Assessing the presence of abnormal regulation of cortisol secretion by membrane hormone receptors: in vivo and in vitro studies in patients with functioning and non-functioning adrenal adenoma.

Regulation of cortisol secretion by aberrant hormone receptors may play a role in the pathogenesis of ACTH-independent Cushing's syndrome. In this study, the topic was evaluated by combining in vivo and in vitro approaches. Cortisol responses to various stimuli (standard meal, GnRH + TRH, cisapride, vasopressin, glucagon) were assessed in 6 patients with clinical or subclinical adrenal Cushing's syndrome, and non-functioning adrenal adenoma in two cases. Abnormal responses were observed in three patients with Cushing's syndrome; one patient showed a gastric inhibitory polypeptide (GIP)-dependent cortisol rise after meal, together with responses after GnRH and cisapride; the second patient showed an LH-dependent cortisol response to GnRH, and in the third cortisol rose after cisapride. The pattern of receptor expression performed by RT-PCR showed that while GIP-R was only expressed in tumor from the responsive patient, 5-hydroxytryptamine type 4 receptor and LH-R were also present in normal adrenal tissues and tissues from non-responsive patients. Interestingly, an activating mutation of Gsalpha gene was identified in one of these tumors. Therefore, cortisol responses to agents operating via Gs protein coupled receptors (in one case associated with Gsalpha mutation) were found in Cushing's patients, while these responses were absent in the others. The finding of receptor expression in normal and non-responsive tumors suggests that different mechanisms are probably involved in inducing in vivo cortisol responses.     

A possible association between aldosterone response to vasopressin and circadian change of aldosterone in the patients with aldosterone-producing adenoma

Vasopressin was reported to stimulate secretion of both cortisol and aldosterone through eutopic V1a receptors in adrenal gland. Recently, adrenal hyper-responsiveness of plasma cortisol to vasopressin with eutopic overexpession of V1a receptors has been reported in Cushing's syndrome, such as a majority of cases of ACTH-independent macronodular adrenal hyperplasia and some cases of Cushing's adenomas. There were a few reports regarding the aldosterone response to vasopressin in aldosterone-producing adenoma. The aim of our study was to investigate the aldosterone response to vasopressin and its pathophysiological roles in the patients with aldosterone-producing adenoma. Vasopressin-loading test was performed in 10 patients with aldosterone-producing adenoma, and in 16 patients with non-functioning adrenal tumors. The roles of the aldosterone response to vasopressin were analyzed in terms of hormonal secretion and the expression of V1a receptor mRNA on the operated adrenal gland in aldosterone-producing adenoma. We found that (1) a varying aldosterone response to vasopressin was observed, (2) absolute response of plasma aldosterone in aldosterone-producing adenoma was significantly higher than that in non-functioning tumor, (3) aldosterone response rate to vasopressin was significantly and negatively correlated with the decline rate (%) in plasma aldosterone from morning to evening in aldosterone-producing adenoma, (4) V1a receptor mRNA was expressed at various values in aldosterone-producing adenoma, and (5) surgical removal of aldosterone-producing adenoma eliminated the aldosterone response to vasopressin observed in patients with aldosterone-producing adenoma. These findings indicated that vasopressin might be involved in the coordination of aldosterone secretion through eutopic expression of V1a receptor in aldosterone-producing adenoma.     

Analysis of interactions responsible for vasopressin binding to human neurohypophyseal hormone receptors-molecular dynamics study of the activated receptor-vasopressin-G(alpha) systems.

