Rat Adrenocortical Carcinoma Research Articles

Ubiquitous and bifunctional 180 kDa atrial natriuretic factor-dependent guanylate cyclase

Original studies with rat adrenocortical carcinoma identified a 180 kDa cell-surface protein which contained both guanylate cyclase and atrial natriuretic factor (ANF) receptor, representing a potentially new type of bifunctional receptor protein. It is both a receptor and a guanylate cyclase. This critical conclusion of bifunctionality was based on the observation that the pure 180 kDa protein, whose purity was established by protein staining of the denatured gels, contained both the ligand binding and guanylate cyclase activities. Utilizing the antibody to 180 kDa membrane guanylate cyclase (180 kDa mGC), we now (i) report the complete purification of 180 kDa mGC from rat testes; (ii) demonstrate by affinity cross-linking studies that the homogeneous 180 kDa protein isolated from rat testes and adrenal gland binds ANF and (iii) show that bovine aortic endothelial cell membranes contain the 180 kDa mGC that is ANF-dependent in the production of cyclic GMP. These results validate the conclusion of the bifunctionality, ubiquity, and the general linkage to the ANF-dependent generation of cyclic GMP signal of this protein.

Molecular comparison of ?2-adrenergic receptors from rat adrenocortical carcinoma and human blood platelet

We have previously described a simple two-step purification technique to isolate alpha 2-adrenergic receptors from the rat adrenocortical carcinoma (Jaiswal, R. K. and Sharma, R. K. (1985) Biochem. Biophys. Res. Commun. 130, 58-64). Utilizing this technique we have now achieved approximately 77,000-fold purification to apparent homogeneity of alpha 2-adrenergic receptors from human platelets. We have compared the biochemical characteristics of these receptors with those from the rat, which were purified approximately 40,000-fold to homogeneity. The [125I] receptor proteins from two sources showed: (a) a single radioactive band with a Mr of 64,000 as evidenced by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE); and (b) a single symmetrical peak with a pI of 4.2 by isoelectric focusing polyacrylamide gel electrophoresis. Both proteins showed typical alpha 2-adrenergic binding characteristics with specific binding activities of 13.85 nmol/mg and 14.17 nmol/mg protein. These values are close to the theoretical binding activity of 15.6 nmol/mg protein for 1 mol of the ligand binding 1 mol of the receptor protein. These results attest to the purity of the receptors, to its Mr of 64,000, and to its acidic nature. However, the peptide maps of the radioiodinated alpha 2-adrenergic receptors from rat adrenocortical carcinoma and human blood platelets reveal some distinct differences which may relate to the differences in the pharmacological specificities between rodent and non-rodent alpha 2-adrenergic receptors.

Purification and characterization of the 180-kDa membrane guanylate cyclase containing atrial natriuretic factor receptor from rat adrenal gland and its regulation by protein kinase C

The original concept that cyclic GMP is one of the mediators of the hormone-dependent process of steroidogenesis has been strengthened by the characterization of a 180-kDa protein from rat adrenocortical carcinoma and rat and mouse testes. This protein appears to have an unusual characteristic of containing both the atrial natriuretic factor (ANF)-binding and guanylate cyclase activities, and appears to be intimately involved in the ANF-dependent steroidogenic signal transduction. In rat adrenal glands we now demonstrate: 1) the direct presence of a 180-kDa ANF-binding protein in GTP-affinity purified membrane fraction as evidenced by affinity cross-linking technique and by the Western blot analysis of the partially purified enzyme; 2) that the enzyme is biochemically and immunologically different from the soluble guanylate cyclase as there is no antigenic cross-reactivity of 180-kDa guanylate cyclase antibody with soluble guanylate cyclase; 3) in contrast to the soluble guanylate cyclase, the particulate enzyme is not stimulated by nitrite-generating compounds and hemin; and 4) protein kinase C inhibits both the basal and ANF-dependent guanylate cyclase activity and phosphorylates the 180-kDa guanylate cyclase. These results reveal the presence of a 180-kDa protein in rat adrenal glands and support the contention that: (a) this protein contains both the guanylate cyclase and ANF receptor; (b) the 180-kDa enzyme is coupled with the ANF-dependent cyclic GMP production; (c) the 180-kDa enzyme is biochemically distinct from the nonspecific soluble guanylate cyclase; and (d) there is a protein kinase C-dependent negative regulatory loop for the operation of ANF-dependent cyclic GMP signal pathway which acts via the phosphorylation of 180-kDa guanylate cyclase.

