosphate accumulation that is consistent with activation of PLC in normal AEC. CF nasal epithelial cells displayed a more rapid metabolism of the inositol phosphates induced with I-epinephrine or methoxamine compared with normal tracheal epithelial cells. This may represent a tissue-specific difference rather than a disease-related difference.

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were corrected as described in 1. Because comparable results were obtained with both methods of inositol phosphate analysis, results were pooled and reported accordingly. Analysis of polyphosphatidylinositides. Pooled lipid extracts were evaporated to dryness under N2, and the residue was dissolved in 50 ~1 of chloroform-methanol (2:1, vol/vol). An aliquot (40 ~1) of each sample was spotted onto oxalate-impregnated silica gel G-25 thin-layer chromatography plates (plastic backed) (15, 17). Phospholipids were developed in a solvent system consisting of chloroform-acetone-methanol-acetic acidHz0 (160:60:52:48:32, vol/vol/vol/vol/vol) (13). The lipids were visualized by exposure to iodine vapor. Phosphoinositides and other phospholipids were identified by comparison of retardation factors to those of authentic lipid standards. To determine the radioactive content of the lipids, spots were cut from the plate and extracted with 1 ml methanol. Scintillation fluid (10 ml) was added, and vials were counted in a liquid scintillation counter. Data analysis. Phosphatidylinositide content and inositol phosphate levels were calculated as total disintegrations per minute per microgram DNA. DNA was quantitated with Hoechst dye with calf thymus DNA as the standard (6, 7). Basal levels were defined as those obtained from cells incubated with carrier, but not drugs, and were determined at each time point indicated in Tables 1-3 and Fig. l-6. For cells incubated with drugs of interest, data were calculated, at each time point, as a comparison or ratio of experimental disintegrations per minute per microgram DNA divided by basal disintegrations per minute per microgram DNA. Levels of significance were determined using Student’s t test for paired samples or for comparison of population means. Materials. M3/0-[1,2-~H(N)l inositol (specific activity 2.2 TBq/ml or 60.8 Ci/mmol) was purchased from New England Nuclear (Boston, MA). Methoxamine HCl was generously supplied by Burroughs Wellcome (Research Triangle Park, NC), and prazosin HCl by Pfizer Pharmaceutical (New York). dlPropranolol HCl, I-epinephrine HCl, pertussis toxin, and thinlayer chromatography plates were purchased from Sigma Chemical (St. Louis, MO). Tissue culture media and supplies were purchased from GIBCO (Cleveland, OH) and Sigma. Formate and chloride resins were obtained from Bio-Rad Laboratories (Richmond, CA). All other chemicals were reagent grade. RESULTS

Effect of agonists on IP2 and IP3 levels. These studies were initiated to ascertain the signaling mechanism linking acl-adrenergic activation of NaCl(K) cotransport to a requirement for increased cytosolic Ca2+. One mechanism that operates in many cells involves mobilization of intracellular nonmitochondrial Ca2+ stores by the intracellular second messenger IPS. The kinetics of inositol phosphate metabolism is shown for normal and CF AEC in Fig. 1 and 2, respectively. In normal AEC, the endogeneous catecholamine l-epinephrine caused a transient 1.7fold increase in IP3 levels that peaked at 20 s (Fig. IA). IP2 reached a maximal 1.6-fold increase at 40 s. Although I-epinephrine interacts with both a- and ,& adrenergic receptors in airway epithelium, previous studies showed selective activation of bumetanide-sensitive chloride transport by a-adrenergic stimulation (23). This also pertains to the formation of IP3 and IP2 as seen in the data of Table 1. In cells pretreated with the ,& adrenergic antagonist dl-propranolol, peak and IP2

