CHARACTERIZATION AND REGULATION BY PROTEIN KINASE C OF RENAL GLOMERULAR ATRIAL NATRIURETIC PEPTIDE RECEPTOR-COUPLED GUANYLATE CYCLASE
Barbara J. Ballermann*, Ravi B. Marala” and Rameshwar K. Sharma ***
*Renal Division and Department of Medicine, Brigham and Women’s Hospital and The Harvard Center for the Study of Kidney Disease, Boston, MA 02115
The Department of Physiology and Biophysics, University of Tennessee Memphis, Memphis TN 38163
** The Section of Regulatory Biology, Department of Brain and Vascular Research, The Cleveland Clinic Research Institute, Cleveland OH 44195
Received October 24, 1988
Summary The nature and regulation of atrial natriuretic peptide (ANP)-sensitive guanylate cyclase in rat renal glomerular membranes was examined. By affinity crosslinking techniques, three bands with apparent molecular masses of 180, 130 and 64 kDa were specifically labeled with [125]]ANP. A specific antibody to the 180 kDa membrane guanylate cyclase of rat adrenocortical carcinoma recognized a 180 kDa band on Western blot analysis of solubilized, GTP-affinity purified glomerular membrane proteins. The same antibody completely inhibited ANP-stimulated guanylate cyclase activity in glomerular membrane fractions. Partially puri- fied protein kinase C inhibited ANP-stimulated guanylate cyclase activity in glomerular mem- brane fractions. It is concluded that a 180 kDa ANP-sensitive guanylate cyclase is present in glomerular membranes, and that this enzyme is inhibited directly by protein kinase C. · 1988 Academic Press, Inc.
Atrial natriuretic peptide (ANP), a hormone synthesized and secreted by atrial myocytes (1), markedly enhances renal salt and water excretion (2,3). In the kidney, ANP increases the glomerular filtration rate (4,5) and decreases collecting duct sodium transport (6,7). In keeping with these biological effects, binding sites for ANP are concentrated in renal glomeruli and renal medulla (8). As in other tissues, ANP stimulates cGMP accumulation in isolated glomer- uli (9-11). However, in glomeruli, ANP-stimulated cGMP generation requires concentrations of ANP 1-2 orders of magnitude greater than those required to saturate ANP receptors (10,11). It has therefore been postulated that glomerular ANP receptors may not be coupled directly to guanylate cyclase (10). Also, Maack et al (12) showed that renal cortical ANP binding sites represent predominantly “silent” receptors which may serve only to clear ANP from the circu- lation, suggesting that the majority of glomerular ANP receptors are not coupled to guanylate cyclase.
Several distinct ANP binding sites have been identified. In a large number of tissues, ANP binds specifically to a 120 kDa disulfide-linked dimeric cell-surface protein not coupled to guanylate cyclase (13-15), which represents the “silent” ANP receptor (12). Monomeric gua- nylate cyclase-coupled 130 kDa ANP receptors have also been identified in a large number of
tissues and cell types (13,15), and were found to co-purify with guanylate cyclase activity (16,17). In addition, a 180 kDa monomeric guanylate cyclase purified to homogeneity from rat adrenocortical carcinoma cells was found to bind ANP with a stoichiometry of 1:1 (18). The same enzyme/ANP receptor has been demonstrated in rat and mouse testes (19).
ANP-stimulated cGMP accumulation in vascular smooth muscle cells is inhibited by activa- tion of V1 vasopressin receptors and angiotensin receptors (20,21) and ANP-stimulated guany- late cyclase activity in adrenocortical carcinoma cells and in aortic smooth muscle cells is in- hibited by phorbol esters (22,23), suggesting that protein kinase C inhibits ANP-receptor cou- pled guanylate cyclase.
The present study was undertaken to characterize guanylate cyclase coupled ANP receptors in glomerular membranes, and to determine possible regulation of this enzyme by protein ki- nase C. The presence of the 180 kDa ANP receptor-guanylate cyclase complex in glomerular membranes and and regulation of this enzyme by protein kinase C is demonstrated.
