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Role of estrogen and photoperiod is well-established in avian reproduction. In addition, the distribution and the expression of arginine vasotocin (AVT) and its receptor VT3 to ensure reproductive/breeding conditions in Japanese quail influenced by them has been the main focus of this review. To consider this aspect the mRNA expression of VT3 receptor and its ligand AVT in the shell gland has been emphasized. In birds, AVT performs a dual role as an anti-diuretic hormone and the functions accomplished by oxytocin in mammals. The physiological actions of AVT in birds are mediated through its diverse receptor subtypes VT1, VT2, VT3 and VT4. Dynamic alteration of VT3 expression during different reproductive and photo-sexual conditions of quail can be modulated by estrogen. In addition to the endocrine effect of AVT, the shell gland is complemented by its paracrine action via its receptors. Evidences indicate that the expression of shell gland AVT modulated by estrogen appears to play a priming role by modulating the availability of VT3 receptor for the required action of neurohypophysial AVT during oviposition.

The neurohormone arginine vasotocin (AVT) in non mammalian vertebrates is homologous to arginine vasopressin (AVP) in mammals. Its actions are mediated via G protein-coupled receptors that belong to the vasotocin/mesotocin family. Because of the known regulatory effects of nonapeptide hormones on anterior pituitary functions, receptor subtypes in that family have been proposed to be located in anterior pituitary cells. Recently, an avian vasotocin receptor subtype designated VT4R has been cloned, which shares 69% sequence homology with a human vasopressin receptor, the V1aR. In the present study, a polyclonal antibody to the VT4R was developed and validated to confirm its specificity to the VT4R. The antibody was used to test the hypothesis that the VT4R is present in the avian anterior pituitary and is specifically associated with certain cell types, where its expression is modulated by acute stress. Western blotting of membrane protein extracts from pituitary tissue, the use of HeLa cells transfected with the VT4R and peptide competition assays all confirmed the specificity of the antibody to the VT4R. Dual-labelling immunofluorescence microscopy was utilised to identify pituitary cell types that contained immunoreactive VT4R. The receptor was found to be widely distributed throughout the cephalic lobe but not in the caudal lobe of the anterior pituitary. Immunoreactive VT4R was associated with corticotrophs. Approximately 89% of immunolabelled corticotrophs were shown to contain the VT4R. The immunoreactive VT4R was not found in gonadotrophs, somatotrophs or lactotrophs. To determine a possible functional role of the VT4R and previously characterised VT2R, gene expression levels in the anterior pituitary were determined after acute immobilisation stress by quantitative reverse transcriptase-polymerase chain reaction. The results showed a significant increase in plasma corticosterone levels (three- to four-fold), a significant reduction of VT4R mRNA and an increase of VT2R mRNA (P < 0.05) in acutely immobilised chicks compared to controls. The data suggest a role of the VT4R in the avian stress response.

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.

Research in mammals has demonstrated a variety of regulatory effects of vasopressin and oxytocin on endocrine functions of the anterior pituitary gland. Less evidence is available regarding the hypophysiotropic action of arginine vasotocin (AVT) comprising vasopressic and oxytocic activities in birds. Some hypophysiotropic effects of AVT may result from its interactions with brain circuits controlling pituitary functions, whereas others are caused by its direct affect on pituitary cells. Use of an antiserum to the vasotocin receptor VT2 (VT2R) has revealed numerous immunoreactive cells in the anterior pituitary gland of the chicken. The objective of the present study has been to identify endocrine phenotypes of chicken pituitary cells containing VT2R by means of immunohistochemical labeling. VT2R immunoreactivity has been found in all cells immunoreactive for adrenocorticotropin and alpha-melanotropin. Approximately 10% of labeled lactotropes are also immunoreactive for VT2R and lie around the anatomical boundary dividing the cephalic and caudal lobes. In both corticotropes/melanotropes and lactotropes, immunoreactive VT2R is present in a narrow layer outlining cell bodies. Immunoreactive VT2R is not found in gonadotropes, thyrotropes, or somatotropes. These results provide evidence for the important role of VT2Rs in mediating effects of AVT on endocrine secretion from corticotropes and, partially, from lactotropes.

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.

In birds, ACTH release from the anterior pituitary gland during stress is controlled by CRH and arginine vasotocin (AVT). Using 5-wk-old male chicks, simultaneous iv injections of CRH and AVT were found to result in a greater than additive increase in plasma corticosterone levels compared with that obtained with individual administration of either peptide hormone. In order to investigate molecular mechanisms underlying this observation, the chicken CRH receptor (CRHR) and vasotocin VT2 receptor (VT2R) were fused to cyan and yellow fluorescent proteins and expressed in HeLa cells. The resulting CRHR and VT2R fusion proteins were expressed appropriately in the plasma membrane and were found to couple to downstream signal transduction pathways. Quantitative fluorescence resonance energy transfer (FRET) analysis was used to determine whether the CRHR and VT2R formed heterodimers. In the absence of CRH and AVT, the FRET efficiency was 15–18%, and the distance between receptors was 5–6 nm. Treatment of the cells that expressed both cyan fluorescent protein-CRHR and yellow fluorescent proteinVT2R with CRH or AVT alone did not lead to a significant change in the FRET efficiency. However, simultaneous addition of these hormones increased the efficiency of the FRET signal and decreased the distance between the two receptors. In HeLa cells expressing both CRHR and VT2R, treatment with CRH and AVT resulted in a significant increase in cAMP production over that with CRH alone, indicating that heterodimer formation may enhance the ability of the CRHR to activate downstream signal transduction. (Molecular Endocrinology 21: 2178–2188, 2007)

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.

