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BACE1 ([beta]-site amyloidogenic cleavage of precursor protein-cleaving enzyme 1) is a [beta]-secretase protein that plays a central role in the production of the [beta]-amyloid peptide in the brain and is thought to be involved in the Alzheimer's pathogenesis. In type 2 diabetes, amyloid deposition within the pancreatic islets is a pathophysiological hallmark, making crucial the study in the pancreas of BACE1 and its homologous BACE2 to understand the pathological mechanisms of this disease. The objectives of the present study were to characterize the localization of BACE proteins in human pancreas and determine their function. High levels of BACE enzymatic activity were detected in human pancreas. In normal human pancreas, BACE1 was observed in endocrine as well as in exocrine pancreas, whereas BACE2 expression was restricted to [beta]-cells. Intracellular analysis using immunofluorescence showed colocalization of BACE1 with insulin and BACE2 with clathrin-coated vesicles of the plasma membrane in MIN6 cells. When BACE1 and -2 were pharmacologically inhibited, BACE1 localization was not altered, whereas BACE2 content in clathrin-coated vesicles was increased. Insulin internalization rate was reduced, insulin receptor [beta]-subunit (IR[beta]) expression was decreased at the plasma membrane and increased in the Golgi apparatus, and a significant reduction in insulin gene expression was detected. Similar results were obtained after specific BACE2 silencing in MIN6 cells. All these data point to a role for BACE2 in the IR[beta] trafficking and insulin signaling. In conclusion, BACE2 is hereby presented as an important enzyme in [beta]-cell function. [beta]-site amyloidogenic cleavage of precursor protein-cleaving enzyme 1; [beta]-secretase; pancreas; insulin receptor [beta]-subunit trafficking doi: 10.1152/ajpendo.00420.2010.

An increasing amount of evidence demonstrates the beneficial role of oxytocin (OT) in the cardiovascular system. Similar actions are attributed to genistein, an isoflavonic phytoestrogen. The treatment with genistein activates the OT system in the aorta of ovariectomized (OVX) Sprague-Dawley (SD) rats. The objective of this study was to determine the effects of low doses of genistein on the OT-induced effects in rat hypertension. The hypothesis tested was that treatment of OVX spontaneously hypertensive rats (SHRs) with genistein improves heart structure and heart work through a mechanism involving the specific OT receptor (OTR). OVX SHRs or SD rats were treated with genistein (in [micro]g/g body wt sc, 10 days) in the presence or absence of an OT antagonist (OTA) [d[(C[H.sub.2]).sub.5], Tyr[(Me).sup.2], [Orn.sup.8]]-vasotocin or a nonspecific estrogen receptor antagonist (ICI-182780). Vehicle-treated OVX rats served as controls. RT-PCR and Western blot analysis demonstrated that left ventricular (LV) OTR, downregulated by ovariectomy, increased in response to genistein. In SHRs or SD rats, this effect was blocked by OTA or ICI-182780 administration. The OTR was mainly localized in microvessels expressing the CD31 marker and colocalized with endothelial nitric oxide synthase. In SHRs, the genistein-stimulated OTR increases were associated with improved fractional shortening, decreased blood pressure (12 mmHg), decreased heart weight-to-body weight ratio, decreased fibrosis, and lowered brain natriuretic peptide in the LV. The prominent finding of the study is the detrimental effect of OTA treatment on the LV of SHRs. OTA treatment of OVX SHRs resulted in a dramatic worsening of ejection fractions and an augmented fibrosis. In conclusion, these results demonstrate that cardiac OTRs are involved in the regulation of cardiac function of OVX SHRs. The decreases of OTRs may contribute to cardiac pathology following menopause. oxytocin receptor; ovariectomy; hypertrophy; genistein doi: 10.1152/ajpheart.00487.2009.

Endothelial caveolin-I (cav-1) is an anchoring protein in plasma membrane caveolae where it binds endothelial nitric oxide synthase (eNOS) and limits its activation, particularly in animals fed a high salt (HS) diet. Cav-1 also interacts with steroid receptors such as the mineralocorticoid receptor (MR). To test the hypothesis that vascular reactivity is influenced by an interplay between MR and cav- 1 during HS diet, we examined the effects of MR blockade on NOS-mediated vascular relaxation in normal and cav-l-deficient mice. Wild-type (WT) and car-1 knockout mice ([cav-1.sup.-/-]) were fed for 14 days a HS (4% NaCl) diet with and without the MR antagonist eplerenone (Epl; 100 mg x [kg.sup.-1] x [day.sup.-1]). After systolic blood pressure (BP) was measured, the thoracic aorta was isolated for measurement of vascular reactivity, and the aorta and heart were used for measurement of eNOS and MR expression. BP was not different between WT + Epl and WT, but was higher in [cav-1.sup.-/-] + Epl than in [cav-1.sup.-/-] mice. Phenylephrine (Pbe)-induced vascular contraction was less in [cav-1.sup.-/-] than WT, and significantly enhanced in [cav-1.sup.-/-] + Epl than in [cav-1.sup.-/-] , but not in WT + Epl compared with WT. Endothelium removal and NOS blockade by [N.sup.[omega]]-nitro-L-arginine methyl ester (L-NAME) enhanced Phe contraction in [car-1.sup.-/-], but not [cav-1.sup.-/-] + Epl. ACh-induced aortic relaxation was reduced in [cav-1.sup.-/-] + Ep1 versus [cav-1.sup.-/-], but not in WT + Epl compared with WT. Endothelium removal, L-NAME, and the guanylate cyclase inhibitor ODQ abolished the large ACh-induced relaxation in [cav-1.sup.-/-] and the remaining relaxation in the [cav-1.sup.-/-] + Epl but had similar inhibitory effect in WT and WT + Epl. Real-time RT-PCR indicated decreased eNOS mRNA expression in the aorta and heart, and Western blots revealed decreased total eNOS in the heart of [cav-1.sup.-/-] + Epl compared with [cav-1.sup.-/-]. Vascular and cardiac MR expression was less in [cav-1.sup.-/-] than WT, but not in [cav-1.sup.-/-] + Epl compared with [cav-1.sup.-/-]. Plasma aldosterone (Aldo) was not different between WT and [cav-1.sup.-/-] mice nontreated or treated with Epl. Thus in cav-1 deficiency states and HS diet MR blockade is associated with increased BP, enhanced vasoconstriction, and decreased NOS-mediated vascular relaxation and eNOS expression. The data suggest that, in the absence of cav-1, MR activation plays a beneficial role in regulating eNOS expression/activity and, consequently, the vascular function during HS diet. caveolae; aldosterone; eplerenone; endothelium; endothelial nitric oxide synthase doi: 10.1152/ajpheart.00661.2009.

