Transcriptional regulation of hypoxia-inducible factor 1alpha by HIPK2 suggests a novel mechanism to restrain tumor growth.

HIPK2 has been implicated in restraining tumor progression by more than one mechanism, involving both its catalytic and transcriptional co-repressor functions. Starting from the finding that HIPK2 knockdown by RNA-interference (HIPK2i) induced significant up-regulation of HIF-1alpha mRNA and of its target VEGF in tumor cells, we evaluated the role of HIPK2 in transcriptional regulation of HIF-1alpha. We found that HIPK2 overexpression downmodulated both HIF-1alpha reporter activity and mRNA levels and showed that HIPK2 was bound in vivo to the HIF-1alpha promoter likely in a multiprotein co-repressor complex with histone deacetylase 1 (HDAC1). Thus, the HIF-1alpha promoter was strongly acetylated following HIPK2 knockdown. The HIF-1alpha-dependent VEGF transcription was evaluated by co-transfection of a dominant negative (DN) construct of HIF-1alpha that inhibited VEGF reporter activity induced by HIPK2 knockdown. HIF-1alpha and VEGF up-regulation in HIPK2i cells correlated with increased vascularity of tumor xenografts in vivo and tube formation in HUVEC in vitro. These findings provide the first evidence of HIPK2-mediated transcriptional regulation of HIF-1alpha that might play a critical role in VEGF expression.

Expression and functional role of urotensin-II and its receptor in the adrenal cortex and medulla: novel insights for the pathophysiology of primary aldosteronism.

CONTEXT: The involvement of urotensin II, a vasoactive peptide acting via the G protein-coupled urotensin II receptor, in arterial hypertension remains contentious. OBJECTIVE: We investigated the expression of urotensin II and urotensin II receptor in adrenocortical and adrenomedullary tumors and the functional effects of urotensin II receptor activation. DESIGN: The expression of urotensin II and urotensin II receptor was measured by real time RT-PCR in aldosterone-producing adenoma (n = 22) and pheochromocytoma (n = 10), using histologically normal adrenocortical (n = 6) and normal adrenomedullary (n = 5) tissue as control. Urotensin II peptide and urotensin II receptor protein were investigated with immunohistochemistry and immunoblotting. To identify urotensin II-related and urotensin II receptor-related pathways, a whole transcriptome analysis was used. The adrenocortical effects of urotensin II receptor activation were also assessed by urotensin II infusion with/without the urotensin II receptor antagonist palosuran in rats. RESULTS: Urotensin II was more expressed in pheochromocytoma than in aldosterone-producing adenoma tissue; the opposite was seen for the urotensin II receptor expression. Urotensin II receptor activation in vivo in rats enhanced (by 182 +/- 9%; P < 0.007) the adrenocortical expression of immunoreactive aldosterone synthase. CONCLUSIONS: Urotensin II is a putative mediator of the effects of the adrenal medulla and pheochromocytoma on the adrenocortical zona glomerulosa. This pathophysiological link might account for the reported causal relationship between pheochromocytoma and primary aldosteronism.

Leptin and the regulation of the hypothalamic-pituitary-adrenal axis.

Leptin, the product of the obesity gene (ob) predominantly secreted from adipocytes, plays a major role in the negative control of feeding and acts via a specific receptor (Ob-R), six isoforms of which are known at present. Evidence has been accumulated that leptin, like other peptides involved in the central regulation of food intake, controls the function of the hypothalamic-pituitary-adrenal (HPA) axis, acting on both its central and peripheral branches. Leptin, along with Ob-R, is expressed in the hypothalamus and pituitary gland, where it modulates corticotropin-releasing hormone and ACTH secretion, probably acting in an autocrine-paracrine manner. Only Ob-R is expressed in the adrenal gland, thereby making it likely that leptin affects it by acting as a circulating hormone. Although in vitro and in vivo findings could suggest a glucocorticoid secretagogue action in the rat, the bulk of evidence indicates that leptin inhibits steroid-hormone secretion from the adrenal cortex. In keeping with this, leptin was found to dampen the HPA axis response to many kinds of stress. In contrast, leptin enhances catecolamine release from the adrenal medulla. This observation suggests that leptin activates the sympathoadrenal axis and does not appear to agree with its above-mentioned antistress action. Leptin and/or Ob-R are also expressed in pituitary and adrenal tumors, but little is known about the role of this cytokine in the pathophysiology.