The beta 2-adrenergic receptor (beta 2AR) signal transduction system regulates many key functions of airway epithelium. In this study, we have pharmacologically characterized the beta 2AR and determined the impact of glucocorticoids on beta 2AR gene transcription in SPOC1 cells, a continuous cell line derived from the tracheal epithelium of rats. [125I]Cyanoiodopindolol assays demonstrated that binding to SPOC1 cell membranes was saturable (Bmax = 62.6 +/- 6 fmol/mg protein) and of high affinity (Kd = 6.3 +/- 0.8 pM). From competition experiments, the rank order of potency of agonists (isoproterenol > epinephrine >> norepinephrine) and the high affinity (Ki = 0.37 +/- 0.05 nM) of the beta 2-selective antagonist ICI 118,551 suggested the predominance of the beta 2AR subtype. Two isoforms of the alpha subunit of Gs (45 and 52 kDa) were identified by Western blot analysis. Isoproterenol-stimulated cyclic AMP levels increased in a dose-dependent manner, confirming that SPOC1 cell beta 2ARs are functionally coupled to adenylyl cyclase. The effect of glucocorticoids on beta 2AR expression was assessed in radioligand and transient transfection assays. Dexamethasone treatment of SPOC1 cells increased both beta 2AR protein and beta 2AR-luciferase fusion gene expression 1.6- to 3.1-fold, with the greatest increase demonstrated in cells cultured at low density compared to cells grown at high density.

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 and hemorrhage increase hypothalamic vasotocin (AVT) gene expression and stimulate the secretion of AVT from the posterior pituitary gland. In this study, c-fos expression was used to identify areas in the forebrain and brainstem of the domestic chicken that are activated following acute osmotic stress and hemorrhage-induced hypotension. Conscious hens were osmotically stimulated by administering a single intraperitoneal injection of 3 M NaCl (5 ml/kg). Urethane-anesthetized hens were bled to a mean systemic arterial pressure of 80-90 mm Hg and maintained at this blood pressure for 1 h with additional bleedings as required. In both studies, the expression of c-fos was determined in control and experimental birds by using Northern blot analysis and in situ hybridization analysis. Osmotic stress and hemorrhage-induced hypotension increased c-fos expression in the same brain regions. Prominent structures in the forebrain that expressed c-fos mRNA following acute osmotic stress and hemorrhage-induced hypotension included the supraoptic nucleus and paraventricular nucleus and nuclei within the hypothalamus that are anterior and ventral to the third ventricle. In the chicken, this region includes the organum subseptale, the o. vasculosum laminae terminalis, and the nucleus septalis medialis. In the brainstem, following either injection of 3 M NaCl or hemorrhage-induced hypotension, increased c-fos expression was observed in the nucleus of the solitary tract, parabrachial nucleus, area postrema, and locus ceruleus. Thus, the chicken central nervous system appears to use shared neuronal circuitry to stimulate hypothalamic AVT release in response to disturbances in body fluid composition and decreases in either systemic blood pressure or volume.

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.

The interactions of catecholamines with alpha 1B-adrenergic receptors (alpha 1B-AR) located on the surface of many cell types are responsible for physiologic and pathologic functions in mammalian systems. Transcription of the alpha 1B-AR gene leads to the expression of multiple alpha 1B-AR mRNAs that are distributed in a tissue-specific fashion. The purpose of this study was to define the 5'-untranslated regions of the multiple alpha 1B-AR gene transcripts. Evidence for a previously unidentified intron in the alpha 1B-AR gene upstream of the receptor open reading frame was obtained via rapid amplification of cDNA ends. A product was amplified and was found to be missing the nucleotide interval from -708 to -194, (+1 is the start of translation). Evidence for tissue-specific alternative intron splicing was obtained from ribonuclease protection assays and RT-PCR experiments. Using an RNA probe extending from -240 to +93 and including 45 nucleotides into the putative intron, a single protected fragment was detected in heart RNA while two protected fragments were detected in liver RNA. RT-PCR amplification of the region spanning the intron resulted in detection of two PCR products in liver RNA and no detectable product in heart RNA. These findings emphasize the complexity of alpha 1B-AR gene regulation and suggest that multiple alpha 1B-AR mRNAs with different 5'-UTRs may play a role in regulating alpha 1B-adrenergic responsiveness.

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.

