Adrenocorticotropin-Dependent Particulate Guanylate Cyclase in Rat Adrenal and Adrenocortical Carcinoma: Comparison of Its Properties with Soluble Guanylate Cyclase and Its Relationship with ACTH-Induced Steroidogenesis1
PONNAL NAMBI, NAMBI V. AIYAR, AND RAMESHWAR K. SHARMA2
Laboratory of Basic Hormone Research, Department of Biochemistry, University of Tennessee Center for the Health Sciences, 894 Union Avenue, Memphis, Tennessee 38163
Received March 8, 1982, and in revised form April 28, 1982
Previously we described the ACTH-dependent particulate guanylate cyclase from rat adrenal gland and from the rat adrenocortical carcinoma that was distinct from the soluble enzyme [P. Nambi and R. K. Sharma (1981) Endocrinology 108, 2025-2027; P. Nambi and R. K. Sharma (1981) Biochem. Biophys. Res. Commun. 100, 508-514]. Herein, we report the detailed kinetic and functional differences between the two enzymes. (i) The particulate guanylate cyclase was stimulated by low concentrations of ACTH1- 39 (10-11 M) and ACTH1-24 (10-13 M). The ACTH-antagonist (ACTH7-38) and 4-methyl-4- aza-5x-cholestane, the compounds that competitively inhibit the steroidogenic activity of ACTH, inhibited the hormonally dependent guanylate cyclase. In contrast, the sol- uble cyclase was not stimulated by ACTH. (ii) The particulate enzyme was not stim- ulated by sodium azide, sodium nitroprusside, excess Mn2+, dithiothreitol (1 mm), and N-ethylmaleimide. On the other hand, all these agents stimulated the soluble enzyme. (iii) The 1/2 V of the soluble enzyme is achieved at 0.06 mM MnGTP whereas the particulate enzyme is not saturable up to 2 mM MnGTP. (iv) Cd2+ did not affect the particulate enzyme but inhibited the soluble enzyme. (v) Tuftsin (10-6-10-5 M) did not stimulate the membrane enzyme, whereas it strongly stimulated the soluble enzyme. These results favor the concept that the adrenal particulate and soluble guanylate cyclases are functionally different and may also be two structurally independent en- tities.
ACTH3 stimulates the production of cyclic GMP in isolated cells derived from the adrenal glands of rat (1-4) and beef (5), as well as in the adrenal glands de- rived from human (6), rat (7), crocodile (8), and Blue and Mako shark (9). An ex- cellent temporal correlation exists be-
tween cyclic GMP formation, phosphory- lation, and corticosterone synthesis when the isolated fasciculata cells are exposed to submaximal steroidogenic concentra- tions of ACTH (10). Calcium is obligatory to the ACTH-stimulated rise of cyclic GMP and the process of adrenal steroido- genesis (11). However, calcium alone can neither cause an increase of cyclic GMP nor induce the production of corticoste- rone (11). These observations indicated that the hormonal response for adrenal steroidogenesis is mediated by cyclic GMP through the cyclic GMP-dependent pro- tein kinase. Subsequently, the presence of
1 This investigation was supported by Grant PCM80- 0873 from the National Science Foundation and Grant CA-16091 from the National Cancer Institute.
2 Author to whom all correspondence should be sent.
3 Abbreviations used: ACTH, adrenocorticotropic hormone.
cyclic GMP-dependent protein kinase in adrenal cortex (12, 13) and the direct stim- ulatory effect of ACTH and cyclic GMP on the transformation of cholesterol to cor- ticosterone in isolated adrenal cells have been demonstrated (14). Despite the fact that these results toegether indicate that both cyclic GMP and calcium are impor- tant mediatory components of the ACTH- induced adrenocortical steroidogenesis, failure of previous attempts to demon- strate a hormone-sensitive guanylate cy- clase (15, 16) in cell-free preparations of the endocrine or nonendocrine tissues to- gether with the observations that guany- late cyclase can be nonspecifically acti- vated by several agents such as sodium nitroprusside, sodium azide, ascorbic acid, and compounds that affect the oxidation- reduction potential of biological reactions, had seriously compromised the mediatory role of cyclic GMP in hormonally induced physiological responses (16).
