Adrenomedullin (ADM) and proadrenomedullin N-terminal 20 peptide (PAMP) are two hypotensive peptides, contained in adrenal medulla, which are able to inhibit aldosterone secretion from zona glomerulosa. In this study we have compared the effects of the two peptides on the production of post-pregnenolone steroids by dispersed rat zona glomerulosa cells. ADM and PAMP did not alter basal steroid secretion. Conversely, they inhibited angiotensin-II (10(-9) M)-stimulated 18-hydroxy-11-deoxycorticosterone, corticosterone, 18-hydroxycorticosterone and aldosterone production, without affecting progesterone and 11-deoxycorticosterone secretion. PAMP was significantly more effective than ADM, their minimal effective concentrations being 10(-10) M and 10(-8)-10(-7) M, respectively. ADM, but not PAMP markedly lowered either basal and agonist-enhanced release of 11-dehydrocorticosterone. Collectively, these findings allow us to draw the following conclusions: (i) ADM and PAMP electively inhibit 11 beta-hydroxylase and aldosterone synthase, PAMP being probably to be considered the proadrenomedullin-derived physiological inhibitor of mineralocorticoid secretion in rats; and (ii) ADM, but not PAMP, also exerts a clear-cut inhibitory action on 11 beta-hydroxysteroid dehydrogenase, the enzyme that converts corticosterone to its inactive form.

The effects of endothelin-l (ET-I) and cyclosporine-A (CSA) on adrenal steroidogenesis were investigated in vitro, using rat capsule-zona glomerulosa (ZG) and zona fasciculata (ZF) preparations. ET-1 concentration-dependently raised aldosterone (ALDO) and corticosterone (B) outputs by capsule-ZG and ZF preparations, respectively. Maximal effective concentrations was 10(-8) M. Two specific antagonists of subtypes A and B of ET-I receptors (BQ-123 and BQ-788, respectively) were employed. BQ-788 impaired the secretory response of our adrenal preparations to 10(-8) M ET-I, while BQ-123 was ineffective. The maximal effective concentration of BQ-788 was 10(-7) M. CSA (10(-8) M) elicited marked ALDO and B secretagogue effects, which were of the same order of magnitude of those evoked by 10(-8) M ET-1 and were abolished by 10(-7) M BQ-788. Autoradiography showed I-125-ET-1 binding in both ZG and ZF. BQ-788 decreased labeling in the ZG and virtually eliminated it in the ZF; CSA effects were analogous to those of BQ-788, as also demonstrated by quantitative densitometry. In light of these findings, the following conclusions can be drawn: 1) ET-1 stimulates in the rat not only the secretion of ALDO from ZG, but also that of B from ZF; 2) the adrenocortical secretagogue effect of ET-1 is exclusively mediated by the B subtype of ET-I receptors; and 3) CSA, at relatively low concentrations, exerts a stimulatory action on adrenal steroidogenesis, probably acting as an agonist of ET-1 receptors of type B.

The effects of interleukin-1beta (IL-1beta) on the secretory activity of Leydig cells were investigated in hypophysectomized/human chorionic gonadotropin (hCG)-treated rats. IL-1beta dose-dependently increased basal and hCG-stimulated plasma testosterone concentration (PTC) and testosterone secretion by isolated Leydig cells. Basal PTC returned to the control level 12 h after the injection of a maximal effective dose of IL-1beta (10 nmol/rat), but after 24 and 36 h it was markedly lower than in control animals; after 48 h PTC again attained the control value. IL-1beta did not affect basal PTC in rats injected 24 h before with 10 nmol of IL-1beta (IL-1beta-pretreated rats), but it dose-dependently decreased hCG-stimulated PTC. However, IL-1beta dose-dependently enhanced both basal and hCG-stimulated testosterone production by dispersed Leydig cells obtained from IL-1beta-pretreated rats. Corticotropin-releasing hormone (CRH) concentration was very low in the testes of control rats, but it did display a striking rise 24 h after the injection of IL-1beta. As expected, CRH (100 pmol/rat) and IL-1beta (10 nmol/rat) elicited a marked decrease in hCG-enhanced PTC in IL-1beta-pretreated rats. (alpha-Helical)-CRH, a competitive inhibitor of CRH, did not alter hCG-stimulated PTC in control IL-1beta-pretreated rats, but it did completely annul the inhibitory effect of both CRH and IL-1beta. In light of the present findings, it seems reasonable to suggest that IL-1beta exerts a two-fold modulatory action on rat testis steroidogenesis: IL-1beta (i) acutely stimulates testosterone secretion by acting directly on Leydig cells, and (ii) induces the intratesticular production of CRH, which in turn inhibits Leydig-cell secretory activity. This last effect, however, requires 24 h to become manifest, since it probably involves the de novo expression of CRH gene.

