Primary Aldosteronism: New Answers, New Questions.

There have been 2, and possibly 3, major questions for primary aldosteronism (PA) answered at least in principle over the past 5 years. The first is that of somatic mutations underlying the majority of aldosterone producing adenomas. The second is the extension of our knowledge of the genetics of familial hypertension, and the third the role of renal intercalated cells in sodium homeostasis. New questions for the next 5 years include a single accepted confirmatory/exclusion test; standardisation of assays and cut-offs; alternatives to universal adrenal venous sampling; reclassification of 'low renin hypertension'; recognition of the extent of 'occult' PA; inclusion of low-dose mineralocorticoid receptor antagonist in first-line therapy for hypertension; and finally, possible resolution of the aldosterone/inappropriate sodium status enigma at the heart of the cardiovascular damage in PA.

Primary aldosteronism: are we missing the wood for the trees?

The prevalence of primary aldosteronism (PA) is around 10% of hypertensives, with markedly increased risk of cardiovascular damage compared with age-, sex- and BP-matched essential hypertension (EH). Currently, if hypertension is present in 20% of the population, PA will account for 2%; of these PA patients only 1% are ever screened, let alone diagnosed and treated, and the remaining 99% suboptimally treated, if at all. Mineralocorticoid receptor (MR) antagonists are effective in lowering BP, uniquely vasoprotective and safe when titrated to effect in EH. In resistant hypertension (BP elevated despite 3 or more conventional agents, including a diuretic), which constitutes 20-30% of EH, addition of a low dose MR antagonist reproducibly produces BP lowering of 20-30 mm Hg. Two thirds of PA is unilateral, and normally treated by MR antagonists; in unilateral PA surgery is recommended, but there are also studies reporting MR antagonist therapy to be noninferior over the longer term. There thus seems to be a very strong case for including a low dose MR antagonist in first-line therapy for new hypertension, given its utility and safety across EH, its particular efficacy in resistant hypertension, and its specific benefits for the 99% of subjects with occult PA. We do not have the resources to diagnose PA, but we do have the wherewithal to treat it.

Mineralocorticoid action: target tissue specificity is enzyme, not receptor, mediated.

Mineralocorticoid receptors, both when in tissue extracts and when recombinant-derived, have equal affinity for the physiological mineralocorticoid aldosterone and for the glucocorticoids cortisol and corticosterone, which circulate at much higher concentrations than aldosterone. Such receptors are found in physiological mineralocorticoid target tissues (kidney, parotid, and colon) and in nontarget tissues such as hippocampus and heart. In mineralocorticoid target tissues the receptors are selective for aldosterone in vivo because of the presence of the enzyme 11 beta-hydroxy-steroid dehydrogenase, which converts cortisol and corticosterone, but not aldosterone, to their 11-keto analogs. These analogs cannot bind to mineralocorticoid receptors.

Rat anterior pituitary. Distinction of an approximately 8S, corticosterone-preferring species from dexamethasone-binding glucocorticoid receptors.

Studies on the feedback inhibition of ACTH release by steroid hormones and on the binding of tritiated steroids by the pituitary have prompted the hypothesis that receptors in addition to or other than classical glucocorticoid receptors may mediate steroid hormone effects in this tissue. Accordingly, we have asked whether more than one glucocorticoid-binding species, distinct from corticosteroid binding globulin, can be found in rat anterior pituitary gland. In our study we have demonstrated high affinity (K(d) 4 degrees C approximately 1 nM) binding sites for tritiated corticosterone ((3)H-B) in rat pituitary cytosol, distinct from classical glucocorticoid receptors and transcortin-like sites. Unlike (3)H-B-transcortin complexes, (3)H-B bound to such sites is adsorbed onto hydroxylapatite and is stabilized by sulphydryl group reducing agents. Sucrose density gradient analysis in low ionic strength buffer under equilibrium conditions ((3)H-B+/-nonradioactive competitors throughout) showed (3)H-B to sediment as a single, approximately 8S peak, from which (3)H-B was consistently better displaced by B than dexamethasone (DM); (3)H-DM similarly bound to an approximately 8S peak, from which it was better displaced by DM than B. The existence of two species of pituitary glucocorticoid receptors is further supported by clear differences in specificity for a range of steroids, and in the differential depletion of cytoplasmic sites after in vivo DM administration. Similar "B-preferring" sites were not found in thymus cytosols. These results demonstrate that there exist in the pituitary high affinity intracellular binding sites for naturally occurring glucocorticoids, distinct from classical glucocorticoid receptors and transcortin-like sites. Physiological roles as glucocorticoid receptors remain to be established for these B-preferring sites.