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levels induced by I-epinephrine did not significantly differ from those observed with cells not treated with dlpropranolol (P c 0.60 and P < 0.95, respectively). This finding indicates that activation of a-adrenergic receptors, but not ,8-adrenergic receptors, induces inositol phosphate formation. Previous studies showed that the aI-adrenergic agonist methoxamine stimulated bumetanide-sensitive chloride transport in normal and CF AEC (23). As seen in Fig. lB, this agonist also induced a transient accumulation of IP, and IP2 in normal AEC with peak levels attained at 20 and 40 s, respectively. In CF cells, l-epinephrine induced IP3 and IP2 accumulation but the kinetics of the response differed from that observed in normal AEC (Fig. 2A). A peak 2.2fold increase in IP3 levels in CF AEC occurred at 10 s after hormone stimulation in contrast with 20 s for normal AEC (compare Fig. 1A and 24). Maximal IP3 accumulation was not significantly higher than that in normal AEC (P < 0.15). Likewise, IP2 levels increased 2.1-fold after 20-s incubation with hormone. Although, peak IP2 levels were observed earlier than with normal cells the maximal IP2 levels attained were not significantly different from that in normal AEC (P < 0.20). Methoxamine induced a similar pattern of rapid inositol phosphate turnover in CF AEC (Fig. ZB). Comparison of maximal inositol phosphate levels, expressed as total disintegrations per minute per microgram DNA, reached by normal and CF AEC showed no significant difference, i.e., P < 0.30 for IP3 and P < 0.10 for IP2. Effects of agonists on inositol lipid metabolism. We next investigated whether receptor-mediated polyphosphoinositide breakdown may serve as a source of IPZ. To test this possibility, we measured polyphosphoinositide metabolism and its stimulation by endogeneous and aadrenergic agonists. Figure 3 shows the kinetics of PIP and PIP2 metabolism in hormone-treated normal AEC. I-Epinephrine maximally decreased PIP2 by 35% and PIP by 30%, and methoxamine lowered PIP2 levels by 30% and PIP by 30%. The decreased PIP and PIP2 levels in stimulated cells indicates metabolism of polyphosphoinositides. Lowest PIP2 and PIP levels occurred after 20s stimulation (Fig. 3, A and B). Although both hormones cause a rapid reduction in PIP2 and PIP levels, the kinetics of subsequent recovery differed. PIP2 recovery precedes PIP recovery in cells treated with 1-epinephrine and vice versa with methoxamine. Inositol lipid breakdown induced by l-epinephrine may include a ,&adrenergic component that is not seen with inositol phosphate metabolism (Table 1). To test this possibility, we pretreated cells with dl-propranolol to block ,&adrenergic receptors. Cells were challenged with l-epinephrine and inositol phosphate, and inositol lipid levels were determined. We found no significant difference in inositol lipid levels in stimulated cells untreated or pretreated with dl-propranolol (Table 1). These results indicate the absence of a ,&adrenergic component in lepinephrine stimulation of polyphosphoinositide breakdown. In CF AEC, I-epinephrine and methoxamine also caused a rapid reduction in PIP2 and PIP levels that did not differ significantly from normal cells in terms of

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Fig. 1. Kinetics of inositol phosphate accumulation in normal airway epithelial cells (AEC) in response to Zepinephrine (A) and methoxamine (B). Cultures were challenged with either HEPES-modified Hanks’ balanced salt solution (basal) or hormone (experimental) for indicated time intervals. Final concentration of hormone was 10 PM. Mean inositol 1,4,5trisphosphate (IPS) values, expressed as disintegrations per minute per microgram DNA, at 20 s were 427 t 93 (n = 12) for basal condition of no drug addition, 566 & 96 (n = 10) for I-epinephrine, and 502 t 143 (n = 8) for methoxamine. Mean inositol 1,4-bisphosphate (IP2) values were 224 t 29 (n = 12) for basal condition of no drug addition, 312 & 52 (n = 9) for I-epinephrine, and 301 t 98 (n = 9) for methoxamine. Variability in radiolabeling was observed from experiment to experiment and, hence, results were normalized to basal condition at each time point. Relative metabolite level was calculated as the ratio of total disintegrations per minute per microgram DNA obtained under experimental conditions compared with basal condition of no drug addition. Each time point represents mean of 4-14 experimental values. Bars represent SE. Circles, IP,; triangles, IP,. Significantly different from 1.0: *P c 0.05, **P c 0.01, ***p < 0.005, ****fcp< 0.001.

