ACTH diazotized to agarose: effects on isolated adrenal cells

ACTH coupled in an azo linkage to an agarose support induces steroidogenesis in free adrenal cells in the same manner as does free ACTH. Observation of incubates of adrenal cells and ACTH-agarose indicates that 1) agarose-ACTH is not adherent to the cell surface, 2) entrance of ACTH into the cell may not be a prerequisite to the initiation of steroidogenesis, 3) the continuous presence of ACTH is not necessary to maintain maximal steroid production in cellular incubates, and 4) induction does not alter the functional integrity of the bound corticotropin.     

Stimulation of phosphatidylinositol turnover by acetylcholine, angiotensin II and ACTH in bovine adrenal fasciculata cells

The effect of acetylcholine, angiotensin II and adrenocorticotropin (ACTH) on phosphatidylinositol (PI) metabolism was examined using bovine adrenocortical fasciculata cell suspensions. The three agents, which acutely stimulate glucocorticoid production by these cells, were all able to increase [32P]Pi incorporation into cellular PI. However, whereas the relative steroidogenic potency (at maximally active concentrations) was ACTH greater than or equal to angiotensin II greater than acetylcholine, the effect on PI labeling was in the order angiotensin II greater than acetylcholine greater than ACTH. The dose-response curves for steroidogenesis and that for PI labeling were superimposable in the case of angiotensin II (ED50 = 1 X 10(-8) M) and of acetylcholine (ED50 = 5 X 10(-7) M), while the two responses were dissociated under graded ACTH challenge. Both steroidogenic response and increased PI labeling elicited by angiotensin II and acetylcholine were respectively inhibited by (Sar1-Ala8)-angiotensin II and muscarinic antagonists. Time-course study showed that in the case of angiotensin II and acetylcholine, the sequence of events was: increased phosphatidic acid labeling, increased PI labeling, activated steroidogenesis. By sharp contrast, under ACTH stimulation, increased steroidogenesis was detected well before activation of PI metabolism. These data suggest that in bovine adrenocortical fasciculata cell, steroidogenesis may be activated by two different pathways. The first one would act mainly through cyclic AMP-dependent intracellular events and is usually accepted in the mechanism of action of ACTH. The other, cyclic AMP-independent pathway, as in the case of angiotensin II and acetylcholine actions, may involve phospholipid-mediated intracellular processes.     

Differential redistribution of protein kinase C in human aldosteronoma cells and adjacent adrenal cells stimulated with ACTH and angiotensin II

We have examined protein kinase C activity and hormone secretion in aldosteronoma cells derived from adrenocortical glomerulosa cells and in adjacent adrenal cells containing adrenocortical fasciculata-reticularis cells. When aldosteronoma cells were stimulated with ACTH or angiotensin II, protein kinase C activity gradually decreased in cytosol whereas it increased in membrane. Coincident with the changes of protein kinase C activity, there was enhancement of secretion of aldosterone. On the other hand, incubation of adjacent adrenal fasciculata-reticularis cells with ACTH induced cortisol secretion and an increase in cytosolic protein kinase C activity, accompanied by a decrease in the enzyme activity in membrane. Upon stimulation with angiotensin II, adjacent adrenal fasciculata-reticularis cells did not secrete cortisol and no significant changes of protein kinase C activities were observed in either cytosolic or membrane fractions. These results indicate that both ACTH and angiotensin II stimulate aldosterone secretion and cause translocation of protein kinase C from cytosol to membranes in aldosteronoma cells, whereas, in fasciculata-reticularis cells, only ACTH stimulates cortisol secretion and this is associated with translocation of protein kinase C in the opposite direction, viz., from membrane to cytosol.     

