Activation of a cGMP-stimulated cAMP phosphodiesterase by protein kinase C in a liver Golgi-endosomal fraction.

The ability of Ca2+/phospholipid-dependent protein kinase (protein kinase C, PKC) to stimulate cAMP phosphodiesterase (PDE) activity in a liver Golgi-endosomal (GE) fraction was examined in vivo and in a cell-free system. Injection into rats of 4 beta-phorbol 12-myristate 13-acetate, a known activator of PKC, caused a rapid and marked increase in PKC activity (+325% at 10 min) in the GE fraction, along with an increase in the abundance of the PKC alpha-isoform as seen on Western immunoblots. Concurrently, 4 beta-phorbol 12-myristate 13-acetate treatment caused a time-dependent increase in cAMP PDE activity in the GE fraction (96% at 30 min). Addition of the catalytic subunit of protein kinase A (PKA) to GE fractions from control and 4 beta-phorbol 12-myristate 13-acetate-treated rats led to a comparable increase (130-150%) in PDE activity, suggesting that PKA is probably not involved in the in-vivo effect of 4 beta-phorbol 12-myristate 13-acetate. In contrast, addition of purified PKC increased (twofold) PDE activity in GE fractions from control rats but affected only slightly the activity in GE fractions from 4 beta-phorbol 12-myristate 13-acetate-treated rats. About 50% of the Triton-X-100-solubilized cAMP PDE activity in the GE fraction was immunoprecipitated with an anti-PDE3 antibody. On DEAE-Sephacel chromatography, three peaks of PDE were sequentially eluted: one early peak, which was stimulated by cGMP and inhibited by erythro-9 (2-hydroxy-3-nonyl) adenine (EHNA); a selective inhibitor of type 2 PDEs; and two retarded peaks of activity, which were potently inhibited by cGMP and cilostamide, an inhibitor of type 3 PDEs. Further characterization of peak I by HPLC resolved a major peak which was activated (threefold) by 5 microM cGMP and inhibited (87%) by 25 microM EHNA, and a minor peak which was insensitive to EHNA and cilostamide. 4 beta-Phorbol 12-myristate 13-acetate treatment caused a selective increase (2.5-fold) in the activity associated with DEAE-Sephacel peak I, without changing the K(m) value. These results suggest that PKC selectively activates a PDE2, cGMP-stimulated isoform in the GE fraction.     

Effects of IGF-I on proliferation and steroidogenesis of cultured adrenal zona glomerulosa cells

In a primary culture of bovine adrenal zona glomerulosa cells, insulin-like growth factor I (IGF-I)/somatomedin C stimulated DNA synthesis, as measured by [3H] thymidine uptake, at concentrations of 10(-9) and 10(-7) M. IGF-I also prevented ACTH-induced suppression of [3H] thymidine uptake. IGF-I in no way affected aldosterone secretion during short-term exposure to cultured cells, however. Our findings suggest that IGF-I plays an important role in the proliferation of adrenal zona glomerulosa cells.     

Specificity of cGMP binding to a purified cGMP-stimulated phosphodiesterase from bovine adrenal tissue

The binding of [3H]cGMP (guanosine 3',5'-monophosphate) to purified bovine adrenal cGMP-stimulated phosphodiesterase was measured by Millipore filtration on cellulose ester filter. [3H]cGMP-binding activity was enhanced when the assay was terminated in buffer containing 70% of saturated ammonium sulfate to dilute the enzyme and wash the filters. The cGMP-binding activity was co-purified with the phosphodiesterase activity. The binding of [3H]cGMP to purified enzyme was measured in the presence or absence of the phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine. 1-Methyl-3-isobutylxanthine showed linear competitive inhibition with respect to cGMP as substrate in the phosphodiesterase reaction but stimulated the [3H]cGMP-binding activity in the binding assay. The stimulatory effect appeared not to be the result of preservation from [3H]cGMP hydrolysis; no cGMP phosphodiesterase activity has been measured under the cGMP-binding assay conditions, in the absence or presence of the inhibitor. Half-maximal stimulation by 1-methyl-3-isobutylxanthine occurred in the 5-7 microM concentration range. The specificity of binding of [3H]cGMP was investigated by adding increasing concentration of unlabeled analogs of cAMP (adenosine 3',5'-monophosphate) and cGMP. The binding of [3H]cGMP (50 nM) was displaced by unlabeled cGMP and cAMP with the following potency: 50% displacement was reached at the 0.1 microM cGMP range and only at a fiftyfold higher cAMP concentration. Our data with comparative series of analogs (e.g. 5'-amino-5'-deoxyguanosine 3',5'-monophosphate and 3'-amino-3'-deoxyguanosine 3',5'-monophosphate) showed that the potencies of stimulation of cAMP phosphodiesterase activity parallels displacement curves or [3H]cGMP binding to purified enzyme with no correlation with phosphodiesterase inhibition sequences. Those experiments suggest that the cGMP-binding activity is directly related to the non-catalytic (allosteric) cGMP-binding site.     