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Fig. 2. Kinetics of inositol phosphate accumulation in cystic fibrosis (CF) AEC in response to I-epinephrine (A) and methoxamine (B). Experimental conditions were same as described in Fig. 1. Mean IP3 values at 10 s were 379 & 68 (n = 10) for basal condition, 517 t 123 (n = 8) for I-epinephrine, and 681 t 263 (n = 9) for methoxamine. Mean IPz values were 285 * 57 (n = 7) for basal condition, 476 t 125 (n = 8) for Z-epinephrine, and 405 of: 116 (n = 9) for methoxamine. Each time point represents mean of 4-10 experimental values from same number of different patient tissue samples. Bars represent SE. Circles, IP,; triangles, IP2. Significantly different from 1.0: *P < 0.05, **P < 0.025.

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magnitude of inositol lipid decrease and in the time frame of the response (Fig. 4). Lowest PIP, and PIP levels were observed after 20-s incubation with either I-epinephrine or methoxamine. As with normal cells, these levels differed significantly from a ratio of 1.0 despite the small change in inositol lipid level. Sensitivity of hormone response to cu-adrenergic antagonist. Prazosine, an al-adrenergic antagonist, blocks

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methoxamine and I-epinephrine activation of NaCl(K) cotransport in human AEC (23) and I-epinephrinemediated potassium channel activation in canine tracheal epithelium (32). The results presented in Fig. 5 show that prazosin also blocks responsesto I-epinephrine in normal and CF AEC. Thus prazosin antagonized the reduction in PIP2 and PIP levels and elevation in IP3 and IP, levels induced by I-epinephrine. This indicates

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1.44t0.31 (7) 1.30t0.27 (4) 0.90&O. 10 (6) 0.93&O. 18 (7)

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AEC, this time point was 20 s. Changes in PIP, PIPZ, IP3, and IP, levels produced by I-epinephrine and methoxamine in the absence of PTX were significantly different from a baseline ratio of 1.0 (Table 2). In PTX-treated cells, the change in inositol lipid and inositol phosphate levels was reversed and significantly different from that observed in PTX-untreated cells. Indeed, metabolite levels observed in PTX-treated cells were not significantly different from unity (Table 2). This is consistent with decreased metabolism of polyphosphoinositides. In CF AEC, maximal hormonal effects occurred at 10 s for IP3 and 20 s for IP2, PIP2, and PIP. PTX reduced I-epinephrine-induced IP3 and IP2 accumulation from 2.15 to 1.11 and 1.77 to 1.06, respectively, at these time points. The lower inositol phosphate levels observed in PTX-treated cells were not significantly different from 1.0 (Table 3). PIP, and PIP breakdown were also decreased from 27 to 14% and from 24 to 5%, respectively (Table 3). The results suggest a pattern of reduced polyphosphoinositide breakdown. PTX also significantly affected the responses to the al-adrenergic agonist methoxamine. IP3 and IP, accumulation decreased from 1.85 to 0.81 and 1.31 to 0.99, respectively (Table 3). Changes in PIP and IP2 levels, although not significant, display a shift that is consistent with a model of PTX-inhibition of PLC activity.