The present study was designed to determine the role of central vasopressin 1 receptors ([V.sub.1]R) in the regulation of cardiovascular parameters in chronically stressed infarcted rats and sham-operated rats under resting conditions and during exposure to acute alarming stress. The experiments were performed on four groups of conscious sham-operated and four groups of infarcted rats subjected to intraventricular infusion of either vehicle or a [V.sub.1]R antagonist ([V.sub.1]RANT). Two groups of infarcted and two groups of sham-operated rats were subjected to mild chronic stressing. Mean arterial blood pressure (MABP) and heart rate (HR) were determined under resting conditions and alter exposure to acute stress (air jet). During vehicle infusion, MABP and HR increases in response to acute stress in the infarcted rats not subjected to chronic stress, and in the infarcted and sham-operated chronically stressed rats, were significantly greater than in the sham-operated rats not exposed to chronic stress. However, MABP and HR responses to acute stress in the chronically stressed infarcted rats and chronically stressed sham-operated rats did not differ. [V.sub.1]RANT abolished differences in cardiovascular responses to acute stress between the experimental groups. Resting cardiovascular parameters were not affected by any of the experimental treatments. It is concluded that chronic stressing enhances the pressor and tachycardic responses to acute stress in the sham-operated rats but does not further intensify these responses in infarcted rats. The results provide evidence that central [V.sub.1]Rs are involved in potentiation of cardiovascular responses to acute stress in chronically stressed rats, infarcted rats, and chronically stressed infarcted rats. vasopressin 1 receptor antagonist; heart failure; tachycardia; depression doi:10.1152/ajpregu.00543.2009

Early life stress is a predisposing factor for the development of chronic intestinal disorders in adult life. Here, we show that stress associated with early weaning in pigs leads to impaired mucosal barrier function. Early weaning (15- to 21-day weaning age) resulted in sustained impairment in intestinal barrier function, as indicated by reductions in jejunal transepithelial electrical resistance and elevations in mucosal-to-serosal flux of paracellular probes [sup.3]H]mannitol and [[sup.14]C]inulin measured at 5 and 9 wk of age, compared with that shown in late-weaned pigs (23- to 28-day weaning age). Elevated baseline short-circuit current was observed in jejunum from early-weaned pigs and was shown to be mediated via enhanced [Cl.sup.-] secretion. Jejunal barrier dysfunction in early-weaned pigs coincided with increased lamina propria immune cell density particularly mucosal mast cells. The mast cell stabilizer drug sodium cromoglycolate ameliorated barrier dysfunction and hypersecretion in early-weaned pigs, demonstrating an important role of mast cells. Furthermore, activation of mast cells ex vivo with c48/80 and corticotrophin-releasing factor (CRF) in pig jejunum mounted in Ussing chambers induced barrier dysfunction and elevations in short-circuit current that were inhibited with mast cell protease inhibitors. Experiments in which selective CRF receptor antagonists were administered to early-weaned pigs revealed that CRF receptor 1 (CRFr1) activation mediates barrier dysfunction and hypersecretion, whereas CRFr2 activation may be responsible for novel protective properties in the porcine intestine in response to early life stress. weaning age; mast cells; corticotropin releasing factor doi:10.1152/ajpgi.00081.2009.

Parathyroid hormone (PTH) restores bone mass to the osteopenic skeleton, but significant questions remain as to the underlying mechanisms. The receptor for advanced glycation end products (RAGE) is a multiligand receptor of the immunoglobulin superfamily; however, recent studies indicate a role in bone physiology. We investigated the significance of RAGE to hormone-induced increases in bone by treating 10-wk-old female Rage-knockout (KO) and wild-type (WT) mice with human PTH-(1-34) at 30 [micro]g x [kg.sup.-1] x [day.sup.-1] or vehicle control, 7 days/wk, for 7 wk. PTH produced equivalent relative gains in bone mineral density (BMD) and bone mineral content (BMC) throughout the skeleton in both genotypes. PTH-mediated relative increases in cortical area of the midshaft femur were not compromised in the null mice. However, the hormone-induced gain in femoral cancellous bone was significantly attenuated in Rage-KO mice. The loss of RAGE impaired PTH-mediated increases in femoral cancellous bone volume, connectivity density, and trabecular number but did not impact increases in trabecular thickness or decreases in trabecular spacing. Disabling RAGE reduced femoral expression of bone formation genes, but their relative PTH-responsiveness was not impaired. Neutralizing RAGE did not attenuate vertebral cancellous bone response to hormone. Rage-null mice exhibited an attenuated accrual rate of bone mass, with the exception of the spine, and an enhanced accrual rate of fat mass. We conclude that RAGE is necessary for key aspects of the skeleton's response to anabolic PTH. Specifically, RAGE is required for hormone-mediated improvement of femoral trabecular architecture but not intrinsically necessary for increasing cortical thickness. receptor for advanced glycation end products; anabolic; high-mobility group box 1 protein; osteoblast; osteoporosis; osteoimmunology doi:10.1152/ajpendo.00564.2009.