Recently, however, the presence of ACTH-responsive particulate guanylate cyclase from rat adrenal and rat adreno- cortical carcinoma has been demonstrated (17, 18). In contrast to the particulate guanylate cyclase, the soluble guanylate cyclase is not stimulated by ACTH; but it is markedly stimulated by sodium nitro- prusside (17, 18). In order to establish the physiological role of the particulate en- zyme in adrenocortical steroidogenesis, it is essential to unequivocally differentiate the two enzymes. The present report com- pares, in detail, the kinetic and molecular properties of the ACTH-dependent adre- nocortical particulate guanylate cyclase with that of the soluble enzyme. The re- sults support the concept (17-19) that the two enzymes are functionally and kineti- cally distinct molecular species and the particulate guanylate cyclase is associated with the ACTH-induced steroidogenesis.
MATERIALS AND METHODS
GTP, creatine kinase, creatine phosphate, sodium nitroprusside, sodium azide, and tuftsin were purchased from Sigma. ACTH1-39 was obtained from USP Corticotropin Reference Standard; and ACTH1-24 was a gift from Ciba-Geigy. All other re- agents were of analytical grade and were obtained commercially.
The source of the particulate and soluble guanylate cyclase (100,000g pellet and supernatant, respec- tively) was from the rat adrenal gland or rat adre- nocortical carcinoma 494 (20). The enzyme prepara- tions used in these studies were as described previ- ously (17, 18). Briefly, the tissues were homogenized in ice-cold buffer (0.25 M sucrose, 1 mM MgCl2, 5 mM Tris-HCI, pH 7.5) for four 30-s periods in a Brinkman Polytron at a setting of 6. After filtration through a double layer of gauze, the homogenate was centri- fuged at 400g for 10 min at 4℃. The supernatant was centrifuged at 100,000g for 60 min at 4°C. The re- sulting pellet was washed twice in ice-cold incubation buffer (10 mM MgCl2, 50 mM Tris-HCI, pH 7.5) by resuspension and centrifugation at 100,000g for 60 min. The final pellet, resuspended in incubation buffer, was used as a source of particulate cyclase and the supernatant as a source of soluble enzyme.
Guanylate cyclase was assayed (15, 16, 21) using GTP and the determination of cyclic GMP was done by radioimmunoassay (22, 23). The sensitivity of the methods was increased by acetylation of cyclic GMP in the samples (24). Generally, the incubation tubes contained an assay mix continaing 10 mM theoph- ylline, 50 mM Tris-HC1, 15 mM creatine phosphate, 20 µg of creatine phosphokinase, and 20 ul of enzyme solution (30-50 µg protein). The reaction in a final volume of 100 ul was initiated by the addition of 20 ul of the substrate containing MnCl2 and GTP, the final concentration in the assay being 4 and 1 mm, respectively. Incubation (37°C, 10 min) was termi- nated by the addition of 0.90 ml of 50 mM sodium acetate buffer, pH 6.2, followed by heating the mix- ture for 3 min in a boiling water bath. Supernatant fractions obtained by centrifugation at 1500 rpm for 20 min were used for the radioimmunoassay. Samples incubated without enzyme or with heated enzyme serve as controls. All assays were performed in trip- licate and were repeated at least three times. The data presented are mean values from one represen- tative experiment. Protein was determined by the Bradford method with the use of bovine serum al- bumin as a standard (25).
RESULTS
Distribution of particulate and soluble guanylate cyclase. Table I shows the dis- tribution of particulate and soluble guan- ylate cyclases in rat adrenal glands and the rat adrenocortical carcinoma. More than 80% of the normal adrenal guanylate cyclase was particulate, whereas only 23% was particulate in the adrenocortical car- cinoma. This situation is in contrast to the neoplasm of the liver where the particu- late guanylate cyclase activity is higher than the parent nonmalignant tissue (26).