Table 1. Independence of I-epinephrine response from ,8-adrenergic receptor Ratio

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Values are means t SE of ratio of experimental to control of no hormone addition for number of experiments in parentheses. IP3, inositol 1,4,5trisphosphate; IP2, inositol 1,4bisphosphate; PIPZ, phosphatidylinositol 4,5bisphosphate; PIP, phosphatidylinositol 4-phosphate. Radiolabeled cell cultures were incubated with 10 PM dl-propranolol for 30 s before addition of I-epinephrine. Cultures were treated with hormone for 20 s. Reaction was terminated and cultures processed as detailedin METHODS AND MATERIALS.

an al-adrenergic mechanism for polyphosphoinositide breakdown and inositol phosphate accumulation. This conclusion is supported by the data in Fig. 6, which demonstrate the sensitivity of methoxamine-induced responsesto prazosin. PTX inhibition of hormone-stimulated polyphosphoinositide metabolism. A common feature of receptormediated polyphosphoinositide turnover is the involvement of GTP-binding proteins, or G proteins, in the activation of PLC (26). One particularly useful tool used to identify a role for G proteins in receptor-PLC interactions is PTX, which ADP ribosylates and functionally inactives G proteins (12, 16). To test the effects of PTX on hormone-stimulated polyphosphoinositide metabolism, cells prelabeled with myo- [3H] inositol were treated with 100 ng PTX/ml culture medium for 2 h before experiments. This treatment did not significantly alter baseline radiolabeled PIP2 levels. Using information from the kinetics of inositol phosphate accumulation and inositol lipid breakdown shown in Figs. 1 and 2, we examined PTX effects at time points of maximal accumulation (IP3, IPZ) and of maximal breakdown (PIPZ, PIP) of inositol metabolite. In normal

DISCUSSION

Previous studies demonstrated activation of bumetanide-sensitive Cl transport in human AEC that is mediated by the endogeneous catecholamine I-epinephrine and the al-adrenergic agonist methoxamine (23). We now show that stimulation of human AEC with the same hormones that activate NaCl cotransport induces a rapid polyphosphoinositide turnover with a corresponding accumulation of IP3 (Figs. l-4) that is sensitive to the cyladrenergic antagonist prazosin (Figs. 5 and 6) and to PTX (Tables 2-3). These results are consistent with a signal transduction mechanism involving al-adrenergic-

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3. Inositol lipid breakdown in norAEC in presence of I-epinephrine (A) and methoxamine (B). Experimental conditions were same as described in Fig. 1. Inositol lipids were extracted and analyzed as described in METHODS AND MATERIALS. Mean phosphatidylinositol 4,5-bisphosphate (PIP2) values, at 20 s were 1,402 & 408 (n = 13) for basal condition, lJ60.9 t 296 (n = 8) for Zepinephrine, and 1,266 & 364 (n = 9) for methoxamine. Mean phosphatidylinosito1 4-phosphate (PIP) values were 3,632 t 1,497 (n. = 13) for basal condition, 3,087 t 870 (n = 10) for I-epinephrine, and 2,301 & 592 (n = 11) for methoxamine. Each time point represents mean of 3-13 experiments. Bars represent SE. Circles, PIP; triangles PIP2. Significantly different from 1.0: *p < 0.05, **P

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A Fig. 4. Inositol lipid breakdown in CF AEC in response to I-epinephrine (A) and methoxamine (B). Experimental conditions were same as described in Fig. 1. Mean PIP:! values, at 20 s were 916 t 163 (n = 8) for basal condition, 703 jr 266 (n = 7) for I-epinephrine, and 881 It 187 (n = 8) for methoxamine. Mean PIP values were 2,822 t 696 (n = 8) for basal condition, 1,906 t 243 (n = 7) for Zepinephrine, and 2,281 t 428 (n = 6) for methoxamine. Each time point represents mean of 3-8 experimental values from same number of different patient tissue samples. Bars represent SE. Circles, PIP; triangles, PIP,. Significantly different from 1.0: *p < 0.05, **p < 0.01.

***p c 0.0005. o.o!

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IP3

IP2

Fig. 5. Prazosin (Praz) blocks I-epinephrine-induced polyphosphoinositide breakdown and inositol phosphate accumulation in normal (A) and CF AEC (B). Cell cultures were preincubated with 1 PM prazosin before addition of I-epinephrine. Reaction was terminated after 20-s incubation and inositol lipids and inositol phosphates analyzed as described in METHODS AND MATERIALS. Each time point represents mean of 4-10 experimental values. Bars represent SE. Significantly different from 1.0: *P < 0.05, **P < 0.025, ***p < 0.01.

