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The avian neurohypophyseal hormone AVT is an important regulatory hormone involved in many physiological processes including oviposition; an age-related phenomenon, through its action on the shell gland. In this study, immunohistochemistry and in situ hybridization was performed to study the expression of immunoreactive (ir) AVT and its oxytocic-like receptor VT3 transcript in the ovary/shell gland of Japanese quail representing sexually immature, mature and old condition. Our results indicate that ir-AVT is present in the ovary of sexually active adult only, but in the shell gland it is observed in both sexually active adult and sexually quiescent old quail. Further, VT3 gene transcript although not detected in the shell gland of sexually immature birds, has been found abundantly in the myometrium of shell gland of sexually active adult quail with a slight decrease in old birds. It is concluded that in addition to the ovarian function and shell gland activity, the expression of AVT and VT3 receptor in the shell gland also varies with the age dependent reproductive/egg laying performance of the Japanese quail. Our findings also suggest that (i) local synthesis of AVT and the expression of its oxytocic-like VT3 receptors is estrogen dependent and (ii) shell gland AVT upregulates its VT3 receptor (paracrine role) in advance to facilitate the role of neurohypophyseal AVT during oviposition.

Avian neurohypophysial hormone arginine vasotocin (AVT) is known to regulate shell gland contractility during oviposition. While studying the role of estrogen in the expression and regulation of AVT and its oxytocic-like receptor VT3, using in situ hybridization and immunohistochemistry, it was observed that the expression of AVT and its receptor was not detected in the shell gland of sexually immature Japanese quail. However, administration of estrogen to these birds not only stimulates the growth and activity (as assessed by increased mucosal fold length, total protein content and alkaline phosphatase level) of the shell gland but also upregulates the expression of AVT and VT3. Further, administration of estrogen antagonist tamoxifen to sexually mature bird shows opposite results. On the other hand, localization of ir-AVT, observed in the ovary of sexually mature bird, was not detected in the estrogen treated sexually immature quail. It is concluded that estrogen not only affects the growth and differentiation of avian oviduct, but also regulates the expression of shell gland AVT and its receptor VT3. Present findings suggest that the locally synthesized AVT acts in a paracrine way to upregulate VT3 receptor and thus facilitates the endocrine function of neurohypophysial AVT during oviposition.

The avian neurohypophysial hormone arginine vasotocin (AVT) is an important regulatory hormone involved in many physiological processes including fluid balance, blood pressure regulation, stress responses and reproductive events including oviposition. The mechanisms by which AVT stimulates myometrial contraction during oviposition are not well established in birds. In the present study, immunohistochemistry and in situ hybridization were used to localize AVT and the oxytocin-like VT3 receptor in the shell gland of Japanese quail (Coturnix coturnix japonica). Using an AVT-specific antibody, immunoreactive AVT (ir-AVT) was observed in the myometrium of both photosensitive and photorefractory birds. Similarly, VT3 receptor gene transcripts were detected in the myometrial layer of the shell gland of both photosensitive and photorefractory birds. Body mass, shell gland mass and length of mucosal folds of the shell gland of photosensitive birds was higher compared to that of photorefractory birds. Treatment of photorefractory birds with estrogen increased shell gland activity (mass and length of mucosal folds) and levels of both AVT and VT3 receptor mRNA, whereas treatment of photosensitive birds with the estrogen antagonist tamoxifen decreased shell gland activity and levels of both AVT and VT3 receptor mRNA. Our results suggest that shell gland contractility in response to AVT may be regulated during the reproductive cycle of the Japanese quail and that, in part, this regulation is estrogen-dependent.

Arginine vasotocin (AVT), a neurohypophysial hormone, has many essential functions in birds including the regulation of salt and fluid balance, blood pressure, the stress response and a variety of behaviors. In addition, AVT controls reproductive functions in birds that are served by oxytocin in mammals. In the following review, we examine the functions of AVT in birds with an emphasis on the present state of knowledge concerning the cloned receptors for this important hormone. Receptor and gene structure, signal transduction mechanisms and expression pattern are all discussed. Finally, we explore the phylogenetic relationships between the cloned avian receptors and other vertebrate and invertebrate neurohypophysial hormone receptors.