Postnatal development and puberty are times of strong physical maturation and require large quantities of energy. The hypothalamic neuropeptide melanin-concentrating hormone (MCH) regulates nutrient intake and energy homeostasis, but the underlying mechanisms are not completely understood. Here we use a novel rat knockout model in which the MCH precursor Pmch has been inactivated to study the effects of loss of MCH on energy regulation in more detail. [Pmch.sup.-/-] rats are lean, hypophagic, osteoporotic, and although endocrine parameters were changed in [pmch.sup.-/-] rats, endocrine dynamics were normal, indicating an adaptation to new homeostatic levels rather than disturbed metabolic mechanisms. Detailed body weight growth and feeding behavior analysis revealed that Pmch expression is particularly important during early rat development and puberty, i.e., the first 8 postnatal weeks. Loss of Pinch resulted in a 20% lower set point for body weight that was determined solely during this period and remained unchanged during adulthood. Although the final body weight is diet dependent, the Pmch-deficiency effect was similar for all diets tested in this study. Loss of Pinch affected energy expenditure in both young and adult rats, although these effects seem secondary to the observed hypophagia. Our findings show an important role for Pmch in energy homeostasis determination during early development and indicate that the MCH receptor 1 system is a plausible target for childhood obesity treatment, currently a major health issue in first world countries. melanin-concentrating hormone; hypothalamus: childhood obesity; rat knockout model doi:10.1152/ajpendo.00154.2009.

Hypocretins/orexins act through two receptor subtypes: [OX.sub.1] and [OX.sub.2]. Outside the brain, orexin receptors are expressed in adrenal glands, where orexins stimulate the release of glucocorticoids. To further address the regulation of steroidogenesis, we analyzed the effect of orexins on the expression of steroidogenic enzymes in human adrenocortical National Cancer Institute (NCI) H295R cells by qPCR. In NCI H295R cells, [OX.sub.2] receptors were highly expressed, as they were in human adrenal glands. After treatment of NCI H295R cells with orexin A for 12-24 h, the cortisol synthesis rate was significantly increased, whereas 30 min of treatment showed no effect. While CYP11B1 and CYP11B2 mRNA levels were increased already at earlier time points, the expression of HSD3B2 and CYP21 mRNA was significantly up-regulated after treatment with orexin A for 12 h. Likewise, orexin B increased CYP21 and HSD3B2 mRNA levels showing, however, a lower potency compared with orexin A. The mRNA levels of CYP11A and CYP17 were unaffected by orexin A. [OX.sub.2] receptor mRNA levels were down-regulated after 12 and 24 h of orexin A treatment. Orexin A increased intracellular [Ca.sup.2+] but not cAMP concentrations in NCI H295R cells. Furthermore, inhibition of PKC and MAPK kinase/ ERK kinase (MEK1/2) prevented the increase of HSD3B2 expression by orexin A. Accordingly, orexin A treatment of NCI H295R cells markedly enhanced ERK1/2 phosphorylation that was prevented by PKC and, in part, PKA inhibition. In conclusion, orexins may influence adrenal steroidogenesis by differential regulation of the expression of steroidogenic enzymes involving [Ca.sup.2+], as well as PKC-ERK1/2 signaling. hypocretin-1 and -2; [OX.sub.1] and [OX.sub.2] receptor; real-time reverse transcriptase-polymerase chain reaction; cortisol doi: 10.1152/ajpregu.91034.2008.

Gonadotropin-releasing hormone (GnRH) acts via seven transmembrane receptors to stimulate gonadotropin secretion. Sustained stimulation desensitizes GnRH receptor (GnRHR)-mediated gonadotropin secretion, and this underlies agonist use in hormone-dependent cancers. Since type I mammalian GnRHR do not desensitize, agonist-induced internalization and downregulation may underlie desensitization of GnRH-stimulated gonadotropin secretion; however, research focus has recently shifted to anterograde trafficking, with the finding that human (h)GnRHR are mostly intracellular. Moreover, there is little direct evidence for agonist-induced trafficking of hGnRHR, and whether or not type 1 mammalian GnRHR show agonist-induced internalization is controversial. Here we use automated imaging to monitor expression and internalization of hemagglutinin (HA)-tagged hGnRHRs, mouse (m) GnRHR, Xenopus (X) GnRHRs, and chimeric receptors (hGnRHR with added XGnRHR COOH tails, h.XGnRHR) expressed by adenoviral transduction in HeLa cells. We find that agonists stimulate downregulation and/or internalization of mGnRHR and XGnRHR, that GnRH stimulates trafficking of hGnRHR and can stimulate internalization or downregulation of hGnRHR when steps are taken to increase cell surface expression (addition of the XGnRHR COOH tail or pretreatment with pharmacological chaperone). Agonist effects on internalization (of h.XGnRHR) and downregulation (of hGnRHR and h.XGnRHR) were not mimicked by a peptide antagonist and were prevented by a mutation that prevents GnRHR signaling, demonstrating dependence on receptor signaling as well as agonist occupancy. Thus agonist-induced internalization and downregulation of type I mammalian GnRHR occurs in HeLa cells, and we suggest that the high throughput imaging systems described here will facilitate study of the molecular mechanisms involved. seven transmembrane receptor; trafficking; G protein-coupled receptor