In the liver, transcript destabilization contributes to the decrease in steady-state levels of beta 2-adrenergic receptor mRNA that occurs during early postnatal development in the rat. From genomic DNA, polymerase chain reaction (PCR) was used to amplify a 718-basepair (bp) fragment of the beta 2-adrenergic receptor gene including the entire 3'-untranslated region. Results from SDS-gel electrophoresis and autoradiography demonstrated a M(r) 85,000 cellular factor present in postnatal day 60, but not fetal day 18 rat liver that was ultraviolet (UV) light-crosslinked to in vitro transcribed beta 2-adrenergic receptor RNA 3'-untranslated region. Unlabeled beta 2-adrenergic receptor RNA 3'-untranslated region, but not mouse beta-actin RNA, competed with labeled beta 2-adrenergic receptor RNA 3'-untranslated region for binding to the M(r) 85,000 protein. Cross-linking of the beta 2-adrenergic receptor RNA 3'-untranslated region to the M(r) 85,000 protein was inhibited by the ribohomopolymer poly(U), with poly(A), poly(C) and poly(G) having little or no effect. Thus, a M(r) 85,000 protein has been identified in adult male rat liver that may interact with U-rich sequences in the 3'-untranslated region of the beta 2-adrenergic receptor mRNA and may account for the decreased stability of hepatic beta 2-adrenergic receptor gene transcripts that occurs during development.

Glucocorticoids increase beta 2-adrenergic responsiveness and receptor density in the lung, but the underlying mechanisms have not been clearly elucidated. To determine whether changes in beta 2-adrenergic receptor gene expression are involved in vivo, we measured beta 2-adrenergic receptor mRNA levels and beta 2-adrenergic receptor density in lungs from Sprague-Dawley rats treated with a daily injection of dexamethasone (1 mg/kg subcutaneously) for 1, 3, or 7 days. Animals were sacrificed either 2 or 24 h after receiving the last injection. beta 2-Adrenergic receptor mRNA levels were significantly (p < .05) elevated compared to saline-treated controls in the lungs of animals sacrificed 2 h after dexamethasone injection for 1 day (174 +/- 12%), 3 days (236 +/- 18%), and 7 days (220 +/- 11%). Receptor mRNA levels measured 24 h after dexamethasone injection did not differ significantly from the control group. Induction of beta 2-adrenergic receptor mRNA by dexamethasone was transient, since no significant cumulative or sustained increase in receptor mRNA levels was observed during the study period. Treatment with dexamethasone increased beta 2-adrenergic receptor density as expected, but no significant increase in receptor density was detected until 24 h after the third daily injection of dexamethasone, when levels reached 2045 +/- 150 fmol/mg protein compared to 1292 +/- 34 fmol/mg protein in the control group. Receptor density then remained at this elevated level through 7 days of treatment. These results show that dexamethasone up-regulates both the beta 2-adrenergic receptor and its mRNA in vivo in the lung. The induction of beta 2-adrenergic receptor mRNA levels indicates that glucocorticoids may regulate receptor density in the lung through modulation of gene expression. However, the difference between the time course of induction for the beta 2-adrenergic receptor and its mRNA suggests that additional translational or post-translational mechanisms may also be involved.

Arginine vasotocin (AVT) and corticotropin-releasing hormone (CRH) are two neuronal regulators in the hypothalamic–pituitary–adrenal (HPA) axis that modulate biological responses to stress in avian species. When AVT and CRH are administered together in vitro or in vivo , levels of adrenocorticotropic hormone (ACTH) or plasma corticosterone (CORT) are released, respectively, in a synergistic manner. The underlying mechanism of this greater than additive stress response was investigated by expressing the vasotocin receptor type 2 (VT2R) and CRH receptor type 1 (CRH-R1), both G-protein coupled receptors, in HeLa cells. Fluorescence resonance energy transfer (FRET) analysis provided the evidence for heterodimerization of the VT2R/CRH-R1 in the presence of their respective ligands, AVT and CRH. The VT2R and CRH-R1 were tagged at the C-terminal ends with either cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP), and a VT2R chimera was constructed by replacing the fourth transmembrane region (TM4) of the VT2R with TM-IV of the β 2 -adrenergic receptor (β 2 AR). When VT2R/β 2 AR chimera and CRH-R1 were expressed in HeLa cells, heterodimerization was partly disrupted. Taken together, these data indicate that TM-IV of the VT2R may provide an important interface for effective receptor dimerization, suggesting that direct molecular interaction between VT2R and CRH-R1 receptors plays a role in mediating an enhanced interaction between these two receptors. Their interaction at the anterior pituitary level may potentiate the endocrine output of the avian HPA system.

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.

Previous studies have revealed two size classes of alpha 1b-adrenergic receptor mRNAs, 3.3 kb and 2.7 kb, in the Sprague Dawley rat that are transcribed from a single gene and are expressed in approximately equal amounts in liver. Only the 2.7 kb mRNA is expressed in heart. Both alpha 1b-adrenergic receptor mRNAs appear to share extensive regions of homology, therefore, we used oligonucleotide-directed ribonuclease H mapping to detect sequence differences between the two transcripts. Initial experiments using oligo (dT)-directed RNase H hydrolysis indicated that the two mRNAs have poly [A+] tails of identical length. By using region-specific cDNA probes, we determined that the sequence difference between the two alpha 1b-adrenergic receptor mRNAs lies in the 5' end, upstream from the known initiator AUG in the 2.7 kb transcript. In addition, results from ribonuclease protection assays and Northern blot analysis in which an oligonucleotide was used as the probe suggested that both alpha 1b-adrenergic receptor mRNAs are transcribed from the same DNA strand.