Effect of atrial natriuretic peptide on ACTH, dibutyryl cAMP, angiotensin II and potassium-stimulated aldosterone secretion by rat adrenal glomerulosa cells

We examined the effect of rat atrial natriuretic peptide (ANP) on ACTH, dibutyryl cAMP, angiotensin II and potassium-stimulated aldosterone secretion by dispersed rat adrenal glomerulosa cells. ANP inhibited ACTH, angiotensin II and potassium-stimulated aldosterone secretion with IC50's between 0.15-0.20 nM. Inhibition by 10 nM ANP could not be overcome with higher concentrations of these stimuli. ANP shifted the dibutyryl cAMP dose-response curve slightly to the right but did not blunt the maximal aldosterone secretory response. The sites of ANP inhibition in the aldosterone biosynthetic pathway for these stimuli were also examined. ANP inhibited activation of the cholesterol desmolase (CD) enzyme complex by ACTH, angiotensin II and potassium. Activation of the corticosterone methyl oxidase (CMO) enzyme complex by potassium was inhibited by ANP, however, activation by ACTH was not blocked. We concluded that: 1) ANP is a potent inhibitor of ACTH, angiotensin II and potassium-stimulated aldosterone secretion; 2) inhibition of ACTH stimulation is primarily due to lower cAMP levels and; 3) inhibition of angiotensin II and potassium stimulation reflects a block in the activating mechanism of the CMO and/or CD enzyme complexes, whereas CD but not CMO activation by ACTH is inhibited by ANP.     

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.     

ACTH regulation of cholesterol movement in isolated adrenal cells

Confluent bovine adrenal cell primary cultures respond to stimulation by adrenocorticotropin (ACTH) to produce steroids (initially predominantly cortisol and corticosterone) at about one-tenth of the output of similarly stimulated rat adrenal cells. The early events of steroidogenesis, following ACTH stimulation, have been investigated in primary cultures of bovine adrenal cortical cells. Steroidogenesis was elevated 4-6-fold within 5 min of exposure to 10(-7) M ACTH and increased linearly for 12 h and declined thereafter. Cholesterol side-chain cleavage (SCC) activity was increased 2.5-fold in mitochondria isolated from cells exposed for 2 h to ACTH and 0.5 mM aminoglutethimide (AMG), even though cytochrome P-450scc only increases after 12 h. Mitochondrial-free cholesterol levels increased during the same time period (16.5-25 micrograms/mg of protein), but then both cholesterol levels and SCC activity declined in parallel. More prolonged exposure to ACTH prior to addition of AMG caused the elevation in mitochondrial cholesterol to more than double, possibly due to enhanced binding capacity. Early ACTH-induced effects on cellular steroidogenesis result from these changes in mitochondrial-free cholesterol. The maximum rate of cholesterol transport to mitochondria in AMG-blocked cells was consistent with the maximum rate of cellular steroidogenesis. Cycloheximide (0.2 mM) rapidly blocked (less than 10 min) cellular steroidogenesis, cholesterol SCC activity, and access of cholesterol to cytochrome P-450scc without affecting mitochondrial-free cholesterol. Exposure of confluent cultures to the potent environmental toxicant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (10(-8) M), for 24 h prior to ACTH addition decreased the rates of ACTH- and cAMP-stimulated steroidogenesis but did not affect the basal rate. In both cases, the effectiveness of TCDD increased with time of exposure to the stimulant. Although cholesterol accumulated in the presence of ACTH and AMG (13-28 micrograms/mg), pretreatment of cells with TCDD caused a decrease in mitochondrial cholesterol (13-8 micrograms/mg). The effect of TCDD was produced relatively rapidly (t1/2 approximately 4 h). Since even in the absence of TCDD, the mitochondria of ACTH-stimulated cells also eventually lose cholesterol (after 2 h) TCDD pretreatment may increase the presence of a protein(s) that cause this mitochondrial-cholesterol depletion following stimulation by ACTH or cAMP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Aldosterone production after short-term culture of rat adrenal capsule: responsiveness to angiotensin II, potassium and ACTH

In vitro studies of aldosterone production have traditionally used freshly isolated adrenal glomerulosa tissue. In the present study we examined the effects of short-term culture of rat adrenal capsule on its secretory capacity by measuring both basal and stimulated aldosterone production. Capsules were maintained in culture for 24 h, and then responses to administered angiotensin II (1 X 10(-7) M), potassium (an increase of 2mM) and ACTH (1 X 10(-8) M) were determined during perifusion. Results were compared with responses by freshly isolated adrenal capsule. Although short-term culture reduced basal aldosterone production, responsiveness to administered stimuli was intact and often was greater than that observed with fresh capsular tissue. The results indicate that short-term culture of zona glomerulosa provides a suitable in vitro preparation for examining aldosterone secretory responsiveness to stimuli.     