Vasopressin (CYFQNCPRG-NH(2), AVP) is a semicyclic endogenous peptide, which exerts a variety of biological effects in mammals. The main physiological roles of AVP are the regulation of water balance and the control of blood pressure and adrenocorticotropin hormone (ACTH) secretion, mediated via three different subtypes of vasopressin receptors: V1a, V1b and V2 receptors (V1aR, V1bR and V2R, respectively). They are the members of the class A, G-protein-coupled receptors (GPCRs). AVP also modulates several behavioral and social functions. In this study, the interactions responsible for AVP binding to vasopressin V1a and V2 receptors versus the closely related oxytocin ([I3,L8]AVP, OT) receptor (OTR) have been investigated. Three-dimensional models of the activated receptors were constructed using multiple sequence alignment, followed by homology modeling using the complex of activated rhodopsin with Gt(alpha) C-terminal peptide of transducin MII-Gt(338-350) prototype as a template. AVP was docked into the receptor-G(alpha) systems. The three lowest-energy pairs of receptor-AVP-G(alpha) (two complexes per each receptor) were selected. The 1-ns unconstrained molecular dynamics (MD) of complexes embedded into the fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) lipid bilayer was conducted in the AMBER 7.0 force field. Six relaxed receptor-AVP-G(alpha) models were obtained. The residues responsible for AVP binding to vasopressin receptors have been identified and a different mechanism of AVP binding to V2R than to V1aR has been proposed.     

Clinical and subclinical ACTH-independent macronodular adrenal hyperplasia and aberrant hormone receptors

ACTH-independent macronodular adrenal hyperplasia (AIMAH) is a very rare cause of endogenous Cushing's syndrome (CS). In this review, the clinical characteristics, the pathophysiology, and the management of AIMAH are described. AIMAH typically presents with overt CS, but subclinical oversecretion of cortisol has been increasingly described. The diagnosis is suspected by adrenal nodular enlargement on conventional imaging following the demonstration of ACTH-independent hypercortisolism. Final diagnosis is established by histological examination of the adrenal tissue. Bilateral adrenalectomy is the treatment of choice but unilateral adrenalectomy has been proposed in selected cases. In patients with subclinical CS, the decision to treat should be individualized. The pathophysiology of this condition has begun to be elucidated in recent years. Diverse aberrant membrane-bound receptors expressed in a non-mutated form in the adrenal gland have been found to be implicated in the regulation of steroidogenesis in AIMAH. When systematically screened, most patients with AIMAH and CS or subclinical CS exhibit an in vivo aberrant cortisol response to one or various ligands suggesting the presence of aberrant adrenal receptors. A protocol designed to screen patients for the presence of these aberrant receptors should be undertaken in all patients with AIMAH. The identification of these receptors provides the potential for novel pharmacological therapies by suppressing the endogenous ligands or blocking the receptor with specific antagonists.     

Vasopressin V2 Receptor/Bioligand Interactions

We predict some essential interactions between the V2 vasopressin renal receptor (V2R) and its agonists [Arg⁸]vasopressin (AVP) and [D-Arg⁸]vasopressin (DAVP), and the non-peptide antagonist OPC-31260. V2R controls antidiuresis and belongs to the superfamily of heptahelical transmembrane (7TM) G-protein-coupled receptors (GPCRs). The receptor was built, the ligands were docked and the structures relaxed using advanced molecular modeling techniques. Docked agonists and antagonists appear to prefer similar V2R compartments. A number of receptor amino acid residues are indicated, mainly in the TM3–TM7 helices, as potentially important in ligand binding. Many of these residues are invariant for either the GPCR superfamily or the subfamily of related (vasopressin V2R, V1aR and V1bR and oxytocin OR) receptors. Moreover, some of the equivalent residues in V1aR have already been found critical for ligand affinity [Mouillac et al., J. Biol. Chem., 270 (1995) 25771].     