Materials and Methods GTP, creatine phosphate, creatine phosphokinase, and 3-[(3-cholami- dopropyl)dimethylammonia-1-1 propane sulfonate] (CHAPS) were purchased from Sigma. GTP-affinity gel was from Pharmacia. Disuccinimidyl suberate was from Pierce. [125]] ANP was from DuPont-New England Nuclear. All other reagents were of analytical grade and ob- tained commercially. ANP consisting of 26-amino acid: H-Arg-Arg-Ser-Ser-Cys-Phe-Gly- Gly-Arg-Ile-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr-OH was generously provided by Dr. Ruth Nutt (Merck Sharpe and Dohme).
Glomeruli were isolated as previously described (10) and subjected to hypotonic shock by incubation for 15 min on ice in ethylenediaminetetraacetic acid (EDTA) 5 mM, phenylmethyl- sulfonylfluoride (PMSF) 1mM and tris(hydroxymethyl)aminomethane (Tris) 10 mM, pH 7.4. Glomeruli were washed twice in 250 mM sucrose containing 0.1 mM PMSF, resuspended in four volumes of sucrose/PMSF and homogenized using three 20-sec bursts of a Polytron (Brinkmann, Westbury NY) at setting 8 with 60-sec cool-down periods between homogenizer bursts. The material was sedimented at 1000 X g for 10 min, and the resultant supernatant at 43,000 X g for 60 min. The final pellet was resuspended in 1.5 ml homogenizing solution and kept frozen at -70℃ until use.
· For affinity crosslinking, glomerular membranes (150-250 µg protein) were incubated at 4ºC for 16 h in ANP binding buffer (125 mM NaCl, 25 mM N-2-hydoxyethylpiperazine-N’-2- ethanesulfonic acid (Hepes), 1 mM Bacitracin, 0.1 mM PMSF, 0.2 g/dl bovine serum albumin (BSA) with 0.2 uCi [125]]ANP in the absence or presence of 1 uM unlabeled ANP in a total volume of 250 ul. The volume was then adjusted to 1.0 ml with binding buffer, and mem- branes were sedimented at 45,700 X g for 30 min. Membranes were resuspended in 100 ul of 50 mM sodium phosphate pH 7.4, 11 ul of 10 mM disuccinimidyl suberate in DMSO were added, and incubated on ice for 15 min. The reaction was quenched with 56 ul 1M Tris, 0.2 M EDTA, pH 6.8, and membranes were sedimented at 45,700 X g for 20 min. The membranes were resuspended in 100 ul of sample buffer for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) containing 2% mercaptoethanol as the reducing agent, boiled for 3 min, and resolved on a 7.5% polyacrylamide gel under constant current conditions. Gels were dried, and exposed to Kodak XAR5 film to prepare the autoradiograms.
Guanylate cyclase activity was determined in particulate glomerular membrane prepara- tions, after solubilization of the membrane material (24), and after partial purification of the ANP-coupled membrane guanylate cyclase by GTP-affinity chromatography (18). The GTP- affinity purified fractions were also used for Western blot analysis (19,25). Guanylate cyclase activity assays were performed in 100 ul reaction mixtures containing 10-20 µg membrane protein, 50 mM Tris-HCL, pH 7.5, 10 mM theophylline, 15 mM creatine phosphate, 20 ng cre- atine phosphokinase, 1 mM GTP, 4 mM MnCl2 and 1 mM CaCl (26). To determine the ef- fects of protein kinase C on guanylate cyclase activity, indicated tubes also contained 1 uM ANP and 20ul partially purified protein kinase C, 1 mM ATP, phosphatidylserine 50 µg/tube, and diolein 8 ug/tube. Control tubes contained all reagents except phosphatidylserine and di- olein, known activators of protein kinase C. The membranes were preincubated for 5 min at 37°C. The reactions were initiated by the addition of Mn-GTP (2 mM MnCl2, 1 mM GTP) and incubated for 10 min at 37℃. Incubations were terminated by the addition of 0.9 ml ice-cold
sodium acetate buffer, pH 6.2, followed by heating in a boiling water bath for 3 min. The amount of cGMP formed was determined as described earlier (26) and expressed as pmol cGMP/mg protein/min.
