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Involvement of Direct Phosphorylation in the Regulation of the Rat Parotid Na+-K+-2Cl− Cotransporter

1995; Elsevier BV; Volume: 270; Issue: 42 Linguagem: Inglês

10.1074/jbc.270.42.25252

1083-351X

Akihiko Tanimura, Kinji Kurihara, Stephan J. Reshkin, R Turner,

Drug Transport and Resistance Mechanisms

We identify a 175-kDa membrane phosphoprotein (pp175) in rat parotid acini whose properties correlate well with the Na+-K+-2Cl− cotransporter previously characterized functionally and biochemically in this tissue. pp175 was the only phosphoprotein immunoprecipitated by an anti-Na+-K+-2Cl− cotransporter antibody and the only membrane protein whose phosphorylation state was conspicuously altered after a brief (45-s) exposure of acini to the β-adrenergic agonist isoproterenol. Phosphopeptide mapping provided evidence for three phosphorylation sites on pp175, only one of which was labeled in response to isoproterenol treatment. The half-maximal effect of isoproterenol on phosphorylation of pp175 (≈20 nM) was in excellent agreement with its previously demonstrated up-regulatory effect on cotransport activity. Increased phosphorylation of pp175 was also seen following acinar treatment with a permeant cAMP analogue and with forskolin, conditions that have likewise been shown to up-regulate the cotransporter. Combined with earlier results from our laboratory, these data provide strong evidence that the up-regulation of the cotransporter by these agents is due to direct phosphorylation mediated by protein kinase A. AlF4− treatment, which results in an up-regulation of cotransport activity comparable with that observed with isoproterenol (∼6-fold), caused a similar increase in phosphorylation of pp175. However, hypertonic shrinkage and treatment with the protein phosphatase inhibitor calyculin A, which also up-regulate the cotransporter (∼3-fold and ∼6-fold, respectively) caused no change in the phosphorylation level. Furthermore, although acinar treatment with the muscarinic agonist carbachol results in a dramatic up-regulation of cotransport activity and a concomitant phosphorylation of pp175, no phosphorylation of pp175 was seen with the Ca2+-mobilizing agent thapsigargin, which is able to fully mimic the up-regulatory effect of carbachol on transport activity. Taken together, these results indicate that direct phosphorylation is only one of the mechanisms involved in secretagogue-induced regulation of the rat parotid Na+-K+-2Cl− cotransporter. We identify a 175-kDa membrane phosphoprotein (pp175) in rat parotid acini whose properties correlate well with the Na+-K+-2Cl− cotransporter previously characterized functionally and biochemically in this tissue. pp175 was the only phosphoprotein immunoprecipitated by an anti-Na+-K+-2Cl− cotransporter antibody and the only membrane protein whose phosphorylation state was conspicuously altered after a brief (45-s) exposure of acini to the β-adrenergic agonist isoproterenol. Phosphopeptide mapping provided evidence for three phosphorylation sites on pp175, only one of which was labeled in response to isoproterenol treatment. The half-maximal effect of isoproterenol on phosphorylation of pp175 (≈20 nM) was in excellent agreement with its previously demonstrated up-regulatory effect on cotransport activity. Increased phosphorylation of pp175 was also seen following acinar treatment with a permeant cAMP analogue and with forskolin, conditions that have likewise been shown to up-regulate the cotransporter. Combined with earlier results from our laboratory, these data provide strong evidence that the up-regulation of the cotransporter by these agents is due to direct phosphorylation mediated by protein kinase A. AlF4− treatment, which results in an up-regulation of cotransport activity comparable with that observed with isoproterenol (∼6-fold), caused a similar increase in phosphorylation of pp175. However, hypertonic shrinkage and treatment with the protein phosphatase inhibitor calyculin A, which also up-regulate the cotransporter (∼3-fold and ∼6-fold, respectively) caused no change in the phosphorylation level. Furthermore, although acinar treatment with the muscarinic agonist carbachol results in a dramatic up-regulation of cotransport activity