Hepatic responsiveness to beta 2-adrenergic stimulation is dynamically regulated during early development as well as following hepatic injury and disease. In the present study, the molecular mechanisms that underlie the decline in the steady-state levels of hepatic beta 2-adrenergic receptor mRNA that occurs during development in the male rat were investigated. As determined by nuclear run-on assays, an age-associated reduction in beta 2-adrenergic receptor gene transcription was observed. The transcription rate of the beta 2-adrenergic receptor gene in postnatal day 18 liver was approximately 50% lower than that of fetal liver. Stability of beta 2-adrenergic receptor gene transcripts was highest (t1/2 approximately 6h) in hepatocytes isolated from fetal rats and was lowest (t1/2 approximately 6h) in hepatocytes from postnatal day 14 rats. In fetal hepatocytes, but not postnatal day 2 hepatocytes, cycloheximide appeared to stabilize beta 2-adrenergic receptor gene transcripts in the presence of actinomycin D. These findings establish the molecular basis of reduced steady-state levels of beta 2-adrenergic receptor mRNA in liver during early postnatal development and suggest multilevel regulatory control of hepatic beta 2-adrenergic receptor gene expression.

Probing total cellular and poly [A+] RNA isolated from various rat tissues with a full-length cDNA encoding the hamster alpha 1-adrenergic receptor results in detection of two transcripts, 3.3 kb and 2.7 kb, which probably both encode the alpha 1b-adrenergic receptor subtype. Both the 3.3 kb and 2.7 kb mRNAs were found to be associated with hepatic polysomes which suggests that these mRNA species are translated into protein. Using non-overlapping 5' and 3' cDNA probes, large sequence differences were not evident between the 3.3 kb and 2.7 kb mRNAs, although the 3'-probe hybridized to a 4.0 kb mRNA in addition to the two smaller transcripts in poly [A+] RNA isolated from renal cortex, but not other tissues. The relative amounts of the 3.3 kb and 2.7 kb mRNAs varied considerably among the five tissues studied. However, the ratio of the two transcripts remained relatively constant in the same tissue taken from animals at different developmental ages. Currently, the physiological significance of multiple alpha 1b-adrenergic receptor gene transcripts is unclear. However, our results suggest that alpha 1b-adrenergic receptor gene expression in the rat is under complex regulatory control that in part is tissue-dependent.

Corticotropin releasing hormone receptor (CRHR) and the VT2 arginine vasotocin receptor (VT2R) are vital links in the hypothalamic-pituitary-adrenal axis that enable a biological response to stressful stimuli in avian species. CRHR and VT2R are both G-protein coupled receptors (GPCRs), and have been shown by us to form a heterodimer via fluorescent resonance energy transfer (FRET) analysis in the presence of their respective ligands, corticotrophin releasing hormone (CRH) and arginine vasotocin (AVT). The dimerization interface of the heterodimer is unknown, but computational analyses predict transmembrane domains (TMs) as likely sites of the interaction. We constructed chimerical VT2Rs, tagged at the C-terminal ends with either cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP), by replacing the fourth transmembrane region (TM4) of VT2R with TM4 of the beta2-adrenergic receptor (beta2AR). The VT2R/beta2AR chimeras were expressed in HeLa cells and proper trafficking is confirmed by observing cell membrane localization using confocal microscopy. VT2R/beta2AR-YFP chimera functionality was confirmed with a Fura-2 acetoxymethyl ester (Fura-2AM) assay. FRET analysis was then performed on VT2/beta2AR-chimera/CRHR pairs, and the calculated distance was observed to be10 nm apart, indicating that heterodimerization was partly disrupted by mutating TM4 of the VT2R. Therefore, TM4 may form one region of the possible dimerization interfaces between the VT2R and CRHR.

The role of arginine vasotocin in the regulation of the pituitary-adrenal axis of domestic fowl was analyzed by studying the expression of its recently cloned pituitary receptor VT2 and adrenal activity following osmotic stress. Four days of water deprivation induced an increase in plasma osmolality-a known stimulator of AVT synthesis and release from hypothalamic magnocellular neurons. Water deprivation also decreased pituitary mRNA levels for both the VT2 receptor and for pro-opiomelanocortin (POMC). Despite a decrease in the expression of VT2 mRNA, immunoreactive-VT2 receptor levels in the pituitary increased, suggesting a possible role for post-transcriptional mechanisms in the regulation of this receptor. Further, adrenal activity (as judged by adrenal weight, cholesterol content, 3beta hydroxysteroid dehydrogenase, cortical cord width and cortico-medullary ratio) showed stimulation in water-deprived chicken as compared to control. On the basis of present findings, it is concluded that water deprivation down regulates the mRNA expression of AVT receptor VT2 as well as POMC but stimulates adrenal function. It is also suggested that in addition to the release of magnocellular AVT into the neurohypophysis to act as antidiuretic hormone following water deprivation, AVT may also modulate HPA axis to cope with the osmotic stress.

