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MM Ho and GP Vinson

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.

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F Xiao, Puddefoot JR and GP Vinson

One controversy in the field of vascular angiotensin generation has surrounded the nature and particularly the source of vascular renin. This study investigated the expression of renin protein and its mRNA in aortic endothelial cells using immunocytochemistry, Western blotting, in situ hybridization and reverse transcription PCR (RT-PCR). Using a monoclonal antibody against human renin, immunocytochemical analysis revealed positive immunoreactivity in the cytoplasm of cultured bovine aortic endothelial cells. Immunoblotting of solubilized proteins separated by SDS-PAGE from cultured aortic endothelial cells identified two immunoreactive species with molecular masses of approximately 37-40 kDa. In situ hybridization showed that renin mRNA was localized in the cytoplasm of these cells. Using RT-PCR of RNA extracted from bovine aortic endothelial cells with primers specific for human renin, a clear single band was detected, which had the predicted size of 142 bp for (pro)renin. Angiotensin II (Ang II) was assayed in conditioned medium (CM) from cultured bovine aortic endothelial cells, and in addition, the effects of Ang II and CM on the proliferation of aorta smooth muscle cells (ASMC) were also studied. The results showed that CM contained Ang II equivalent to 15.05+/-4.67 pg/10(6) cells. Assay of smooth muscle cell proliferation by cell number, and by tritiated thymidine uptake, showed that proliferative responses in the presence of Ang II at a concentration of 10(-6)M were evident within 1 day of subculture, and cell numbers were nearly twice those of controls after 2 days. Thymidine incorporation into ASMC was also increased by Ang II in a dose-dependent manner and by endothelial cell CM. In both cases, stimulated proliferation was inhibited by the Ang II type 1 (AT1) receptor selective antagonist, losartan. These findings suggest that these vascular endothelial cells are a source of locally synthesized renin that may thus be involved in vascular Ang II generation. They also suggest that Ang II produced by the endothelial cells may be secreted and stimulate ASMC proliferation via the AT1 receptor.

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F Xiao, Puddefoot JR and GP Vinson

Aldosterone, possibly locally generated, has been suggested to have a role in potentiating angiotensin II (AII)-stimulated hypertrophy of cultured vascular smooth muscle cells. To examine the possibility that aldosterone may mediate the proliferative actions of AII, rat aortic smooth muscle cells (RASMCs) in culture were treated with AII in the presence and absence of the specific AII type 1 receptor (AT1) antagonist, losartan, and aldosterone was assayed in culture medium extracts by radioimmunoassay. AII significantly enhanced aldosterone formation (at 10(-8) M: 123.8+/-14.85 vs control 71. 28+/- 8.71 fmol/10(5) cells, P<0.05; at 10(-7) M: 172.38+/-33.44, P<0.05), but not in the presence of losartan (at 10(-8) M: 53. 71+/-18.73, P>0.05; at 10(-7) M: 89.68+/-25.05, P>0.05). In other studies, the reverse transcriptase-polymerase chain reaction, performed on RNA extracted from RASMCs using aldosterone synthase (CYP11B2) specific primers, gave a single band of about 268 bp, consistent with that expected for the enzyme. Finally, using [(3)H]methylthymidine uptake as an index of cellular proliferation, tritium incorporation was increased in the AII-treated group at concentrations greater than 10(-10) M. The aldosterone antagonist, spironolactone (10(-5) M), inhibited the incorporation of [(3)H]thymidine into RASMCs stimulated by AII. These results suggest that locally generated aldosterone may mediate the effects of AII, acting via the AT1 receptor, in stimulating RASMC proliferation.

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GP Vinson, R Teja, MM Ho, JP Hinson and Puddefoot JR

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 fasciculata-generated 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.

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A Muscella, S Marsigliante, MA Carluccio, GP Vinson and C Storelli

Cultured human umbilical vein endothelial cells (HUVECs) at passage 4 specifically bound 70 +/- 12 fmol [3,5-3H]Tyr4-Ile5-angiotensin (Ang) II/mg protein, with a Kd of 0.9 +/- 0.36 nM. Binding was eliminated in cells preincubated with a monoclonal antibody (6313/G2) raised against the subtype AT1 of the Ang II receptor. Freshly seeded HUVECs were positive for 6313/G2 antibody by immunocytochemistry, and such immunoreactivity was still retained at passage 4. Incubation of HUVECs for 20 min with different concentrations of Ang II provoked a significant increment in Na+/K+ ATPase activity compared with controls, in a dose- and time-dependent manner. Maximal response was obtained with 1000 pM Ang II after 20 min stimulation and resulted in a 2.2-fold increment in Na+/K+ ATPase activity. This stimulation was abolished when cells were incubated with 1000 pM Ang II in the presence of 1 microM of the specific AT1 subtype inhibitor, DuP753. Moreover, preincubation of HUVECs with 6313/G2 or with 1 mM dithiothreitol also inhibited the stimulatory effect of Ang II. These results suggest that the AT1 receptor subtype mediates the Na+/K+ ATPase response to Ang II in these cells.