Studies of the secretion of corticotropin-releasing factor and arginine vasopressin into the hypophysial-portal circulation of the conscious sheep. II. The central noradrenergic and neuropeptide Y pathways cause immediate and prolonged hypothalamic-pituitary-adrenal activation. Potential involvement in the pseudo-Cushing's syndrome of endogenous depression and anorexia nervosa.

Studies were performed to determine the effects of intracerebroventricular norepinephrine (NE) or neuropeptide Y (NPY) on the ovine hypothalamic-pituitary-adrenal (HPA) axis. NE (50 micrograms) increased mean hypophysial-portal corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) levels (1 h, 1.3- and 2.9-fold; 4 h, 2.2- and 5.7-fold) and caused acute and sustained increases in mean plasma ACTH and cortisol. NPY (50 microgram) also increased mean CRF and AVP levels (1 h, 1.4- and 4.2-fold; 4 h, 1.1- and 1.9-fold), increased pituitary-adrenal activity at 1 h, and caused ACTH hypersecretion at 4 h. When added to cultured ovine anterior pituitary cells, NPY neither increased basal ACTH release nor augmented CRF- or AVP-induced ACTH release. We conclude that: (a) activation of either the central noradrenergic or NPY pathways causes an acute and sustained stimulation of the ovine HPA axis; (b) such activation increases the AVP/CRF ratio, suggesting a dominant role for AVP in the ovine stress response; and (c) the central noradrenergic or NPY systems may cause sustained HPA activation by attenuating or disrupting the glucocorticoid negative feedback on those brain areas concerned with regulation of the HPA axis. The possible roles of the central noradrenergic and NPY systems in the etiology of the hypercortisolemia of endogenous depression and anorexia nervosa are discussed.

Defective nuclear accumulation of androgen receptors in disorders of sexual differentiation.

Nuclear transfer of androgen receptors (AR) and glucocorticoid receptors (GR) was determined in cultured genital skin fibroblasts from 10 normal controls and eight patients with abnormalities of the external genitalia. In whole cell studies, cultures were incubated for 20 min at 37 degrees C with [3H]methyltrienolone (3H-R1881) or tritiated dexamethasone, and specific binding was determined in whole cell, cytoplasmic, and crude nuclear fractions. Between normal and affected fibroblasts no difference was seen in cellular levels of GR, or in cytoplasmic and nuclear distribution of GR. In normal fibroblasts, cytoplasmic binding of 3H-R1881 represented 56%, and crude nuclear binding 44%, of total binding; in fibroblasts from five of the eight patients similar values (cytoplasmic 55% and nuclear 44%) were seen for 3H-R1881 binding. In fibroblasts from the other three patients no decrease in total cellular levels of AR were seen; nuclear compartmentalization, however, was much lower (approximately 20%) than in other cultures. In vitro reconstitution studies, combining 3H-R1881-loaded cytosol with naive nuclei, lead us to suggest that the defect in nuclear compartmentalization lies at the level of the nuclear acceptor site rather than the cytoplasmic binder in affected cells. We interpret the data to suggest that defective nuclear binding of AR complexes may be involved in a proportion of cases of abnormal development of the external genitalia.

Mineralocorticoid and glucocorticoid effects on 31,000- and 29,000-dalton proopiomelanocortin in rat anterior pituitary and neurointermediate lobe.

The effects of adrenal steroids on proopiomelanocortin (POMC) levels in rat pituitary have been studied by two-dimensional gel electrophoresis. In intact rats the relative abundance of POMC was much higher in the neurointermediate lobe (N-IL) than in anterior pituitary (AP); in both tissues the predominant species appeared to be of 29,000-dalton (29K) molecular mass, with lesser amounts of a 31K form. In both tissues, the 31K and 29K forms showed multiple spots, consistent with different degrees of sialoglycosylation. Adrenalectomy was followed by a marked increase in AP levels of POMC, and a marked decrease in N-IL levels. In adrenalectomized rats, dexamethasone administration did not affect N-IL levels of POMC, but suppressed 35S incorporation into POMC in AP in a dose-related manner; deoxycorticosterone showed minimal effects on AP levels of POMC, but progressively elevated N-IL levels; 9 alpha fluorocortisol (9 alpha fF) progressively both suppressed AP levels, and raised N-IL levels of POMC. Estimation of immunoreactive (ir) ACTH and ir-beta-endorphin in parallel samples showed an elevation of N-IL levels in response to mineralocorticoids (deoxycorticosterone, 9 alpha fF), and a paradoxical elevation of AP levels in response to glucocorticoids (dexamethasone, 9 alpha fF) compared with oil-injected adrenalectomized controls. We conclude (a) that glucocorticoids suppress the secretion of ir-ACTH and ir-beta-endorphin to a greater extent than they inhibit the synthesis of POMC; (b) that mineralocorticoids specifically elevate the N-IL levels of both POMC and its immunoreactive product (beta-endorphin).