Role of angiotensin II, endothelin-1 and L-type calcium channel in the development of glomerular, tubulointerstitial and perivascular fibrosis.

OBJECTIVE: Fibrosis is a hallmark of renal damage in several diseases, including arterial hypertension. We, therefore, investigated the role of angiotensin II, endothelin-1 and of L-type calcium channels in the development of the glomerular, vascular, and tubulointerstitial fibrosis in a model of severe angiotensin II-dependent hypertension. METHODS: Five-week-old Ren-2 transgenic rats (TGRen2) received for 4 weeks a placebo, bosentan (100 mg/kg body weight), irbesartan (50 mg/kg body weight), the ETA-selective endothelin receptor antagonist BMS-182874 (BMS; 52 mg/kg body weight), the combination of irbesartan (50 mg/kg body weight) plus BMS (52 mg/kg body weight), and nifedipine (30 mg/kg body weight). RESULTS: Glomerular volume, tubulointerstitial fibrosis, glomerular, and perivascular fibrosis were accurately quantified by histomorphometry in four-to-six sections per kidney. Glomerular fibrosis was lowered by BMS (P < 0.001), whereas tubulointerstitial fibrosis was blunted by bosentan (P < 0.001) and irbesartan (P < 0.005). Perivascular fibrosis was reduced by nifedipine and BMS. As only irbesartan and irbesartan plus BMS decreased blood pressure (P < 0.001 vs. placebo), these effects on fibrosis were independent of blood pressure. CONCLUSION: Angiotensin II and L-type calcium channels modulate fibrosis selectively in the tubulointerstitial and in the perivascular compartments, respectively. The prevention of fibrosis with ET-1 receptor antagonism in all three compartments supports a major role of ET-1 in the development of renal fibrosis.

Tryptase- and leptin-positive mast cells correlate with vascular density in uterine leiomyomas.

OBJECTIVE: In vitro and in vivo studies have linked mast cell (MC) degranulation and activation with angiogenesis and neovascularization. This assumption is partially supported by the close anatomical association between MC and the vasculature and the recruitment of these cells during tumor growth. The aim of this study was to correlate the extent of angiogenesis with the number of MC expressing tryptase and leptin in human leiomyomas. STUDY DESIGN: Tissues from human leiomyomas and control specimens were investigated immunohistochemically, using murine monoclonal antibodies against the endothelial cell marker CD31, leptin, and the MC marker tryptase. RESULTS: Angiogenesis, measured as microvessel counts, was highly correlated with MC tryptase- and leptin-positive cell counts. CONCLUSION: These data suggest that angiogenesis in leiomyomas is correlated to expression of tryptase in MC granules and provide for the first time evidence of a putative role of leptin, also contained in MC secretory granules, in MC-dependent angiogenesis.

Galanin stimulates cortisol secretion from human adrenocortical cells through the activation of galanin receptor subtype 1 coupled to the adenylate cyclase-dependent signaling cascade.