and a concomitant phosphorylation of pp175, no phosphorylation of pp175 was seen with the Ca2+-mobilizing agent thapsigargin, which is able to fully mimic the up-regulatory effect of carbachol on transport activity. Taken together, these results indicate that direct phosphorylation is only one of the mechanisms involved in secretagogue-induced regulation of the rat parotid Na+-K+-2Cl− cotransporter. INTRODUCTIONBecause of its experimental accessibility, relative homogeneity and rich hormonal responsiveness, the rat parotid gland is rapidly becoming one of the more popular mammalian experimental models for the study of the mechanism(s) and regulation of epithelial fluid and electrolyte secretion(1Nauntofte B. Am. J. Physiol. 1992; 263: G823-G837PubMed Google Scholar, 2Turner R.J. Ann. N. Y. Acad. Sci. 1993; 694: 24-35Crossref PubMed Scopus (57) Google Scholar). Work from a number of laboratories has established that salt and water secretion by the acinar cells, which comprise the bulk of this gland, is due to transepithelial Cl− movement(1Nauntofte B. Am. J. Physiol. 1992; 263: G823-G837PubMed Google Scholar, 2Turner R.J. Ann. N. Y. Acad. Sci. 1993; 694: 24-35Crossref PubMed Scopus (57) Google Scholar, 3Petersen O.H. Maruyama Y. Nature. 1984; 307: 693-696Crossref PubMed Scopus (475) Google Scholar). The active step in this process is Cl− entry across the acinar basolateral membrane, a large component of which has been shown to be due to Na+-K+-2Cl− cotransport(4Melvin J.E. Kawaguchi M. Baum B.J. Turner R.J. Biochem. Biophys. Res. Commun. 1987; 145: 754-759Crossref PubMed Scopus (53) Google Scholar, 5Melvin J.E. Turner R.J. Am. J. Physiol. 1992; 262: G393-G398PubMed Google Scholar).Consistent with its important role in secretion, we have shown that the activity of the rat parotid Na+-K+-2Cl− cotransporter is regulated by a number of physiological and other potentially physiologically relevant stimuli. We first demonstrated a substantial (∼6-fold) up-regulation of cotransporter activity following β-adrenergic stimulation and provided good evidence that this was due to a phosphorylation event mediated by cyclic AMP-dependent protein kinase(6Paulais M. Turner R.J. J. Clin. Invest. 1992; 89: 1142-1147Crossref PubMed Scopus (58) Google Scholar). This up-regulation is paralleled in vivo by an increase in salivary flow seen when sympathetic (adrenergic) stimulation, arising, for example, from mastication, is superimposed on parasympathetic (muscarinic) stimulation(7Johnson D.J. Sreebny L.M. The Salivary System. CRC Press, Inc., Boca Raton, FL1987: 135-155Google Scholar), the main fluid secretory stimulus for the gland. In a later publication (8Paulais M. Turner R.J. J. Biol. Chem. 1992; 267: 21558-21563Abstract Full Text PDF PubMed Google Scholar) we demonstrated that the rat parotid Na+-K+-2Cl− cotransporter is up-regulated (again ∼6-fold) by aluminum fluoride (AlF4−), an activator of G-proteins, and by calyculin A, a protein phosphatase inhibitor. Based on several factors, including diverse sensitivity to blockade of up-regulation by protein kinase inhibitors and the observation that AlF4− does not induce cAMP generation in the rat parotid, we have argued that the mechanisms of action of AlF4− and calyculin A on the cotransporter are different from that of β-adrenergic stimulation and from one another(8Paulais M. Turner R.J. J. Biol. Chem. 1992; 267: 21558-21563Abstract Full Text PDF PubMed Google Scholar).More recently (9Evans R.L. Turner R.J. Mol. Biol. Cell. 1994; 5 (abstr.): 116aGoogle Scholar) 1C. Ferri, R. L. Evans, M. Paulais, A. Tanimura, and R. J. Turner, submitted for publication. we have shown that Na+-K+-2Cl− cotransport activity in these cells is also increased by muscarinic stimulation (>15-fold) and by hypertonic shrinkage (∼3-fold). Our data suggest that these latter effects are also unrelated to one another and unrelated to the effect of β-adrenergic stimulation (see "Discussion"). At this time our understanding of these up-regulatory events is still incomplete, and the physiological significance of some of these stimuli remains to be determined. However, our results clearly demonstrate that the rat parotid Na+-K+-2Cl− cotransporter is under tight regulatory control, in all likelihood by multiple intracellular signaling