Structure and function studies of the cGMP-stimulated phosphodiesterase.

Studies of cGMP binding to both the native cyclic GMP-stimulated phosphodiesterase and to two unique isolated chymotryptic fragments lacking the catalytic domain suggest that the enzyme contains two noncatalytic cGMP-binding sites/homodimer. In the presence of high concentrations of ammonium sulfate, 2 mol of cGMP are bound/mol of cGMP-stimulated phosphodiesterase homodimer. Under these conditions, linear Scatchard plots of binding are obtained that give an apparent Kd of approximately 2 microM. The inclusion of 3-isobutyl-1-methylxanthine produces a curvilinear plot. In the absence of ammonium sulfate, the dissociation of cGMP from the holoenzyme is rapid, having a t1/2 of less than 10 s, and addition of ammonium sulfate to the incubation greatly decreases this rate of dissociation. The native enzyme is resistant to degradation by chymotrypsin in the absence of cGMP; however, in its presence, chymotrypsin treatment produces several discrete fragments. Similarly, in the presence but not in the absence of cGMP, dicyclohexylcarbodiimide causes an irreversible activation of the enzyme without cross-linking the nucleotide to the phosphodiesterase. Both observations provide evidence that a different conformation in the enzyme results from cGMP binding. Only the conformation formed upon cGMP binding is easily attacked by chymotrypsin or permanently activated by treatment with dicyclohexylcarbodiimide. One major chymotryptic cleavage site exposed by cGMP binding is at tyrosine 553, implying that this region takes part in the conformational change. Limited proteolysis experiments indicate that these noncatalytic binding sites are located within a region of internal sequence homology previously proposed to include the cGMP-binding site(s) and that they retain a high affinity and specificity for cGMP independent of the catalytic domain of the enzyme. The products formed by partial proteolysis can be separated into individual catalytically active and cGMP-binding fractions by anion exchange chromatography. Gel filtration and electrophoresis analysis of the isolated fractions suggest that the cGMP-binding peak has a dimeric structure. Moreover, it can be further resolved by polyethyleneimine high performance liquid chromatography into two peaks (Peaks IIIA and IIIB). Peak IIIA binds 2 mol of cGMP/mol of dimer with an apparent Kd of 0.2 microM. Peak IIIB, however, has greatly reduced cGMP binding. Further digestion of these fragments with cyanogen bromide show that the differences between Peaks IIIA and IIIB are due to one or more additional proteolytic nicks in IIIB that remove a few residues near its C terminus, most probably residues 523-550 or 534-550. This in turn suggests that this region is essential for cGMP-binding activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inositol phosphate release and steroidogenesis in rat adrenal glomerulosa cells. Comparison of the effects of endothelin, angiotensin II and vasopressin.

Endothelin has steroidogenic activity in adrenal glomerulosa cells, as do two other vasoconstrictor peptides, angiotensin II and vasopressin. The steroidogenic activities of angiotensin II and vasopressin are probably mediated via the phosphatidylinositol-turnover pathway and associated changes in cytosolic Ca2+ concentration. Endothelin caused a steroidogenic response, which was small compared with that to angiotensin II and quantitatively similar to the vasopressin response. Cytosolic free Ca2+ responses were similarly higher to angiotensin II than to either of the other two peptides. However, total inositol phosphate responses to endothelin and angiotensin II were similar when these were measured over 20 min, and were quantitatively greater than the vasopressin response. A detailed study has been made of the phosphatidylinositol-turnover response to endothelin in comparison with responses to angiotensin II and vasopressin. Each of the three peptides produced a rapid and transient rise in Ins(1,4,5)P3 (max. 5-15 s), followed by a slow sustained rise. Ins(1,4,5)P3 was metabolized by both dephosphorylation and phosphorylation pathways, but the relative importance of the two metabolic pathways was different under stimulation by each of the three peptides. These findings show that adrenal glomerulosa cells can distinguish between the stimulation of phosphatidylinositol turnover by three different effectors. These differences in the pathway may be associated with the observed different steroidogenic and Ca2+ responses to the three peptides.     

