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It has been recently suggested that 16α,18-dihydroxydeoxycorticosterone (16α,18-diOHDOC) may act as a “positive allosteric effector” of the binding of aldosterone to mineralocorticoid receptors. To test this hypothesis, a series of in vitro and in vivo studies examining the effect of 16α,18-diOHDOC on tritiated aldosterone ( 3 HA) binding to mineralocorticoid receptors was performed. Using kidney slices from adrenalectomized rats, in vitro incubations were made for 20′ at 37C, over a range of concentrations of 3 HA plus tenfold dexamethasone to confine tracer binding to mineralocorticoid receptors. At no concentration of 3 HA did 16α,18-diOHDOC enhance binding; at all tracer concentrations a slight competing effect was observed. When 3 HA was injected into rats in vivo with and without 16α,18-diOHDOC, a similar insignificant displacement of 3 HA binding was seen in renal cytoplasmic fractions from adrenalectomized test rats. Additional in vitro studies were performed in an attempt to elucidate the mechanism of postulated action of 16α,18-diOHDOC. Neither renal cytoplasmic binding of oestradiol, postulated as a secondary pathway for steroid influenced Na + retention, nor the binding of dexamethasone to renal glucocorticoid receptors, was altered by 16α,18-diOHDOC. Binding of tritiated 16α,18-iOHDOC in renal cyto-plasmic fractions was shown to be non specific, in that it could not be displaced by excess unlabelled 16α,18-diOHDOC. Finally, in a series of in vivo experiments using adrenalectomized rats, we could not show any effect of 16α,18-diOHDOC on urinary electrolyte excretion, either alone or in combination with low doses of aldosterone. Accordingly, we can find no evidence for 16α-diOHDOC having a direct effect on the kidney: in particular, there would appear as yet no molecular evidence for 16α,18-diOHDOC being a positive allosteric effector of aldosterone.

Specificity in mineralocorticoid versus glucocorticoid action. The mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR) share considerable structural and functional homology. Overlapping effects on epithelial sodium transport are observed in vivo; in vitro, both are able to bind and transactivate through a common hormone response element. This has led several investigators to suggest that specificity is conferred primarily by prereceptor mechanisms, and we have addressed this question using both in vitro and in vivo approaches. Although the MR has been regarded as less transcriptionally active than the GR in vitro, significant differences are observed when epithelial rather than fibroblast cell lines are used. These differences are mediated by the N-termini of the receptors. Activation of intracellular signaling pathways differentially modulates MR- versus GR-mediated transactivation. Although these studies identify mechanisms by which specificity may be achieved, they do not prove that this occurs in vivo. Such studies have been limited by an absence of MR-regulated genes. Known candidate aldosterone-responsive genes have been examined in the rat distal colon; the time course and the specificity of the response to a single parenteral dose of corticosteroid has been characterized. The epithelial sodium channel β and γ subunit genes are both up-regulated within 60 minutes by either MR or GR activation. Similar responses are observed for the serum and glucocorticoid-regulated kinase and channel-inducing factor genes. All four genes show clear and rapid up-regulation of their mRNA levels by aldosterone, which is paralleled by GR-mediated up-regulation of expression. While they are indeed aldosterone-responsive genes, genes that are uniquely aldosterone-regulated remain to be identified.

In two patients with severe diabetic ketoacidosis, electrocardiography showed transient anterior changes suggestive of acute transmural infarction without subsequent evidence of myocardial necrosis. While the mechanism of these and other temporary electrocardiographic changes in diabetic ketoacidosis remains unclear, appreciation of their transient nature is essential if misdiagnosis of myocardial infarction and possible inappropriate delay in intravenous fluid administration are to be avoided. When electrocardiographic abnormalities are present early in diabetic ketoacidosis, the full 12-lead electrocardiogram should be repeated after adequate resuscitation.