Lack of renal effects of DOCA, ACTH, spironolactone, and angiotensin II in Squalus acanthias

Angiotensin II was infused intravenously in spiny dogfish sharks (Squalus acanthias). There were no significant effects on arterial blood pressure, glomerular filtration rate, urine flow, or Na excretion either in comparison with pre- and postinfusion values or in comparison with values measured in a control group of fish given elasmobranch saline intravenously. In other dogfish, glomerular filtration rate, urine flow, and Na and K excretory rates were measured for 3 days following implantation of desoxycorticosterone (DOCA), adrenocorticotropin (ACTH), or spironolactone; a control group was given no drug. There were no significant differences between these four groups of fish with respect to any of the measured parameters. These results suggest that the dogfish kidney is not a target organ for several substances known to affect renal function, either directly or indirectly, in other animals.     

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.     

Cyclic AMP-independent effects of ACTH on glomerulosa cells of the rat adrenal cortex

The aim of the present paper is to point out the complexity of ACTH action in glomerulosa cells of the adrenal cortex. We demonstrate that the increase in cAMP production induced by ACTH is the result of a balance between activation of adenylyl cyclase and direct modulation of a PDE2 phosphodiestease activity, an effect mediated by inhibition of cGMP content. Moreover, Ca²⁺ is essential for cAMP production and aldosterone secretion, but its exact primary action is not clearly determined. We recently described that ACTH activated a chloride channel, via the Ras protein, which can be involved in steroidogenesis. ACTH also increases tyrosine phosphorylation of several proteins. These data, together with those of phospholipase C activation, indicate that ACTH action in the adrenal is complex, and most certainly not limited to cAMP production, in particular for the low concentrations of the hormone.

Some years ago, cAMP was considered to be the unique second messenger of ACTH action; now it becomes more and more evident that ACTH triggers complex signaling pathways using several second messengers in a closely interacting way. The most predominant point is that these signals are observed for low concentrations of ACTH.     

Characteristics of angiotensin II-, K+- and ACTH-induced calcium influx in adrenal glomerulosa cells. Evidence that angiotensin II, K+, and ACTH may open a common calcium channel

The characteristics of angiotensin II-, K+-, and adrenocorticotropin (ACTH)-induced calcium influx were studied in isolated adrenal glomerulosa cells. Basal calcium influx rate is 0.64 +/- 0.09 nmol/min/mg of protein. Addition of angiotensin II (1 nM) causes a rapid 230% increase in calcium influx rate. This angiotensin II-induced calcium influx is sustained and is rapidly reversed by angiotensin II antagonist, [Sar1,Ala8]angiotensin II. Addition of either K+ or ACTH (1 nM) causes a 340 or 160% increase, respectively, in the rate of calcium influx. The effect of either angiotensin II, K+, or ACTH on calcium influx is dependent on extracellular calcium. The apparent Km for calcium is 0.46, 0.35, and 0.32 mM, respectively. When the extracellular concentration of K+ is 2 mM, neither angiotensin II nor ACTH stimulates calcium influx. Conversely, when extracellular K+ is increased to 6 mM, both angiotensin II and ACTH cause a greater stimulation of calcium influx than at 4 mM K+. When extracellular K+ is increased to 10 mM, calcium influx is 360% of the basal influx seen at 4 mM K+, and neither angiotensin II nor ACTH further stimulates the influx rate. Nitrendipine (1 microM) blocks both angiotensin II- and K+-induced calcium influx completely. In contrast, 10 microM nitrendipine does not completely block ACTH-induced calcium influx. The calcium channel agonist, BAY K 8644, also stimulates calcium influx; 10 nM BAY K 8644 leads to a rate of calcium influx which is 185% of basal. This BAY K 8644-induced increase in calcium influx and that caused by either angiotensin II or ACTH are additive. In contrast, BAY K 8644 has more than an additive effect on the calcium influx when paired with 6 mM K+. These results suggest that angiotensin II, K+, and ACTH stimulate calcium influx via a common calcium channel but act by different mechanisms to alter its function.     