During hormonally induced ovarian follicle growth, granulosa cell proliferation increases and returns to baseline prior to the administration of an ovulatory stimulus. Several key genes appear to follow a similar pattern, including the luteinizing hormone receptor (LHCGR), suggesting an association between cell cycle progression and gene expression. The expression of LHCGR mRNA in granulosa cells isolated from immature rats and treated in culture with FSH increased in a time-dependent manner, whereas administration of the cell cycle inhibitor mimosine completely suppressed expression. Although forskolin was able to induce luteinization in cells treated with mimosine, human chorionic gonadotropin had no effect, indicating the functional loss of LHCGR. The effects of mimosine on cell cycle progression and LHCGR mRNA expression were reversible within 24 h of mimosine removal. Cell cycle inhibition did not alter the stability of LHCGR mRNA, indicating that the primary effect was at the transcriptional level. To determine whether the relationship between LHCGR expression and cell cycle were relevant in vivo, immature rats were given a bolus of PMSG, followed by a second injection of either saline or PMSG 24 h later to augment levels of proliferation. The expression of LHCGR mRNA was elevated in the ovaries of animals receiving a supplement of PMSG. Mimosine also blocked cell cycle progression and LHCGR mRNA expression in macaque granulosa cells isolated following controlled ovarian stimulation cycles and in two different mouse Leydig tumor lines. These data collectively indicate that LHCGR mRNA is expressed as a function of the passage of cells across the G1-S phase boundary. granulosa cell; gene expression; ovary; proliferation

Recent studies have documented various roles of adrenal corticosteroid signaling in cardiac physiology and pathophysiology. It is known that glucocorticoids and aldosterone are able to bind glucocorticoid receptor (GR) and mineralocorticoid receptor, and these ligand-receptor interactions are redundant. It, therefore, has been impossible to delineate how these nuclear receptors couple with corticosteroid ligands and differentially regulate gene expression for operation of their distinct functions in the heart. Here, to particularly define the role of GR in cardiac muscle cells, we applied a ligand-based approach involving the GR-specific agonist cortivazol (CVZ) and the GR antagonist RU-486 and performed microarray analysis using rat neonatal cardiomyocytes. We indicated that glucocorticoids appear to be a major determinant of GR-mediated gene expression when compared with aldosterone. Moreover, expression profiles of these genes highlighted numerous roles of glucocorticoids in various aspects of cardiac physiology. At first, we identified that glucocorticoids, via GR, induce mRNA and protein expression of a transcription factor Kruppel-like factor 15 and its downstream target genes, including branched-chain aminotransferase 2, a key enzyme for amino acid catabolism in the muscle. CVZ treatment or overexpression of KLF15 decreased cellular branched-chain amino acid concentrations and introduction of small-interfering RNA against KLF15 cancelled these CVZ actions in cardiomyocytes. Second, glucocorticoid-GR signaling promoted gene expression of the enzymes involved in the prostaglandin biosynthesis, including cyclooxygenase-2 and phospholipase A2 in cardiomyocytes. Together, we may conclude that GR signaling should have distinct roles for maintenance of cardiac function, for example, in amino acid catabolism and prostaglandin biosynthesis in the heart. endocrinology; cardiovascular system; KLF15; cylooxygenase-2; phospholipase A2

Postnatal inactivation of epithelial androgen receptor (AR) in prostate epithelial AR knockout (PEARKO) mice results in hindered differentiation but enhanced proliferation of epithelial cells. As this resembles the precancerous proliferative atrophy of human prostates with undifferentiated but intensively replicating epithelial cells, we utilized the PEARKO mice to characterize the epithelial response to castration-induced involution with a focus on identifying the potential role of stromal AR and responsiveness of the androgen-deprived epithelia to the aromatizable androgen testosterone (T) or its nonaromatizable metabolite dihydrotestosterone (DHT). PEARKO and littermate control mice were orchidectomized at 8 wk of age and treated 2 wk later with subdermal implantation of 1-cm Silastic tubing filled with T or DHT for a week. Following castration, the prostatic involution and epithelial apoptosis did not significantly differ between control (intact AR) and PEARKO (only stromal AR) males, demonstrating that prostate epithelial involution following castration is mediated primarily via stromal AR-dependent apoptotic signals. Androgen replacement (T/DHT) for 7 days induced significant growth and epithelial proliferation in all prostate lobes in both control and PEARKO, but full regrowth was observed only in controls treated with T. In PEARKO, prostate androgen (T and DHT) treatment induced significant epithelial cell 'shedding' into the lumen, with T treatment resulting in acinar disorganization, cyst formation, and aberrant epithelial structures, described as a 'gland within a gland.' These data suggest that epithelial AR inactivation during postnatal prostate development sensitizes prostate epithelial cells to paracrine signaling mediated by stromal AR activity leading to indirectly androgen-induced epithelial hyperproliferation and formation of epithelial hyperplastic cysts by aromatizable androgens. gene targeting; Cre-loxP technology; genetic mouse model

The molecular mechanisms by which bone cells transduce mechanical stimuli into intracellular biochemical responses have yet to be established. There is evidence that mechanical stimulation acts synergistically with parathyroid hormone PTH(1-34) in mediating bone growth. Using picosecond time-resolved fluorescence microscopy and G protein-coupled receptor conformation-sensitive fluorescence resonance energy transfer (FRET), we investigated conformational transitions in parathyroid hormone type 1 receptor (PTH1R). 1) A genetically engineered PTH1R sensor containing an intramolecular FRET pair was constructed that enabled detection of conformational activity of PTH1R in single cells. 2) The nature of ligand-dependent conformational change of PTH1R depends on the type of ligand: stimulation with the PTH(1-34) leads to conformational transitions characterized by decrease in FRET efficiency while [NH.sub.2]-terminal truncated ligand PTH(3-34) stimulates conformational transitions characterized by higher FRET efficiencies. 3) Stimulation of murine preosteoblastic cells (MC3T3-E1) with fluid shear stress (FSS) leads to significant changes in conformational equilibrium of the PTH1R in MC3T3-E1 cells, suggesting that mechanical perturbation of the plasma membrane leads to ligand-independent response of the PTH1R. Conformational transitions induced by mechanical stress were characterized by an increase in FRET efficiency, similar to those induced by the [NH.sub.2]-terminal truncated ligand PTH(3-34). The response to the FSS stimulation was inhibited in the presence of PTH(1-34) in the flow medium. These results indicate that the FSS can modulate the action of the PTH(1-34) ligand. 4) Plasma membrane fluidization using benzyl alcohol or cholesterol extraction also leads to conformational transitions characterized by increased FRET levels. We therefore suggest that PTH1R is involved in mediating primary mechanochemical signal transduction in MC3T3-E1 cells. mechanosensor; fluorescence resonance energy transfer; fluid shear stress; G protein-coupled receptor; mechanotransduction