A single exposure to a low concentration (10(-10) mol/L) of several tumor promoters, namely 12-O-tetradecanoylphorbol-13-acetate (TPA), phenobarbital (PB), nafenopin, saccharin, teleocidin, benzoyl peroxide, butylated hydroxytoluene (BHT), dichlorodiphenyltrichloroethane (DDT), lindane, clofibrate, and melittin significantly stimulated DNA synthesis of neonatal rat hepatocytes in 4-day-old primary cultures. These cultures were kept in low-calcium (0.01 mmol/L) HiWoBa2000 synthetic medium, thereby evoking a neoplastic phenotype in otherwise normal (i.e., non-initiated) cells. The simultaneous addition of a single dose of alpha-tocopherol (10(-4) mol/L) or selenous acid (10(-5) mol/L), just as that of exogenous superoxide dismutase (SOD) (4), together with each of the above agents fully suppressed the stimulation of hepatocytic DNA synthesis by the xenobiotics. Hence, these findings strengthen the view that superoxide anions (or some other oxidizing compounds) act as the common mediators of the mitogenic effects of various tumor promoters in hepatocytes. Inhibition kinetics studies, in which TPA in a single dose (10(-10) mol/L) was used as the paradigmatic compound together with several kinds of inhibitors of its activity showed that the early mitogenic effects of TPA, i.e., the commitment of quiescent (G0) hepatocytes and the reentry into active cycling of hepatocytes spontaneously poised at the G1/S boundary, required oxidizing compounds, arachidonate metabolism derivatives, and plasmalemmal calcium-binding sites and transmembrane calcium fluxes. Instead, a later TPAs effect, the flow into DNA synthesis of hepatocytes previously committed to cycle, was shown to be controlled by retinoid-modulable activities, by some product(s) of the lipoxygenase pathway, and again by plasmalemmal calcium-binding sites and transmembrane calcium fluxes. Such results reveal that in the neonatal rat hepatocyte the ability to answer to a single mitogenic stimulus and the metabolic pathways by which this answer is enacted depend upon the mitotic cycle setting of the hepatocytes at the moment of the experimental treatment.

A single exposure to a wide range of concentrations (10(-15)-10(-4) mol/l) of the tumour promoters 12-O-tetradecanoylphorbol-13-acetate (TPA) and phenobarbital (PB) significantly stimulated the flow into DNA synthesis and mitosis of neonatal rat hepatocytes in 4-day-old primary cultures kept in high-calcium (1.8 mmol/l) Eagle's FBS-MEM. Maximal effects were observed in the dose range 10(-12)-10(-6) mol/l. Moreover, both xenobiotics retained their full mitogenic effectiveness when given to hepatocytes incubated in calcium-deficient (0.01 mmol/l) FBS-MEM, thereby evoking a neoplastic phenotype in otherwise normal (i.e., non-initiated) cells. Proliferation kinetic studies showed that TPA and PB acted according to the actual cell cycle setting of the cells: they committed a fraction of the quiescent (GO) hepatocytes to grow, and enabled hepatocytes previously poised at the G1/S and G2/M boundaries to start cycling again, but exerted no influence on liver cells already engaged by themselves in active cycling. These diverse activities of TPA and PB were independent of serum (growth) factors; they were also fully elicited in hepatocytes grown in synthetic media (Eagle's MEM or HiWoBa2000), and were not changed by the addition of a mitogenically effective dose (10(-10) mol/l) of epidermal growth factor/urogastrone (EGF) with or without FBS. Conversely, the addition of the specific plasmalemmal calcium chelator, EGTA, or displacer, La3+, or of the anticalmodulin drugs, W-13 and calmidazolium (or R24571: an agent hardly entering cells) fully inhibited the mitogenic activities of tumour promoters in quiescent and intra-cycle-blocked hepatocytes, while having no effect on untreated or xenobiotic-treated liver cells already cycling by themselves. Such inhibitory effects were in all instances independent of the actual extracellular calcium concentration and took place according to time-related double kinetics. Hence, xenobiotic-activated processes taking place at the plasmalemmal level and involving the activation of calcicalmodulin-dependent enzymes were of critical importance for both the G0/G1 and G1/S transitions in primary hepatocytes.

Low concentrations (e.g. 10(-12) and 10(-11) mol/l) of imidazole and indomethacin strongly stimulated DNA synthesis and mitosis of hepatocytes in 4-day-old primary cultures of neonatal rat liver. These agents seem to have acted by inducing quiescent hepatocytes to begin cycling rather than by affecting already cycling cells, because they did not shorten the total cell cycle time. Neither compound stimulated DNA synthesis by hepatocytes cultured in low (0.010 mol/l) calcium medium. Nevertheless, hepatocytes in calcium-deficient medium must have been mitogenically activated by these compounds and, hence, been able to reach a late stage of prereplicative development because they did initiate DNA synthesis very soon after the addition of calcium.