Differences in proteins synthesized by fibroblasts from normal individuals and patients with complete testicular feminization.

Patterns of protein synthesis by genital skin fibroblasts from three unrelated normal individuals and three unrelated patients with complete testicular feminization were compared to two-dimensional gel electrophoresis. cell lines were maintained in monolayer culture and pulse labeled with [35S]methionine. Cells were lysed in 9 M urea, and aliquots of 20 microliters subjected to isoelectric focussing and polyacrylamide gel electrophoresis followed by autoradiography. Gels of control fibroblasts showed two proteins (mol wt approximately 45,000, approximately 85,000; pKi approximately 5.0) markedly more prominent than on gels from affected fibroblasts. This pattern was unaltered by prior exposure to dihydrotestosterone, suggesting differences in constitutive proteins of the fibroblast cells. Parallel studies demonstrated a marked reduction in the ability of fibroblasts from patients with complete testicular feminization to bind androgens in vitro compared with those of normal individuals. The relationship between these proteins, androgen receptors, and androgen insensitivity requires further investigation.

Glucocorticoid and mineralocorticoid effects on adrenocorticotropin and beta-endorphin in the adrenalectomized rat.

Immunoreactive ACTH (ir-ACTH) and immunoreactive beta-endorphin (ir-betaEP) were determined in plasma, anterior pituitary, neuro-intermediate lobe, and hypothalamus of sham-adrenalectomized rats, and adrenalectomized rats given six daily injections of vehicle (oil), dexamethasone, 9alpha-fluorocortisol or deoxycorticosterone. 6 d after adrenalectomy, anterior pituitary ir-ACTH and ir-betaEP were double, and plasma levels approximately fivefold those in controls. Adrenalectomy did not alter hypothalamic levels of either peptide, or ir-betaEP in neuro-intermediate lobe, in which tissue ir-ACTH was below detection limit at routine dilutions. Dexamethasone (0.2-200 mug/d) concurrently suppressed plasma ir-ACTH and ir-betaEP, with a near maximal effect at 20 mug, and a half-maximal effect between 2 and 6 mug; similar dose-response characteristics were found for thymolysis. Step-wise increases in anterior pituitary content of both peptides were found, with no change in hypothalamic levels of either peptide, or neuro-intermediate lobe ir-betaEP. 9alpha-fluorocortisol (0.2-200 mug/d) produced plasma, anterior pituitary, and hypothalamic effects equivalent to dexamethasone, but with one-tenth the potency. Unlike dexamethasone, higher doses of 9alpha-fluorocortisol significantly elevated neuro-intermediate lobe ir-betaEP. Deoxycorticosterone (2-2,000 mug/d) produced no significant changes in plasma, anterior pituitary or hypothalamic levels of either peptide; like 9alpha-fluorocortisol, doses of >60 mug/d significantly elevated neuro-intermediate lobe ir-betaEP. Whereas ir-ACTH and ir-betaEP synthesis in and release from the anterior pituitary are under complex negative feedback glucocorticoid control, there exists a mineralocorticoid-specific effect on neuro-intermediate lobe content of ir-betaEP.

Induction by glucocorticoids of angiotensin converting enzyme production from bovine endothelial cells in culture and rat lung in vivo.