pathways, and thus that it provides a particularly rich experimental system for the study of transport regulation by hormonal and other stimuli.In the present paper we explore these phenomena further by studying the effects of these various up-regulatory stimuli on the phosphorylation state of the Na+-K+-2Cl− cotransport protein itself. Although it is generally accepted that phosphorylation events play an important role in cellular signaling, relatively few studies have actually directly explored their possible involvement in the regulation of facilitative membrane transport proteins. We show here that there is a good correlation between increased transport activity and increased transporter phosphorylation following β-adrenergic stimulation and AlF4− treatment of rat parotid acini, suggesting that the regulation of the cotransporter by these stimuli is due to direct phosphorylation. Somewhat surprisingly, however, this was not the case for the other stimuli studied, in spite of the fact that some of these agents have been shown to increase both the transport activity and the phosphorylation state of Na+-K+-2Cl− cotransporters in lower species(11Lytle C. Forbush III, B. J. Biol. Chem. 1992; 267: 25438-25443Abstract Full Text PDF PubMed Google Scholar, 12Torchia J. Yi Q. Sen A.K. J. Biol. Chem. 1994; 269: 29778-29784Abstract Full Text PDF PubMed Google Scholar). These observations indicate that the Na+-K+-2Cl− cotransporter in the rat parotid is regulated both via direct phosphorylation and via other, as yet unidentified, mechanisms.EXPERIMENTAL PROCEDURESMaterials and MediaMale Wistar strain rats, weighing 250-300 g, were purchased from Harlan Sprague-Dawley (Indianapolis, IN). Carrier-free 32Pi (10 mCi/ml) and [3H]bumetanide (80.8 Ci/mmol) were obtained from Amersham Corp. Collagenase P, protease inhibitors, and dibutyryl cAMP were from Boehringer Mannheim.(-)Isoproterenol, phorbol 12-myristate 13-acetate (PMA), 2The abbreviations used are: PMAphorbol 12-myristate 13-acetatePAGEpolyacrylamide gel electrophoresisPSSphysiological salt solution. V8 protease, and bovine serum albumin (number A6003) were purchased from Sigma. Calyculin A and forskolin were from Calbiochem. Phosphatidylserine (number 840032, from bovine brain; supplied in chloroform) was from Avanti Polar Lipids (Birmingham, AL). Molecular weight standards, prepoured 4-20% SDS-PAGE gels, and prepoured 16% Tricine gels were obtained from Novel Experimental Technology (San Diego, CA). Protein G-Sepharose beads were from Pierce. All other chemicals were from standard commercial sources and were reagent grade or the highest purity available.The digestion medium was Earle's minimum essential medium (Biofluids, Rockville, MD) containing 0.22 units/ml collagenase P, 2 mM glutamine, and 1% bovine serum albumin. The physiological salt solution (PSS) contained 135 mM NaCl, 5.8 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4, 0.73 mM NaH2PO4, 11 mM glucose, 20 mM HEPES (pH 7.4 with NaOH), 2 mM glutamine, and 1% bovine serum albumin. The digestion medium and PSS were continuously gassed with 95% O2, 5% CO2 and 100% O2, respectively. The stop solution for the 32Pi labeling studies contained 100 mM NaCl, 20 mM HEPES (pH 7.4 with NaOH), 10 mM Na2ATP, 50 mM NaF, 15 mM sodium pyrophosphate, 100 μM sodium orthovanadate, 5 mM EDTA, 300 μM phenylmethylsulfonyl fluoride, 100 μML-tosylamido-2-phenylethyl chloromethyl ketone, 1.5 μM pepstatin, and 1.5 μM leupeptin.Protein concentration was measured with the BCA protein assay system (Pierce).Preparation of Parotid AciniRats were anesthetized with diethyl ether and killed by cardiac puncture. The parotid glands were removed, dissected free of fat, and finely minced in a small volume of ice-cold digestion medium. The glands from two rats were suspended in 10 ml of digestion medium and incubated at 37°C with continuous agitation. The mince was dispersed by gently pipetteting 10 times with a 10-ml plastic pipette after 30 min and again after 45 min of digestion. The mince was then centrifuged (400 × g, 15 s), the supernatant was discarded and replaced with the same volume of fresh digestion medium, and incubation was continued. After a total of 60 min of digestion the mince was pipetted 10 times with a 10-ml pipette fitted with a Rainin