Rabbit anti-membrane guanylate cyclase antibody was prepared by immunizing rabbits with purified rat adrenocortical carcinoma 180-kDa particulate guanylate cyclase (18). IgG frac- tions were prepared from serum pools by 3 X precipitation with (NH4)SO4 followed by DEAE- cellulose chromatography (27). These IgG fractions from immune and normal rabbit serum pools were used for all experiments. Bovine brain protein kinase C was partially purified through the phenyl-Sepharose chromatography step as described by Walton et al (28).
Western blot analysis was performed as described in (25), by subjecting the GTP-affinity purified fraction to 7.5% SDS-PAGE, followed by electrophoretic transfer of the proteins to ni- trocellulose membrane. The protein binding sites were blocked by incubating the membrane with 2% bovine serum albumin for 2 h at room temperature. The membrane was then incubat- ed for 2 h at room temperature with the anti-180 kDa rat adrenocortical membrane guanylate cyclase polyclonal antibody at a 1:200 dilution, followed by 2 h incubation at room tempera- ture with peroxidase-conjugated anti-rabbit IgG at a 1:1000 dilution. The color was developed by soaking the blot in peroxidase color reagent: 600 µg 4-choro-1-naphthol/0.015% H2O2/20 mM Tris, pH 7.6. The reaction was terminated by extensive washing with water.
Results In glomerular membranes, ANP significantly stimulated guanylate cyclase activity (Fig. 1A). Preincubation of the membranes for 1 h with polyclonal anti-membrane guanylate cyclase antibody (1:200) inhibited ANP-stimulated, but not baseline guanylate cyclase activity. Guanylate cyclase activity in solubilized membranes was not stimulated by ANP, however, ba-
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Guanylate Cyclase Activity (pmol/mg protein/min)
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seline guanylate cyclase activity was significantly inhibited by the anti-membrane guanylate cyclase antibody (Fig. 1B). ANP also failed to stimulate partially purified membrane guany- late cyclase, but again, baseline guanylate cyclase activity was significantly inhibited by the 180 kDa membrane guanylate cyclase antibody (Fig. 1C). Nonimmune rabbit IgG fractions failed to inhibit baseline or ANP-stimulated guanylate cyclase activity in these fractions.
Solubilization of membrane proteins followed by GTP-affinity purification resulted in a nearly 5-fold enrichment of guanylate cyclase activity (Fig. 1). Coomassie blue staining of the SDS-PAGE resolved proteins from the crude membranes, the solubilized membranes and the GTP-affinity purified preparations are shown in Figure 2 (lanes 1-3), and revealed a large num- ber of bands. However, Western blotting of the GTP-affinity purified preparations (Figure 2, lane 5) with the anti-membrane guanylate cyclase antibody revealed a single band, with an ap- parent molecular mass of 180 kDa.
Affinity crosslinking followed by SDS-PAGE under reducing conditions demonstrated three bands specifically labeled with [125]]ANP, at 64, 130 and 180 kDa, respectively (Figure 3). The relative abundance of the binding sites was 54, 24, and 22%, respectively, as deter- mined by densitometry.
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Guanylate cyclase activity pmol / mg protein / mg
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1 = Memb. only
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2 = Memb. + ANF
3 = Memb. + PKC
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4 = Memb. + ANF + PKC
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In the presence of diolein and phosphatidylserine, partially purified protein kinase C had no significant effect on baseline guanylate cyclase activity, but completely inhibited stimulation of membrane guanylate cyclase activity by ANP in particulate fractions of glomerular membranes (Fig. 4). This effect of protein kinase C was not observed in the absence of the protein kinase C activators diolein and phosphatidylserine. Protein kinase C purified to homogeneity (28) also inhibited ANP-stimulated guanylate cyclase activity in particulate glomerular membrane fractions (data not shown).