TABLE I DISTRIBUTION OF PARTICULATE AND SOLUBLE GUANYLATE CYCLASES IN NORMAL RAT ADRENAL GLAND AND ADRENOCORTICAL CARCINOMA
| Tissue | Guanylate cyclase (cyclic GMP pmol/ mg/10 min) | |
|---|---|---|
| Particulate | Soluble | |
| Normal rat adrenal gland | 480 | 100 |
| Adrenocortical carcinoma | 90 | 300 |
Note. The particulate and soluble fractions were assayed for guanylate cyclase activity as indicated under Materials and Methods. The incubation was at 37°℃ for 10 min in a total volume of 0.1 ml con- taining assay mix and enzyme (30-50 µg protein). Reaction was started with MnGTP (4:1) and cyclic GMP was quantitated by the radioimmunoassay.
Particulate guanylate cyclase as a func- tion of time and protein concentration. The adrenocortical particulate guanylate cy- clase activity was linear up to 30 min at
(pmoi/mg PROTEIN)
CYCLIC GMP (pmol/ml/10min)
A
B
1200
60
800
40
400
20
CYCLIC GMP
0
0
10
20
TIME (min)
30
0
PROTEIN (ug)
40
80
0
( pmol/mg PROTEIN/10min)
C
600
400
200
0
0
1
2
GTP (mM)
3
4
5
CYCLIC GMP (pmol/mg PROTEIN/10min)
80
60
40
TO
4
DEO
20
0
0
CATION (mM)
5
10
37°C (Fig. 1A) and up to 100 µg protein (Fig. 1B).
Effect of GTP. Figure 1C depicts the ef- fect of varying concentrations of GTP on the adrenocortical particulate guanylate cyclase at a constant 4 mM concentration of MnCl2. At 100 uM GTP the half-maximal activation of the enzyme was observed.
Effect of divalent cations. It has been shown earlier (17, 18) that the particulate and soluble guanylate cyclase activities have an absolute requirement for Mn2+ as the divalent cation for their optimum ac- tivities. The activities with Mg2+ are ap- proximately 5% of those with Mn2+ (17). Figure 2 indicates that the substitution of Mn2+ with other divalent cations such as Ca2+, Ba2+, Sr2+, and Co2+ in the incubation medium markedly decreased the adreno- cortical particulate guanylate cyclase. They were less than 10% as effective as Mn2+ in restoring the basal guanylate cyclase activity (Fig. 2).
Effect of ACTH 1-99 and ACTH 1-21. Pre- viously (17, 18) it has been shown that the low concentrations of ACTH, 10-13 to 10-11 M, that are submaximally steroidogenic in isolated adrenal cells activate particulate adrenal guanylate cyclase. ACTH1-24 is a synthetic corticotropin that is as equipo-
00
A
Q
CYCLIC GMP (% ABOVE CONTROL)
60
40
o
0
20
o
0
O
+
o
.
o
o
80
0
60
B
40
20
O
o
1
o
0
9
9
0
t2
ACTH (M)
10
10
tent (27) as ACTH1-39 in its steroidogenic activity in the isolated adrenal cells. ACTH7-38 (28) and 4-methyl-4-aza-5x-cho- lestane (29) are competitive steroidogenic inhibitors of ACTH. In order to compare the activity of guanylate cyclase in re- sponse to ACTH1-39 with ACTH1-24, and to evaluate whether these activities are in- hibited by ACTH7-38 and 4-methyl-4-aza-
5a-cholestane, the particulate guanylate cyclase was incubated with ACTH1-39 or ACTH1-24 in the presence or absence of these inhibitors. ACTH1-24, like ACTH1-39 (17, 18) activated the particulate enzyme over a narrow concentration range of the hormone (Figs. 3A and B) and their stim- ulatory effect was inhibited by ACTH7-38 and 4-methyl-4-aza-5x-cholastane (Table II). These compounds did not have any effect on the basal activity of the enzyme. Since the ACTH-responsive guanylate cy- clase activity is inhibited by the ACTH antagonist, it appears that ACTH recep- tors are coupled to guanylate cyclase and at physiological concentrations of the hor- mone, cGMP mediates the process of ad- renal steroidogenesis.