On the basis of both clinical observations and experimental studies it has been proposed that renal kallikrein is a mineralocorticoid regulated protein. In other studies, changes in renal kallikrein activity have been implicated in the genesis of, and/or response to, hypertension. Using a cloned complementary DNA (cDNA) to rat pancreatic kallikrein (pcXP39) for hybridization histochemistry, and both Northern and dot blot analysis, we studied expression of the kallikrein gene in steroid-treated control animals, and in three strains of genetically hypertensive rats. No differences in renal kallikrein messenger RNA (mRNA) levels were found between adrenalectomized rats and those treated for 5-14 days with 9 alpha-fludrocortisone, corticosterone or dexamethasone, or between hypertensive rats and their appropriate controls. Since mRNA levels appear essentially invariant under such circumstances, the change in renal kallikrein activity/immunoreactivity after chronic mineralocorticoid elevation, or in hypertensive rats, presumably reflects modulation at the post-transcriptional level.

By using 32P-labeled oligonucleotide probes for in situ DNA-RNA hybridization, we have defined the distribution of messenger RNA (MRNA) for arginine vasopressin-neurophysin II (AVP-NPII) in the rat hypothalamus. With a specific pentadecamer nucleotide probe, strong specific hybridization is seen in supraoptic, suprachiasmatic, and paraventricular nuclei in both Sprague-Dawley and Brattleboro rats. This finding, of equivalent distribution of AVP-NPII mRNA in Brattleboro and control hypothalami, confirms and extends the recent report of equivalent levels of AVP-NPII mRNA by Northern blot analysis. (Endocrinology 116: 2366–2368, 1985)

Anterior pituitary kallikrein-like enzyme activity, immunoreactivity and mRNA levels have previously been shown to be regulated by estrogen, in parallel with prolactin. In this study, we have examined the relationship between kallikrein and prolactin mRNA levels in estrogen-induced pituitary tumors. Treatment of Fischer 344 rats with diethylstilbestrol implants for 3, 5 and 7 weeks produced a dramatic increase in kallikrein mRNA levels and a modest increase in prolactin mRNA levels. These changes were partially reversed by bromocriptine treatment, and completely reversed by bromocriptine plus estrogen withdrawal. Using a panel of oligonucleotide probes specific for various members of the rat kallikrein gene family, we have shown that the kallikrein-like gene expressed appears to be true kallikrein.

A right adrenal androgen-producing adenoma was identified by CT scanning in a 33-year-old woman after delivery of an otherwise-normal, virilized female infant. Despite clinical evidence of mild chronic androgen excess, maternal reproductive function was normal. Post-partum studies showed 2-5-fold excess in maternal plasma testosterone, androstenedione and dehydroepiandrosterone-sulphate; urinary androgen metabolites were increased 3-10-fold. Androgen production from the tumour increased acutely in response to hCG administration; after removal of the tumour, androgen levels were normal and showed negligible responses to hCG. These findings lead us to speculate that endogenous chorionic gonadotrophin may have led to augmented androgen production from the adrenal tumour during pregnancy, causing fetal virilization as previously described in patients with ovarian tumours. This study demonstrates that CT scanning can be valuable in distinguishing between adrenal and ovarian androgen-producing tumours, a distinction which is often unreliable when based on physiological manipulations of hormone secretion.

We have investigated the domain of adrenal steroid action in a variety of mammalian cells and tissue by high resolution two-dimensional gel electrophoresis and autoradiography. Following in vivo or in vitro treatment with steroids, minced tissue or cells were pulsed with [35S]methionine, and they newly synthesized polypeptides compared with controls by visual inspection of partial protein maps. We have found a similar protein (Mr approximately 41-44K, pI approximately 6.3-6.5) to be consistently increased by dexamethasone in 3 rat tissues and 9 human, bovine and rat cell lines. The protein was constitutively synthesized in all targets; quantitative measurements on the magnitude of response show a 2-fold induction by dexamethasone in all systems, in vivo and in vitro. Glucocorticoid-specific hormones but not sex steroids increase the rate of synthesis; deoxycorticosterone has agonist or antagonist effects on 43K synthesis in different systems studied. Co-electrophoresis experiments indicated that 43K proteins co-migrate in at least 3 rat cell lines from tissues with diverse functions. The functional significance of this common glucocorticoid-induced protein is unknown; its ubiquity and electrophoretic properties suggest a highly conserved, common indicator of glucocorticoid action in diverse target cells and tissues, rather than a tissue-specific response.