Previous studies showed that galanin receptors are expressed in the rat adrenal, and galanin modulates glucocorticoid secretion in this species. Hence, we investigated the expression of the various galanin receptor subtypes (GAL-R1, GAL-R2 and GAL-R3) in the human adrenocortical cells, and the possible involvement of galanin in the control of cortisol secretion. Reverse transcription-polymerase chain reaction detected the expression of GAL-R1 (but not GAL-R2 and GAL-R3) in the inner zones of the human adrenal cortex. The galanin concentration dependently enhanced basal, but not ACTH-stimulated secretion of cortisol from dispersed inner adrenocortical cells (maximal effective concentration, 10(-8) M). The cortisol response to 10(-8) M galanin was abrogated by GAL-R1 immunoneutralization, and unaffected by GAL-R2 or GAL-R3 immunoneutralization. Galanin (10(-8) M) and ACTH (10(-9) M) enhanced cyclic-AMP production from dispersed cells, and the response was suppressed by the adenylate cyclase inhibitor SQ-22536 (10(-4) M). Galanin did not affect inositol triphosphate release, which, in contrast, was raised by angiotensin-II (10(-8) M). SQ-22536 and the protein kinase (PK)A inhibitor H-89 (10(-5) M) abolished the cortisol response to 10(-8) M galanin, while the phospholipase C inhibitor U-73122 and the PKC inhibitor calphostin-C were ineffective. Preincubation with pertussis toxin (Ptx) (0.5 microg/ml) partially inhibited the cortisol response to galanin. We conclude that galanin stimulates cortisol secretion from human inner adrenocortical cells, acting through GAL-R1 coupled to the adenylate cyclase/PKA-dependent signaling cascade via a Ptx-sensitive Galpha protein.

The renal antifibrotic effects of angiotensin-converting enzyme inhibition involve bradykinin B2 receptor activation in angiotensin II-dependent hypertension.

OBJECTIVE: The renoprotective action of angiotensin I-converting enzyme inhibitors (ACE-Is) is well established, but the role played by bradykinin (BK) remains unclear. We therefore investigated whether an enhanced BK effect on B2 receptor subtype mediated the antifibrotic effect of ACE-Is and whether neutral endopeptidase (NEP) inhibition, which can blunt BK degradation more effectively than ACE inhibition, provided further renoprotection in a rat model of angiotensin (Ang) II-dependent renal damage. METHODS: Five-week-old Ren-2 transgenic rats (TGRen2) received, for 8 weeks, a placebo, ramipril (5 mg/kg body weight) or the dual ACE + NEP inhibitor MDL 100,240 (MDL) (40 mg/kg body weight). After 4 weeks, the B2 receptor antagonist icatibant (0.5 mg/kg body weight) was administered on top of active treatment for 4 weeks to 50% of the TGRen2 rats. Blood pressure was measured weekly by a tail-cuff method and, after sacrifice, kidney weight, glomerular volume, density of glomerular profiles were measured; tubulo-interstitial fibrosis, glomerular and perivascular fibrosis were quantified by histomorphometry. RESULTS: The development of hypertension and tubulo-interstitial fibrosis was prevented by both ramipril and MDL (P = 0.0001 versus placebo); icatibant annulled the latter effect. Glomerular and perivascular fibrosis were unaffected by either ramipril or MDL alone; however, combined treatment with icatibant enhanced glomerular fibrosis (P = 0.0001 versus placebo). CONCLUSION: Enhanced BK effect on B2 subtype receptors is essential for the prevention of tubulo-interstitial fibrosis with ACE or dual ACE + NEP inhibition in TGRen2 rats.

Real-time PCR analysis of leptin and leptin receptor expression in the rat prostate, and effects of leptin on prostatic acid phosphatase release.

We have recently demonstrated the expression of leptin and leptin receptor (Ob-R) isoforms a, b, c, e and f in the rat seminal vesicles and prostate. The aim of the present study was to provide a semiquantitative real-time PCR estimation of leptin/Ob-R isoform mRNA expression in the seminal vesicles and individual components of rat prostate, and to ascertain the in vitro effects of leptin on prostate acid phosphatase release. The highest expression of the leptin and Ob-R genes was in the seminal vesicles and lateral prostate lobe, respectively. Of the various isoforms, Ob-Rb displayed the highest and Ob-Re the lowest expression. Leptin (10(-8) and 10(-6) M) enhanced acid phosphate release from seminal vesicles, and (10(-6) M) decreased it from the coagulating lobe. Taken together, our findings support the contention that leptin may be involved in the autocrine-paracrine functional regulation of rat seminal vesicles and prostate. The physiological relevance of the marked heterogeneity of the different prostate lobes in both their leptin/Ob-R expression and functional response to leptin remains to be addressed.