BackgroundThe β2-adrenergic receptor (β2AR) is a primary target for medications used to treat asthma. Due to the low abundance of β2AR, very few studies have reported its localization in tissues. However, the intracellular location of β2AR in lung tissue, especially in airway smooth muscle cells, is very likely to have a significant impact on how the airways respond to β-agonist medications. Thus, a method for visualizing β2AR in tissues would be of utility. The purpose of this study was to develop an immunofluorescent labeling technique for localizing native and recombinant β2AR in primary cell cultures.MethodsA panel of six different antibodies were evaluated in indirect immunofluorescence assays for their ability to recognize human and rat β2AR expressed in HEK 293 cells. Antibodies capable of recognizing rat β2AR were identified and used to localize native β2AR in primary cultures of rat airway smooth muscle and epithelial cells. β2AR expression was confirmed by performing ligand binding assays using the β-adrenergic antagonist [3H] dihydroalprenolol([3H]DHA).ResultsAmong the six antibodies tested, we identified three of interest. An antibody developed against the C-terminal 15 amino acids of the human β2AR (Ab-Bethyl) specifically recognized human but not rat β2AR. An antibody developed against the C-terminal domain of the mouse β2AR (Ab-sc570) specifically recognized rat but not human β2AR. An antibody developed against 78 amino acids of the C-terminus of the human β2AR (Ab-13989) was capable of recognizing both rat and human β2ARs. In HEK 293 cells, the receptors were predominantly localized to the cell surface. By contrast, about half of the native rat β2AR that we visualized in primary cultures of rat airway epithelial and smooth muscle cells using Ab-sc570 and Ab-13989 was found inside cells rather than on their surface.ConclusionAntibodies have been identified that recognize human β2AR, rat β2AR or both rat and human β2AR. Interestingly, the pattern of expression in transfected cells expressing millions of receptors was dramatically different from that in primary cell cultures expressing only a few thousand native receptors. We anticipate that these antibodies will provide a valuable tool for evaluating the expression and trafficking of β2AR in tissues.

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.

The avian homologs of arginine vasopressin (AVP) and oxytocin (OT) are arginine vasotocin (AVT) and mesotocin (MT), respectively. In birds, AVT shares many of the functions of AVP including regulation of fluid balance, blood pressure regulation and the stress response. AVT also plays an oxytocin-like reproductive role in birds by stimulating uterine (shell gland) contraction during oviposition. The role of MT in avian reproduction is not clear. Here, we report the cloning of a third neuropeptide receptor in the chicken (Gallus gallus). Parsimony analysis reveals that the new receptor has highest homology to mammalian OT receptors and the MT receptors of non-mammalian vertebrates. Moreover, the receptor bears far less homology to the two avian VT receptors that have been cloned. Reverse transcription-polymerase chain reaction and in in situ hybridization analyses reveal the receptor is expressed in both the endometrium and myometrium of the shell gland. The expression pattern and high homology to OT receptors suggest that the receptor may stimulate myometrial contraction and therefore play a critical role in oviposition.

Regulation of beta2-adrenergic receptor (beta2AR) levels by glucocorticoids is a physiologically important mechanism for altering beta2AR responsiveness. Glucocorticoids increase beta2AR density by increasing the rate of beta2AR gene transcription, but the cis-elements involved have not been well characterized. We now show that one of six potential glucocorticoid response elements (GREs) in the 5'-flanking region of the rat beta2AR gene is necessary for glucocorticoid-dependent stimulation of receptor gene expression. Using a nested set of deletion fragments of the rat beta2AR gene 5'-flanking region fused to a luciferase reporter gene, glucocorticoid-dependent induction of reporter gene expression in HepG2 cells was localized to a region between positions -643 and -152, relative to the transcription initiation site. In electrophoretic mobility shift assays, a double-stranded oligonucleotide incorporating a near-consensus GRE from this region (positions -379 to -365) formed complexes with the human recombinant glucocorticoid receptor, as well as with nuclear protein from dexamethasone-treated HepG2 cells. Mutation of a single base within this GRE sequence greatly diminished interaction of the mutated oligonucleotide with the human recombinant glucocorticoid receptor. The functional activity of the GRE was characterized using a luciferase reporter construct driven by a minimal thymidine kinase promoter. In HepG2 cells transfected with constructs containing the GRE, dexamethasone increased reporter gene expression approximately 3-fold, whereas a dexamethasone effect was not observed with constructs lacking the GRE. Taken together, these findings show that a GRE located at positions -379 to -365 in the 5'-flanking region of the rat beta2AR gene mediates glucocorticoid stimulation of beta2AR gene transcription.