Arginine vasotocin is a neurohypophysial hormone in lower vertebrates including birds. Its actions are mediated through G-protein coupled receptors that belong to the vasopressin/oxytocin receptor family. Our laboratory recently cloned a vasotocin receptor, designated the VT2 receptor (VT2R), which shares high sequence identity at both the nucleotide and amino acid level with the mammalian V1b vasopressin receptor (V1bR). In the present study, we report development and use of an antibody to the VT2R to obtain anatomical evidence for testing the hypothesis that the VT2R is the avian homolog of the mammalian V1bR. Results verified the specificity of the antibody and demonstrated a receptor distribution occurring predominantly in the cephalic lobe of the pars distalis and co-localizing with adrenocorticotropin in corticotrophs. With respect to VT2R distribution and cell-type localization in pituitary gland, evidence presented support its similarity with the mammalian V1bR. In contrast to the mammalian V1bR, VT2R expression was not observed in chicken brain. Further research will be required to determine which receptor/s in the arginine vasotocin/mesotocin family are expressed in brain and mediate regulatory functions of vasotocin in the central nervous system.

The neurohypophysial hormone arginine vasotocin (AVT) stimulates adrenocorticotropin hormone (ACTH) secretion from the avian anterior pituitary gland resulting in increased adrenal secretion of corticosterone in response to stress. Here, we report molecular cloning and functional characterization of a gene encoding an AVT receptor subtype, designated the VT2 receptor, that may mediate the stimulatory effect of AVT on ACTH secretion in birds. The open reading frame predicts a 425 amino acid polypeptide that includes seven segments of 19 to 24 hydrophobic amino acids, typical of guanine nucleotide-protein coupled receptors. Phylogenetic analysis revealed that the VT2 receptor shares highest identity with the mammalian V1b-vasopressin receptor subtype. Expressed VT2 receptors in COS7 cells mediate AVT-induced phosphatidylinositol turnover and Ca(2+) mobilization. In the domestic chicken, expression of VT2 receptor gene transcripts is limited to the pituitary gland. Based on similarities in sequence, site of expression and coupled signal transduction pathways, we conclude that the VT2 receptor is the avian homolog of the mammalian V1b-vasopressin receptor, and therefore may play an important role in the avian stress response.

To examine a possible sexual dimorphism in the osmotic control of arginine vasotocin (AVT) release in birds, age-matched male and female Japanese quail were subjected to water deprivation. The observed increased plasma osmolalities were accompanied by increased plasma AVT levels. Plasma mesotocin levels did not change with water deprivation. The sensitivity of the osmotic control of AVT release as determined by the slope of the relationship between plasma AVT levels and plasma osmolalities was significantly (P0.05) higher in males than in females. By Northern blot analysis, levels of hypothalamic AVT gene transcripts were increased 2.3 +/- 0.14- and 3.5 +/- 0.13-fold in water-deprived male and female Japanese quail, respectively, compared to normally hydrated birds. Our data suggest gender-related differences in the osmotic control of AVT release and in hypothalamic AVT gene expression in the Japanese quail.

In chickens, hyperosmolality stimulates the secretion of vasotocin (AVT) and up-regulates hypothalamic AVT gene expression. Hemorrhage, on the other hand, has not been considered an effective stimulus for AVT release in this species. The effects of acute osmotic stress and prolonged hemorrhagic hypotension on AVT gene expression and secretion were studied in White Leghorn hens. Conscious hens were osmotically stimulated by administering a single ip injection of 3 M NaCl (5 ml/kg). Urethane-anesthetized hens were bled to a mean arterial pressure of 80-90 mm Hg and the pressure was maintained within this range by additional bleeding. A total of about 30% of the estimated blood volume was removed. Both experiments were terminated after 1 hr of stimulation. Plasma AVT levels in the hyperosmotic and hypovolemic hens were 4- and 2-fold higher, respectively, compared to controls. Hypothalamic AVT mRNA levels, detected by Northern blot analysis, were 2.5- and 2-fold higher in the osmotically stimulated and hypotensive groups, respectively, compared to control groups. As determined by in situ hybridization, both osmotic stimulation and hypovolemia resulted in an increase in the number of AVT mRNA-containing neurons in the supra-optic and paraventricular nuclei. Our results indicate that, under the conditions used, hypotension and hyperosmolality are equally effective in stimulating AVT gene expression and secretion of AVT.

Osmotic stress stimulates the release of the avian hypothalamic neuropeptide arginine vasotocin (AVT) into the peripheral circulation. We conducted the present study to investigate the effects of salt-loading on AVT secretion and AVT gene expression in specific hypothalamic nuclei in chickens. White Leghorn chickens were provided food ad lib and either water or 2% NaCl to drink. Both plasma osmolality and plasma AVT levels were significantly increased in chickens that drank 2% NaCl for either two or four days compared to that in chickens that drank water. Results from in situ hybridization analysis demonstrated an increase in the number of neurons expressing AVT mRNA in the supraoptic (SON) and paraventricular nuclei (PVN) in chickens provided 2% NaCl to drink compared to chickens that were provided water to drink. The number of grains per neuron increased in the PVN, but not in the SON of osmotically stimulated birds. Thus, increased osmolality resulting from ingestion of hypertonic saline is an effective stimulus to increase hypothalamic AVT mRNA content in chickens.