The effect of corticosteroids on angiotensin converting enzyme was investigated in endothelial cell cultures and intact rat lung. Cultured endothelial cells from bovine aorta showed net production of angiotensin converting enzyme (ACE) over 2 d culture in serum-free medium. Dexamethasone (DM) increased cell ACE activity six- to sevenfold at 100 nM with a threshold effect at 0.3 nM. The effect of DM on ACE production was completely inhibited by actinomycin D or cycloheximide. Deoxycorticosterone (DOC) and aldosterone were markedly less active, with a threshold near 100 nM and significant (two to threefold) stimulation of ACE activity at 1 muM. In cells incubated in the presence of 10 nM DM, DOC (10 muM) significantly inhibited ACE production compared with 10 nM DM alone, suggesting that DOC is a partial agonist/partial antagonist in this enzyme system. Protein content of cells or medium was unchanged by steroids at all doses used. In vivo, adrenalectomized rats showed lower pulmonary ACE compared with intact controls, and when injected with DM (40 mug/d for 4 d) showed a significant (twofold, P < 0.002) increase in lung ACE over oil-injected, adrenalectomized controls; serum ACE did not change. Injection with DOC (40 mug/d) or aldosterone (10 mug/d) had no effect on lung or serum ACE. Over a range (0.6 to 2,000 mug) of concentrations of DM administered daily for 7 d, the dose-response curve of DM for induction of pulmonary ACE mirrored that for thymolysis; for both, half-maximal effects were seen at approximately 6 mug DM/d, and plateau levels at 60 mug/d. We conclude that glucocorticoids are potent inducers of ACE activity in endothelial cells in culture and in rat lung in vivo, and that the action of aldosterone and DOC reflects occupancy of glucocorticoid receptors. This effect may be of (patho)physiological relevance in regulating levels of ACE in local vascular beds, and thereby modulating local levels of the vasoactive peptides angiotensin II and bradykinin.

Immunoreactive beta-endorphin in a subpopulation of mouse spleen macrophages.

Using radioimmunoassay and immunofluorescence with antibodies to beta-endorphin (beta EP) and ACTH, we have shown that a subpopulation of mouse spleen cells, expressing Mac-1, a marker of macrophage differentiation, contains immunoreactive (ir)-beta EP, ir-ACTH, and smaller amounts of presumptive higher molecular weight forms of both. Neither nonadherent spleen cells, nor adherent or nonadherent cells from peripheral blood, contained detectable levels of these peptides. These findings suggest that beta EP and ACTH may be synthesized in a subpopulation of spleen macrophages, and are consistent with the possibility that these or related peptides may modulate lymphocyte function in the specific microenvironment of the spleen.

Pro-opiomelanocortin messenger ribonucleic acid and posttranslational processing of beta endorphin in spleen macrophages.

We have previously demonstrated low levels of immunoreactive (ir)-beta-endorphin (beta-EP) and ir-ACTH in a subpopulation of mouse spleen macrophages, which is consistent with an involvement of opioid peptides in modulation of immune responses. Gel chromatography studies suggested the presence of an approximately 3.5,000-molecular weight (mol wt) species, putatively beta-EP, an approximately 11.5,000-mol-wt species, putatively beta-lipotropin, and a higher molecular weight species (putative beta-EP precursor, pro-opiomelanocortin (POMC). In this study we have extended our original findings by demonstrating the presence of messenger RNA for POMC by the use of a complementary DNA probe and Northern blot analysis of extracts of mouse and rat spleen. In addition, using high performance liquid chromatography (HPLC), we have shown that the major endorphin species in mouse spleen macrophages is beta-EP1-31, and that there are smaller amounts of each of the acetylated forms, N-acetyl-beta-EP1-16 (alpha-endorphin), N-acetyl-beta-EP1-17 (gamma-endorphin), N-acetyl-beta-EP1-27, and N-acetyl-beta-EP1-31. We interpret these studies as showing that (a) the spleen is an organ of POMC synthesis and that (b) the predominant COOH-terminal product of macrophage POMC is the opiate-receptor active species beta-EP1-31.

N-acetyl endorphin in rat spermatogonia and primary spermatocytes.

In previous reports modest levels of beta-endorphin have been found by radioimmunoassay in rat testis, and localized by immunofluorescence to the interstitial cells. We have confirmed these previous reports and extended them by showing that the majority of testicular endorphins are acetylated forms, N-acetyl gamma-endorphin, N-acetyl alpha-endorphin, and N-acetyl beta-endorphin1-27. In addition, N-acetylated endorphins are not found in interstitial cells, but are confined to spermatogonia and primary spermatocytes.

A new human breast carcinoma cell line (PMC42) with stem cell characteristics. III. Hormone receptor status and responsiveness.