Vasopressin V2 Receptor/Bioligand Interactions

Summary We predict some essential interactions between the V2 vasopressin renal receptor (V2R) and its agonists [Arg⁸]vasopressin (AVP) and [D-Arg⁸]vasopressin (DAVP), and the non-peptide antagonist OPC-31260. V2R controls antidiuresis and belongs to the superfamily of heptahelical transmembrane (7TM) G-protein-coupled receptors (GPCRs). The receptor was built, the ligands were docked and the structures relaxed using advanced molecular modeling techniques. Docked agonists and antagonists appear to prefer similar V2R compartments. A number of receptor amino acid residues are indicated, mainly in the TM3-TM7 helices, as potentially important in ligand binding. Many of these residues are invariant for either the GPCR superfamily or the subfamily of related (vasopressin V2R, V1aR and V1bR and oxytocin OR) receptors. Moreover, some of the equivalent residues in V1aR have already been found critical for ligand affinity [Mouillac et al., J. Biol. Chem., 270 (1995) 25771].     

Identifying neuropeptide and protein hormone receptors in Drosophila melanogaster by exploiting genomic data.

Most neuropeptide and protein hormone receptors belong to the large superfamily of G-protein-coupled receptors (GPCRs). These cell membrane proteins steer many important processes such as development, reproduction, homeostasis and behaviour when activated by their corresponding ligands. The first insect genome, that of the fruitfly Drosophila melanogaster, was sequenced in 2000, and about 200 GPCRs have been annnotated in this model insect. About 50 of these receptors were predicted to have neuropeptides or protein hormones as their ligands. Since 2000, the cDNAs of most of these candidate receptors have been cloned and for many receptors the endogenous ligand has been identified. In this review, we will give an update about the current knowledge of all Drosophila neuropeptide and protein hormone receptors, and discuss their phylogenetic relationships.     

Renal vasopressin receptor expression and function in rats following spaceflight.

It has been suggested there is a decreased renal responsiveness to vasopressin following spaceflight and that this may be the mechanism for the increased urine flow that is observed following return to normal gravity. In the present study, we have therefore measured vasopressin receptor expression and activity in kidneys taken from rats 1 and 14 days following spaceflight of 15 days duration. Measurements of renal vasopressin V(2) and V(1a) receptor mRNA expression by quantitative RT-PCR demonstrated little difference at either 1 day or at 14 days following return from space. Evaluation of (3)H-labeled arginine vasopressin binding to membranes prepared from kidneys indicated that the majority of the vasopressin receptors were V(2) receptors. Furthermore, the data suggested that binding to vasopressin V(2) or V(1a) receptors was unaltered at 1 day and 14 days following spaceflight. Similarly, the ability of vasopressin to stimulate adenylate cyclase suggested no change in vasopressin V(2) receptor activity in these animals. These data suggest that, whatever changes in fluid and electrolyte metabolism are observed following spaceflight, they are not mediated by changes in vasopressin receptor number or vasopressin-induced stimulation of adenylate cyclase.     

Is there convergence in the molecular pathways underlying the repeated evolution of sociality in African cichlids?

Despite wide variation in the complexity of social interactions across taxa, the basic behavioral components of sociality appear to be modulated by conserved hormone pathways. Specifically, the nonapeptide hormones oxytocin and vasopressin and their receptors have been implicated in regulating diverse social behaviors across vertebrates. Here, we took advantage of the repeated evolution of cooperative breeding in African cichlids to investigate whether there are consistent brain gene expression patterns of isotocin and arginine vasotocin (teleost homologues of oxytocin and vasopressin), as well as their receptors, between four closely related pairs of social (cooperative) and non-social (non-cooperative) species. We first found that the coding sequences for the five genes studied were highly conserved across the eight species. This is the first study to examine the expression of both isotocin receptors, and so we performed a phylogenetic analysis that suggests that these two isotocin receptors are paralogues that arose during the teleost genome duplication. When we then examined brain gene expression patterns relative to social system, we found that there were whole-brain gene expression differences between the social and non-social species in many of the species pairs. However, these relationships varied in both the direction and magnitude among the four species pairs. In conclusion, our results suggest high sequence conservation and species-specific gene expression patterns relative to social behavior for these candidate hormone pathways in the cichlid fishes.     