ACTH does not stimulate the soluble guanylate cyclase (17, 18).
Effect of sodium nitroprusside and so- dium azide. Nitric oxide generating com- pounds stimulate soluble and most of the particulate guanylate cyclases (16, 30). With only two exceptions, particulate bo- vine tracheal smooth muscle (30) and par- ticulate BALB 3T3 fibroblasts (31), sodium nitroprusside has so far activated guany- late cyclase in all tissues examined (30). Sodium azide and sodium nitroprusside did not activate the adrenocortical carci- noma particulate guanylate cyclase. On the other hand, soluble guanylate cyclase
| Conditions | Cyclic GMP pmol/mg/10 min | +4-Methyl-4- aza-cholestane (100 µM) | |
|---|---|---|---|
| Control | +ACTH7-38 (10-8 M) | ||
| Normal rat adrenal glands | |||
| Basal | 523 | 523 | 489 |
| ACTH1-39, 10-11 M | 853 | 589 | 589 |
| Adrenocortical carcinoma | |||
| Basal | 117 | 117 | 128 |
| ACTH1-39, 7 X 10-11 M | 219 | 128 | |
| 1.4 × 10-10 M | 198 | 128 | |
| ACTH1-24, 10-18 M | 200 | 117 | |
Note. Conditions for assays were the same as described in Table I.
CYCLIC GMP (% ABOVE CONTROL)
A
B
400
300
200
100
o
0
O
o
o
0
SODIUM NITROPRUSSIDE (M)
0
10
-5
10-4
10-3
0
10-5
104
SODIUM AZIDE (M)
10-3
was stimulated by these agents in a con- centration-dependent manner (Figs. 4A and B).
Effect of Cd2+. CdCl2 is a dithiol reagent (32) which inhibits the rat liver-soluble guanylate cyclase but has no effect on the particulate enzyme at low concentrations (19, 33). In adrenocortical carcinoma Cd2+ is 200-fold more potent in causing 50% inhibition of the soluble guanylate cyclase activity than that of the particulate en- zyme activity (Fig. 5).
Effect of tuftsin. Tuftsin (34) is a four amino acid peptide with the sequence of L-Thr-L-Lys-L-Pro-L-Arg (35, 36). At low concentrations, between 10-6 to 10-5 M, this peptide did not stimulate the partic- ulate guanylate cyclase but stimulated the soluble enzyme (Fig. 6). At 10-4 M tuftsin stimulated the particulate adrenocortical enzyme by only 30% but stimulation of the soluble guanylate cyclase was over 200%.
Effect of excess Mnº+. Both particulate and soluble adrenocortical guanylate cy- clases (17, 18), like guanylate cyclases from other sources (15, 16), show specific requirement for the substrate MnGTP. The soluble guanylate cyclase from other sources requires Mn2+ in excess of near saturating concentrations of MnGTP for its optimum activity, whereas such is not the case for the particulate enzyme (19, 37, 38). The adrenocortical-soluble guanylate cyclase in the presence of 1 mM MnGTP was maximally stimulated at 3 mM Mn2+.
CYCLIC GMP (% CHANGE OF CONTROL)
100
50
o
+
o
0
o
-
50
o
100
10-7
10-6
10-5
Cd(NO312 (M)
10-4
10-3
At higher concentrations of the ion the peak activity of the enzyme was markedly decreased (Fig. 7). In contrast, the partic- ulate enzyme was not affected by excess Mn2+ (Fig. 7).
Effect of MnGTP. In response to increas- ing MnGTP concentration, the soluble ad- renocortical guanylate cyclase showed a 1/2 V of 0.06 mM similar to the soluble guanylate cyclase from other sources (37- 40); in contrast, the particulate enzyme did not achieve saturation up to 2 mM MnGTP (Fig. 8).