Characterization and regulation by protein kinase C of renal glomerular atrial natriuretic peptide receptor-coupled guanylate cyclase

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 [125I]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 purified protein kinase C inhibited ANP-stimulated guanylate cyclase activity in glomerular membrane 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.

Inhibition of α2-adrenergic receptor-mediated cyclic GMP formation by a phorbol ester, a protein kinase C activator

alpha 2-adrenergic receptor-mediated signal transduction in rat adrenocortical carcinoma cells occurs through the opposing regulation of two second messengers, cyclic GMP and cyclic AMP, in which guanylate cyclase is coupled positively and adenylate cyclase negatively to the receptor signal. We now show that in these cells phorbol 12-myristate 13-acetate (PMA), a known activator of protein kinase C, inhibits the alpha 2-agonist (p-aminoclodine)-dependent production of cyclic GMP in a dose-dependent and time-dependent fashion. The half-maximal inhibitory concentration of PMA was 10(-10) M. A protein kinase C inhibitor, 1-(5-isoquinolinyl-sulfonyl)-2-methyl piperazine (H-7), caused the release of the PMA-dependent attenuation of p-aminoclodine-stimulated cyclic GMP formation. These results suggest that protein kinase C negatively regulates the alpha 2-receptor coupled cyclic GMP system in these cells, a feature apparently shared with the other cyclic GMP-coupled receptors such as those of muscarine, histamine, and atrial natriuretic factor.

Characterization of atrial-natriuretic-factor-receptor-coupled membrane guanylate cyclase from rat and mouse testes

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.

Negative regulation of atrial natriuretic factor receptor coupled membrane guanylate cyclase by phorbol ester Potential protein kinase C regulation of cyclic GMP signal in isolated adrenocortical carcinoma cells of rat

Rat adrenocortical carcinoma cells possess a high density of atrial natriuretic factor (ANF) receptors which are coupled with membrane guanylate cyclase and corticosterone production. Herein we show that pretreatment of these cells with phorbol 12-myristate 13-acetate (PMA), a known activator of protein kinase C, attenuates the ANF-stimulated cyclic GMP accumulation in a dose-dependent manner. The half maximum inhibitory concentration of PMA was 10 −10 M. When these cells were incubated with PMA in the presence of 1-(5-isoquinolinyl-sulfonyl)-2-methyl piperazine, a protein kinase C inhibitor, the PMA-mediated attenuation of ANF-stimulated cyclic GMP formation is blocked. These results suggest that protein kinase C negatively regulates the ANF-receptor coupled membrane guanylate cyclase system in these cells.

Coexistence of guanylate cyclase and atrial natriuretic factor receptor in a 180-kD protein.

Atrial natriuretic factor (ANF) is a peptide hormone that is released from atria and regulates a number of physiological processes, including steroidogenesis in adrenal cortex and testes. The parallel stimulation of membrane guanylate cyclase and corticosterone production in isolated fasciculata cells of rat adrenal cortex has supported the hypothesis of a mediatory role for cyclic guanosine monophosphate (cyclic GMP) in signal transduction. A novel particulate guanylate cyclase tightly coupled with ANF receptor was purified approximately 273,000-fold by two-step affinity chromatography. The enzyme had a molecular size of 180 kilodaltons and was acidic in nature with a pI of 4.7. Its specific activity was 1800 nanomoles of cyclic GMP formed per minute per milligram of protein. The purified enzyme bound ANF with a specific binding activity of 4.01 nanomoles per milligram of protein, a value that is close to the theoretical binding activity of 5.55 nanomoles per milligram of protein for 1 mole of the ligand binding 1 mole of the receptor protein. These results indicate that the guanylate cyclase-coupled ANF receptor exists in a 180-kilodalton protein of rat adrenocortical carcinoma and represent a step toward the elucidation of the basic mechanism of cyclic GMP-mediated transmembrane signal transduction in response to a hormone.

Expression of autophosphorylating protein kinase 500 in normal and neoplastic rat cells.