PIP

PIP2

IP3

IP2

Fig. 6. Prazosin sensitivity of methoxamine-stimulated polyphosphoinositide breakdown and inositol phosphate accumulation in normal (A) and CF AEC (B). Cell cultures were preincubated with 1 PM prazosin before addition of methoxamine. Reaction was terminated after 20-s incubation with indicated hormone and inositol lipids and inositol phosphates analyzed as described in METHODS AND MATERIALS. Each time point represents mean of 3-7 experimental values. Bars represent SE. Significantly different from 1.0: *p < 0.05, **P < 0.01,

***p < 0.005.

mediated activation of PLC with a role for PTX-sensitive G proteins in PIP, hydrolysis. These studies were done with normal and CF AEC in in vitro culture to allow labeling with myo- [3H]inositol, a precursor of polyphosphoinositides and inositol phosphates, reach radioisotopic eouilibrium. Cultured AEC provide an excellent

experimental system for study because they retain and express hormone receptors (11, 35, 36) and ion transporters (28, 32, 33, 35, 37) of intact tissue. Addition of L-epinephrine to normal human cells produced a rapid, yet transient, increase in IP3 and IP2

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Table 2. Sensitivity of hormonal response in non-CF cells to PTX

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PLC to IP,. This finding is particularly important because, potentially, direct hydrolysis of PIP to IP2 allows for regulation of IP3 and DAG levels. Additional inforRatio (Experimental/Basal) mation on the metabolism of PIP is necessary to deterInositol Metabolite mine the source of IP2. - PTX + PTX The kinetics of PIP, and PIP breakdown provide new I-Epinephrine information on inositol lipid metabolism in human AEC PIP 0.74t0.10 (6)" 1.41t0.29 (5)" (Figs. 3 and 4). Lowest PIP2 and PIP levels were attained PIP2 0.64t0.12 (5)' 1.45rto.19 (qb after 20-s incubation with I-epinephrine or methoxamine. 1.68t0.19 (13)" 1.05kO.24 (5)b IP3 Recovery, however, is characterized by increased radio1.33t0.16 (12)" 1.03t0.31 (5)b I& labeling of PIP2 and PIP to levels >l.O, suggesting that Methoxamine I-epinephrine stimulates resynthesis of PIP, and PIP PIP 0.64kO.05 (8)e 1.43kO.17 (4)b" (Figs. 3 and 4). Pinpointing the exact synthetic pathway PIP2 0.70t0.10 (5)d 1.12t0.10 (4)b is difficult because of the intricate cycle of phosphatases 1.59t0.22 (6)" 0.72kO.27 (3)b IP3 and kinases that link polyphosphoinositides and inositol 1.49kO.33 (10) 0.83t0.26 (5)b I& phosphates. One can conjecture that direct conversion of Values are means t SE of ratio of experimental to basal for number IP, to PIP is followed by phosphorylation to PIP2 to of experiments in parentheses. CF, cystic fibrosis. Pertussis toxin (PTX) was added to cell cultures prelabeled with myo-[3H]inositol to regain steady-state levels of both polyphosphoinositides. a final concentration of 100 rig/ml. Cell cultures were incubated for 2 This scheme may involve a pathway that circumvents a h at 37°C in humidified atmosphere of 5% COz. Culture medium was Li+-sensitive enzyme myo-inositol 1-phosphatase. discarded and cell cultures prepared for experiments as described in In CF AEC, I-epinephrine and methoxamine also inMETHODS AND MATERIAL. Airway epithelial cells (AEC) were incubated duce a rapid, yet transient, polyphosphoinositide breakwith HEPES-modified Hanks’ balanced salt solution (control) or hormone at 10 PM final concentration for 20 s. a P < 0.05, b P < 0.0005, down (Fig. 4) that is sensitive to prazosin, an crl-adrecomparison of PTX-untreated with PTX-treated AEC. ’ P < 0.05, d P nergic antagonist (Fig. 5B and 6B). However, IP3 and < 0.025, e P < 0.0025, compared with ratio of 1.0. IP, accumulation induced by either agonist peaks at least 10 s earlier than in normal