PMC42 is a new human breast carcinoma cell line. In this report the content of estrogen, progesterone, and glucocorticoid receptors in PMC42 has been determined. The estrogen receptor content (1,750 cytoplasmic sites and 350 nuclear sites) was lower than that described for MCF7 and T47D. The cells would not proliferate in serum-free medium without the addition of beta-estradiol (optimum concentration 10(-8) M) or progesterone (5 X 10(-8) M). The addition of both hormones induced a more than additive increase in proliferation (P less than .005, n = 18). Similarly, addition of insulin or hydrocortisone induced proliferation; however, in this case, the effect of the hormones together was only additive. The addition of tamoxifen (10(-6) M) led to a significant decrease in cell numbers and inhibited the stimulatory effects of 10(-8) M beta-estradiol.

Frequency of 1,25-dihydroxyvitamin D3 receptor in human breast cancer.

Receptors for 1,25-dihydroxyvitamin D3 have been shown to exist in cultured breast cancer cells and in primary breast cancers. It is reported here that 1,25-dihydroxyvitamin D3 receptor (1,25-DR) was present in 80% of 54 unselected breast cancers. The concentration of 1,25-DR in the 43 receptor-positive tumors was 1.9 +/- 0.4 fmol/mg protein (S.E.). There was no correlation between 1,25-DR presence or concentration and the age of the patient or the concentration of estrogen, progesterone, androgen, or glucocorticoid receptors. 1,25-DR was also found in two of three renal cortical carcinomas but only in three of 14 gastrointestinal tract carcinomas. The relatively low concentration of 1,25-DR in these breast cancers, compared with that found in cultured breast cancer cells, is partially explained by incomplete "exchange" with occupied receptors. Since the serum vitamin D-binding protein is not precipitated from serum itself or from tissue homogenates using the polyethylene glycol method, artifactual 1,25-DR levels due to the inevitable contamination of tissue specimens with this protein can be excluded. These findings indicate that 1,25-DR is not a nonspecific marker of cancer. The high frequency of 1,25-DR in the breast cancers may be related to the calcium-transporting ability of breast cancer cells which allows them to grow as osteolytic metastases.

Molecular interactions between telomerase and the tumor suppressor protein p53 in vitro.

The telomere DNA polymerase (telomerase) and the tumor suppressor protein p53 are frequently associated with human cancers, and activation of telomerase and inactivation of p53 involved in cancer cell immortalization. In this report, we demonstrate a direct interaction of telomerase with p53 in the nuclear lysates of human breast cancer cells, and with recombinant human p53, by affinity chromatography and immunoprecipitation. On activity criteria, the interaction is between the carboxyl-terminal region of p53 and a region close to the amino-terminus of human telomerase-associated protein 1 (hTEP1). Incubation of recombinant p53 with nuclear telomerase extracts results in inhibition of telomerase activity, with the C-terminal region of p53 being essential for inhibition. This effect is not mediated by binding to telomerase substrate DNA, but requires the region near the N-terminus of hTEP1, in that a synthetic peptide derived from this region of hTEP1 similarly inhibits telomerase activity. Together, these in vitro interactions between telomerase and p53 suggest that the activity of telomerase may be regulated by p53, down-regulation of which in turn would favor up-regulation of telomerase activity in cancer cell development.

Target tissue specificity of mineralocorticoids.

Mineralocorticoid receptors have equivalent, high affinity for aldosterone and for the physiological glucocorticoids cortisol and corticosterone, which circulate at much higher concentrations. In physiologic mineralocorticoid target tissues, glucocorticoids are excluded from mineralocorticoid receptors by conversion to their receptor-inactive 11-keto metabolites, by the enzyme 11beta hydroxysteroid dehydrogenase. Aldosterone, in contrast, escapes metabolism by cyclizing its 11-OH group with the reactive -CHO at position 18. In most endocrine systems, specificity is conferred in large part by the ability of the receptor to distinguish between signal and noise. Why, for mineralocorticoid action, specificity is prereceptor-a nonmetabolized signal, and an enzyme that excludes noise-remains to be explored.

Apparent mineralocorticoid excess, 11beta hydroxysteroid dehydrogenase and aldosterone action Closing one loop, opening another.

The recent cloning of the human enzyme 11beta hydroxysteroid dehydrogenase type 2 (11betaHSD2), and the demonstration of point mutations or deletions in both familial and apparently sporadic cases of apparent mineralocorticoid excess (AME), underlines the importance of this enzyme in excluding glucocorticoids from mineralocorticoid receptors (MR). Although the sodium retention characteristic of AME can thus be explained by absent or very reduced (< 10%) levels of renal 11 betaHSD2 activity, whether or not the enzymatic defect contributes to the elevated blood pressure by mechanisms other than sodium retention remains to be determined.