Recent evidence suggests that mineralocorticoid receptor (MR) antagonism has beneficial effects on tissue oxidative stress and insulin metabolic signaling as well as reducing proteinuria. However, the mechanisms by which MR antagonism corrects both renin-angiotensin-aldosterone system (RAAS) impairments in renal insulin metabolic signaling and filtration barrier/podocyte injury remain unknown. To explore this potential beneficial interactive effect of MR antagonism we used young transgenic (mRen2)27 (Ren2) rats with increased tissue RAAS activity and elevated serum aldosterone levels. Ren2 and age-matched Sprague-Dawley (SD) control rats (age 6-7 wk) were implanted with a low dose of the MR antagonist spironolactone (0.24 mg/day) or vehicle, both delivered over 21 days. Albuminuria, podocyte-specific proteins (synaptopodin, nephrin, and podocin), and ultrastructural analysis of the glomerular filtration barrier were measured in relation to RAAS activation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, reactive oxygen species (ROS), and the redox-sensitive Rho kinase (ROK). Insulin metabolic signaling was determined via measurement of insulin receptor substrate-1 (IRS-1) phosphorylation, IRS-1 ubiquitin/proteasomal degradation, and phosphorylation of Akt. Ren2 rats exhibited albuminuria, loss of podocytespecific proteins, and podocyte foot process effacement contemporaneous with reduced renal IRS-1 and protein kinase B/Akt phosphorylation compared with SD control rats (each P < 0.05). Ren2 kidneys also manifested increased NADPH oxidase/ROS/ROK in conjunction with enhanced renal tissue levels of angiotensin II (ANG II), ANG(1-12), and angiotensin type 1 receptor. Low-dose spironolactone treatment reduced albuminuria and tissue RAAS activity and improved podocyte structural and protein integrity with improvements in IRS-1/Akt phosphorylation. Thus, in this model of RAAS activation, MR antagonism attenuates glomerular/podocyte remodeling and albuminuria, in part through reductions in redox-mediated impairment of insulin metabolic signaling. renal mineralocorticoid receptor; reduced nicotinamide adenine dinucleotide phosphate oxidase; oxidative stress; podocyte

Exogenous glucocorticoids act within the hindbrain to enhance the arterial pressure response to acute novel stress. Here we tested the hypothesis that endogenous glucocorticoids act at hindbrain glucocorticoid receptors (GR) to augment cardiovascular responses to restraint stress in a model of stress hyperreactivity, the borderline hypertensive rat (BHR). A 3- to 4-mg pellet of the GR antagonist mifepristone (Mif) was implanted over the dorsal hindbrain (DHB) in Wistar-Kyoto (WKY) and BHRs. Control pellets consisted of either sham DHB or subcutaneous Mif pellets. Rats were either subjected to repeated restraint stress (chronic stress) or only handled (acute stress) for 3-4 wk, then all rats were stressed on the final day of the experiment. BHR showed limited adaptation of the arterial pressure response to restraint, and DHB Mif significantly (P [less than or equal to] 0.05) attenuated the arterial pressure response to restraint in both acutely and chronically stressed BHR. In contrast, WKY exhibited a substantial adaptation of the pressure response to repeated restraint that was significantly reversed by DHB Mif. DHB Mif and chronic stress each significantly increased baseline plasma corticosterone concentration and adrenal weight and reduced the corticosterone response to stress in all rats. We conclude that endogenous corticosterone acts via hindbrain GR to enhance the arterial pressure response to stress in BHR, but to promote the adaptation of the arterial pressure response to stress in normotensive rats. Endogenous corticosterone also acts in the hindbrain to restrain corticosterone at rest but to maintain the corticosterone response to stress in both BHR and WKY rats. corticosterone; brain; nucleus of the solitary tract; hypothalamic-pituitary-adrenal axis; chronic stress

The study was designed to investigate the effect of estradiol on the excitatory effect of oxytocin (OT) on colon motility. Female Wistar rats were used, and some of them were ovariectomized (OVX) and treated with vehicle or estradiol (Ez). A plastic balloon made of condom was inserted into colon to monitor the change of colonic pressure in vivo. Longitudinal muscle strips of distal colon were prepared to monitor the spontaneous contraction of colon in vitro. Expression of OT receptor (OTR) was investigated by Western blot analysis. Expression of OTR mRNA was detected by RT-PCR. Immunohistochemistry was used to locate OTR. In OVX rats, pretreatment of [E.sub.2] (4-100 [micro]g/kg sc) dose-dependently increased the excitatory effect of OT on colon motility both in vivo and in vitro and increased the expression of OTR and OTR mRNA in colon. Systemic administration of OT excited the colon motility in vivo in rats at perioda of proestrus and estrus but did not influence it at diestrus period, when the concentration of plasma [E.sub.2] was lowest in the estrous cycle. Pretreatment of atosiban, the specific OTR antagonist, and TTX, the blocker of voltage-dependent sodium channel on nerve fiber, attenuated the excitatory effect of OT on colon motility. OTR was located in myenteric plexus of colon. These results suggested that [E.sub.2] increased the excitatory effect of OT on colon motility by upregulating the expression of OTR in myenteric plexus. colon; motility