CYCLIC GMP (% ABOVE CONTROL)
200
160
120
80
40
o
0
6
o
0
10
6
10
5
10
6
TUFTSIN (M)
CYCLIC GMP (pmol/mg PROTEIN/10min)
600
500
400
300
200
100
0
0
2.0
EXCESS MnCl2 (mM)
4.0
6.0
8.0
10.0
Effect of dithiothreitol and N-ethylmal- eimide. The sulfhydryl reagents, dithio- threitol and N-ethylmaleimide, have very different types of effects on the particulate and soluble adrenocortical guanylate cy- clases (Fig. 9). The low concentrations of dithiothreitol (up to 2 mM) that markedly stimulated the soluble enzyme had no ef- fect on the particulate enzyme (Fig. 9A). Higher concentrations of dithiothreitol (4-8 mM) caused inhibition of the peak activity of the soluble enzyme. N-Ethyl- maleimide inhibited the particulate en- zyme and stimulated the soluble enzyme with inhibition occuring at higher concen- trations (>0.025 mM), (Fig. 9B).
CYCLIC GMP {pmol/mg PROTEIN/10min)
600
400
200
0
0
MnGTP (mM)
1.0
2.0
CYCLIC GMP 1% CHANGE OF CONTROL)
150
8
300
100
150
50
0
-40
0
80
0
DITHIOTHREITOL (mM)
2
4
6
8
0
N-METHYLMALEIMIDE (2.5 *M)
10-6
10-5
10%
10-3
10°2
DISCUSSION
Original studies (39) implicated cyclic GMP as a biologic effector molecule me- diating the effect of acetylcholine through adrenergic receptors. The “Yin Yang” hy- pothesis (42) postulated that the biologic regulation of a cell is governed by the op- posing biological activities of cyclic AMP and cyclic GMP. However, the biologic role of cyclic GMP became seriously compro- mised since (a) the attempts to demon- strate a hormonally dependent guanylate cyclase failed in every tested system (15, 16); (b) the guanylate cyclase activity was nonspecifically stimulated by polyunsatu- rated fatty acids, peroxides, hydroperox- ides, free radicals, ascorbic acid, sodium nitroprusside, and several other agents that presumably affect the oxidation-re- duction potential of the biochemical re- actions (16); and (c) there was a general consensus that cyclic GMP-dependent pro- tein kinase does not phosphorylate a spe- cific protein clearly distinct from that of the cyclic AMP-dependent protein kinase (43). Some of these reservations regarding the biological role of cyclic GMP appear to be overcome since specific substrates for cyclic GMP-dependent protein kinase in the particulate fractions of smooth muscle have been shown (44) and the evidence has been provided in support of the two dis- tinct types of guanylate cyclase, particu-
| Conditions | Particulate | Soluble |
|---|---|---|
| ACTH | Stimulation | No effect |
| Sodium nitroprusside | No effect | Stimulation |
| Sodium azide | No effect | Stimulation |
| Tufstin | No effect | Stimulation |
| Cd2+ | Inhibition at high concentration (EC50 ~ 400 AM) | Inhibition at low concentration (EC50 ~ 2 uM) |
| Dithiothreitol | No effect | Strong stimulation |
| N-Ethylamleimide | Inhibition | Stimulation at low concentration and inhibition at high concentrations |
late and soluble, in rat adrenal glands (17), the rat adrenocortical carcinoma (18), and rat liver (19). Although the original stud- ies indicated the existence of soluble and particulate fractions of guanylate cyclase in almost all eukaryotic tissues tested (39, 45-47) and they usually could be distin- guished by their physical and certain ki- netic characteristics (39, 45, 46), the view persisted that the apparent differences could be attributable to “assay conditions, other constituents in crude preparations, or possibly other factors (16).” In spite of the fact that the antibody raised against sea urchin sperm particulate guanylate cyclase cross-reacted with mammalian particulate but not soluble enzyme (48), indicating their antigenic differences, the possibility existed that the invertebrate guanylate cyclase might not be represen- tative of the mammalian system. The studies with rat liver showed that the membrane bound guanylate cyclase activ- ity was markedly increased (49) by pro- teolysis. This led to the attractive hy- pothesis that the cytosolic form of the guanylate cyclase originates from the membrane bound by a simple endogenous proteolysis (50), implying that there is a direct relationship between the two nat- urally occurring guanylate cyclases. How- ever, based on the kinetic evidence, this hypothesis has been recently revised in
support of the concept that the liver sol- uble and particulate guanylate cyclases are distinct molecular species (19).