RC-200 blue pipette tip (Rainin Instruments, Emeryville, CA). Finally at times 75 and 85 min the mince was pipetted five times with a 10-ml pipette fitted with a Rainin RT-96 yellow tip. The resulting suspension was centrifuged and resuspended in 10 ml of PO4−-free-PSS (PSS without NaH2PO4) and passed through a 450-μm nylon screen (PGC Scientific, Gaithersburg, MD). The material in the filtrate was collected by centrifugation, resuspended in 10 ml of fresh PO4−-free PSS, and recentrifuged, then resuspended in 3-4 ml of PO4−-free PSS.32PiLabeling and StimulationAcini were incubated at 37°C in PO4−-free-PSS in the presence of 50 μCi/ml 32Pi for 30 min. The suspension was then placed on the bench for 1-2 min to allow the acini to settle, and the supernatant was carefully removed and discarded. The remaining material was diluted to 10 ml with PSS, washed once in the same volume, and finally resuspended in 3-4 ml of PSS at room temperature.Aliquots (200 μl) of labeled cells were incubated with the agents indicated at 37°C in siliconized glass tubes (Sigmacote number SL-2; Sigma). The incubation was terminated by the addition of 800 μl of ice-cold stop solution and disruption by immersion in a Branson B-12 sonicator bath (Shelton, CT) as follows. Each sample was first sonicated for 30 s and then placed on ice. After disruption of all samples in this way, each sample was subsequently sonicated to clarity.Preparation of Membrane ExtractSonicated samples (1 ml total volume) were centrifuged at 1000 × g for 10 min, and the resulting pellet was discarded. The supernatant was centrifuged at 100,000 × g for 1 h. We refer to the supernatant and pellet from this high speed spin as the "cytosolic fraction" and the "particulate fraction," respectively. The particulate fraction was resuspended in 0.7 ml of extraction buffer (stop solution containing 0.3% Triton X-100 and no NaCl) and kept on ice for 30 min. This sample was then centrifuged at 100,000 × g for 30 min. The supernatant and pellet from this second high speed spin are referred to as the "Triton extract" and the "Triton-insoluble fraction" respectively. As shown below (see "Results") the Triton extract was the only fraction that contained the phosphoprotein of interest (the Na+-K+-2Cl− cotransporter), and thus only this fraction was usually retained and analyzed.Gel Electrophoresis and AutoradiographySDS-PAGE was performed essentially as described by Laemmli (13Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205955) Google Scholar) using a 4% polyacrylamide stacking gel and a 4-20% (continuous gradient) polyacrylamide separating gel. Tricine-SDS (16%) electrophoresis was carried out according to Schagger and von Jagow(14Schagger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10434) Google Scholar). Samples were heated at 100°C for 2 min in sample buffer containing 2.5% SDS, 50 mM Tris-HCl (pH 6.8), 4% glycerol, 100 mM dithiothreitol, and 0.04% bromphenol blue and centrifuged before electrophoresis. Gels were stained with Coomassie Blue, dried, and visualized by autoradiography using Kodak X-Omat AR film (Eastman Kodak, Rochester, NY). Gels used in phosphopeptide mapping studies were washed twice for 15 min in 20% ethanol before drying to remove acetic acid. Autoradiographs were scanned using a Molecular Dynamics computing densitometer (Molecular Dynamics, Sunnyvale, CA) to quantitate 32Pi labeling. Linearity of the densitometric scans was confirmed using autoradiographic 14C Micro-Scales (Amersham Corp.). Samples of Triton extract electrophoresed for quantitation of 32Pi labeling typically contained ∼15 μg of protein (the protein concentration of the Triton extract was ∼0.5 mg/ml).Production of Antiserum against Parotid Bumetanide Binding ProteinThe following method was used to produce sheep antiserum directed against a rabbit parotid bumetanide binding protein previously identified in our laboratory as the bumetanide moiety (and perhaps all) of the Na+-K+-2Cl− cotransporter in this tissue(15Reshkin S.J. Lee S.L. George J.N. Turner R.J. J. Membr. Biol. 1993; 136: 243-251Crossref PubMed Scopus (13) Google Scholar). Deglycosylated bumetanide binding protein (Mr∼135,000) was purified from rabbit parotid basolateral membranes as described previously(15Reshkin S.J. Lee S.L. George