We have characterized the in vivo actions of human wild-type FSH receptor (FSHR) overexpressed in Sertoli cells of transgenic (Tg) mice (TgFSHRwt) compared with transgenic overexpression of the human activated mutant FSHR*D567G (TgFSHR*D567G). Testicular TgFSHRwt expression significantly elevated specific FSH binding (>2-fold, P < 0.0 i) relative to nontransgenic testes, similar to increased FSH binding in TgFSHR*D567G testes. Isolated TgFSHRwt Sertoli cells exhibited higher FSH-stimulated cAMP levels compared with non-Tg or TgFSHR*D567G cells but did not display the elevated FSH-independent basal cAMP levels found in TgFSHR*D567G Sertoli cells. Furthermore, Sertoli cell overexpression of TgFSHR*D567G but not TgFSHRwt allowed promiscuous cAMP responses to human chorionic gonadotropin (300 IU/ml) and TSH (30 mIU/ml), demonstrating increased constitutive signaling and altered glycoprotein hormone specificity via the intracellular D567G substitution rather than FSHR overexpression. Despite elevating Sertoli cell FSH sensitivity, overexpression of TgFSHRwt had no detectable effect upon normal testis function and did not stimulate Sertoli and germ cell development in testes of gonadotropin-deficient hypogonadal (hpg) mice, in contrast to the increased meiotic and postmeiotic germ cell development in TgFSHR*D567G hpg testes. Increased steroidogenic potential of TgFSHR*D567G hpg testes was demonstrated by elevated Cypllal and Star expression, which was not detected in TgFSHRwt hpg testes. Androgen-regulated and Sertoli cell-specific Rhox5 gene expression was increased in TgFSHR*D567G but not TgFSHRwt hpg testes, providing evidence of elevated LH-independent androgen activity due to mutant FSHR*D567G. Hence, transgenic FSHR overexpression in Sertoli cells revealed that the D567G mutation confers autonomous signaling and steroidogenic activity in vivo as well as promiscuous glycoprotein hormone receptor activation, independently of FSHR overexpression alone. follicle-stimulating hormone receptor; mutation; transgenic; Sertoli cell; mouse

Relaxin induces sustained physiological responses, which brings into question the deactivation processes typical of most G protein-coupled receptors (GPCR) for its receptor, relaxin family peptide receptor 1 (RXFPI). Here, we examined relaxin-dependent phosphorylation of RXFP1 and the related insulin-like peptide 3 (INSL3) receptor, RXFP2, as well as the capacity of these receptors to recruit [beta]-arrestins and internalize in response to ligand stimulation. We confirmed in human embryonic kidney (HEK)-293T cells, expressing RXFP1 or RXFP2, that both receptors elicit prolonged cAMP responses up to 6 h after stimulation. Receptors immunoprecipitated from [sup.32]P metabolically labeled cells were used to investigate the agonist-specific phosphorylation. Rapid and robust receptor phosphorylation was not observed for either RXFP1 or RXFP2, although some [sup.32]P-incorporation was observed at 30 min; however, this was not statistically significant. In accord with this result, RXFP1 and RXFP2 demonstrated poor internalization in response to relaxin or INSL3, as compared with the angiotensin II type 1 receptor ([AT.sub.1]R), which undergoes rapid and robust phosphorylation and internalization in response to angiotensin II. Additionally, coexpression of GPCR kinases has no effect on the rate of internalization for either RXFP1 or RXFP2. Confocal microscopy was used to follow the trafficking of green fluorescent proteinlabeled [beta]-arrestins after receptor activation. Neither RXFP1 nor RXFP2 activation results in recruitment of [beta]-arrestins to the cell surface, whereas [AT.sub.1]R rapidly recruits both [beta]-arrestins-1 and -2. The apparent lack of classical regulation for RXFPI and RXFP2 provides the molecular basis for the prolonged signaling and physiological actions of relaxin and related peptides. G protein-coupled receptor; relaxin; insulin-like peptide 3

The present study investigated the effects of diabetes and high glucose on GHRH receptor (GHRH-R) mRNA and protein levels in the pituitary of diabetic rats 2, 21, and 60 days post-streptozotocin (post-STZ) administration. Two days post-STZ, the 2.5-kb GHRH-R mRNA transcript was increased. Twenty-one days post-STZ, both the 2.5- and 4-kb transcripts and a 72-kDa [sup.125]I-GHRH-GHRH-R complex were elevated. Sixty days post-STZ, the 4-kb transcript remained increased and the 45-kDa [sup.125]I-GHRH-GHRH-R complex (functional receptor) was decreased. Hypothalamic GHRH mRNA and serum total IGF-I levels were reduced at all three time points. To better understand the role of high glucose on GHRH-R regulation, time-course effects of 33 compared with 6 mM D-glucose (DG) were examined in cultured anterior pituitary cells from 2-mo-old healthy rats. Membrane lipoperoxidation was present in 33 mM DG, and GHRH-R mRNA levels were diminished after 24 h, Fluo-GHRH internalization was marginal after 16-24 h, and GHRH-induced cAMP levels were decreased after 24 and 48 h. Altogether, these results indicate that the increase of the 2.5-kb GHRH-R mRNA transcript in vivo could be a consequence of a decrease of hypothalamic GHRH mRNA levels in STZ rats. Since it does not affect primarily functional GHRH-R levels, the initial diminution of circulating IGF-I levels could result from a decreased GHRH-R stimulation by GHRH. Thus, the effect of glucotoxicity would be related to a decrease of functional GHRH-R protein, as observed in rats 60 days post-STZ and in cultured pituitary cells from healthy rats exposed to a high-glucose environment. insulin-like growth factor I; oxidative stress; hypothalamus; cyclic adenosine monophosphate