The results of the present study (sum- marized in Table III) clearly indicate that the particulate adrenocortical guanylate cyclase is different from the soluble form of the enzyme by the following criteria: (a) the physiological concentrations of ACTH that stimulate submaximal corti- costerone production in isolated adrenal cells also activate the particulate guany- late cyclase. Similar results are obtained with the synthetic ACTH1-24 which is as potent in steroidogenic activity as ACTH1-39 (27). Of particular significance are the results with ACTH7-38 and 4- methyl-4-aza-cholestane, the two agents which are known to antagonize the ster- oidogenic activity of ACTH in a competi- tive manner (28, 29) also inhibit the ACTH- dependent guanylate cyclase activity. In contrast to these results, the soluble guan- ylate cyclase is unresponsive to ACTH (17, 18); (b) nitric oxide generating compounds which stimulate soluble and most of the particulate guanylate cyclases (16, 30), do not activate the adrenocortical particulate guanylate cyclase. On the other hand, they markedly stimulate the soluble guanylate cyclase; (c) Cd2+, a dithiol reagent (32), is 200-fold more potent in inhibiting the 50% activity of the adrenocortical soluble
guanylate cyclase than that of the partic- ulate guanylate cyclase; (d) Tuftsin (34), a four-amino acid peptide (35, 36), at lower concentrations does not affect the activity of adrenal particulate guanylate cyclase but stimulates the soluble cyclase; (e) ex- cess Mn2+, over and above near-saturating concentrations of the substrate MnGTP, does not stimulate the adrenocortical par- ticulate guanylate cyclase but markedly activates the soluble enzyme at low con- centrations; (f) in response to MnGTP con- centration, the adrenocortical particulate guanylate cyclase shows a curve which is kinetically very different than that ob- tained with the soluble enzyme; (g) the adrenocortical particulate guanylate cy- clase activity is unaffected by all concen- trations of dithiothreitol tested whereas low concentrations of this compound stim- ulate the soluble guanylate cyclase and higher concentrations inhibit the peak ac- tivity of the enzyme.
These results (see above) clearly dem- onstrate that the particulate adrenocor- tical guanylate cyclase is different from the soluble enzyme. Our preliminary stud- ies4 with the solubilized adrenocortical carcinoma particulate enzyme indicate that, except for the hormonal dependence, the solubilized form of the enzyme retains all the properties of the native particulate enzyme. In addition, the molecular mass of the solubilized enzyme is between 270,000 and 300,000, a value close to that of the particulate guanylate cyclase from renal medulla (51); in contrast the molec- ular mass of the soluble enzyme4 is 144,000.
In contrast to the proteolytic activation (49) of the liver particulate guanylate cy- clase, the adrenal particulate enzyme is not stimulated by proteolysis (data not shown). These results, therefore, do not support the concept (50) that the only dif- ference between the soluble and the par- ticulate enzyme is that the hypothetical peptide anchors the membrane bound en- zyme in the lipid bilayer. The present re- sults taken together indicate, on the other hand, that the adrenal particulate and sol-
uble enzymes are distinct molecular spe- cies, a concept also proposed for the liver guanylate cyclase (19). These results also provide the evidence that in the adrenal cortex only the particulate form is coupled to the ACTH-induced steroidogenesis. However, only when the particulate and soluble enzymes are purified to homoge- neity, will it become possible to chemically determine their structural differences.
ACKNOWLEDGMENTS
We thank Dr. J. Ramachandran, Hormone Re- search Laboratory, University of California, San Francisco, for the generous gift of ACTH7-89, and Dr. Eva J. Neer, Department of Biochemistry, Harvard Medical School, for critical comments and the review of this manuscript. We also thank Ms. Helga Ahrens for the art work.
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