Endocrine disruptors and rat adrenocortical function: studies on freshly dispersed and cultured cells.

The effects of four endocrine disruptors: resveratrol, diphenylolpropane (bisphenol-A; BSP), benzophenone-3 (BP3) and silymarin on the secretory and proliferative activity of rat adrenocortical cells were investigated in vitro. Resveratrol and BP3 acutely increased basal corticosterone secretion from freshly dispersed adrenocortical cells, and resveratrol and BSP enhanced ACTH-stimulated cells. The 24-h exposure to resveratrol and BP3 increased basal corticosterone production from cultured adrenocortical cells, while ACTH-stimulated secretion was increased only by resveratrol. BSP was ineffective, while silymarin decreased basal, but not ACTH-stimulated secretion. The proliferative activity of the cultured adrenocortical cells was unaffected by the tested disruptors. In conclusion, the in vitro direct effect of endocrine disruptors on adrenocortical steroidogenesis displays a great variability, which seems to depend not only on their chemical nature, but also on their dose and the duration of the exposure of the studied cells.

Identification and localization of adrenomedullin-storing cardiac mast cells.

Adrenomedullin (AM), a potent vasodilatory hypotensive peptide, is expressed in the heart, where it is known to play a protective action. Light-microscopy immunocytochemistry (ICC) demonstrated the presence of AM immunoreactivity not only in the coronary-vessel wall and ventricular myocytes of the human and rat heart, but also in sparse voluminous cells located in the perivascular space. These cells displayed the same location of toluidine blue-positive mast cells, and electron microscopy ICC showed AM-immunogold staining over the granules of rat cardiac mast cells. The incubation of rat left ventricle fragments with the mast-cell histamine releaser compound 48/80 evidenced groups of AM-positive cells undergoing degranulation and caused an increase of approximately 50% in the AM concentration in the incubation medium. Collectively, our findings provide evidence that at least a subset of cardiac mast cells are able to synthesize and store AM, and upon stimulation to release it near coronary arterioles and venules.

Expression of neuropeptides B and W and their receptors in endocrine glands of the rat.

Neuropeptides B and W (NPB and NPW) have been identified as endogenous ligands of the G protein-coupled receptors (GPR) 7 and 8, which in humans are expressed in the hypothalamus and probably involved in the regulation of energy homeostasis and feeding behavior. GPR8 is absent in the rat, where the GPR8-like receptor (GPR8-LR) has been described. Reverse transcription-polymerase chain reaction detected the expression of NPB, NPW, GPR7 and GPR8-LR mRNAs in the hypothalamus, anterior pituitary, thyroid and parathyroid glands, pancreatic islets, adrenal glands, ovary and testis of the rat. Immunocytochemistry demonstrated the presence of NPB and NPW immunoreactivities in these same glands. Radioimmune assay showed that the bolus intraperitoneal injection of 2 nmol/100 g NPB or NPW raised the plasma levels of parathyroid hormone, corticosterone and testosterone. NPB also increased the blood concentration of thyroxine, and NPW that of ACTH and estradiol. Taken together, these findings allow us to suggest that NPB and NPW play a role in the autocrine-paracrine functional regulation of the endocrine system in the rat.

Estradiol and resveratrol stimulating effect on osteocalcin, but not osteonectin and collagen-1alpha gene expression in primary culture of rat calvarial osteoblast-like cells.