The effects of pineal indoles and a crude aqueous pineal extract on ACTH mediated corticosterone release by isolated adrenal cells.

An aqueous extract of bovine pineal tissue contained a principle which caused a potent inhibition of ACTH mediated corticosterone release by isolated adrenal cells, whereas a similar extract of cortical tissue did not have any effect on this process. Two pineal indoles were tested. Melatonin (10^?8 M) did not have any significant effect, whereas 5-methoxytryptophol (10^?8 M) caused a variable but significant additive effect on ACTH mediated corticosterone release. We are uncertain at the present time if this principle is similar to the reported pinealantigonadotropin.     

Responses of rat adrenal glomerulosa and inner zone cells to synthetic ACTH analogs and proopiomelanocortin-derived peptides

A comparison of the responsiveness of isolated rat adrenal decapsular and glomerulosa cells to corticotrophin 1-39 (ACTH 1-39), synthetic ACTH analogs (characterized by a shorter amino acid chain length, the substitution of certain amino acids in the natural sequence by other amino acid residues, the replacement of the C-terminal carboxyl group by an amide), and proopiomelanocortin-derived peptides was performed by measuring corticosterone and aldosterone production, respectively. The potencies of the synthetic ACTH analogs correlated closely with the length of the peptides, similarly in both zones. No activity was observed with the proopiomelanocortin-derived peptides in either zone, with the exception of beta-LPH and alpha-MSH.     

Inhibitory effects on steroid production from isolated adrenal cells of rhesus monkey (Macaca mulatta) of pro-opiomelanocorticotrophic peptides

Adrenal glands from Rhesus monkeys (Macaca mulatta) of 160 days gestation, newborn, 2 months-old infants or 6 months-old infants were excised and prepared, by a collagenase digestion, as a cell suspension. The cells were incubated with 10 pg/ml, 100 pg/ml or 1 ng/ml of a peptide of the ACTH/pro-opiomelanocortin 'family', 57K, 31K, 20K, alpha MSH, ovine-CLIP or gamma LPH either in the presence or absence of 166 pg/ml ACTH1-39. The production by cortisol and androstenedione was measured by radioimmunoassay. Using the steroid production by aliquots of the cell suspension with either no stimulating agent or ACTH1-39 alone as controls, the net influence of these different peptides on basal or ACTH1-39-stimulated production was observed. alpha MSH, ovine-CLIP and gamma LPH had no influence on either basal or stimulated cortisol or androstenedione production. Corticotrophic peptides of 57K, and 20K and pro-opiomelanocortin each had a steroidogenic activity alone, in all age groups. In the fetal and newborn monkeys' adrenal cells, peptides of 57K and 20K at 1 ng/ml had an inhibitory influence on ACTH1-39 stimulated cortisol and androstenedione production. The influence of the 20K peptide is partially inhibitory as the steroidogenic potential of this peptide is not additive with that of ACTH1-39. These results show that, as observed in other species, that the ACTH/pro-opiomelanocortin range of peptides are inhibitory to the action of ACTH1-39 in the developing adrenal.     