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M Tahmasebi, Puddefoot JR, ER Inwang and GP Vinson

Evidence exists for the presence of a discrete tissue renin-angiotensin system (RAS) in mouse and rat pancreas that is thought largely to be associated with the vasculature. To investigate this in the human pancreas, and to establish whether the cellular sites of RAS components include the islets of Langerhans, we used immunocytochemistry to localise the expression of angiotensin II (AT1) receptors and (pro)renin, and non-isotopic in situ hybridisation to localise transcription of the (pro)renin gene. Identification of cell types in the islets of Langerhans was achieved using antibodies to glucagon and insulin. The results show the presence of the AT1 receptor and (pro)renin both in the beta cells of the islets of Langerhans, and in endothelial cells of the pancreatic vasculature. Transcription of (pro)renin mRNA, however, was confined to connective tissue surrounding the blood vessels and in reticular fibres within the islets. These findings are similar to those obtained in other tissues, and suggest that renin may be released from its sites of synthesis and taken up by possible cellular sites of action. The results presented here suggest that a tissue RAS may be present in human pancreas and that it may directly affect beta cell function as well as pancreatic blood flow.

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P Vendeira, D Pignatelli, D Neves, MM Magalhaes, MC Magalhaes and GP Vinson

Adrenocortical regeneration after adrenal autotransplantation provides a model for the study of local autocrine/paracrine mechanisms involved in the growth and differentiation of the adrenal cortex. To study the possible involvement of some growth factors, namely basic fibroblast growth factor (bFGF, FGF-2) and insulin-like growth factor I (IGF-I), in cell differentiation, immunohistochemical and ultrastructural studies were carried out on adrenal autotransplants in adult male rats. To distinguish between fasciculata and glomerulosa-like cells with accuracy, tissue sections were immunostained with IZAb, which recognizes the inner zone antigen (IZAg) present in fasciculata and reticularis cells but absent from the glomerulosa, and by electron microscopy. IGF-I-treated animals exhibited a clear glomerulosa-like zone that was devoid of IZAb immunostaining. In this outer subcapsular area, ultrastructural examination showed cells containing mitochondria with irregular cristae resembling those of the fetal or immature glomerulosa cells. In contrast, no significant morphological differences were observed in bFGF-treated animals when compared with those from saline-treated controls, in both of which, IZAb immunostaining occurred in almost all adrenocortical cells, with no clear zonation or glomerulosa, as seen in the intact animal. Plasma aldosterone and corticosterone concentrations were lower in autotransplanted control animals than in intact controls, although plasma renin activities were similar. IGF-I treatment significantly increased aldosterone concentrations, whereas corticosterone and plasma renin activity were reduced. bFGF infusion further reduced plasma aldosterone, although plasma renin activity and corticosterone were unaffected. These results suggest that the two growth factors have different effects on zonal differentiation and function in the autotransplanted gland. In particular, bFGF, by reducing glomerulosa function, appears partly to replicate the actions of ACTH in normal animals. In contrast, IGF-I enhances the glomerulosa secreting phenotype and diminishes that of the fasciculata/reticularis, possibly replicating the actions of angiotensin II or a low sodium diet.

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A Janossy, E Orso, KS Szalay, Z Juranyi, M Beck, ES Vizi and GP Vinson

Using histochemical and immunocytochemical methods, cholinergic nerve fibres were demonstrated in the rat adrenal cortex, primarily in the capsule and zona glomerulosa, and in the medulla. Some terminated among the glomerulosa cells or around blood vessels. Occasional fibres were also seen in the fasciculata, ending in islets of chromaffin tissue without ramifications on cortical cells. To clarify the role of cholinergic innervation, a microvolume perifusion system was used to study steroid production by the rat adrenal capsule-glomerulosa. Acetylcholine (ACh) itself had no reproducible effects; however, since variable amounts of endogenous ACh were present, the actions of antagonists were also studied. The M1 muscarinic receptor antagonist pirenzepine (10 and 100 microM) stimulated aldosterone secretion. This stimulation was abolished by co-incubation with carbachol, the M1 agonist McN A-343 and by atropine. We found that the action of pirenzepine was blocked by nifedipine (Ca2+ channel blocker), suggesting that pirenzepine (through release of endogenous ACh) provides an acute stimulus by enhancing Ca2+ inflow. Hemicholine, a choline uptake blocker, reduced the stimulatory effect of pirenzepine on steroid secretion, confirming that stimulation was of neural origin. Neither the non-selective muscarinic receptor antagonist atropine, the selective M1-M3 muscarinic receptor antagonist 4-DAMP, nor the selective M2 muscarinic receptor antagonist methoctramine influenced aldosterone output. Receptor-binding studies revealed the existence of M3 receptors in capsule-glomerulosa homogenates. We conclude that pirenzepine acts on presynaptic M1 autoreceptors to increase spontaneous ACh release from varicose axon terminals that lie in close proximity to the glomerulosa cells. In turn ACh may thus stimulate steroidogenesis acutely through M3 receptors. These results support the concept of a direct cholinergic influence on zona glomerulosa function in the rat.