J.N. Turner R.J. J. Membr. Biol. 1993; 136: 243-251Crossref PubMed Scopus (13) Google Scholar). A suitable quantity of protein (100 μg for the first injection and 30-40 μg for subsequent injections) was diluted to 1 ml with phosphate-buffered saline and combined with 1 ml of complete (first injection) or incomplete (subsequent injections) Freund's adjuvant. Injections were carried out at the NIH Animal Care Center (Ungulate Section) in Poolesville, Maryland. The primary injection was subcutaneous, and subsequent injections (3, 8, 19, and 36 weeks later) were intramuscular; all injections were done at multiple sites. Anti-bumetanide binding protein antibody titer in serum samples was monitored by Western blot analysis against purified bumetanide binding protein and rabbit parotid basolateral membranes. In these samples the antiserum strongly labeled proteins of Mr 135,000 and 160,000-175,000, respectively. 3S. J. Reshkin, S. J. Tessler, M. L. Moore, and R. J. Turner, unpublished results. The range of molecular weights observed for the labeling of native membranes (160-175 kDa) was related to the gel system used. With the large format Bio-Rad gels used in our previous publication (15Reshkin S.J. Lee S.L. George J.N. Turner R.J. J. Membr. Biol. 1993; 136: 243-251Crossref PubMed Scopus (13) Google Scholar) we observed an Mr of 160,000 (in this same publication we showed that the parotid bumetanide binding protein had a native Mr of 160,000 and a deglycosylated Mr of 135,000), while with the prepoured Novex minigels used in the present work we consistently observed a Mr of ∼175,000.Immunoprecipitation of 32Pi-Labeled ProteinsProtein G-Sepharose beads were prewashed twice with washing buffer (extraction buffer titrated to pH 8.6 with Tris and containing 0.1% SDS and 300 mM NaCl), once in low pH buffer (50 mM glycine-HCl, pH 2.3, containing 150 mM NaCl and 0.5% Triton X-100), and once in extraction buffer and then suspended in extraction buffer containing 1% ovalbumin.A 400-μl aliquot of Triton extract was incubated overnight at 4°C with immune or non-immune sheep serum (7 μl/100 μg of extract protein). Prewashed protein G-Sepharose beads (10 μl of beads/μl of serum) were then added. After 30 min of additional incubation, the beads were collected by centrifugation and washed six times with washing buffer. The tube was changed for the last spin. Protein retained by the washed beads was then eluted with 100 μl of electrophoresis sample buffer.Immunoprecipitation of [3H]Bumetanide Binding ActivityTriton extracts for bumetanide binding studies were prepared by a modification of the procedure given above. This modification was based on our previous observation that the bumetanide binding activity of the Na+-K+-2Cl− cotransporter could be preserved in detergent solutions by the addition of suitable exogenous lipids(16Turner R.J. George J.N. J. Membr. Biol. 1990; 113: 203-210Crossref PubMed Scopus (8) Google Scholar, 17Corcelli A. Turner R.J. J. Membr. Biol. 1991; 120: 125-130Crossref PubMed Scopus (1) Google Scholar). The procedure was as follows. A "particulate fraction" was prepared as described above from cells that had not been labeled with 32Pi. However, instead of extraction buffer, the particulate fraction was resuspended in Buffer K (100 mM mannitol, 10 mM HEPES, 1 mM EDTA, 195 mM potassium gluconate, and 5 mM KCl buffered to pH 7.4 with Tris). This material was centrifuged again at 100,000 × g for 1 h, and the resulting pellet was resuspended in Buffer K at a protein concentration of 2.5-3.5 mg/ml, fast frozen in aliquots, and stored above liquid nitrogen. On the day of the bumetanide binding experiment, frozen samples were thawed and diluted with Buffer K to a protein concentration of 2 mg/ml. This suspension was mixed with the same volume of 0.6% Triton X-100 in Buffer K, left on ice for 10 min, and then transferred to a glass tube in which a suitable volume of phosphatidylserine (final concentration 0.15%) had been evaporated. This mixture was sonicated to clarity in a Branson B-12 bath sonicator (∼60-s immersion) and centrifuged at 100,000 × g for 1 h. The resulting supernatant, which is analogous to the "Triton extract" described above for 32Pi labeling studies, is referred to here as the "lipid-stabilized Triton extract."Immunoprecipitation