We have analyzed 3.4 kb of DNA from the 5′-flanking region of the rat β 2 -adrenergic receptor gene and assessed its promoter activity in A549 cells, a human lung adenocarcinoma cell line. A single transcription start site was identified approx. 223 bp upstream of the ATG start codon. A549 cells were transfected with luciferase reporter plasmids containing segments of the rat β 2 -adrenergic receptor 5′-flanking region. Our results suggest that both positive and negative cis -acting regulatory sequences are present in the 5′-flanking region of the rat β 2 -adrenergic receptor gene.

The distribution of immunoreactive vasotocin (IR-AVT) and AVT mRNA in the hypothalamus of White Leghorn cocks was determined by immunohistochemistry and in situ hybridization, respectively. In control birds that were provided with water ad lib, AVT mRNA was distributed in the periventricular and lateral regions of the hypothalamus in clusters of neurons that correspond structurally with the mammalian paraventricular (PVN) and supraoptic (SON) nuclei. Although the distribution of AVT, identified by immunohistochemistry of adjacent serial sections within the hypothalamus, was similar to the distribution of AVT mRNA, the possibility that some positive staining was due to mesotocin neurons was not excluded. Water deprivation for 2 and 4 days resulted in both an increase in levels of AVT mRNA per neuron and the number of AVT mRNA-containing cells. Additionally, water deprivation resulted in a decrease in the amount of IR-AVT per neuron. The results indicate that osmotic stimulation increases AVT gene expression not only in individual neurons but also by activating subpopulation of neurons that are not observed in normally hydrated birds.

Several investigators have reported a diminished responsiveness of senescent cardiac muscle to norepinephrine and beta-adrenoceptor agonists. In contrast, relatively little is known regarding the effects of aging on myocardial actions mediated specifically by alpha-adrenoceptor stimulation. Thus, the current study examined aging-dependent changes in: (a) the inotropic response to methoxamine, an alpha-adrenoceptor agonist; (b) characteristics of myocardial alpha 1-adrenoceptors as monitored by specific [3H]prazosin binding; and (c) steady state levels of alpha 1-adrenoceptor mRNA as determined by Northern blot analysis. Cardiac preparations were isolated from 4-, 14-, and 25-month-old F344 rats. An aging-associated decline was observed in the maximum positive inotropic effect elicited by methoxamine in right ventricular strips (160 +/- 23, 134 +/- 13 and 79 +/- 26% increase above control developed tension in 4, 14 and 25 months, respectively) with no change in ED50 values. [3H]Prazosin binding to ventricular sarcolemmal membranes revealed a reduction in receptor number (82 +/- 7, 69 +/- 6 and 59 +/- 5 fmol/mg protein in 4, 14 and 25 months, respectively); the apparent dissociation constant was not affected. Steady state levels of alpha 1-adrenoceptor mRNA decreased progressively between 4 and 25 months of age (14- and 25-month levels were approximately 71 and 38% of 4 months, respectively), while steady state levels of beta-actin mRNA did not change with age. These results suggest that the aging-related decline in alpha 1-adrenergic responsiveness in rat ventricular muscle is mediated, at least in part, by a decrease in cardiac alpha 1-adrenoceptor density.(ABSTRACT TRUNCATED AT 250 WORDS)

In Northern blots of total cellular and poly(A+) RNA isolated from rat liver, renal cortex, spleen, and brain probed with a full-length cDNA encoding the hamster alpha 1-adrenergic receptor, hybridization was observed to two distinct mRNAs, at approximately 3.3 kb and approximately 2.7 kb. Only the approximately 2.7 kb mRNA species was visualized in Northern blots of total cellular and poly(A+) RNA isolated from cardiac ventricular muscle. From screening a rat heart cDNA library with the full-length hamster alpha 1-adrenergic receptor cDNA, a 632 base pair cDNA was isolated. Based upon its high degree of identity, 86% at the nucleotide level, with the hamster alpha 1-adrenergic receptor cDNA, this cDNA was considered to include the 3' end of the rat alpha 1-adrenergic receptor. When used as a probe in Northern blots of liver RNA, both the approximately 3.3 kb and approximately 2.7 kb mRNAs were visualized. Both mRNA species were expressed in fetal as well as adult liver, but steady-state levels of each gene transcript were approximately 3-fold higher in adult compared to fetal liver. Finally, results from Southern analysis of restriction enzyme fragments of genomic DNA suggest that the two gene transcripts may be products of a single gene.