Stimulus-secretion coupling in angiotensin-stimulated adrenal glomerulosa cells

Adrenal glomerulosa cell is a suitable model for a comparative study of signal transducing mechanisms since its secretory activity is regulated by at least three different mechanisms: the adenylate cyclase-cAMP system (for ACTH), the voltage-dependent Ca2+ channel (for K+ and perhaps for angiotensin II) and the inositol 1,4,5-trisphosphate-Ca2+ system (for angiotensin II and vasopressin). The role of inositol phosphates, extracellular Ca2+ and protein kinase C in the induction and sustaining of aldosterone production by cells exposed to angiotensin II is critically reviewed.     

The effects of extracellular K+ and angiotensin II on cytosolic Ca++ and steroidogenesis in adrenal glomerulosa cells

We evaluated changes in cytosolic calcium concentration (Ca++) and steroidogenesis in rat adrenal glomerulosa cells (GC) stimulated with potassium (K+) or angiotensin II (AII). Cytosolic Ca++ concentration was determined using the Ca++-sensitive, fluorescent dye QUIN 2. Raising extracellular K+ increased cytosolic Ca++ from 267 +/- 23 nM at 3.7 mM K+ to a maximum of 377 +/- 40 nM at 8.7 mM K+ (p less than 0.01, N = 23). AII also increased cytosolic Ca++ from 238 +/- 20 nM to a maximum of 427 +/- 42 nM at 10(-7) M (p less than 0.01, N = 16). In parallel studies, K+ and AII stimulated aldosterone secretion from QUIN 2-loaded GC at concentrations similar to those which raised cytosolic Ca++. QUIN 2-loaded cells were as responsive steroidogenically as unloaded cells and showed trypan blue exclusion of 98% suggesting that QUIN 2 did not compromise cellular viability. These results provide direct support for a role of cytosolic Ca++ as a second messenger during stimulation of aldosterone secretion by both K+ and AII.     

Control of phosphatidylinositol turnover in adrenal glomerulosa cells

The purpose of the present experiments was to compare the effects on phosphatidylinositol metabolism of agents stimulating aldosterone secretion. Glomerulosa cells, isolated from rat adrenals, were incubated in the presence of one of the following stimuli: angiotensin II, elevated potassium concentration, corticotropin, dibutyryl cyclic AMP and prostaglandin E2. Of all these substances, only angiotensin II stimulated the incorporation of [32P]phosphate into phosphatidylinositol. The effect was already detected 2.5 min and was still maintained 60 min after the onset of stimulation. A slight enhancement of the incorporation into other phospholipids was observed in the first minutes of stimulation. Cycloheximide abolished the effect of angiotensin II on aldosterone production, but not on phosphatidylinositol synthesis. In cells prelabelled with [32P]phosphate, radioactivity in phosphatidylinositol relative to that in other phospholipids decreased in response to angiotensin II within 5 min. This indicates that angiotensin II induces a specific breakdown of phosphatidylinositol. Corticotropin failed to enhance the incorporation of [32P]phosphate into phosphatidylinositol and other phospholipids in isolated fasciculate-reticularis cells. The results suggests that although both angiotensin II and potassium are presumed to act through changes in calcium metabolism, angiotensin alone generates the calcium signal by increased phosphatidylinositol turnover.     