Evaluation of FLIPR Calcium 3 Assay Kit--a new no-wash fluorescence calcium indicator reagent

We have evaluated the FLIPR Calcium 3 Assay Kit (Calcium 3), a new no-wash fluorescence calcium indicator dye reagent, for the measurement of agonist-stimulated calcium signaling in cells expressing the serotonin 2C (5-HT(2C)), metabotropic glutamate receptor 5 (mGluR5) and the vasopressin 2 (V2) G-protein-coupled receptors. Calcium 3 yielded equivalent (5-HT(2C)) or superior (mGluR5 and V2) sensitivity to FLUO-4 as indexed by the change in fluorescence counts following agonist application. Assay variability, indexed by CV, using Calcium 3 or FLUO-4 was equivalent with 5-HT(2C) receptor responses although CVs were reduced using Calcium 3 in the examples of the mGluR5 and V2 receptors. Receptor pharmacologies based on agonist EC(50) values were identical when either Calcium 3 or FLUO-4 were utilized. Our results validate Calcium 3 as a compelling alternative to FLUO-4 in the choice of fluorescent dye reagent for studying G-protein-coupled receptors, providing the advantage of a homogenous, no-wash assay format.     

Vasopressin V2-receptor mobile fraction and ligand-dependent adenylate cyclase activity are directly correlated in LLC-PK1 renal epithelial cells

The role of hormone receptor lateral mobility in signal transduction was studied using a cellular system in which the receptor mobile fraction could be reversibly modulated to largely varying extents. The G-protein-coupled vasopressin V2-type receptor was labeled in LLC-PK1 renal epithelial cells using a fluorescent analogue of vasopressin, and receptor lateral mobility measured using fluorescence microphotolysis (fluorescence photobleaching recovery). The receptor mobile fraction (f) was approximately 0.9 at 37 degrees C and less than 0.1 at 10 degrees C, in accordance with previous studies. When cells were incubated for 1 h at 4 degrees C without hormone, and then warmed up to 37 degrees C and labeled with the vasopressin analogue, f increased from approximately 0.4 to 0.8 over approximately 1 h. The apparent lateral diffusion coefficient was not markedly affected by temperature pretreatment. Studies with radiolabeled vasopressin indicated that temperature pretreatment influenced neither receptor number nor binding/internalization kinetics. F-actin staining revealed that temperature change resulted in reversible changes of cytoskeletal structure. The maximal rate of in vivo cAMP production at 37 degrees C in response to vasopressin, but not to forskolin (receptor-independent agonist), was also markedly influenced by preincubation of cells at 4 degrees C, thus paralleling the effects of temperature preincubation on f. A linear correlation between f and maximal cAMP production was observed, suggesting that the receptor mobile fraction is a key parameter in hormone signal transduction in vivo. We conclude that mobile receptors are required to activate G-proteins, and discuss the implications of this for signal transduction mechanisms.     

Transgenic and Transcriptional Studies on Neurosecretory Cell Gene Expression

1. Studies of the regulation of neurosecretory cell gene expression suffer from the lack of suitable cell lines. Two approaches have been used to overcome this deficit: transfection of neuropeptide genes into heterologous cell lines and generation of transgenic animals.2. Studies with heterologous cell lines have revealed the potential involvement of nuclear hormone receptors, POU proteins, and fos/jun/ATF family members in the regulation of the vasopressin and oxytocin genes. Although limited in their scope, these studies have contributed greatly to the dissection of basic properties of elements in the vasopressin and oxytocin gene promoters.3. Transgenic mice, and more recently rats, have been used to elucidate genomic regions governing cell specificity and physiological regulation of neurosecretory gene expression. The genes encoding the neuropeptides vasopressin and oxytocin have been used in many transgenic studies, due to the well-defined expression patterns and physiology of the endogenous neuropeptides. Cell-specific and physiologically regulated expression of these transgenes has been achieved, demonstrating the action of putative represser elements and regulation of the expression of one gene by sequences present in the other gene.4. Appropriate expression and translation of transgenes have resulted in the production of several useful systems. Expression of oncogene sequences in gonadotropin-releasing hormone neurons has allowed the development of cell lines from the resulting tumors, overproduction of corticotropin-releasing factor has produced animal models of anxiety and obesity, and directed ectopic expression of growth hormone has generated a potentially useful rat model of dwarfism. These and other animal models of human disease will provide important avenues for the development of therapeutic strategies.     