of [3H]bumetanide binding activity from the above lipid-stabilized Triton extracts was carried out using immune and nonimmune IgG preabsorbed onto protein G-Sepharose beads. This was done in order to avoid any possible interference of serum with the [3H]bumetanide binding assay and to allow quantitation of protein remaining after immunoprecipitation (see "Results"). Protein G beads were washed twice with Buffer K containing 0.3% Triton X-100 and 0.15% phosphatidylserine (sonicated to clarity as above) and then resuspended in the same buffer containing 1% ovalbumin and incubated for 40 min with immune or nonimmune sheep serum (10 μl of beads/μl of serum; total volume ∼300 μl). The beads were then washed three times in Buffer K plus 0.3% Triton X-100 and 0.15% phosphatidylserine, added to the lipid-stabilized Triton extract (70 μl of beads/100 μg of extract protein), and incubated for 2 h at 4°C. After removal of the beads by centrifugation, [3H]bumetanide binding activity remaining in the resulting supernatant was determined by the method given below.[3H]Bumetanide Binding AssayEquilibrium bumetanide binding was measured using a nitrocellulose filtration assay as described previously(16Turner R.J. George J.N. J. Membr. Biol. 1990; 113: 203-210Crossref PubMed Scopus (8) Google Scholar, 18Turner R.J. George J.N. J. Membr. Biol. 1988; 102: 71-77Crossref PubMed Scopus (20) Google Scholar). Briefly, a 20-μl aliquot of sample was combined with 20 μl of incubation medium consisting of either Buffer K containing 10 μCi/ml [3H]bumetanide or the same medium with all potassium replaced by Na+. After a 15-min incubation the reaction was terminated by the addition of ice-cold stop solution followed by Millipore filtration (HAWP 0.45 μm). Other procedures were as described previously(16Turner R.J. George J.N. J. Membr. Biol. 1990; 113: 203-210Crossref PubMed Scopus (8) Google Scholar, 18Turner R.J. George J.N. J. Membr. Biol. 1988; 102: 71-77Crossref PubMed Scopus (20) Google Scholar). [3H]bumetanide binding observed in the absence of Na+ was subtracted from that observed in its presence to yield the Na+-dependent component of binding. In previous studies (16Turner R.J. George J.N. J. Membr. Biol. 1990; 113: 203-210Crossref PubMed Scopus (8) Google Scholar, 18Turner R.J. George J.N. J. Membr. Biol. 1988; 102: 71-77Crossref PubMed Scopus (20) Google Scholar) we have demonstrated that this Na+-dependent component of binding represents the specific binding of bumetanide to its inhibitory site on the Na+-K+-2Cl− cotransporter.Phosphopeptide MappingTriton extracts for phosphopeptide mapping studies were prepared as described above for 32Pi-labeling studies, except that the extraction buffer contained 1% Triton X-100 and the protein concentration of the extract was ∼3 mg/ml. Following SDS-PAGE (∼90 μg of extracted protein/lane) the band corresponding to the Na+-K+-2Cl− cotransporter (pp175, see "Results") was identified using autoradiography, cut from the dried gel, and swollen in 1 ml of a buffer containing 50 mM NH4HCO3 (pH 8.0), 1 mM dithiothreitol, and 20 μg/ml V8 protease. After 6 or 12 h of incubation at 37°C protease digestion was terminated by heating the sample to 100°C for 5 min. The liquid was set aside, and the gel slice was then sequentially incubated in 500 μl of distilled water for 2 h, 500 μl of distilled water for 1 h, 500 μl of 0.1% SDS for 1.5 h, and 500 μl of 0.1% SDS for 1 h. All of these samples were combined (total volume 3 ml) and dried in a Savant DNA Speed Vac (Savant Instruments Inc., Farmingdale, NY). This final V8 protease digest was then taken up in sample buffer and subjected to Tricine-SDS gel electrophoresis.Data AnalysisAll experiments were repeated three or more times with similar results. Data are given as means ± S.E.RESULTSEvidence for Isoproterenol-dependent Phosphorylation of the Rat Parotid Na+-K+-2Cl−CotransporterAs already mentioned, in a previously published report from our laboratory (6Paulais M. Turner R.J. J. Clin. Invest. 