Characterization of atrial-natriuretic-factor-receptor-coupled membrane guanylate cyclase from rat and mouse testes
Ravi B. MARALA and Rameshwar K. SHARMA* Department of Biochemistry, University of Tennessee, Memphis, Center of Health Sciences, Memphis, TN 38163, U.S.A.
Studies with isolated adrenal cells and mouse testicular cells have supported a mediatory role of cyclic GMP in ANF (atrial natriuretic factor)-dependent steroidogenic signal transduction. This concept has been strengthened by the purification and biochemical characterization of a 180 kDa protein, which appears to contain both ANF receptor and guanylate cyclase activities, from rat adrenocortical carcinoma cells. Utilizing the antibody to 180 kDa membrane guanylate cyclase as a probe, we now demonstrate the direct presence of ANF-dependent membrane guanylate cyclase in mouse and rat testes. The antibody blocks the ANF-dependent guanylate cyclase activity in isolated membranes, and Western-blot analysis of the partially purified enzyme reveals a single 180 kDa protein. The presence of this enzyme in mouse and rat testes, together with its previous demonstration in rat adrenocortical carcinoma, represent an important potential biochemical role for this enzyme in receptor-mediated steroidogenic signal transduction.
INTRODUCTION
Atrial natriuretic factor (ANF) is a peptide hormone that is released from atria and regulates sodium excretion, water balance and blood pressure (Cantin & Genest, 1985; deBold, 1985; Schwartz et al., 1985; Atlas & Laragh, 1986). The putative ‘second messenger’ of certain ANF-signal transductions is cyclic GMP (Win- quist et al., 1984). In a number of steroid-secreting cells, ANF inhibits basal and hormone-stimulated steroido- genesis, which is accompanied by an elevation in cyclic GMP level and by a decline in cyclic AMP. For instance, aldosterone secretion in rat adrenoglomerulosa cells (Atarashi et al., 1984; Chartier et al., 1984; DeLean et al., 1984; Kudo & Baird, 1984), both cortisol and aldosterone secretions in human adrenal tissue in vitro (Naruse et al., 1987) and gonadotropin-stimulated progesterone production in murine tumour cells (Pandey et al., 1985) are decreased. These results implicate cyclic GMP in the modulation of steroidogenesis, but the mechanism by which the cyclic nuclotide mediates ANF action is now known. It is noteworthy that, to date, the sole example of a link between any metabolic activity and the ANF-receptor-mediated signal is the process of steroidogenesis.
In direct constrast with the above results, infusion studies in vivo with rat adrenal gland (Nakamura et al., 1985) and studies in situ with mouse interstitial cells (Bex & Corbin, 1985) show that ANF stimulates the produc- tion of corticosterone and testosterone in the respective systems. The process of steroidogenesis is accompanied both by an elevation of membrane guanylate cyclase activity and cyclic GMP levels in isolated fasciculata cells of rat adrenal cortex (Jaiswal et al., 1986) and by an elevation of cyclic GMP levels in mouse Leydig cells (Mukhopadhyay et al., 1986a; Pandey et al., 1986), supporting the mediatory role of this nucleotide in both adrenocortical and testicular steroidogenesis. In further
support of this concept, we have demonstrated the presence of ANF-dependent membrane guanylate cyclase in rat adrenocortical carcinoma and have purified a 180 kDa protein from this tissue that contains guanylate cyclase activity and is coupled to an ANF receptor (Paul et al., 1987). This provides a potential biochemical link between the ANF-receptor steroidogenic signal and cyclic GMP. In support of the close coupling of the ANF signal with testicular steroidogenesis (Bex & Corbin, 1985; Mukhopadhyay et al., 1986b), in which cyclic GMP is a potential second messenger (Mukhopadhyay et al., 1986a; Pandey et al., 1986), we have extended these studies by directly demonstrating, in mouse and rat testis, an ANF-dependent membrane guanylate cyclase which is biochemically and immunologically indisting- uishable from the rat adrenocortical carcinoma 180 kDa protein. Implications of these results for the plausible mechanism by which cyclic GMP mediates steroidogenic signal transduction have been explored.
