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The hypothalamic-pituitary-adrenal (HPA) axis is a major part of the neuroendocrine system that controls responses to stress, and has an important function in the regulation of various body processes. We previously created a mouse line deficient in the melanocortin 2 receptor (MC2R). MC2R-deficient mice (MC2R−/− mice) have high adrenocorticotropic hormone (ACTH) levels because of undetectable corticosterone levels. Increased neuromedin B (NMB) expression was recently reported in the pituitary gland of adrenalectomized mice, a model for acute adrenal insufficiency. To investigate gene expression in the pituitary gland under chronic adrenal deficiency, we examined the pituitary gland of MC2R−/− mice, a model of chronic adrenal insufficiency. To understand the molecular background of pituitary cells under chronic adrenal deficiency, we first performed DNA microarray analyses using the pituitary glands of the MC2R−/− mice. The DNA microarray analysis and real-time polymerase chain reaction showed that NMB expression was higher in the MC2R−/− than in the wild-type (WT) mice. We detected NMB expression in the MC2R−/− pituitary corticotrophs by immunohistochemistry using the specific antibodies for ACTH and NMB. In addition, the plasma NMB concentration was significantly higher in the MC2R−/− mice than in the WT mice. Subcutaneous implantation of a sustained-release corticosterone pellet decreased the expression of NMB mRNA as well as pituitary proopiomelanocortin mRNA. In isolated anterior pituitary cells, NMB mRNA expression was increased by the administration of corticotropin-releasing hormone (CRH) and was suppressed by dexamethasone treatment. In this study, we first demonstrate NMB expression in corticotrophs and its regulation by CRH and glucocorticoids. Furthermore, corticotrophs seemed to secrete NMB into the systemic circulation.

Specific receptors for angiotensin II (A II) were demonstrated in membrane fractions and collagenase-dispersed cells from the zona glomerulosa of the rat adrenal gland. The equilibrium association constant (Ka) of the A II binding sites was similar in particulate fractions (2.0 +/- 0.4 (SE) X 10(9) M-1) and intact glomerulosa cells (1.8 +/- 0.3 X 10(9) M-1). Specific binding of [125I]iodo-A II was enhanced by increasing sodium concentration, and in the presence of dithiothreitol, EDTA, and EGTA. Plasma membrane fractions prepared by density gradient centrifugation showed increased binding of [125I]iodo-A II, and were correspondingly enriched in adenylate cyclase and sodium-potassium-dependent ATPase. Steroid production by collagenase-dispersed adrenal glomerulosa cells was highly responsive to A II and ACTH. Significant increases in aldosterone and corticosterone production were elicited by A II concentrations as low as 3 X 10(-11) M, equivalent to normal blood levels of A II in rats (5 X 10(-11) M). The maximum increase in aldosterone production, of 6--7 times the basal value, was obtained at 10(-9) M A II. Dispersed capsular cells were also highly sensitive to ACTH, responding to concentrations down to 3 X 10(-12) M with increased aldosterone production, reaching a maximum aldosterone response of 20-fold above the basal value. The magnitudes of the aldosterone and corticosterone responses to A II in capsular and fasciculata-reticularis cells were commensurate with the distribution of A II receptors, which were 11-fold more concentrated in capsular cells. The ability of A II to evoke aldosterone production at physiological concentrations, and the correspondence between A II binding and steroidogenesis in capsular cells, demonstrate the functional importance of A II receptor sites in the zona glomerulosa of the rat adrenal cortex.

The influence of extracellular potassium concentration on adrenal sensitivity to angiotensin II and ACTH was studied in isolated canine adrenal glomerulosa cells. When potassium was absent from the incubation medium, the aldosterone response to angiotensin II or ACTH was completely abolished. At physiologic angiotensin II concentrations (2.5 x 10(-11) M), aldosterone formation increased 4-fold when potassium concentration was increased from 2.5 to 5.0 mM, and rose 6-fold as potassium was increased from 2.5 to 7.5 mM. In the absence of angiotensin II, the same changes in potassium concentration increased aldosterone production only to 2-fold and 3.5-fold, respectively. The effect of potassium concentration upon the aldosterone response to ACTH was similar but less marked. The concentration and binding affinity of angiotensin II receptor sites in glomerulosa cells were not changed by increasing potassium concentrations from 0 to 7.5 mM. These observations demonstrate that the aldosterone response to the glomerulosa cell to angiotensin II is potassium-dependent within the physiological range for each of these stimuli. Such an interaction suggests that the in vivo effect of potassium upon aldosterone secretion includes a significant modulating action upon adrenal sensitivity to angiotensin II, as well as a direct action of potassium upon the adrenal glomerulosa cell.