Hormone-sensitive cAMP phosphodiesterase in liver and fat cells

Summary The enzymatic characteristics and the mode of hormone-dependent stimulation of cAMP phosphodiesterase are reviewed. The hormone-sensitive phosphodiesterase is a low Km enzyme, which has been found in liver and fat cells. The fat cell enzyme is mostly associated with the endoplasmic reticulum. The liver cell enzyme is also associated with certain subcellular structures.The hormone-sensitive phosphodiesterase appears to have catalytic and regulatory domains and is thought to be attached to subcellular structures at the regulatory portion of the enzyme. The catalytic domain of the fat cell enzyme can be obtained in a soluble form from the microsomal preparation by mild proteolysis or by dithiothreitol treatment at 0–4 °C. The catalytic domain of the liver enzyme can be solubilized by either hypotonic treatment or mild trypsin digestion. The catalytic domains solubilized from the basal and hormonally activated forms of the enzyme are apparently identical.The membrane-bound basal enzyme from adipocytes is activated in a concentrated salt solution without being solubilized. On the other hand, the plus-insulin activity is deactivated in a low salt solution or by a short dithiothreitol treatment at 37°, apparently without suffering any changes in the catalytic domain. In contrast, p-chloromercuriphenyl sulfonate seems to inactivate the enzyme by interacting with SH-groups in the catalytic domain. Although the liver enzyme is not similarly affected by salt concentrations, its catalytic activity is blocked by p-chloromercuribenzoate.The adipocyte enzyme can be solubilized with a mixture of Lubrol WX and Zwittergent 3–14. The apparent Stokes radius of the basal enzyme is approximately 87 A, while that of the hormone-stimulated enzyme is approximately 94 A.Apparently, the same species of phosphodiesterase is activated by both insulin and epinephrine in fat cells and by insulin and glucagon in liver, possibly being mediated by reactions involving phosphorylation. However, it is yet to be ascertained how phosphorylation is involved and how the apparent Stokes radius of the adipocyte enzyme is increased as a result of stimulation.     

Dual effects of dopamine in rat adrenal glomerulosa cells

The effects of dopamine (DA) on cAMP production and aldosterone secretion were compared in freshly isolated cells and in primary cultures of rat adrenal glomerulosa cells. Under isolated conditions, glomerulosa cells exhibited dopamine receptors from DA-1 and DA-2 subclass, whereas in cultured conditions, where cells are very sensitive to their known stimuli, cells only exhibited dopamine receptors from the DA-1 subclass. Moreover, unlike freshly isolated cells, dopamine stimulated both cAMP production and aldosterone secretion in 3-day cultured preparations. These effects were receptor specific since they were completely suppressed by Scherring 23390 (a specific DA-1 antagonist) and were unaffected by a beta-adrenergic antagonist. As in vivo rat adrenal cortex contains DA, we discuss a possible involvement of this neurotransmitter in the regulation of aldosterone secretion.     

Effects of ACTH and angiotensin II on cytosolic calcium in cultured adrenal glomerulosa cells. Role of cAMP production in the ACTH effect

We have used microspectrofluorometry and video imaging techniques in order to study and compare the changes in intracellular calcium concentrations [( Ca2+]i) of individual Fura-2 loaded glomerulosa cells cultured for three days and stimulated either with angiotensin II (AT), K+, or adrenocorticotropin (ACTH). As previously demonstrated for freshly isolated cells, K+ ion induces an immediate increase in [Ca2+]i, although AT induces a biphasic response, characterized by an initial transient spike, followed by a sustained plateau. In this study, we demonstrate, for the first time, that ACTH is able to induce a [Ca2+]i increase in cultured glomerulosa cells from rat and bovine sources. Moreover, it is clear that the pattern of [Ca2+]i increase elicited by ACTH is different from that observed with AT. In most cases, addition of ACTH leads to a slow increase in [Ca2+]i after a long latency period ranging from 10-15 min, which could be correlated to cAMP time-production. The present results show that: (a) in the absence of extracellular Ca2+, ACTH does not increase [Ca2+]i; (b) the response develops slowly and cases immediately after [Ca2+]e depletion or addition of calcium channel blockers, such as nifedipine or omega-conotoxin; (c) the addition of the calcium channel agonist Bay K 8644 enhances the ACTH response; (d) the cAMP analog, 8-Br-cAMP, induces an increase in [Ca2+]i similar to that observed with ACTH, which is also dependent of the presence of calcium in the extracellular medium; (e) time-production of ACTH-induced cAMP follows quite well the increase in [Ca2+]i; (f) Bay K 8644 also enhances the 8-Br-cAMP induced increase in [Ca2+]i; and (g) ACTH-induced Cai response is inhibited by the specific protein kinase A blocker, HA1004. These observations, combined with previous results obtained on the effects of ACTH on calcium currents and action potentials, suggest that the [Ca2+]i increase induced by ACTH results from a calcium influx through dihydropyridine and omega-conotoxin sensitive calcium channels, which need to be phosphorylated by cAMP for full activation. The use of video-imaging techniques has allowed us to examine the spatial distribution of changes in [Ca2+]i in single cells. The ability to simultaneously record images of a number of cells confirm the heterogeneity of cellular responses, and corroborate results obtained through photocounting only. Our results indicate that ACTH initially increases [Ca2+]i locally beneath the cell membrane and throughout the cell thereafter, whereas angiotensin II elicits a more prominent effect in certain regions of the cell and eventually extends to the entire cell surface.     