Evidence is available that some endocrine disruptors, acting as selective estrogen receptor modulators (SERMs), interfere with osteoblast differentiation and function. Therefore, we investigated whether 17beta-estradiol, bisphenol-A (BSP), silymarin, genistein, resveratrol, procymidone, linurone and benzophenone-3 (BP3) modulate differentiation of rat calvarial osteoblast-like (ROB) cells in primary in vitro culture. Disruptors were added at day 18 of culture and cells were harvested 48 h later. Real time-PCR revealed that estradiol and resveratrol enhanced osteocalcin mRNA expression in ROB cells, while other disruptors were ineffective. The expression of osteonectin and collagen-1alpha was not affected by any disruptor. Estradiol, resveratrol, genistein and BSP stimulated the proliferative activity of ROB cells. In contrast, procymidone and linurone inhibited the proliferative activity, and silymarin and BP3 were ineffective. The conclusion is drawn that i) only resveratrol is able, like estradiol, to stimulate the specialized functions of ROB cells, and ii) the proliferative activity of ROB cells is more sensitive to endocrine disruptors, some of which could probably act via a mechanism independent of their SERM activity.

Endothelin-1 Drives Epithelial-Mesenchymal Transition in Hypertensive Nephroangiosclerosis.

BACKGROUND: Tubulointerstitial fibrosis, the final outcome of most kidney diseases, involves activation of epithelial mesenchymal transition (EMT). Endothelin-1 (ET-1) activates EMT in cancer cells, but it is not known whether it drives EMT in the kidney. We therefore tested the hypothesis that tubulointerstitial fibrosis involves EMT driven by ET-1. METHODS AND RESULTS: Transgenic TG[mRen2]27 (TGRen2) rats developing fulminant angiotensin II-dependent hypertension with prominent cardiovascular and renal damage were submitted to drug treatments targeted to ET-1 and/or angiotensin II receptor or left untreated (controls). Expressional changes of E-cadherin and α-smooth muscle actin (αSMA) were examined as markers of renal EMT. In human kidney HK-2 proximal tubular cells expressing the ETB receptor subtype, the effects of ET-1 with or without ET-1 antagonists were also investigated. The occurrence of renal fibrosis was associated with EMT in control TGRen2 rats, as evidenced by decreased E-cadherin and increased αSMA expression. Irbesartan and the mixed ET-1 receptor antagonist bosentan prevented these changes in a blood pressure-independent fashion (P < 0.001 for both versus controls). In HK-2 cells ET-1 blunted E-cadherin expression, increased αSMA expression (both P < 0.01), collagen synthesis, and metalloproteinase activity (P < 0.005, all versus untreated cells). All changes were prevented by the selective ETB receptor antagonist BQ-788. Evidence for involvement of the Rho-kinase signaling pathway and dephosphorylation of Yes-associated protein in EMT was also found. CONCLUSIONS: In angiotensin II-dependent hypertension, ET-1 acting via ETB receptors and the Rho-kinase and Yes-associated protein induces EMT and thereby renal fibrosis.

Cardiac fibrosis occurs early and involves endothelin and AT-1 receptors in hypertension due to endogenous angiotensin II.

OBJECTIVES: We investigated if endothelin (ET)-1 and the renin-angiotensin-aldosterone system play a role in cardiac fibrosis. BACKGROUND: Angiotensin II (Ang II) can induce cardiac fibrosis, but the underlying mechanisms are incompletely understood. METHODS: Four-week-old transgenic (mRen2)27 rat (TGRen2) received for four weeks a placebo, the mixed ET(A)/ET(B) endothelin receptor antagonist bosentan, the angiotensin II type I receptor (AT-1) antagonist irbesartan, the ET(A) endothelin receptor antagonist BMS-182874, and a combined treatment with irbesartan plus BMS-182874. We measured collagen density on Sirius red-stained serial sections of the left ventricle (LV) with a photomicroscope equipped with specific software and assessed the gene expression of procollagen alpha1(I), atrial natriuretic peptide (ANP), transforming growth factor-beta 1 (TGFbeta1), endothelin converting enzyme, and ET(B) receptor. RESULTS: In the placebo group, hypertension was associated with LV hypertrophy and cardiac fibrosis (LV weight: 4.0 +/- 0.3 mg/g body weight; collagen density: 2.21 +/- 0.16%), which were all prevented with irbesartan (2.3 +/- 0.1, 1.30 +/- 0.13, p < 0.001), but not with BMS-182874 (4.0 +/- 0.2, 2.41 +/- 0.22). Bosentan also prevented fibrosis (1.39 +/- 0.18) but not hypertension and LV hypertrophy (3.38 +/- 0.27). Combined irbesartan and BMS-182874 treatment prevented LV hypertrophy (2.9 +/- 0.1) but not fibrosis (2.52 +/- 0.16). Collagen density correlated (r = 0.414, p < 0.05) with plasma aldosterone levels. In TGRen2 with LV hypertrophy, the gene expression of ANP and ET(B) but not that of TGFbeta1 and procollagen alpha1(I) was increased. CONCLUSIONS: In Ang II-dependent hypertension, cardiac fibrosis was associated with LV hypertrophy and was hindered by both mixed ET(A)/ET(B) blockade and AT-1 blockade. Only the latter treatment prevented both hypertension and LV hypertrophy. Thus, there is a dissociation between the mechanisms of cardiac fibrosis and hypertension, which do and do not entail ET-1, respectively.