EXPERIMENTAL
Materials and methods
Membranes isolated from rat and mouse testes were solubilized as described by Nambi et al. (1982a). The ANF-coupled membrane guanylate cyclase was purified to the GTP-affinity step as described in Paul et al. (1987).
Rabbit anti-(membrane guanylate cyclase) antibody was prepared by immunizing the rabbits with purified rat adrenocortical-carcinoma 180 kDa membrane guanylate cyclase (Paul et al., 1987). IgG fractions were prepared from serum pools by a threefold precipitation with (NH4)2SO4, followed by DEAE-cellulose chromato- graphy (Garvey et al., 1977). These IgG fractions from immune and normal serum pools were used for all experiments.
Abbreviation used: ANF, atrial natriuretic factor.
* To whom correspondence and reprint requests should be sent.
In these studies, synthetic ANF consisting of the 26- amino-acid peptide 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 used.
RESULTS AND DISCUSSION
ANF stimulates crude membrane guanylate cyclase isolated from both rat and mouse testes, and the hormonal stimulation is blocked by the antibody to the 180 kDa rat adrenocortical-carcinoma membrane guanylate cyclase (Table 1), indicating the hormonal dependence of the 180 kDa enzyme. The hormonal dependence is lost upon solubilization and further purification of the enzyme (Table 2). These results are similar to those obtained for the rat adrenocortical carcinoma enzyme (Paul et al., 1987). Since the loss in response to ANF stimulation and elevation of basal activity occurs during the detergent solubilization of the receptor, as speculated previously (Paul et al., 1987), it is possible that a lipid component or an accessory protein necessary for hormonal stimulation is lost in this purification step. This loss would also account for the
release of otherwise-constrained enzyme activity in the non-solubilized membranes.
The basal guanylate cyclase activity in rat testis particulate fraction is ~ 7-fold less than that in mouse testis, but the extent of stimulation is the same in both species (Table 1). This may provide an explanation for the enigmatic behaviour of the rat and mouse cells in the production of steroids in response to ANF signal (Mukhopadhyay et al., 1986b), although both cell types contain the hormonally dependent membrane guanylate cyclase. In the case of rat (in contrast with the mouse), the level of cyclic GMP produced in response to the hormone may be below the threshold to cause steroido- genesis. Our results support this previous interpretation, which was based on the observation that 8-bromo cyclic GMP stimulates testosterone production in rat as well as in mouse testicular cells (Mukhopadhyay et al., 1986a).
There was no significant inhibition of rat or mouse basal guanylate cyclase activity by the 180-kD membrane guanylate cyclase antibody (Table 1). One possible explanation for this is that the basal activity of the crude membrane guanylate cyclase comprises the accumulative activity of as-yet-unknown multiple multiple isoenzyme
Table 1. Stimulation of rat and mouse testis membrane (non-solubilized) guanylate cyclases by ANF, and its inhibition by anti-(180 kDa membrane guanylate cyclase) polyclonal antibody
Membranes were preincubated with or without antibody, as appropriate, for 1 h on ice. The assay system contained 10 mm- theophylline, 15 mm-phosphocreatine, 20 µg of creatine kinase, 1 mM-CaCl2, 50 mM-Tris/HCI, pH 7.5. The total assay volume was 100 ul. The assay mixture was preincubated with or without 1 AM-ANF for 10 min on ice. The reaction was initiated by the addition of substrate solution containing 1 mM-MnCl2 and 4 mm-GTP. Incubation (37 ℃, 10 min) was terminated by addition of 0.9 ml of 50 mm-sodium acetate buffer, pH 6.2, followed by boiling in a water bath for 3 min. The amount of cyclic GMP was quantified by radioimmunoassay. Results are shown as means ± S.E.M. (n = 3).