Inhibition of steroidogenesis in rat adrenal cortex cells by a threonine analogue.

We have investigated the ability of amino acid analogues of serine and threonine to inhibit the increase in steroidogenesis elicited by addition of ACTH or cAMP in cells isolated from the rat adrenal cortex. We have found that the serine analogues, D, L-isoserine, alpha-methyl-D, L-serine and L-homoserine, are almost totally ineffective in inhibiting this process but that the threonine analogue, D, L-beta-hydroxynorvaline, at a concentration of 300 microM inhibits stimulated steroid hormone biosynthesis by ca 95%, while inhibiting overall protein synthesis by only ca 40%. This inhibition was found to occur in a dose-dependent manner and to be reversible by a stoichiometric concentration of threonine. These studies suggest that beta-hydroxynorvaline is functioning as a threonine analogue in our experimental system. Both the onset of inhibition by analogue and reversal of this inhibition by the natural amino acid occurred rapidly, without detectable lag. Since results obtained using cAMP as stimulant parallel those obtained using ACTH, the inhibitory effect of the analogue seems to occur subsequent to the synthesis of cAMP. Additionally, the analogue does not inhibit the conversion of pregnenolone to corticosterone, suggesting the site of action of analogue occurs prior to the synthesis of pregnenolone from cholesterol. Thus, the analogue may be exerting its effect on a protein that is synthesized subsequent to ACTH addition and is important in the acute phase of stimulated steroid hormone biosynthesis. Further, since ACTH action on adrenal cortex cells causes the activation of protein kinase A, which phosphorylates serine and threonine residues, it is possible that the effect of the analogue is to prevent the phosphorylation of a newly-synthesized protein.     

Na-Ca exchanger as a calcium influx pathway in adrenal glomerulosa cells

When aequorin-loaded glomerulosa cells were incubated in isotonic Na2+-free medium containing N-methyl-D-glucamine instead of NaCl, there was an increase in cytoplasmic free calcium concentration, [Ca2+] c, which was not observed when extracellular calcium concentration was reduced to 1 microM. Upon removal of extracellular sodium, there was nearly five-fold increase in fractional efflux ratio of calcium. The reduction of extracellular sodium resulted in a stimulation of calcium influx rate, the magnitude of which was dependent on extracellular sodium concentration. Similar stimulation of calcium influx was observed when extracellular sodium was replaced with lithium. Nitrendipine did not affect the calcium influx induced by the reduction of extracellular sodium while a derivative of amiloride 3',4'-dichlorobenzamil, which inhibits Na-Ca exchange, attenuated calcium influx observed in sodium-free medium. These results indicate that removal of extracellular sodium leads to an increase in [Ca2+] c by stimulating calcium influx and that calcium enters the cell via Na-Ca exchanger.     

Serotonin stimulates calcium influx in isolated rat adrenal zona glomerulosa cells.

To investigate the role of calcium as a second messenger in serotonin-stimulated aldosterone secretion, radiolabelled calcium influx studies were carried out in purified rat adrenal zona glomerulosa cells using 45CaCl2. The results show that serotonin caused calcium influx within 45 seconds of addition and this continued for up to 105 seconds. Angiotensin II also caused calcium influx; however, the effect was significantly smaller than that of serotonin. Serotonin-stimulated calcium influx could be inhibited by the calcium antagonist verapamil and by methysergide, a selective serotonin receptor type-1/2 antagonist. The data indicate that serotonin directly stimulates calcium uptake in zona glomerulosa cells via calcium channels which are coupled to specific serotonin receptors.     