Bioinformatics and type II G-protein-coupled receptors

The best known family B, or Type II, G-protein-coupled receptors (GPCRs) recognize peptides as ligands. The receptors for corticotrophin-releasing factor, parathyroid hormone and secretin typify this group. However, there are only 15 such GPCRs. Many other receptors share sequence homology and have been assigned to this family. The ten 'Frizzled' and one 'Smoothened' receptors show the lowest sequence homology and are not necessarily G-protein coupled. Drosophila genetics have enabled our understanding of their biology. In contrast, relatively little is known about the largest group with family B, the 33 'large amino termini' or large N-terminal family B seven-transmembrane (LNB 7TM) receptors. This review highlights the similarities found between family B receptors and provides a classification of LNB 7TM receptors.     

Activation of p42/p44 mitogen-activated protein kinase by angiotensin II,vasopressin, norepinephrine,and prostaglandin F2α in hepatocytes is sustained,and like the effect of epidermal growth factor,mediated through pertussis toxin-sensitive mechanisms

Several agents that act through G-protein-coupled receptors and also stimulate phosphoinositide-specific phospholipase C (PI-PLC), including angiotensin II, vasopressin, norepinephrine, and prostaglandin (PG) F_2α, activated the ERK1 (p44^mapk) and ERK2 (p42^mapk) members of the mitogen-activated protein (MAP) kinase family in primary cultures of rat hepatocytes, measured as phosphorylation of myelin basic protein (MBP) by a partially purified enzyme, immunoblotting, and in-gel assays. All these agonists induced a peak activation (two to threefold increase in MBP-phosphorylation) at 3–5 min, followed by a brief decrease, and then a sustained elevation or a second increase of the MAP kinase activity that lasted for several hours. Although all the above agents also stimulated PI-PLC, implicating a G_q-dependent pathway, the elevations of the concentration of inositol (1,4,5)-trisphosphate did not correlate well with the MAP kinase activity. Furthermore, pretreatment of the cells with pertussis toxin markedly reduced the MAP kinase activation by angiotensin II, vasopressin, norepinephrine, or PGF_2α. In addition, hepatocytes pretreated with pertussis toxin showed a diminished MAP kinase response to epidermal growth factor (EGF). The results indicate that agonists acting via G-protein-coupled receptors have the ability to induce sustained activation of MAP kinase in hepatocytes, and suggest that G_i-dependent mechanisms are required for full activation of the MAP kinase signal transduction pathway by G-protein-coupled receptors as well as the EGF receptor. J. Cell. Physiol. 175:348–358, 1998. © 1998 Wiley-Liss, Inc.     

Nonpeptidic ligands for peptide and protein receptors.

The first potent nonpeptidic ligands for somatostatin, luteinizing hormone-releasing hormone, glucagon and bradykinin receptors have been reported. Nonpeptidic clinical candidates have been identified or are currently under study for substance P, bradykinin, endothelin, growth hormone secretagogue, angiotensin, vasopressin, motilin and cholecystokinin. Design, screening, combinatorial chemistry and classical medicinal chemistry all played important roles in these advances.     

The erbA oncogene represses the actions of both retinoid X and retinoid A receptors but does so by distinct mechanisms.