Autophosphorylating protein kinase 500 (AUT-PK 500) is a unique serine protein kinase that was originally purified and characterized from the rat adrenocortical carcinoma. A specific RIA with an assay sensitivity of 10 ng (0.02 pmol) was developed for AUT-PK 500 and applied to normal, embryonic, fetal, neonatal, immortal, and neoplastic tissues and cultured cells. As compared to normal rat tissues, the expression of AUT-PK 500 is elevated 100-fold in spontaneously occurring adrenocortical carcinoma 494, 50- to 60-fold in four chemically induced, rapidly growing hepatomas, 30-fold in the chemically induced mammary carcinoma, 20-fold in the cultured hepatoma cell line, and 4-fold in the Rat I and Rat II established tissue culture cell lines. There was also a 5-fold increase in the enzyme when freshly cultured rat skin epithelial-like cells were established. Furthermore, in vivo studies showed that when the rat liver was chemically transformed into its premalignant altered foci, there was a 7-fold elevation of AUT-PK 500. Embryonic cells and fetal and neonatal tissues contained barely detectable (less than 0.22 micrograms/mg of protein) amounts of the protein kinase. These results suggest that AUT-PK 500 is not involved in the differentiation process during fetal development but may be elevated during early steps of carcinogenesis and is further elevated during later stages.

Purification and biochemical characterization of α 2-adrenergic receptor from the rat adrenocortical carcinoma

The α 2-adrenergic receptor was purified from rat adrenocortical carcinoma 494 by an affinity chromatographic step using a novel para-aminoclonidine-sepharose resin followed by a gel-permeation high performance liquid chromatographic step. The iodinated receptor protein was homogeneous as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by high performance liquid chromatography. Both SDS-PAGE and high performance liquid chromatographic studies revealed that Mr of the protein was 64,000, suggesting the monomeric nature of the receptor protein. The purified protein showed the typical binding characteristics of α 2-adrenergic receptor.

Rat adrenocortical carcinoma 494 autophosphorylating protein kinase, autophosphorylating protein kinase 500. Purification, biochemical and immunological characterization, and substrate specificity.

A novel autophosphorylating protein kinase, autophosphorylating protein kinase 500, independent of cyclic AMP, cyclic GMP, calcium, and calmodulin was purified from rat adrenocortical carcinoma 494 by ammonium sulfate fractionation followed by the chromatographic steps of DEAE-cellulose, gel filtration, cyclic AMP-epoxy Sepharose, and phosphocellulose. Sometimes two additional chromatographic purification steps of chromatofocusing and gel filtration were necessary for complete purification. The enzyme was homogeneous as evidenced by one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sucrose density sedimentation studies indicated that Mr of the enzyme was 490,000, while ultracentrifugal analysis demonstrated a value of 481,400 (+/-7%). The protein was composed of two identical subunits each with Mr = 250,000. The enzyme molecule was slightly asymmetric with frictional and sedimentation coefficients of 1.28 and 18.20, respectively, and a Stokes radius of 66 A. Isoelectric focusing electrophoresis revealed a single peak with pI 4.6, indicating acidity of the protein. The enzyme self phosphorylated one or more of its serine residues. The reaction utilized the terminal phosphate of ATP; GTP was inactive. Divalent cations (5 mM Mn2+ or 10 mM Mg2+) were essential for optimum activity. Autophosphorylating protein kinase 500 did not phosphorylate the commonly used exogenous substrates such as histones, casein, phosvitin, or protamine. Analysis of autophosphorylating protein kinase 500 with rabbit anti-autophosphorylating protein kinase 500 IgG by immunoelectrophoresis and crossed immune electrophoresis demonstrated single arcs of precipitation, confirming the biochemical demonstration of enzyme purification and homogeneity. Indirect immunofluorescence studies revealed an intracytoplasmic localization of the enzyme in cultured and freshly isolated adrenocortical carcinoma 494 cells. Both cell types revealed an intensity of perinuclear enzyme fluorescence, but an absence of the enzyme in the nuclei or nucleoli. The anti-autophosphorylating protein kinase 500 IgG blocked the self-catalyzed phosphorylation of autophosphorylating protein kinase 500, providing immunological support of the biochemical results that autophosphorylation is an intrinsic characteristic of the enzyme. When autophosphorylating protein kinase 500 was incubated with membrane-bound ribosomes, it phosphorylated a Mr = 31,000 protein. This phosphorylation was blocked by the anti-autophosphorylating protein kinase 500 IgG.(ABSTRACT TRUNCATED AT 400 WORDS)

Fine structural effects of a DDT derivative on a rat adrenocortical carcinoma.

Fine structural effects of a DDT derivative on a rat adrenocortical carcinoma.