The effects of water deprivation on the secretion of vasotocin (AVT) and expression of the AVT gene were studied in White Leghorn cockerels. Animals deprived of water for 4 days were compared with normally hydrated controls. Blood samples were obtained for measurements of plasma osmolality and AVT levels, and the hypothalamus was collected for extraction of total cellular RNA. A 519-bp AVT cDNA was prepared by reverse transcriptase–polymerase chain reaction and a 209-bpPstI/EcoRI restriction fragment from the 3′ region of the fowl AVT cDNA was used as a probe for Northern blot analysis. Plasma osmolality and AVT levels in dehydrated birds were about 30 and 350% greater, respectively, than those in normally hydrated controls. The quantity of hypothalamic AVT mRNA was 2.3-fold greater in water-deprived birds compared to controls. The size of the hypothalamic AVT transcript was about 100-bp longer in the water-deprived birds. As determined by RNase H treatment in the presence and absence of oligo(dT)12–18, the increase in mean size of the AVT mRNA in dehydrated animals was due to a longer poly(A) tract. Our results indicate that osmotic stress up-regulates expression of the AVT gene and increases the accumulation of AVT mRNA in the hypothalamus. This accumulation may, in part, be due to lengthening of the AVT mRNA poly(A) tail which is a general mechanism associated with stabilization of vertebrate mRNAs.

α 1b -Adrenergic receptor gene expression was investigated in two rat hepatic cell lines, Clone 9 and McA-RH7777 cells. By Northern blot analysis, Clone 9 cells expressed a 2.7 kb α 1b -adrenergic receptor gene transcript whereas two transcripts, 3.3 kb and 2.7 kb, were observed in total cellular RNA isolated from rat liver. A binding site for the α 1 -adrenergic antagonist [ 3 H]prazosin was observed in Clone 9 cell membrane preparations ( B max = 47 ± 7 fmol/mg protein and K d = 0.11 ± 0.02 nM, n = 5. In contrast, α 1b -adrenergic receptor gene transcripts could not be detected in total cellular RNA prepared from McA-RH7777 cells by either Northern blot analysis or ribonuclease protection assays. However, results from nuclear run-off assays indicated that the α 1b -adrenergic receptor gene was transcribed in McA-RH7777 cells and α 1b -adrenergic receptor gene transcripts were observed in McA-RH7777 cell nuclear RNA. These results suggest that α 1b -adrenergic receptor gene expression in liver may be regulated in part post-transcriptionally and that this level of regulation may be altered or disrupted in the Clone 9 and McA-RH7777 cell lines.

Arginine vasotocin gene transcripts in various tissues of the domestic fowl were detected by reverse transcriptase polymerase chain reaction followed by Southern blot analysis using a 209 bp fragment from the 3'-region of a cDNA encoding chicken arginine vasotocin as the probe. Relatively strong signals were observed with hypothalamic, adenohypophysial and proventricular RNA as the starting material. Lesser signals were obtained from RNA isolated from shell gland, adrenal gland, post-ovulatory follicles and ovarian thecal cells. Arginine vasotocin gene transcripts were undetectable in the posterior pituitary gland, small intestine and large intestine. These results suggest that in addition to its well-known antidiuretic and oxytocic actions, arginine vasotocin may act as a local neuromodulator or mediator and have other important autocrine or paracrine actions in non-hypothalamic tissues.

Metabolic events stimulated by epinephrine and norepinephrine in hepatocytes isolated from fetal and early postnatal male rats are largely mediated through the beta 2-adrenergic receptor-/cyclic AMP dependent-system, whereas the same stimuli are transduced through the alpha 1-adrenergic receptor-/phosphatidylinositol dependent-system in hepatocytes isolated from young adult male rats. This developmental transition was investigated by correlating hepatic alpha 1- and beta 2-adrenergic receptor gene transcript levels with receptor levels as determined with selective radioligands in livers from late fetal to postnatal day 120 male Sprague-Dawley rats. beta 2-Adrenergic receptor concentration, initially high in membrane preparations isolated from fetal livers (203 +/- 21 fmol/mg protein), dropped precipitously in postnatal day 6 livers (14 +/- 2 fmol/mg protein) and remained low throughout development out to postnatal day 90. beta 2-Adrenergic receptor mRNA levels were highest in fetal livers, were decreased somewhat in postnatal day 6 livers and were undetectable in livers beyond postnatal day 15. In contrast, hepatic alpha 1-adrenergic receptor concentration was relatively low in fetal livers (86 +/- 25 fmol/mg protein) and remained low until postnatal day 18. Thereafter, a steady increase in alpha 1-adrenergic receptors was observed until adult levels. (270 +/- 24 fmol/mg protein) were achieved at postnatal day 27. alpha 1-Adrenergic receptor mRNA levels increased approximately 3-fold, reaching a peak at postnatal day 24. Surprisingly, at postnatal day 30 hepatic alpha 1-adrenergic receptor mRNA levels dropped to fetal levels; but, gradually increased with continued development. Thus, hepatic alpha 1- and beta 2-adrenergic receptors appear to be under complex regulatory control which may include transcriptional, as well as post-transcriptional, mechanisms.