1992; 89: 1142-1147Crossref PubMed Scopus (58) Google Scholar) we demonstrated that the Na+-K+-2Cl− cotransport activity of rat parotid acini is markedly (∼6-fold) and rapidly (within 40 s) up-regulated following β-adrenergic stimulation. In addition, our data indicated that this effect was mediated by protein kinase A. The first series of experiments presented below were undertaken in order to determine whether this effect could be attributed to a direct phosphorylation of the cotransport protein. In the experiment illustrated in Fig. 1 we compare protein phosphorylation patterns in 32Pi-labeled rat parotid acini incubated for 45 s in the presence (+) or absence(-) of the β-adrenergic agonist isoproterenol (1 μM). Following isoproterenol treatment the cells were disrupted, cytosolic (Cy) and particulate fractions were isolated, and the latter were further separated into Triton extracts (TE) and Triton-insoluble (TI) fractions (see "Experimental Procedures" for details). Fig. 1 shows an autoradiograph of an SDS-PAGE gel on which these various fractions were run. After this short period of incubation with isoproterenol, clear differences in the phosphorylation patterns of only two proteins were seen. The first corresponds to a relatively sharp band at Mr∼95,000 in the autoradiographs of the cytosolic fractions (small arrow). This protein, whose phosphorylation is increased with isoproterenol treatment, is not considered further here. The second phosphoprotein appears as a rather diffuse band centered at Mr≈ 175,000 in the autoradiographs of the Triton extracts (large arrow); its phosphorylation state is likewise markedly increased by isoproterenol, and its presence in the Triton extract indicates that it is an integral membrane protein.A number of factors discussed in the remainder of the paper provide strong evidence that the 175-kDa phosphoprotein (pp175) identified above is (a major part or all of) the rat parotid Na+-K+-2Cl− cotransporter.Demonstration of a Strong Correlation between Phosphorylation of pp175 and cAMP-dependent Up-regulation of the Parotid Na+-K+-2Cl−CotransporterIn our previous report (6Paulais M. Turner R.J. J. Clin. Invest. 1992; 89: 1142-1147Crossref PubMed Scopus (58) Google Scholar) we demonstrated that the half-maximal effect of isoproterenol for up-regulation of the rat parotid Na+-K+-2Cl− cotransporter was seen at ∼20 nM, measured after 37.5 s of agonist incubation at 37°C. The dose response of isoproterenol for the phosphorylation of pp175, measured under essentially identical experimental conditions, is illustrated in Fig. 2A. The half-maximal effect of isoproterenol for phosphorylation is also seen at ∼20 nM, in excellent agreement with its effect on the up-regulation of cotransport activity.Figure 2Effects of isoproterenol concentration and cAMP on the phosphorylation of pp175. 32Pi-Labeled acini were exposed to the concentrations of stimuli indicated for 45 s at 37°C and then Triton-extracts were isolated and analyzed by SDS-PAGE and autoradiography as in Fig. 1. The phosphorylation of the 175-kDa phosphoprotein (pp175) identified in Fig. 1 was quantified by scanning densitometry of the resulting autoradiographs. The pp175 phosphorylation determined in this way for each experimental condition has been normalized to the pp175 phosphorylation determined from a control (untreated) sample from the same preparation run on the same gel. The results shown are the means ± S.E. of three or more independent experiments. A, phosphorylation of pp175 versus isoproterenol (ISO) concentration. B, phosphorylation of pp175 after acinar treatment with isoproterenol, the permeant cAMP analogue dibutyryl cAMP (DBcAMP), and the activator of adenylate cyclase forskolin (FOR).View Large Image Figure ViewerDownload Hi-res image Download (PPT)In parotid acinar cells, cAMP is thought to be the major intracellular messenger mediating the effects of β-adrenoreceptor stimulation. In our earlier work (6Paulais M. Turner R.J. J. Clin. Invest. 1992; 89: 1142-1147Crossref PubMed Scopus (58) Google Scholar) we also demonstrated that significant up-regulation of cotransport activity was seen following acinar treatment with permeant analogues of cAMP and with forskolin, which increases intracellular cAMP by direct activation of the catalytic subunit of adenylate cyclase. Consistent with the effects of these agents on transport, in Fig. 2B we show that increased phosphorylation of pp175 is likewise seen when acini are treated with the permeant cAMP analogue dibutyryl cAMP and with forskolin.The stron