Discussion This study demonstrates the presence of a 180 kDa ANP-sensitive membrane gua- nylate cyclase in rat renal glomerular membranes. This conclusion is drawn from the findings that ANP-sensitive guanylate cyclase activity in glomerular membranes is completely inhibited by antibody raised against purified 180 kDa membrane guanylate cyclase of rat adrenocortical carcinoma, and that the antibody recognized a 180 kDa band on Western blots of partially puri- fied glomerular membrane proteins. In addition, specific labeling of a 180 kDa band by [125]]ANP in affinity crosslinking studies suggests that the 180 kDa membrane guanylate cy- clase in glomerular membranes contains ANP binding activity, as is also the case for the 180 kDa guanylate cyclase of rat adrenocortical carcinoma (18).
The presence of ANP-sensitive guanylate cyclase has previously been demonstrated in a large number of tissues (29). However, in most studies, the predominant molecular species containing guanylate cyclase and ANP binding activity appears to be a 130 kDa protein (13,15). Indeed, 130 kDa ANP receptors from bovine adrenocortical cells and rat lung co-puri- fied with guanylate cyclase activity (16,17), suggesting that ANP binding sites and guanylate cyclase activity reside on the same trans-membrane protein. By contrast, purification of mem- brane guanylate cyclase from rat adrenocortical carcinoma yielded a 180 kDa protein, which bound ANP with a near 1:1 stoichiometry (18). In the present study, ANP was found to bind to both, 130 and 180 kDa species, although the antibody to the 180 kDa membrane guanylate cyclase from rat adrenocortical carcinoma only recognized a 180 kDa band on Western blot- ting. These findings, and the inhibition by the antibody of ANP-stimulated guanylate cyclase activity, are consistent with the view that the 180 kDa membrane guanylate cyclase in glomer- ular membranes is functionally and immunologically indistinguishable from the membrane guanylate cyclase in rat adrenocortical carcinoma cells. The possibility that the 130 kDa pro-
tein observed by ANP affinity crosslinking also expresses guanylate cyclase activity was not excluded by the present study.
The loss of ANP-sensitivity upon solubilization of membrane guanylate cyclase is common- ly observed (17-19 ). Since the loss of response to ANP in this study was associated with an increase in baseline activity after detergent solubilization (Fig. 1B), we postulate that a lipid component or accessory protein necessary for hormonal regulation is lost in the solubilization step. The findings that the anti-180 kDa guanylate cyclase antibody inhibited ANP-stimulated, but not baseline guanylate cyclase activity in crude membranes, whereas baseline guanylate cy- clase activity was inhibited by the antibody after solubilization and after partial purification are consistent with the hypothesis that a constraining influence on guanylate cyclase activity was lost during enzyme solubilization.
Hormones known to activate protein kinase C and phorbol esters have been shown to inhib- it ANP-stimulated cGMP accumulation, suggesting a negative influence of protein kinase C on guanylate cyclase (20-23). The present study provides evidence that protein kinase C inhibits guanylate cyclase activity in particulate glomerular membrane fractions. A previous study sug- gested that inhibition of cGMP accumulation by angiotensin II results from stimulation of cGMP phosphodiesterase (21). Since the experiments in the present study were performed in membrane fractions and in the presence of high concentrations of a phosphodiesterase inhibi- tor, the findings are more consistent with direct inhibition of guanylate cyclase activity by pro- tein kinase C. Whether protein kinase C inhibits guanylate cyclase activity through a direct phosphorylation of the enzyme remains to be determined.
In conclusion, this study demonstrates the presence of a 180 kDa ANP-sensitive membrane guanylate cyclase in glomerular membranes. In addition, our findings suggest a novel mecha- nism of regulation of this enzyme, namely direct inhibition by protein kinase C. This study therefore provides further evidence that these two receptor-signalling pathways interact at the level of the plasma membrane.
Acknowledgements We thank Dr. Ruth F. Nutt of Merck Sharpe and Dohme Research Laboratories for the sample of synthetic ANP. We also thank Mary M. McNamara for techni- cal assistance. This research was supported by NSF Grant DCB-83-00500, NIH grant NS- 23744, NIH grant 35930 and NIH grant DK40445.
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