Although (3H)-spiperone has been demonstrated to interact at both dopamine (D2) and serotonin (S2) receptors, it remains a popular choice for characterization of the D2-receptor using both in vitro and in vivo and in vivo assay techniques. Using a clonal smooth muscle cell culture line (DDT1 MF-2), which has previously viously been characterized as possessing alpha 1- and beta 2-adrenergic receptors, we have found that (3H)-spiperone also has a significant affinity for alpha 1-adrenergic receptors. Our results are consistent with other literature reports which have suggested that spiperone may interact at alpha 1-receptors and we have characterized this interaction. We have also found an additional, high affinity binding site for spiperone on these cells which may represent a D2 - receptor. Characterization of this high-affinity site has been difficult since it is present in very low density. We conclude that (3H)-spiperone binds with high affinity to at least three known neurotransmitter receptor sites: D2-dopamine, S2-serotoninn and alpha 1-adrenergic. The binding to the alpha 1-adrenergic receptor exhibits stereospecificity and a considerable degree of similarity in pharmacological profile to the D2-dopamine receptor.

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 compared [3H] arginine8 vasopressin (AVP) and [3H] 1-beta-mercapto-beta, beta-cyclopentamethylene propionic acid, O-methyl tyrosine2, arginine8 vasopressin (d(CH2)5 Tyr(Me)AVP), a selective V1 receptor antagonist, as radioligands for the rat hepatocyte V1 receptor. Both radioligands bound with high affinity to a site in partially purified membranes prepared from Long Evans rat hepatocytes. The binding site concentrations obtained with either radioligand, 608 +/- 101 fmol/mg protein (n = 5) with [3H] AVP and 603 +/- 62 fmol/mg protein (n = 5) with [3H]d(CH2)5 Tyr(Me)AVP, were not significantly (p greater than 0.5) different. Furthermore, the rank order of potency of a series of synthetic vasopressin analogs and related peptides were identical in competition studies using either radioligand and were consistent with a V1 receptor interaction. Our results demonstrate that [3H] d(CH2)5 Tyr(Me)AVP is a suitable radioligand to study the V1 receptor subtype.

Using [3H]-dihydroergocryptine, we have identified in membranes prepared from the DDT1 MF-2 smooth muscle cell line a binding site with characteristics of the alpha 1-adrenergic receptor. Specific binding (90-95% of total binding) was saturable with a binding site concentration of 197 +/- 44 fmol/mg protein and was of high affinity with a dissociation constant of 1.7 +/- 0.4 nM. The order of agonist competition for the binding site was epinephrine (Ki = 2.3 +/- 0.5 microM) greater than or equal to norepinephrine (Ki = 4.4 +/- 1.3 microM) much greater than isoproterenol (Ki = 195.5 +/- 27.6 microM), consistent with an alpha-adrenergic interaction. Computer modelling of competition curves obtained with prazosin (alpha 1-selective) and yohimbine (alpha 2-selective) indicated that the DDT1 cell alpha-adrenergic receptor was predominantly (greater than 95%) of the alpha 1-subtype. Guanine nucleotides, either GTP or 5'-guanylylimidodiphosphate, did not reduce the affinity of either epinephrine of phenylephrine for the [3H]-dihydroergocryptine binding site.

Capacitation and the acrosome reaction of mammalian spermatozoa are essential for fertilization. In vitro results are presented that demonstrate that catecholamines stimulate activation (a whiplash flagellar movement characteristic of capacitated hamster spermatozoa) and the acrosome reaction. Protein-free ultrafiltrates of bovine adrenal cortex and medulla preparations stimulated motility, activation, and acrosome reactions of hamster spermatozoa in the presence of bovine serum albumin. The medulla preparation was more effective than the cortex preparation in the stimulation of activation and acrosome reactions. Epinephrine (0.5-50 μM) and norepinephrine (50.0 μM) in the presence of bovine serum albumin and a partially purified protein-free cortex preparation also stimulated activation and the acrosome reactions. Both activation and acrosome reactions in the presence of epinephrine were inhibited by the adrenergic antagonists phentolamine and propranolol, suggesting the involvement of α- and β-adrenergic receptors in the stimulation of capacitation and the acrosome reaction. In addition, phenylephrine, an α-adrenergic agonist, was as potent as epinephrine in the stimulation of acrosome reactions, but activation was reduced. Isoproterenol, a β-adrenergic agonist, was as potent as epinephrine in the stimulation of activation, but acrosome reactions were reduced. High percentages of both activation and acrosome reactions were observed only in the presence of epinephrine, norepinephrine, or phenylephrine and isoproterenol together.

