Using labelled ligand-binding methods, previous studies have identified specific angiotensin II receptors (Ang II-Rs) in eel liver, kidney and intestine membranes. Isoelectric focusing on polyacrylamide gels also showed that there are two Ang II-R isoforms in eel liver, focusing at isoelectric points (pI) 6.5 and 6.7. These may have different functions. In contrast, eel enterocyte plasma membrane and renal brush border membranes contain only the pI 6.5 form. To characterize the eel receptors more fully, a newly developed monoclonal antibody (6313/G2) which selectively recognizes the AT1 subtype of mammalian Ang II-R was used. In ligand-binding experiments, the preincubation of eel liver membranes with 6313/G2 antibody eliminated the specific [3,5-3H]Tyr4-Ile5-Ang II binding. Moreover, Ang II-receptor complexes from solubilized liver membranes, which were immunoprecipitated by 6313/G2-coated beads, had a pI of 6.5. In immunoblotting experiments, the antibody recognized the isoform focusing at pI 6.5 in eel intestine and liver preparations, but not the liver pI 6.7 isoform. Immunoblotting of SDS gels showed that the antibody bound to a single protein of molecular mass of 75 kDa in eel liver, gill and kidney and to a doublet of molecular mass of about 74 and 75 kDa in intestinal membrane preparations. Immunocytochemistry of paraffin-embedded and cryostat sections of eel liver, kidney, intestine and gill showed that antibody 6313/G2 bound to uniformly distributed intracellular sites and cell surface membranes in proximal tubular cells, absorptive intestinal cells, hepatocytes and chloride cells. It also stained endothelium and both the longitudinal and circular layers of smooth muscle cells in the intestine. The data suggest that the previously described Ang II-R from eel liver, kidney and intestine may be similar to the mammalian AT1 subtype.

Several lines of experimentation suggest that a tissue-sequestered pool of 18-hydroxydeoxycorticosterone (18-OH-DOC) in the rat adrenal may be mobilized as an aldosterone precursor. We show here that this steroid is maintained in a non-extractable form in the membranes of collagenase-dispersed fasciculata/reticularis cells. Because of this stability, the complex can be identified by immunocytochemistry and also, in IEF gels of solubilized inner adrenocortical zone membrane preparations, by immunoblotting. However, the complexed steroid cannot be extracted from the gels into organic solvent unless first treated with trypsin. Preincubation of viable whole glandular tissue with trypsin significantly enhanced aldosterone output and eliminated the trypsin-releasable 18-OH-DOC pool in IEF gels of solubilized inner zone membranes. Both prior sodium depletion and acute trypsin stimulation of whole glands enhanced extractable 18-OH-DOC in glomerulosa tissue membranes. Other experiments using in situ hybridization show that mRNA coding for 11 beta-hydroxylase (which generates 18-OH-DOC) is confined to the inner adrenocortical zones, whereas aldosterone synthase (which does not) is transcribed exclusively in the glomerulosa. The data suggest that a pool of 18-OH-DOC in inner zone membranes can be mobilized for utilization as an aldosterone precursor in the glomerulosa. The results also indicate the existence of an entirely novel tightly binding steroid carrier from which steroid cannot be extracted by organic solvent unless first subjected to proteolytic degradation.

The tissue renin-angiotensin systems (RAS) may have specific roles that complement those of the systemic RAS. In the adrenal, the tissue RAS has been implicated in the regulation of glomerulosa tissue growth and function, and in mediating the response of the tissue to stimulation by ACTH and potassium ions.To examine the role of the rat adrenal tissue RAS in its response to angiotensin II stimulation, adrenals were incubated either as bisected glands or as separated capsular glands (largely glomerulosa) under control conditions, or in the presence of the angiotensin-converting enzyme inhibitor captopril, or of angiotensin II, or both.Captopril inhibited the two different tissue preparations in different ways. In the capsular gland it inhibited basal aldosterone output, but facilitated its response to angiotensin II. In the bisected gland, captopril inhibited the response of aldosterone to angiotensin II.Other data suggest that one way in which captopril functions is by preventing the conversion of fasciculatagenerated 18-hydroxydeoxycorticosterone (18-OH-DOC) to aldosterone in the glomerulosa. Immunolocalisation of 18-OH-DOC in perfused rat adrenal confirms that one function of angiotensin II is to mobilise tissue-sequestered 18-OH-DOC.The results illustrate the importance of tissue RAS in the synthesis of aldosterone and the response to angiotensin II.

Drug resistant infections represent one of the most challenging medical problems of our time. Dcycloserine is an antibiotic used for decades without appearance and dissemination of antibiotic resistant strains, making it an ideal model compound to understand what drives resistance evasion. We investigated why Mycobacterium tuberculosis fails to become resistant to Dcycloserine. To address this question we employed a combination of bacterial genetics, genomics, biochemistry and fitness analysis in vitro, in macrophages and in mice. Altogether, our results suggest that the ultra-low mutation frequency associated with D-cycloserine

Transcription of the (pro)renin gene in the adult rat adrenal gland was studied by non-isotopic in situ hybridization.In glands from control (untreated) animals, transcription was relatively sparse, and occurred mostly in the outer zona fasciculata. Treatment with ACTH increased the apparent signal in both the glomerulosa and in fasciculata zones.A low sodium diet initially enhanced the transcription signal specifically in the glomerulosa, but as the regime was extended from 5 days to more than 2 weeks, the signal was also increased dramatically in the zona reticularis.The results emphasize the potential importance of the intraglandular renin-angiotensin system, particularly under conditions of chronic stimulation. They also suggest that angiotensin II, as well as being the major regulator of the glomerulosa, may also have some role in inner adrenocortical zone functions.