Administration of chemically synthesized ghrelin (Ghr) peptide has been shown to increase food intake and body adiposity in most species. However, the biological role of endogenous Ghr in the molecular control of energy metabolism is far less understood. Mice deficient for either Ghr or its receptor (the growth hormone secretagogue receptor, GHS-R1a) seem to exhibit enhanced protection against high-fat diet-induced obesity but do not show a substantial metabolic phenotype on a standard diet. Here we present the first mouse mutant lacking both Ghr and the Ghr receptor. We demonstrate that simultaneous genetic disruption of both genes of the Ghr system leads to an enhanced energy metabolism phenotype. Ghr/Ghr receptor double knockout (dKO) mice exhibit decreased body weight, increased energy expenditure, and increased motor activity on a standard diet without exposure to a high caloric environment. Mice on the same genetic background lacking either the Ghr or the Ghr receptor gene did not exhibit such a phenotype on standard chow, thereby confirming earlier reports. No differences in food intake, meal pattern, or lean mass were observed between dKO, Ghr-deficient, Ghr receptor-deficient, and wild-type (WT) control mice. Only dKO showed a slight decrease in body length. In summary, simultaneous deletion of Ghr and its receptor enhances the metabolic phenotype of single gene-deficient mice compared with WT mice, possibly suggesting the existence of additional, as of yet unknown, molecular components of the endogenous Ghr system. growth hormone secretagogue receptor, constitutive receptor activity; locomotion

Experiments were conducted on rainbow trout to determine the impact of dietary salt on arterial blood pressure. After 4-6 wk, fish fed a salt-enriched diet exhibited a 37% elevation of dorsal aortic pressure (from 23.8 [+ or -]1.2 to 32.6 [+ or -] 1.4 mmHg) and an 18% increase in ventral aortic pressure (from 33.0 [+ or -] 1.5 to 38.9 [+ or -] 1.3 mmHg). The hypertension presumably reflected the increase in cardiac output (from 31.0 [+ or -] 0.8 to 36.4 [+ or -] 2.2 ml.[min.sup.-1]. [kg.sup.-1]) because systemic and branchial resistances were statistically unaltered by salt feeding. The chronic hypertension was associated with a decrease in the pressor responses of the systemic vasculature to catecholamines and hypercapnia in the salt-fed fish. The reduction in [alpha]-adrenergic responsiveness of the systemic vasculature is consistent with desensitization or loss of functional [alpha]-adrenoceptors ([alpha]-ARs). In support of this idea, the salt-fed fish exhibited significantly decreased levels of [[alpha].sub.1D]-AR mRNA in the dorsal aorta and the afferent (ABA) and efferent branchial arteries (EBA). In contrast, however, the results obtained from norepinephrine doseresponse curves for EBA and ABA vascular rings in vitro did not provide evidence for loss of function of branchial artery [[alpha].sub.1]-ARs in the salt-fed fish. Indeed, the [EC.sub.50] for the EBA norepinephrine doseresponse curve was significantly reduced (from 3.75 x [10.sup.-7] to 2.12 x [10.sup.-7] M) in the salt-fed fish, indicating an increase in the binding affinity of the [[alpha].sub.1]-ARs. norepinephrine; epinephrine; blood pressure; systemic resistance; salt feeding; hypercapnia; efferent branchial artery; afferent branchial artery

Bovine adrenal zona fasciculata cells express background [K.sup.+] channels ([I.sub.AC] channels) whose activity is potently inhibited by ACTH. In whole cell patch clamp recordings, it was discovered that the trivalent lanthanides ([Ln.sup.3+]s) lanthanum and ytterbium interact with two binding sites to modulate [K.sup.+] flow through these channels. Despite large differences in ionic radii, these [Ln.sup.3+]s inhibited [I.sub.AC] channels half-maximally with [IC.sub.50] values near 50 [micro]M. In addition, these [Ln.sup.3+]s blocked and reversed ACTH-mediated inhibition Of [I.sub.AC] [K.sup.+] channels at similar concentrations. The [Ln.sup.3+]s did not alter inhibition of [I.sub.AC] by angiotensin II or cAMP. [Ln.sup.3+]-induced uncoupling of ACTH receptor activation from [I.sub.AC] inhibition was prevented by raising the external [Ca.sup.2+] concentration from 2 to 10 mM. The divalent cation [Ni.sup.2+] (500 [micro]M) also blocked ACTH-dependent inhibition of [I.sub.AC] through a [Ca.sup.2+]-sensitive mechanism. The results are consistent with a model in which [Ln.sup.3+]s produce opposing actions on [I.sub.AC] [K.sup.+] currents through two separate binding sites. In addition to directly inhibiting [I.sub.AC], [Ln.sup.3+]s (and [Ni.sup.2+]) bind with high affinity to a [Ca.sup.2+]-selective site associated with the ACTH receptor. By displacing [Ca.sup.2+] from this site, [Ln.sup.3+]s prevent ACTH from binding and accelerate its dissociation. These results identify [Ln.sup.3+]s as a relatively potent group of noncompetitive ACTH receptor antagonists. Allosteric actions of trivalent and divalent metal cations on hormone binding, mediated through [Ca.sup.2+]-specific sites, may be common to a variety of peptide hormone receptors. [K.sup.+] channels; nickel; calcium; adrenocorticotropic hormone receptor

Human gallbladders with cholesterol stones show impaired muscle contraction caused by agonists that act on transmembrane receptors, increased membrane cholesterol content and abnormal cholesterol-to-phospholipid ratio compared to those with pigment stones. The functions of CCK receptor of gallbladder membranes were studied by radioreceptor assay and cross-linking. Results showed that human gallbladders with cholesterol stones have impaired CCK receptor binding compared to those with pigment stones.