In vitro studies of the adrenal metabolism of halogenated side-chain analogues of cholesterol

The cholesterol side-chain cleavage enzyme system of rat adrenal mitochondria and rat adrenocortical carcinoma cells was found to metabolize three halogenated side-chain cholesterol analogues to pregnenolone. The analogues were 26-bromocholesterol, 26-nor-25( RS)-bromocholesterol and 26-iodocholesterol. The addition of Ca 2+ to rat adrenal mitochondria did not produce an increase in the rate of metabolism of the halogenated sterol to pregnenolone. The brominated sterols suppressed the de novo sterol biosynthesis of rat adrenocortical carcinoma cells. The experimental findings are supportive of the notion that a halogen atom at such a position in a sterol is analogous to a hydroxyl group but unlike a proton. ACTH, therefore, may not be a requirement for the uptake and utilization of such sterols. The halogenated sterols may have a use as probes in the study of sterol transfer into and within cells.

The synthesis and metabolism of [6- 3H]-25-hydroxycholesterol in rat adrenal tumor cells

The synthesis of [6- 3H]-25-hydroxycholesterol from 26-norcholest-5-en-3β-ol-25-one is described. The metabolism of the radiolabeled hydroxysterol in rat adrenocortical carcinoma cells and in mitochondria-enriched preparations of rat adrenal tissue was investigated. We found that [6- 3H]-25-hydroxycholesterol was metabolized efficiently to [ 3H]-pregnenolone in both preparations of rat adrenal tissue.

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 steroidogenesis

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 ACTH 1–39 (10 −11 m) and ACTH 1–24 (10 −13 m). The ACTH-antagonist (ACTH 7–38) and 4-methyl-4-aza-5α-cholestane, the compounds that competitively inhibit the steroidogenic activity of ACTH, inhibited the hormonally dependent guanylate cyclase. In contrast, the soluble cyclase was not stimulated by ACTH. (ii) The particulate enzyme was not stimulated by sodium azide, sodium nitroprusside, excess Mn 2+, 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 m m MnGTP whereas the particulate enzyme is not saturable up to 2 m m MnGTP. (iv) Cd 2+ 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 entities.

The influence of the side chain on sterol side-chain cleavage in rat adrenal glands

The cholesterol side-chain cleavage enzyme system of rat adrenal cortex, the enzyme catalyzing a rate-limiting step of adrenal steroidogenesis, was shown to metabolize a series of cholesterol analogues to pregnenolone. In the presence of Ca 2+, rat adrenocortical mitochondria converted the analogue with two less methylene groups (C 25) than cholesterol into pregnenolone at a faster rate than cholesterol. The analogues with one or three less methylene groups ( 26 or C 24) were metabolized at a similar rate to cholesterol. Lengthening the non-polar side chain produced analogues that did not appear to be metabolized. Studies of the metabolism of these analogues in isolated rat adrenocortical carcinoma cells showed that the C 24 and C 25 analogues were converted into pregnenolone much more efficiently than was cholesterol or the C 26 sterol. The experimental findings are explained in terms of the differing ability of each exogenously added sterol to gain access to the active site of the sterol side-chain cleavage enzyme by passage through the membranes of the adrenal cell.

Fine structural and biochemical effects of aminoglutethimide and o,p'-DDD on rat adrenocortical carcinoma 494 and adrenals.

Rats bearing adrenocortical carcinoma 494 were injected daily for 7, 14, or 21 days with aminoglutethimide (AG) or o,p'-DDD. Reversibility of these steroidogenic inhibitors was determined by injecting other animals for either 14 or 21 days and sacrificing them 14 days later. While the drugs had little effect on body or tumor growth, plasma corticosterone levels were reduced a maximum of 88% in normal and 95% in tumor-bearing rats during AG chemotherapy. These levels were unaltered in normal rats by o,p'-DDD and reduced a maximum of 64% in tumor-bearing animals. Relative adrenal weights generally increased during chemotherapy and then returned to control levels. These changes were mainly due to alterations in the lipid and mitochondrial volume fractions. Lipid increased with both drugs while mitochondria increased with o,p'-DDD and decreased with AG. Cholesterol ester levels paralleled the lipid stereology more closely with AG than o,p'-DDD. With both drugs the most notable changes in tumor fine structure was a decrease in mitochondrial internal membranous vesicles and matrical density. Adrenal mitochondria had the irregular, elongated forms characteristic of tumor-bearing animals and were vacuolated (AG) or had internal rings (o,p'-DDD). The large lipid droplets observed during chemotherapy with both drugs were replaced by numerous small droplets in recovery periods.