Adrenocorticotropin regulates angiotensin II receptors in bovine adrenal cells in vitro

The role of ACTH-(1–24) on angiotensin II receptors has been studied in bovine adrenal glomerulosa cells in primary culture. Angiotensin II receptors were measured in cells pretreated or not by ACTH-(1–24) on day 4 of culture. ACTH-(1–24) decreased angiotensin II binding sites in a time and a dose-dependent manner. After 24 hours of treatment the minimal effective dose of ACTH-(1–24) was 10^?11M and the maximal effect was obtained with 10^?8M. Moreover, ACTH-(1–24) 10^?8M decreased significantly angiotensin II receptors after 6 hours of treatment. Scatchard plot analysis showed that ACTH-(1–24) treatment did not modify the affinity of angiotensin II receptors (Ka = 0.42 and 0.44 × 10⁹M^?1 in control and treated cells respectively) but reduced by about half the number of angiotensin II sites per cell. Like ACTH-(1–24), 8-Bromo-cAMP, forskolin and cholera toxin decreased angiotensin II receptors. Factors such as prolactin, somatostatin, ACTH-(11–24) and dopamine which are bound to adrenal membranes without increasing cAMP production had no effect. In conclusion, these studies $\text{in vitro}$ demonstrate for the first time that ACTH decreases angiotensin II receptors by a direct mechanism acting on glomerulosa cells, and they also suggest that this effect could be mediated by cAMP.     

Short-term effects of ACTH on protein synthesis in adrenal cortex cells of young rats

Two units of ACTH were administered intraperitoneally to young 20 gm-rats which received an intravenous injection of L-leucine-3H thirteen min later. ACTH-injected rats, and control rats which received the isotope alone, were killed at 2-, 10-, 30- and 60-min intervals. Electron microscope autoradiographs in control animals showed strong amino-acid uptake at pulse time (2-min) in the cytoplasm of adrenal zona fasciculata cells. Label was shared between the endoplasmic reticulum (ER) and mitochondria, and a lower but still considerable uptake was seen in nucleoli. At first chase time interval (10-min) cytoplasmic labelling declined, while nuclear and nucleolar labelling increased, both changing little thereafter, and there was a 10-30 min Golgi peak. ACTH administration provoked an overall increase in amino-acid incorporation into cytoplasm, nucleus and nucleolus at pulse time, with no changes in the distribution of the reactions among organelles. Intensification of labelling was most evident over nucleoli, the grain density of which was four-times as high as in controls. The short-term increase in ER and mitochondrial protein synthesis observed after ACTH injections was considered to be consistent with the hypothesis that most newly-formed proteins in these cells may be involved in the regulation of steroidogenesis. The marked increase in nucleolar labelling suggested the presence of proteins involved in RNA synthesis.     

A comparison of bradykinin,angiotensin II and muscarinic stimulation of cultured bovine adrenal chromaffin cells

Bradykinin, angiotensin II and a mascarnic agonist, acetyl-B-methacholine (methacholine) were all found to elict catecholamine release from cultured bovine adrenal chromaffin cells. Bradykinin was the most potent of these secretagogues and methacholine the weakest, with angiotenin II intermediate in efficacy. All three secretagogues were much less effective than nicotinic stimulation. The three secretagogues all produced a rise in cytoplasmic free calcium concentration ([Ca²⁺]_i), measured with the fluorescent indicator fura2, which was partially independent of external calcium. In the case of bradykinin the full rise in ([Ca²⁺]_i) may involve a component of calcium entry in addition to release of calcium from an internal store. Secretion was also found to be partially independent of external calcium. The different efficacies of the three secretagogues in elicting secretion were correlated with the rise in ([Ca²⁺]_i) produced. The differeing efficacies of the three secretagogues may be due to the extent of release of calcium from an intracellular store which itself is less effective in eliciting secretion than a rise in [Ca²⁺]_i following calcium entry due to nicotine. Bradykinin also stimulates calcium entry, and this may increase the efficacy of the initial rise in [Ca²⁺]_i. Treatment with pertussis toxin resulted in an enhancement of secretion in response to all of the secretagogues.Abbreviations ([Ca²⁺]_i) cytoplasmic free calcium concentration - EGTA ethylene glycol bis (-amino ethyl ether)-N,N,N,N,-tetraacetic acid - Hepes 4-(2-hydroxy ethyl)-1-piperazinethanesulphonic acid - TPA 12-O-tetradecanoylphorbol-13-acetate - DAG diacyl glycerol - IP₃ inositol-1,4,5-trisphosphate - PIP₂ phosphatidylinositol-4,5-bisphosphate