Angiotensin II receptors and mechanisms of action in adrenal glomerulosa cells

The plasma-membrane receptors, coupling mechanisms, and effector enzymes that mediate target-cell activation by angiotensin II (AII) have been characterized in rat and bovine adrenal glomerulosa cells. The AII holoreceptor is a glycoprotein of Mr approximately 125,000 under non-denaturing conditions. Photoaffinity labeling of AII receptors with azido-AII derivatives has shown size heterogeneity among the AII binding sites between species and target tissues, with Mr values of 55,000 to 79,000. Such variations in molecular size probably reflect differences in carbohydrate content of the individual receptor sites. The adrenal AII receptor, like that in other tissues, is coupled to the inhibitory guanine nucleotide inhibitory protein (Ni). However, studies with pertussis toxin have shown that stimulation of aldosterone production by AII is not mediated by Ni but by a pertussis-insensitive nucleotide regulatory protein of unidentified nature. Although Ni is not involved in the stimulatory action of AII on steroidogenesis, it does mediate the inhibitory effects of high concentrations of AII upon aldosterone production. The actions of AII on adrenal cortical function are thus regulated by at least two guanine nucleotide regulatory proteins that are selectively activated by increasing AII concentrations. The principal effector enzyme in AII action is phospholipase C, which is rapidly stimulated in rat and bovine glomerulosa after AII receptor activation. AII-induced breakdown of phosphatidylinositol bisphosphate (PIP2) and phosphatidylinositol phosphate (PIP) leads to formation of inositol 1,4,5-trisphosphate (IP3) and inositol 1,4-bisphosphate (IP2). These are metabolized predominantly to inositol-4-monophosphate, which serves as a marker of polyphosphoinositide breakdown, whereas inositol-1-phosphate is largely derived from phosphatidylinositol hydrolysis. The AII-stimulated glomerulosa cell also produces inositol 1,3,4-trisphosphate, a biologically inactive IP3 isomer formed from Ins-1,4,5-trisphosphate via inositol tetrakisphosphate (IP4) during ligand activation in several calcium-dependent target cells. The Ins-1,4,5-P3 formed during AII action binds with high affinity to specific intracellular receptors that have been characterized in the bovine adrenal gland and other AII target tissues, and may represent the sites through which IP3 causes calcium mobilization during the initiation of cellular responses.     

Angiotensin-induced formation and metabolism of inositol polyphosphates in bovine adrenal glomerulosa cells

The actions of angiotensin II (AII) on inositol polyphosphate production and metabolism were analyzed in cultured bovine adrenal glomerulosa cells. In cells labeled for 24 hr with [3H]inositol, AII caused a rapid and prominent rise in formation of Ins-P3 (mainly the Ins-1,3,4,-P3 isomer) and of Ins-P4, with marked increases in two isomers of Ins-P2 and Ins-P. These findings are consistent with rapid formation and turnover of Ins-1,4,5-P3, partly via conversion to Ins-1,3,4,5-P4 with subsequent metabolism to Ins-1,3,4-P3 and lower inositol phosphates. The demonstration of a cytosolic Ins-P3-kinase gave further evidence for the presence of the tris/tetrakisphosphate pathway and Ins-P4 synthesis during AII action in the bovine adrenal cortex.     

Immunocytochemical localization of ACTH-like immunoreactivity in rat adrenal glomerulosa and fasciculata cells

The role of ACTH in the synthesis of the adrenocortical hormones has been largely described. In order to investigate the localization of this peptide at the subcellular level of the adrenal glomerulosa and fasciculata cells, an immunocytological method was used. Rat adrenals were fixed and frozen. Ultrathin sections obtained by cryoultramicrotomy, were incubated with anti-beta (1-24) ACTH or anti-alpha (17-39) ACTH sera. The antigen-antibody reaction was detected by PAP complexes (revealed by 4-chloro-1-naphthol) or with protein A-colloidal gold or IgG-colloidal gold. The results obtained were the same whatever the antisera of the technique employed. All the cells of the adrenal zona glomerulosa and zona fasciculata were labelled. ACTH-like immunoreactivity in zona glomerulosa and zona fasciculata cells was observed at the plasma membrane level, in cytoplasmic matrix, mitochondria and nucleus (in the euchromatin close to the heterochromatin aggregations and, occasionally, associated with the nucleolus). No immunoreactivity was observed when non-immune serum or anti-ACTH serum preincubated with ACTH were used, nor there was any modification of the immunocytochemical reaction when anti-ACTH serum incubated with heterologous antigens was employed. These data, demonstrate the presence of endogenous ACTH in both adrenal glomerulosa and fasciculata cells, and suggest that the peptide is internalized after binding to the plasma membrane.     

Prostacyclin: a potent stimulator of adrenal steroidogenesis.

The relative potencies of various prostaglandins were investigated in trypsin-dispersed cat adrenocortical cells. Prostacyclin proved to be the most potent steroidogenic prostaglandin, being 100-1000 times more potent than PGE2. This stimulant effect of prostacyclin was only partially dependent upon the presence of extracellular calcium and was associated with increased levels of cyclic AMP. These data suggest a possible role for prostacyclin in corticosteroidogenesis.