| Additions to membranes | Guanylate cyclase activity (pmol of cyclic GMP/min per mg of protein) | |
|---|---|---|
| Rat testis | Mouse testis | |
| Basal | 40.36±1.45 | 269.05±6.10 |
| Anti-(180 kDa guanylate cyclase) antibody | 40.75±1.45 | 270.32±4.60 |
| ANF (1 µM) | 72.20±6.70 | 487.46±21.70 |
| ANF (1 µM) + anti-(180 kDa guanylate cyclase) antibody | 42.50±3.10 | 349.88 ±18.90 |
Experimental conditions were identical with those described in Table 1. Results are means ±S.E.M. (n = 3).
| Additions to: | Guanylate cyclase activity (pmol of cyclic GMP/min per mg of protein) | |
|---|---|---|
| Rat testis | Mouse testis | |
| (a) Solubilized fraction | ||
| Basal | 301.88 ±12.26 | 406.35±9.41 |
| ANF (1 M) | 301.71 ±10.40 | 405.68±7.10 |
| Anti-(180 kDa guanylate cyclase) antibody | 302.16± 12.72 | 316.31 ±2.60 |
| (b) GTP-affinity-purified fraction | ||
| Basal | 516.99±19.60 | 515.55±5.13 |
| ANF (1 µM) | 517.51±19.30 | 514.81±6.20 |
| Anti-(180 kDa guanylate cyclase) antibody | 245.99 ±24.24 | 226.6±14.20 |
forms, among which the 180 kDa enzyme represents a minor component. This interpretation is supported by the fact that antibody to the 180 kDa protein blocks almost all of the guanylate cyclase activity of the purified enzyme (Paul et al., 1987).
Biochemical authenticity of the rat and mouse mem- brane guanylate cyclase with the 180 kDa membrane guanylate cyclase is shown by the following criteria: (i) the antibody to the 180 kDa membrane guanylate cyclase (Paul et al., 1987) blocks up to 60% of the partially purified guanylate cyclase activity from both sources (Table 2). It is noteworthy that this antibody is highly specific for particulate guanylate cyclase, since it blocks neither soluble guanylate cyclase nor adenylate cyclase activities (Paul et al., 1987); (ii) Western-blot analysis of the GTP-affinity-purified enzyme shows a single 180 kDa band, although the SDS/polyacrylamide-gel electro- phoresis of the GTP-affinity proteins shows multiple Coomassie Blue-stained bands (Figs. la and 1b). These results demonstrate that the 180 kDa rat adrenocortical- carcinoma membrane guanylate cyclases from rat and mouse testis are biochemically and immunologically indistinguishable.
Subsequent to our original report (Paul & Sharma, 1985) on the complete purification of the 180 kDa adrenocortical-carcinoma membrane guanylate cyclase, we have demonstrated that this protein is tightly coupled to an ANF receptor (Paul et al., 1987). Although the receptor and guanylate cyclase activities are inseparable, antibody to the 180 kDa protein blocks guanylate cyclase activity but does not inhibit the binding of ANF to the protein, which indicates that either the antibody reacts solely against the guanylate cyclase epitope of the protein or that there are two tightly coupled 180 kDa proteins which are inseparable by the present techniques (Paul et al., 1987).
It is noteworthy that lung guanylate cyclase, which apparently is also tightly coupled to an ANF receptor (Kuno et al., 1986), is strikingly different both bio- chemically and kinetically from the 180 kDa membrane guanylate cyclase (Paul et al., 1987): the subunit molecular mass of the lung protein is 120 kDa; in contrast with the 180 kDa guanylate cyclase, the lung enzyme is stimulated by haemin (Waldman et al., 1985); the pI of the lung enzyme is 6, whereas that of the 180 kDa protein is 4.7. Finally, in contrast with the near 1:1 stoichiometry of the binding of ANF to the tumour enzyme, the lung enzyme binds only 14.5% of ANF at the noted theoretical value.