Two peptides with vasodilatory properties consisting of amino acids 1–30 and 31–67 of the 98 a.a. N-terminal end of the prohormone of atrial natriuretic factor (proANF) which circulates in man were investigated to determine if they have specific binding sites on membranes isolated from DDT 1 MF-2 smooth muscle cells. Smooth muscle is a known biologic target of these peptides. Competitive binding experiments revealed that proANFs (1–30), (31–67), and (99–126) (i.e., C-terminus; ANF) each had specific and separate binding sites. The dissociation constants for proANFs (1–30), (31–67), and (99–126) binding were 0.11 nM, 4 nM, and 7.3 nM, respectively. The binding site concentrations for proANFs (1–30), (31–67), and ANF were 2.57, 59.91 and 40 fmols/10 6 cells, respectively. The number of binding sites per cell were 1548, 36,087, and 24,090, respectively, for proANFs (1–30), (31–67), and (99–126) (ANF). Each peptide bound to DDT 1 MF-2 membranes between 10 −8 to 10 −11 M but could only bind to the other peptides' receptors at concentrations of 10 −6 and 10 −7 M. These results suggest that proANF(1–30) and proANF(31–67) do not work through the ANF receptor but rather have their own separate and distinct receptors that mediate their biologic effects.

The experimental evidence reviewed in this article suggests that the kidneys may have an additional function in regulating blood pressure besides their role in controlling both blood volume by urine formation and the relative state of vasoconstriction by the reninangiotensin system. That is, the kidneys may have an additional influence upon the vasculature of a hormonal vasodilating system. The interstitial cells of the renal medulla appear to be mediating this activity and lipid compounds have been extracted from the renal medulla which display depressor activity. One such compound, the anti-hypertensive polar renomedullary lipid (APRL), has been demonstrated to consists of specific alkyl ether analogues of phosphatidylcholine. The vascular responses to these compounds include vasodilation of both arterioles and venules, rapid lowering of arterial blood pressure with little or no tachycardia, increased depressor activity in hypertensive animals, and blockade of vascular smooth muscle α1-adrenergic receptors. Most recently, APRL and a synthetic analogue, 1-0-octadecyl-2-acetyl-sn-glycero-3-phosphorylcholine, have been used to demonstrate α-adrenergic receptor blockade on a smooth muscle cell line (DDT 1 ) by radioligand assays. This action may be due to the insertion of these compounds into cell membranes causing subsequent steric interactions and blockade of the α-adrenergic receptor.

We have investigated the ability of a series of synthetic vasopressin analogues and related peptides to compete with (3H)-arginine8 vasopressin for binding sites in rat renal medulla and dorsal hindbrain. In renal medulla, arginine8 vasopressin and deamino arginine8 vasopressin, a selective antidiuretic, were equipotent while two antagonists of the pressor action of arginine vasopressin were less potent. In the dorsal hindbrain, arginine8 vasopressin and the pressor antagonists were more potent than the synthetic antidiuretic. Potency profiles of these and other analogues suggest that the renal medulla and dorsal hindbrain vasopressin receptors represent different subtypes.

We have identified in the DDT1 smooth muscle cell line a [3H]dihydroergocryptine-binding site having the characteristics of an alpha 1-adrenergic receptor. Specific binding of [3H]dihydroergocryptine to DDT1 cells grown either in monolayer or suspension culture was reversible, saturable, and of high affinity, and the binding site demonstrated stereoselectivity. [3H]Dihydroergocryptine dissociation constants of 1.4 +/- 0.2 nM and 1.4 +/- 0.3 nM were observed for suspension and monolayer cells, respectively. However, the concentration of binding sites in suspension-cultured cells (65,100 +/- 8,300 sites/cell) was significantly greater (p less than 0.001) than that found in monolayer cells (27,900 +/- 4,300 sites/cell). The order of agonist competition for the binding site was epinephrine (Ki = 0.92 +/- 0.32 microM) greater than or equal to norepinephrine (Ki = 2.2 +/- 1.0 microM) greater than isoproterenol (Ki = 137 +/- 17 microM), consistent with an alpha-adrenergic interaction. Results of competition experiments with specific antagonists prazosin (alpha 1-selective) or yohimbine (alpha 2-selective) and a computer modeling technique indicated that the alpha-adrenergic receptor of the DDT1 cell was predominantly (greater than 95%) the alpha 1-subtype.