Adrenomedullin, ANP and BNP are colocalized in a subset of endocrine cells in the rat heart.

We investigated by immunocytochemistry (ICC) the distribution in the rat heart of adrenomedullin (AM), a potent and long-lasting hypotensive peptide which is expressed in the cardiovascular system, where it is known to play a major regulatory role. Hearts were collected from adult male Sprague-Dawley rats, and were perfused for 20 min, according to the Langendorff technique, with endothelin-1 (ET-1) or the mast cell-degranulator compound 48/80. Hearts were frozen, and ICC was performed using standard techniques and a specific anti-rat AM1-50 antibody. We confirmed the presence of a low AM-immunoreactivity in cardiomyocytes and cardiac fibroblasts, as well as in endothelial and smooth muscle cells of coronary vessels. Moreover, we provided evidence of the presence in both atria and ventricles of sparse voluminous AM-positive cells, mainly located near coronary vessels. These cells had the same juxtavascular location of toluidine blue-positive mast cells and their number decreased upon acute exposure to the 48/80 compound. However, ICC showed that in these cells AM was always colocalized with atrial and brain natriuretic peptides. Moreover, AM-storing cells were also positive to MyHC-Apla2, indicating that they share some phenotypic features with immature smooth muscle cells. The number of AM-storing cells underwent a dramatic decrease in response to the potent vasoconstrictor ET-1, suggesting an acute release of stored vasodilatory AM aimed at counteracting coronary constriction. Taken together, our present findings support the hypothesis that these cells may represent a novel subset of endocrine cells, strategically located near blood vessels in the mammalian heart, where they can release vasoactive peptides.

Aortic smooth muscle cell phenotypic modulation and fibrillar collagen deposition in angiotensin II-dependent hypertension.

BACKGROUND: We investigated the effect of nifedipine, AT-1 and ET-1 receptor blockade on arterial smooth muscle cell phenotypes and collagen deposition in TGRen2 transgenic rat (TGR). METHODS: Four-week-old TGR were blood pressure (BP)-matched and allocated to receive a placebo (n=8), the calcium antagonist nifedipine (n=6), the AT-1 specific receptor antagonist irbesartan (n=6), the ET(A)/ET(B) antagonist bosentan (n=6) or the ET(A)-selective antagonist BMS-182874 (n=5). Sprague-Dawley normotensive rats served as controls (n=6). After 4 weeks of treatment animals were euthanized and the left ventricle (LV) and the structural changes in intracardiac arterioles and aorta were assessed histomorphometrically. Smooth muscle cell phenotypes and fibrillar collagen content of the aortic wall were evaluated by immunostaining, using differentiation markers-specific antibodies and Syrius red staining, respectively. The changes in ET(A) and ET(B) receptor density were also assessed with quantitative autoradiography. RESULTS: Compared to placebo, only irbesartan lowered BP (P<0.001) and prevented LV and small resistance artery hypertrophy. The aorta of placebo-treated TGR showed an increase in foetal-type smooth muscle cell content and fibrillar collagen staining, compared to controls. These changes were blunted by irbesartan, which increased ET(A) receptors in the arterial wall, enhanced by BMS-182874 and unaffected by bosentan. Nifedipine also blunted both the VSMC and collagen changes despite having no effect on BP and ET(A) receptors. CONCLUSIONS: In TGRen2, vascular hypertrophy entails both smooth muscle cell phenotypic modulation and collagen deposition. These alterations do not follow closely the BP changes and seem to imply the dihydropyridine-sensitive calcium channels.