Very recently Takayanagi et al. (1987) and Carrier et al. (1987) have also purified and characterized a guanylate cyclase-coupled ANF receptor from bovine adrenal cortex. The enzyme and the receptor activities occur in a single subunit of 135 kDa. Biochemical similarity of this protein either to lung or to the 180 kDa membrane guanylate cyclase is not known, but the molecular size is closer to that of the lung enzyme.
Previous studies have shown that 8-bromo cyclic GMP stimulates steroidogenesis in testicular (Mukhopadhyay et al., 1986b) and isolated fasciculata cells of rat adrenal cortex (Ahrens et al., 1982), and that, in the latter cells, (i) cyclic GMP stimulates cyclic GMP-dependent protein kinase and corticosterone production (Sharma et al., 1976), (ii) cyclic GMP directly stimulates the trans- formation of cholesterol to corticosterone (Sharma & Sawhney, 1978) and (iii) the steroidogenic potential of
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A 20 µg portion of the crude enzymes was subjected to SDS/7.5%-(w/v)-polyacrylamide-gel electrophoresis. (a) Rat testes : lane 1, Coomassie Blue-stained M, markers; lane 2, Coomassie Blue-stained GTP-affinity-purified fraction; lane 3, immunogenic band of the GTP-affinity- purified fraction as revealed by Western-blot analysis utilizing the 180 kDa rat adrenocortical-carcinoma mem- brane guanylate cyclase antibody. (b) Mouse testes : lane 1, Coomassie Blue-stained M, markers; lane 2, Coomassie Blue-stained GTP-affinity-purified fraction; lane 3, im- munogenic band of the GTP-affinity-purified fraction as revealed by Western-blot analysis utilizing the 180 kDa rat adrenocortical-carcinoma membrane guanylate cyclase antibody. Western-blot analysis was performed as des- cribed by Towbin et al. (1979), by subjecting GTP-affinity- purified fraction to SDS/7.5%-polyacrylamide-gel electro- phoresis, followed by electrophoretically transferring the proteins to a nitrocellulose membrane. The protein-binding sites were blocked by nitrocellulose membrane with 3% bovine serum albumin. The membrane was incubated for 2 h at room temperature with 180 kDa rat adrenocortical- carcinoma membrane guanylate cyclase polyclonal anti- body (dilution 1:200), followed by 2 h incubation at room temperature with conjugated anti-(rabbit IgG)-horse- radish peroxidase. The colour was developed by soaking the blot in horseradish peroxidase (600 µg/ml)/0.015% H2O2/10 mM-Tris/HCI, pH 7.4. The reaction was termin- ated by extensive washing with water. The standard M, markers used were myosin (200000), ß-galactosidase (116000), phosphorylase b (94000), bovine serum albumin (68 000) and ovalbumin (43000). Abbreviation: DF, dye front.
cyclic GMP and its analogues correlates closely with their ability to stimulate cyclic GMP-dependent protein kinase (Ahrens et al., 1982). In addition, cyclic GMP- dependent protein kinase has been purified to homo- geneity and biochemically characterized in bovine adrenal cortex and isolated rat adrenal cells (Ahrens et al., 1982). Although all these results provided strong evidence for the mediatory role of cyclic GMP in steroidogenic signal transduction, the crucial support for this concept came with the first demonstration of a hormonally dependent membrane guanylate cyclase which was different from the non-specific soluble guanylate cyclase (Nambi & Sharma, 1981a,b; Nambi et
al., 1982a,b). Our present results on the characterization of the 180 kDa membrane guanylate cyclase, which is tightly coupled to an ANF receptor in mouse and rat testis, as well as in the adrenal gland (Paul et al., 1987), provide the identification of an important missing biochemical link which transduces the transmembrane ANF steroidogenic signal.
We thank Dr. R. Nutt, of Merck, Sharp and Dohme, for the sample of synthetic ANF, and Dr. J. M. Sharma for pre- submission editing of the manuscript. This work was supported by National Science Foundation grant 8609867 and National Institutes of Health grant NS 23744.
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Received 1 September 1987/18 December 1987; accepted 11 January 1988