Pregnenolone biosynthesis in isolated cells of snell rat adrenocortical carcinoma 494

Adrenocorticotrophin (ACTH) produced an insignificant stimulation of pregnenolone biosynthesis from endogenous precursors in isolated cells prepared from the rat Snell adrenal carcinoma 494. On the addition of 25-hydroxycholesterol, the rate of pregnenolone synthesis increased 10-fold. These results, noting also the very low cholesterol content of the tumor cells, suggested that lack of cholesterol was responsible for the poor steroidogenic response of the cells to ACTH. Endogenous pregnenolone production was sensitive to cytochalasin B as well as cycloheximide. However, pregnenolone synthesis after the addition of 25-hydroxycholesterol was not affected by these inhibitors. Removal of cycloheximide from the cells resulted in the immediate restoration of the initial rate of pregnenolone synthesis from endogenous precursors. This suggested that cycloheximide was interfering with the action of a stable activated intracellular messenger.

Rat adrenocortical carcinoma 494: an integrated structural, stereological, and biochemical analysis.

Snell adrenocortical tumor 494 was implanted into male Sprague-Dawley rats and recovered 7, 14, 21, 28 or 35 days following initial detection by palpation (7-10 days following transplantation). Electron microscopic, stereological and biochemical analyses of the tumor were compared to adrenals of normal animals to serve as a baseline for further studies of the effects of chemotherapeutic agents on tumor cells. Tumor cells possessed oval or elongated mitochondrial profiles with tubular cristae, one or two very large (greater than 5 micrometer) lipid droplets, abundant ribosomes and coated vesicles, and sparse rough and smooth endoplasmic reticulum. Stereologic evaluation revealed that tumor lipid volume was 41% and mitochondrial volume 29% that of the normal adrenal controls. Tumor nuclei were 2.5 times larger than adrenocortical nuclei while cellular volumes were similar. On a net weight basis, tumor cholesterol was 55%, cholesterol ester 2.2%, and lipid phosphate 25% of respective mean values for normal adrenal glands. The tumor cholesterol: cholesterol ester ratio progressively decreased with time but remained 18-fold greater than the normal adrenal. Plasma corticosterone levels in tumor-bearing rats were elevated 3-fold by 14 days and initial detection. The adrenals of the tumor-bearing host exhibited marked involution, the extent of which was directly related to tumor size.

Abnormal regulation of adenosine 3',5'-monophosphate and corticosterone formation in an adrenocortical carcinoma.

A spontaneously occurring rat adrenocortical carcinoma which produces corticosterone was maintained by transplantation. The carcinoma appeared to utilize corticosterone biosynthetic steps similar to those of the normal adrenal, but the tumor produced only about 1-10% as much corticosterone per unit tissue weight as nontumorous adrenal glands. The tumor demonstrated little or no increase in corticosterone production in response to adrenocorticotropic hormone (ACTH) either in vivo or in vitro. In normal adrenals, ACTH increases the activity of adenyl cyclase which catalyzes the conversion of adenosine triphosphate (ATP) to adenosine-3',5'-monophosphate (cyclic AMP), the latter then serving as an intracellular regulator of steroidogenesis. ACTH failed to increase cyclic AMP levels in the tumor in vivo or in slices in vitro, conditions under which there were 50- and 20-fold increases in nontumorous adrenals. However, in homogenates fortified with exogenous ATP, adenyl cyclase activity was comparable in the tumor and adrenals, and cyclic AMP formation was increased 3-fold by ACTH in each. As measured in homogenates, the tumor did not possess a greater ability to destroy cyclic AMP than did normal adrenals. Although ATP levels in the carcinoma were found to be considerably lower than those in normal adrenals, it was not clear that this finding can explain the inability of ACTH to increase cyclic AMP levels in intact tumor cells. While the failure to normally influence cyclic AMP levels in the carcinoma cells could be an important factor in the lack of a steroid response to ACTH, several lines of evidence suggest that the tumor possesses one or more additional abnormalities in the regulation of steroidogenesis. First, in the absence of ACTH stimulation, the tissue concentrations of cyclic AMP were comparable in the tumor and in nontumorous adrenals, but these cyclic AMP levels were associated with a lower level of steroidogenesis in the tumor. Second, tumor slices failed to increase corticosterone production when incubated with cyclic AMP, in contrast to 5-fold increases observed with nontumorous adrenals.