Altered regulation of endothelin A receptor subtype in the cerebral arterioles in response to a Japanese-style diet, in stroke-prone hypertensive rats.

OBJECTIVE: To investigate the expression of endothelin (ET)-1 and its receptors in the cerebral arterioles of stroke-prone (spSHR) and control spontaneously hypertensive rats (SHRs) and the changes in endothelin receptor subtypes A and B density elicited by a stroke-permissive diet, before the development of stroke. METHODS: Six-week-old SHRs (n=11) and spSHRs (n=11) were assigned to either a regular or a "Japanese"-style diet, in addition to 1% NaCl in the drinking water, for 4 weeks. Cryosections (10 microm thick) of rat brain were assessed for endothelin receptor distribution and density by autoradiography with [125I]ET-1 (10(-10) mol/l) in the presence of cold ET-1 (10(-6) mol/l) or the peptide antagonists BQ-123 (10(-6) mol/l) or BQ-788 (10(-6) mol/l). Reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry were used to detect specific mRNAs and localize immunoreactive ET-1 and ET(A) and ET(B). RESULTS: In both strains, immunoreactive ET-1 was detected in the endothelium of cerebral arterioles, and RT-PCR and autoradiography demonstrated the coexistence of both receptor subtypes in brain homogenates and the cerebral arteriole walls, respectively. With the regular diet, the ET(A) receptor density was lower in SHRs than in spSHRs (P = 0.007), whereas the ET(B) receptor density was similar (P = NS). The Japanese-style diet increased the density of ET(A) receptors (P = 0.006) in SHRs, but decreased it (P = 0.019) in spSHRs. No effect was seen on ET(B) receptor density. CONCLUSIONS: ET(A) and ET(B) receptor subtypes are expressed in the wall of cerebral arterioles of SHRs and spSHRs. The latter strain showed a marked increase in ET(A) receptor density under a regular diet, in addition to an altered regulation in response to a stroke-permissive diet.

Adrenomedullin stimulates proliferation and inhibits apoptosis of immature rat thymocytes cultured in vitro.

Adrenomedullin (AM) is a hypotensive peptide, which derives from the proteolytic cleavage of pro(p)AM, and acts through two subtypes of receptors, named L1-receptor (L1-R) and calcitonin receptor-like receptor (CRLR). CRLR functions as either a calcitonin gene-related peptide (CGRP) receptor or a selective AM receptor depending on which member of a family of receptor-activity-modifying proteins (RAMPs) is expressed: RAMP1 generates CGRP receptors, while RAMP2 and RAMP3 produce AM receptors. Reverse transcription (RT)-polymerase chain reaction (PCR) consistently allowed the detection of pAM and peptidyl-glycine alpha-amidating monooxygenase (the enzyme converting immature AM to the mature peptide) mRNAs in the thymus cortex of immature (10-day-old) rats. Accordingly, radioimmune assay (RIA) measured low but sizeable AM concentrations in this tissue. RT-PCR also demonstrated the presence of the specific mRNAs of L1-R, CRLR and RAMPs. AM (from 10(-9) to 10(-7)M) increased proliferation index and lowered apoptotic index of cultured immature rat thymocytes, and the effects were annulled by the AM receptor antagonist AM(22-52). In conclusion, our study demonstrated that (1) immature rat thymus cortex expresses AM and the AM receptors L1-R and CRLR/RAMP; and (2) AM, acting via AM(22-52)-sensitive receptors, exerts a potent growth promoting effect on immature rat thymus, by enhancing proliferation and lowering apoptotic death of thymocytes. Taken together, these findings could suggest that AM may play a role in the development of immunity.