cells (Fig. 2). The rapid Table 3. PTX sensitivity of hormonal response hormone-induced inositol phosphate metabolism in CF in CF airway epithelial cells AEC may be more apparent than real as the source of Ratio (Experimental/Basal) cells from CF and normal tissue differed. Clearly, studies Inositol on normal nasal epithelium or CF tracheal epithelium Metabolite - PTX + PTX are needed to clarify the difference in kinetic response. I-Epinephrine We report the first observation that, in human AEC, al-adrenergic-stimulated breakdown of polyphosphoinPIP 0.76t0.13 (8) 0.9520.07 (5) PIP, 0.73t0.14 (7) 0.86t0.08 (5) ositides involves a PTX-sensitive GTP-dependent mech2.15t0.34" (7) l.ll+0.14b (5) I& anism. Observations on I-epinephrine and methoxaminel.77+0.34d (7) 1.06t0.18" (6) I& induced responses in normal AEC preincubated with Methoxamine PTX show a significant reduction in inositol phosphate PIP 0.73t0.13 (6) 1.12kO.17 (4) accumulation and inositol lipid breakdown (Table 2). PIP, 0.68t0.08" (7) 1.30t0.12" (4) These findings suggest that PTX-sensitive GTP-binding l.85+0.3gd (6) 0.81+0.02b (6) I& proteins, or G proteins, serve to couple the al-adrenergic 1.3lt0.18 (8) 1.06kO.18 (6) I& receptor to PLC and thus promote activation of this Values are means & SE of ratio of experimental to basal for number enzyme. In CF AEC, PTX did not uniformly affect all of experiments in parentheses. Cell cultures were treated with 100 ng inositol metabolites (Table 3). This can be explained by pertussis toxin (PTX) per milliliter culture medium as described in the variability in metabolite levels in PTX untreated legend to Table 2. Data are presented for inositol lipids after 20-s incubation with buffer or hormone and for inositol phosphates after cells. As seen in Table 3, in the absence of PTX, metab10-s incubation. a P < 0.05, b P < 0.007, ’ P < 0.0005, comparison of olites with a high standard error, i.g., PIP and PIP, with PTX-untreated with PTX-treated AEC. d P < 0.05, e P < 0.01, comI-epinephrine and PIP and IP, with methoxamine, did pared with ratio of 1.0. not have ratios significantly different from unity. Neveraccumulation that coincides with metabolism of the polytheless, PTX generally shifted polyphosphoinositide mephosphoinositides PIP2 and PIP (Figs. 1 and 3). This tabolism, causing decreased inositol lipid breakdown and result and the similarity in the magnitude of cell re- reduced inositol phosphate accumulation (Table 3). sponsesare consistent with hormonal activation of PLC Overall, metabolite levels in PTX-treated cells were not through a membrane receptor. Peak accumulation of IP3 significantly greater than unity. occurred -10-20 s before maximal IPB accumulation in Alternative explanations for the lack of effect of PTX both CF and normal cells, indicating a more rapid accu- in CF AEC include the presence of tissue-specific submulation of IP3 (Fig. 1). This shift in time frame suggests types of a-adrenergic receptors or the interaction of the that IP, is a metabolite of IP3. However, an alternative intracellular second messengers linked to al- and ,8explanation comes from studies that indicate direct hy- adrenergic receptors, in the regulation of polyphosphoidrolysis of three phosphoinositides (PI, PIP, PIP& by nositide breakdown. The first casebears analogy to musPLC (25). The data of Figs. l-4 show that the breakdown carinic receptor subtypes Ml-M5 in which subtypes M1, MS, and MS produce PTX-insensitive responses and M, of PIP generally precedes maximal accumulation of IP, by 20 s. This may occur if PIP is hydrolyzed directly by and Mq PTX-sensitive responses (5). CF nasal epithelial Downloaded from www.physiology.org/journal/ajplung at Karolinska Institutet University Library (130.237.122.245) on February 12, 2019.