Gastrin biosynthesis involves a complex series of posttranslational modifications; their elucidation requires a knowledge of the structure of the gastrin precursor. The complete structure of rat preprogastrin was deduced from the nucleotide sequence of a full length cDNA clone isolated from a rat antral cDNA library. Northern blot hybridization analysis of rat antral RNA together with human antral RNA, reveals a single mRNA species of approximately 670 bases. Comparison of this sequence with those of porcine and human gastrin reveals extensive (73%) homology in the gastrin coding region as well as short regions of conserved nucleotides in the noncoding regions. The rat sequence encodes a preprogastrin of 104 amino acids which consists of a signal peptide, a 37 amino acid prosegment; and the gastrin 34 sequence, followed by a glycine (the amide donor), and flanked by pairs of arginine residues. Cleavage at an internal pair of lysine residues yields gastrin 17. Unlike the human and porcine sequences, rat preprogastrin contains a 9 amino acid carboxy-terminal extension peptide (-Ser-Ala-Glu-Glu-Glu-Asp-Gln-Tyr-Asn) which is homologous to the midportion of gastrin 17 including the site of tyrosine sulfation.

The serine proteinase glandular kallikrein has been demonstrated in the gastrointestinal tract, although there is some doubt as to whether it is synthesized there or derives from exocrine-gland secretions. Using a rat pancreatic kallikrein cRNA probe we have demonstrated kallikrein-like gene expression in the corpus, duodenum, jejunum, ileum, caecum and colon, and compared the pattern of expression with that of the gastrointestinal peptides somatostatin, gastrin and glucagon. In addition, using a panel of oligonucleotide probes specific for various members of the rat kallikrein-gene family, we have shown that the kallikrein-like gene expressed appears to be expressed as true kallikrein.

Glucocorticoids and mineralocorticoids have distinct in vivo roles despite close structural homology and similarities in vitro. Known mechanisms of specificity focus on factors extrinsic to the receptor; interactions that directly regulate the receptor to confer specificity are less well understood, particularly for the mineralocorticoid receptor (MR). To examine relative MR vs. glucocorticoid receptor (GR) function in a more physiological context, we compared transactivation by GR and MR in the standard experimental fibroblast CV-1 cell line, the renal epithelial LLC-PK1 line, and neuronal medullary raphe RN33B cells. Maximal transactivational activity mediated by MR, relative to that mediated by GR, is enhanced in both of these cell lines and is primarily conferred by an N-terminal-mediated enhancement of the MR response. In addition, the ligand concentration required for maximal transcriptional activity of the GR varies significantly between cell lines. This is independent of binding affinity or 11beta-hydroxysteroid dehydrogenase-mediated inactivation and may contribute to in vivo tissue-specific differences in responses to the GR. Although ligand binding affinity is clearly conferred by the LBD, receptor-specific variations between cell lines in transcriptional sensitivity to ligand appear, rather, to be associated with the N-terminus. These studies demonstrate that the specificity of the MR vs. the GR response may be mediated via unique cellular factors, as well as suggesting a novel means of expanding the cellular response to cortisol.

Nesidioblastosis is a well-recognized cause of persistent hypoglycaemia in neonates. We describe the case of a 24-year-old woman who presented with hyperinsulinaemic hypoglycaemia in whom an insulinoma could not be identified at operation which resulted in her undergoing a subtotal distal pancreatectomy. Histological examination revealed the presence of nesidioblastosis. The finding of nesidioblastosis in adults has been reported previously, albeit rarely. We report the use of in situ hybridization to characterize the patterns of insulin and proglucagon gene expression in the resected pancreas. Insulin expression was observed in both the islets and also the isolated nesidioblasts. The alpha-cells of the islets had lost their usual peripheral distribution suggesting that not only is insulin gene expression dysregulated but that the islets are structurally abnormal.

There is now overwhelming evidence--radio-immunoassay, immunofluorescence, Northern blot analysis with highly specific cDNA probes--for the production of vasopressin and its congeners in the rat ovary as well as the hypothalamus. Ontogeny studies and studies over the oestrous cycle suggest that such production is controlled by different mechanisms in the two tissues. Studies in Brattleboro rats suggest that the production of vasopressin and its congeners involves different post-transcriptional mechanisms in these tissues. In Brattleboro rats hypothalamic mRNA is either not translated, or yields a very short-lived product, whereas ovarian levels of immunoreactive vasopressin are indistinguishable from those in control animals. The mechanism of this tissue specificity, and its implications in terms of potential paracrine roles for vasopressin and, or its congeners awaits explanation.