Investigation of the effect of different regulatory peptides on adrenocortical cell proliferation in immature rats: evidence that endogenous adrenomedullin exerts a stimulating action.

Compelling evidence indicates that the active growth of immature rat adrenal glands is sustained not only by an increased release of pituitary ACTH, but also by other ancillary mechanisms. We investigated whether vasoactive intestinal peptide (VIP), atrial natriuretic peptide (ANP), adrenomedullin (ADM) and proadrenomedullin N-terminal 20 peptide (PAMP) play a relevant role in these mechanisms. These four regulatory peptides were chosen because previous studies demonstrated that they are expressed in rat adrenals and are able to modulate the secretory activity and growth of zona glomerulosa (ZG), i.e., the adrenal layer. Groups of immature (20-day old) rats were given three subcutaneous injections (28, 16 and 4 h before sacrifice) of 2 nmol/100 g of the four peptides and/or selective antagonists of their receptors (VIP-A, ANP-A, ADM-A and PAMP-A), and 0.1 mg/100 g vincristin 3 h before autopsy. Adrenal glands were collected, processed for light microscopy, and the mitotic index (MI; percentage of metaphase-arrested cells) was evaluated in the subcapsular ZG. Neither VIP nor VIP-A affected MI. Both ANP and ANP-A decreased MI and their effects displayed additivity. ADM and PAMP raised MI and the effect was abolished by ADM-A and PAMP-A, respectively. When administered alone ADM-A, but not PAMP-A, significantly lowered MI. Collectively, our findings suggest that: i) neither VIP nor PAMP are involved in the regulation of immature rat adrenals; ii) ANP exerts a non-receptor-mediated inhibitory action, whose physiological relevance remains to be investigated; and iii) endogenous ADM system plays a relevant role in the mechanisms underlying the maintenance of high growth rate during adrenal maturation.

Differential expression of adrenomedullin and its receptors in newborn and adult rat thymus.

Adrenomedullin (ADM) is a hypotensive peptide, which derives from the proteolytic cleavage of pro(p)ADM and acts via two main subtypes of receptors, referred to as L1-receptor (L1-R) and calcitonin-receptor-like receptor (CRLR). While L1-R is selective for ADM, CRLR may bind either calcitonin gene-related peptide (CGRP) or ADM depending on the expression of the subtype 1 or the subtypes 2 and 3 of a family of chaperones, named receptor-activity-modifying proteins (RAMPs). There is evidence that ADM, in addition to regulating blood pressure and water and electrolyte balance, may be also involved during embryogenesis in the growth and differentiation of organs and tissues, especially of those where strong mesenchymal-epithelial interactions take place. Thymus is a linfo-epithelial organ, which undergoes a very rapid prenatal and postnatal growth, playing a pivotal role in the development of immunological defense. Hence, it appeared worthwhile to investigate the expression of ADM system in the newborn (3-day-old) rat thymus as compared to adult (3-month-old) animals. Reverse transcription (RT)-polymerase chain reaction (PCR) allowed the detection of the specific mRNAs of pADM and peptidyl-glycine alpha-amidating monooxigenase (PAM), the enzyme which converts immature ADM to the mature peptide, in both newborn and adult rat thymuses. PAM expression was markedly higher in newborn animals, which accords well with the more elevated concentrations of ADM measured by RIA in newborn than adult rat thymuses. L1-R, CRLR, RAMP1 and RAMP2 mRNA were detected in both groups of rats, and with the exception of RAMP1, the expression was markedly higher in newborn than adult rat thymus. RAMP3 mRNA was present only in the thymus of newborn animals. Collectively, the present findings indicate that ADM system is up-regulated in newborn rat thymus, thereby making it likely that ADM may be involved in the thymus growth and in the development of immunological defense mechanisms.