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cells may express an cul-adrenergic receptor subtype with the sensitivity to PTX as a distinguishing characteristic. In the second case, changes in CAMP content in cells as diverse as tracheal smooth muscle, platelets, and ,&cell of islets of Langerhans (27) are thought to either provide positive or negative modulation of protein kinases and protein phosphatases. This may have relevance to CF, a genetic disease characterized by expression of an abnormal nucleotide-binding protein (29). Incomplete inhibition of I-epinephrine or methoxamine responses in CF AEC may also reflect an alternative pathway for hydrolysis of polyphosphoinositides by a PTX-insensitive mechanism. One possible mechanism involves the formation of phosphatidic acid from DAG with subsequent activation, or continued activation, of PLC (18). This suggests increased DAG kinase activity in CF AEC. Within this scenario, agonist-stimulated PLC induces the breakdown of polyphosphoinositides to IP3 and DAG. IP3 release mobilizes intracellular Ca’+, which, in turn, activates Ca2+-dependent enzymes, such as protein kinase C. DAG kinase metabolizes DAG to phosphatidic acid. If DAG kinase activity is increased in CF AEC, phosphatidic acid accumulates as DAG is metabolized and provides continued PLC hydrolysis of polyphosphoinositides. Protein kinase C may also inhibit PLC through negative feedback (30). Regulation of PLC may then involve a balance between DAG accumulation and degradation. W ‘hether there is an imbalance in DAG metabolism in CF AEC has yet to be determined. Because inositol phosphates are metabolized to IP, which accumulates in the presence of Li+, we focused here on determining the kinetics of polyphosphoinositide breakdown and accumulation of the intracellular messenger IP3 and its breakdown product IP2. These studies provide the first evidence for hydrolysis of polyphosphoinositides as the mechanism of signal transduction of aladrenergic receptors in human AEC. Reports of hormone-induced inositol phosphate accumulation in canine tracheal epithelium did not take into account the species of inositol phosphate affected by agonists (2); hence, very limited information on the kinetics of IP3 and IP2 metabolism in canine AEC is available. Nevertheless, cxadrenergic-induced accumulation of inositol phosphates did not correlate with L-epinephrine-induced chloride secretion but may instead indirectly regulate basolateral potassium channels (32). These reports plus our results linking al-adrenergic-induced polyphosphoinositide breakdown and activation of NaCl cotransport suggest that ac-adrenergic receptors regulate two basolateral transporters required for chloride secretion: NaCl(K) cotransport and potassium channels. Activation of these basolateral transporters depends on intracellular Ca2+ (23, 34), which is regulated, in many cell types, by an IP3-dependent mechanism. Whether IP3 increases cytosolic Ca2+ in human AEC and thereby activates NaCl cotransport has yet to be addressed. The answer to this and other questions concerning the regulation of polyphosphoinositide breakdown, the mechanism of IP3mediated Ca2+ mobilization, and the mechanism of Ca2+dependent transporter activation may have implications in developing therapeutics for CF that circumvent the

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basic . . defect and thus restore ride.

the ability

to secrete chlo-

The author acknowledges stimulating discussions with Dr. George Dubyak. This work was done during the tenure of an Established Investigatorship from the American Heart Association. This work was supported by National Heart, Lung, and Blood Institute Grant HL-43907, National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-27651, and the National Cystic Fibrosis Foundation Grant Z-0338. Address for reprint requests: Dept. of Pediatrics, Case Western Reserve Univ., 2101 Adelbert Rd., Cleveland, OH 44106. Received

11 July

1991; accepted

in final

form

30 August

1991.

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