Estradiol apparently enhances MT(sub 2) melatonin-receptor function in the thermoregulatory caudal artery of the female rat, leading to increased vasodilatation in response to melatonin. This was the conclusion in a study of whether estrogen modulates the function of vascular melatonin receptors, which investigated the influence of female gonadal hormones on functional responses to melatonin in the rat caudal artery. Results also offered the possibility that estradiol may also modulate the function of MT(sub 2) melatonin receptors in other tissues.

Strain-dependent differences in the melatonin profiles of rats were investigated. The inbred rat strains BH/Ztm, ACI/Ztm, LEW/Ztm were analyzed for diurnal (12:12-h light-dark) wheel running activity, melatonin levels of pineal and plasma glands, urinary 6-sulphatoxymelatonin excretion and melatonin receptor density in the suprachiasmatic nuclei. Results show that melatonin has a long-term stimulating effect on its own receptor density in the suprachiasmatic nuclei.

The regulation of the natriuretic peptide receptor A (NPR-A) in inner medullary collecting duct cells by exposure to a variety of natriuretic peptide ligands was examined in cell cultures. Results reveal that prolonged exposure to these ligands depresses the translation of the NPR-A gene. This reduction is directly related to the capacity of these ligands to depress the intracellular cGMP levels.

The elaboration of adrenomedullin (ADM) mRNA and that of its receptor (ADM-R) in the rat kidney is investigated by identifying the sites where they are expressed and the regulation of their synthesis. Results reveal that kidney glomeruli express the highest levels of ADM mRNA followed by the cortex and the medulla. On the other hand, the highest level of expression of ADM-R mRNA was found in the medulla. Increasing the dietary salt intake of the subjects did not change the rate of expression of these mRNAs.

The water retention and vasoconstriction observed in congestive heart failure (CHF) is attributed in part to arginine vasopressin (AVP). Water retention is modulated via the V2 receptor of AVP. The effects of chronic blocking of this receptor is investigated using the infarction-induced CHF model in rats. Results reveal that selective blocking of V2 leads to an increase in the excretion of water. The implications of these findings on diuretic therapy are discussed.

The cellular handling of cholecystokinin (CCK) receptor molecules during CCK-stimulated signaling was analyzed with polyclonal rabbit antiserum directed toward the peptide domain within the COOH terminus of CCK receptor. Immunolocalization of CCK receptor molecules with the CCK antiserum exhibited the recycling of large proportion of intenalized CCK receptors involving induced conformational changes. Furthermore, the internalization of CCK receptors was not affected by receptor phosphorylation.

The RaKCaR Ca2+/polyvalent cation-sensing receptor was cloned to determine changes in the concentrations of extracellular Ca2+/polyvalent cations in kidney cells. Glomeruli cells exhibited RakCaR messenger ribonucleic acid and parathyroid hormone/parathyroid hormone-related protein coexpression. The coexpression of the two receptors in the glumeruli regulate the reabsorption of divalent cations and inhibited the stimulatory effects of parathyroid hormone.

Studies have suggested that the presence of corticosteroids may induce a downregulation of their respective receptors in various organs. Herein, animals that were depleted of cortiosteroid hormones by adrenalectomy, and received aldosterone or dexamethasone at physiological doses, showed no significant changes in the levels of both mineralocorticoid and glucocorticoid receptor mRNA expression in the kidney, heart and colon. It was concluded that the regulation of hormone receptor expression does not depend on hormonal status.

The mineralocorticoid receptor (MR) antagonist, RU-28318, suppresses the enhanced rate of energy gain produced by ovariectomy (OVX) in rats. The suppression of OVX-related changes is due to an increase in the energetic efficiency, rather than a decrease in food intake or increase in energy consumption. A similar effect for surgery and RU-28318 is evident on body energy gain, energetic efficiency, and fat gain. The glucocorticoid receptor antagonist, RU-486, has no effect on the energy changes induced by OVX.

The possibility that cholecystokinin (CCK) receptor can be a multiple kinase substrate and a receptor-specific kinase substrate was examined. Permeabilization experiments were conducted on dispersed rat pancreatic acinar cells. The existence of the two kinases that function on the CCK receptor and its analogy to enzymes in the beta-andrenergic receptor family of kinases were established.

Downregulation of aquaporin-2 and -3 in aging kidney is independent of [V.sub.2] vasopressin receptor. Am J Physiol Renal Physiol 279: F144-F152, 2000.--The mechanisms underlying age-related polyuria were investigated in 10- and 30-mo-old female WAG/Rij rats. Urinary volume and osmolality were 3.9 [+ or -] 0.3 ml/24 h and 2,511 [+ or -] 54 mosmol/kg[H.sub.2]0 in adult rats and 12.8 [+ or -] 0.8 ml/24 h and 1,042 [+ or -] 44 mosmol/kg[H.sub.2]0 in senescent animals. Vasopressin [V.sub.2] receptor mRNA did not significantly differ between 10 and 30 mo, and [[sup.3]H] vasopressin binding sites in membrane papilla were reduced by 30%. The cAMP content of the papilla was unchanged with age, whereas papillary osmolality was significantly lowered in senescent animals. The expression of aquaporin-1 (AQP1) and -4 was mostly unaltered from 10 to 30 mo. In contrast, aquaporin-2 (AQP2) and -3 (AQP3) expression was downregulated by 80 and 50%, respectively, and AQP2 was markedly redistributed into the intracellular compartment, in inner medulla of senescent animals, but not in renal cortex. These results indicate that age-related polyuria is associated with a downregulation of AQP2 and AQP3 expression in the medullary collecting duct, which is independent of vasopressin-mediated cAMP accumulation. aquaporins