Genetic lesions that function as dominant negative mutations in microbial systems have long been recognized. It is only relatively recently, however, that similar dominant negative mutations have been implicated as a basis for genetic and neoplastic disorders in vertebrates. We describe here a dissection of the actions of the erbA oncogene protein, an aberrant form of thyroid hormone receptor that acts as a dominant negative inhibitor of other nuclear hormone receptors. We demonstrate that the ErbA oncoprotein interferes with thyroid hormone and trans-retinoic acid receptors by competing for binding to the corresponding response elements. Heterodimerization of the ErbA oncoprotein with these receptors does not play an observable role in repression. In contrast, however, the ErbA oncoprotein does efficiently form a heterodimer with the retinoid X receptor (RXR) class of nuclear hormone receptors; complex formation enhances the DNA-binding properties of the ErbA protein but dramatically interferes with the ability of the RXR component to activate gene expression. Our results indicate that the erbA oncogene may play a previously unanticipated role in neoplasia by interfering with RXR function.     

Melanin-concentrating hormone receptor subtypes 1 and 2: species-specific gene expression

To assess the contribution of potential central nervous system pathways implicated in the control of appetite regulation and energy metabolism, it is essential to first identify appropriate animal models. Melanin-concentrating hormone (MCH), a conserved cyclic neuropeptide implicated in the modulation of food intake, has been shown to bind and activate two G-protein-coupled receptors, called GPR24 and MCHR2, expressed in human brain and other tissues. Here we show that several non-human species (rat, mouse, hamster, guinea pig, and rabbit) do not have functional MCHR2 receptors, or encode a nonfunctional MCHR2 pseudogene while retaining GPR24 expression. We identified three species for further evaluation that express both MCH receptor subtypes. We cloned and functionally characterized dog, ferret, and rhesus GPR24 and MCHR2 in mammalian cells and studied their brain distribution patterns by in situ hybridization. The homology, expression profile, and functional similarity of the receptors in the dog, ferret, and rhesus to that of human support the potential use of these species as preclinical animal models in the development of therapeutic agents for obesity or other MCH-mediated disorders.     

Prenatal exposure to dexamethasone alters hippocampal drive on hypothalamic-pituitary-adrenal axis activity in adult male rats

Glucocorticoids are essential for normal hypothalamic-pituitary-adrenal (HPA) axis activity; however, recent studies warn that exposure to excess endogenous or synthetic glucocorticoid during a specific period of prenatal development adversely affects HPA axis stability. We administered dexamethasone (DEX) to pregnant rats during the last week of gestation and investigated subsequent HPA axis regulation in adult male offspring in unrestrained and restraint-stressed conditions. With the use of real-time PCR and RIA, we examined the expression of regulatory genes in the hippocampus, hypothalamus, and pituitary, including corticotropin-releasing hormone (CRH), arginine vasopressin (AVP), glucocorticoid receptors (GR), mineralcorticoid receptors (MR), and 11-beta-hydroxysteroid dehydrogenase-1 (11beta-HSD-1), as well as the main HPA axis hormones, adrenal corticotropic hormone (ACTH) and corticosterone (CORT). Our results demonstrate that the DEX-exposed group exhibited an overall change in the pattern of gene expression and hormone levels in the unrestrained animals. These changes included an upregulation of CRH in the hypothalamus, a downregulation of MR with a concomitant upregulation of 11beta-HSD-1 in the hippocampus, and an increase in circulating levels of both ACTH and CORT relative to unrestrained control animals. Interestingly, both DEX-exposed and control rats exhibited an increase in pituitary GR mRNA levels following a 1-h recovery from restraint stress; however, the increased expression in DEX-exposed rats was significantly less and was associated with a slower return to baseline CORT compared with controls. In addition, circulating levels of ACTH and CORT as well as hypothalamic CRH and hippocampal 11beta-HSD-1 expression levels were significantly higher in the DEX-exposed group compared with controls following restraint stress. Taken together, these data demonstrate that late-gestation DEX exposure in rats is associated with persistent changes in both the modulation of HPA axis activity and the HPA axis-mediated response to stress.