In this study the mechanisms involved in ..cap alpha../sub 1/-adrenergic receptor-mediated Ca/sup 2 +/ mobilization at the level of the plasma membrane were investigated. Stimulation of /sup 45/Ca/sup 2 +/ efflux from saponin-permeabilized DDT/sub 1/ MF-2 cells was observed with the addition of either the ..cap alpha../sub 1/-adrenergic agonist phenylephrine and guanosine-5'-triphosphate or the nonhydrolyzable guanine nucleotide guanylyl-imidodiphosphate. In the presence of (/sup 32/P) NAD, pertussis toxin was found to catalyze ADP-ribosylation of a M/sub r/ = 40,500 (n = 8) peptide in membranes prepared from DDT/sub 1/, MF-2 cells, possibly the ..cap alpha..-subunit of N/sub i/. However, stimulation of unidirectional /sup 45/Ca/sup 2 +/ efflux by phenylephrine was not affected by previous treatment of cells with 100 ng/ml pertussis toxin. These data suggest that the putative guanine nucleotide-binding protein which couples the ..cap alpha../sub 1/-adrenergic receptor to Ca/sup 2 +/ mobilization in DDT/sub 1/ MF-2 cells is not a pertussis toxin substrate and may possibly be an additional member of guanine nucleotide binding protein family.

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.

With the development of radioligand binding assays for identifying hormone receptors directly, it has become evident that the number of receptors in a particular cell or tissue does not remain constant but can in fact, be regulated by a variety of agents1. Receptor concentrations decrease with exposure to homologous hormone (called densitization or down-regulation) but may be affected differently by heterologous hormones. Thus in the adrenergic contractile response of rabbit myometrium, mediated by α-adrenergic receptors2, the concentration of α-adrenergic receptors is regulated by sex steroids3,4. This oestrogen treatment increases the concentration of myometrial α-adrenergic receptors approximately threefold over that measured in myometrium from immature rabbits, and, furthermore, is accompanied by an increased contractile response to catecholamines5. We report here that, after 24 h in organ culture. rabbit myometrium is more sensitive to α-adrenergic stimulation in vitro, and that particulate fractions prepared from cultured myometrium have a threefold increase in αadrenergic receptors but not β-adrenergic receptors when compared with myometrium that has not been cultured. Our results suggest that the myometrium α-adrenergic receptor, in addition to being regulated by oestrogen, seems to be under tonic inhibitory control in vivo and that this control may only in part be accounted for by catecholamines.

We have previously shown that the hamster sperm acrosome reaction, an event essential for fertilization, is stimulated by beta-adrenergic agonists and inhibited by beta-adrenergic antagonists. In the present report, we describe attempts to use (+/-)-9-aminoacridylpropranolol (9-AAP), a fluorescent derivative of a potent beta-adrenergic antagonist, to microscopically detect beta-adrenergic receptors on cauda epididymal hamster spermatozoa in vitro. 9-AAP binding to washed hamster spermatozoa was localized primarily in the acrosomal region, but we were unable to consistently displace the 9-AAP with the biologically active (-)-stereoisomers of several beta-adrenertic agonists and antagonists. Such displacement is necessary in order to separate binding to receptors from "nonspecific binding." Thus we did not detect sperm beta-adrenertic receptors by this method. Failure to detect the receptors with 9-AAP may be due to their presence in numbers too low for detection by this compound or to the masking of the receptors in these uncapacitated sperm. However, we could displace 9-AAP with either 5.0 mM NH4Cl or 1.2 microgram/ml nigericin, compounds capable of discharging pH gradients across cell membranes. These compounds have also been previously reported to displace the fluorescent portion of 9-AAP, 9-aminoacridine from the acrosome by such mechanism. The present results suggest that 9-AAP fluorescence does not always represent binding to beta-adrenergic receptors or "nonspecific binding," but may also represent the concentration of 9-AAP in acidic compartments within a cell.

To establish a model of airway smooth muscle function we studied binding of [3H]dihydroalprenolol [( 3H]DHA), a beta-adrenergic antagonist, to membrane preparations of porcine trachealis muscle and investigated the response of adenylate cyclase to l-isoproterenol in tissue and plasma membranes. [3H]DHA binding was of high affinity (Kd = 1.0 +/- 0.1 nM), was saturable (Bmax = 87.6 +/- 13.2 fmol/mg protein), and was 90% beta 2 and 10% beta 1. Adenylate cyclase activity in the membrane preparation was (in pmol.10 min-1.mg protein-1 +/- SE): basal 420 +/- 74, guanosine 5'-triphosphate (GTP) (10 micron) 600 +/- 45, GTP (10 microM) + l-isoproterenol (100 microM) 660 +/- 63, NaF (10 mM) 1,500 +/- 134, and forskolin (100 microM) 3,000 +/- 410. Guanosine 5'-diphosphate (GDP) and GTP were active cofactors; l-isoproterenol appeared to function as an effector exchanging GTP for GDP on the guanine nucleotide regulatory protein. There was close agreement of the effective dose (ED50) of the l-isoproterenol-induced relaxation (0.95 +/- 0.45 microM) and the inhibitory constant of l-isoproterenol binding (0.39 +/- 0.10 microM). l-Isoproterenol (100 microM) induced a 100% increase in adenosine 3',5'-cyclic monophosphate (cAMP) levels in tissue strips over basal activity. Investigation of the difference in adenylate cyclase activity between tissue and plasma membranes revealed that l-isoproterenol responsive adenylate cyclase was diminished after initial homogenization. Electron microscopy demonstrated disruption of all cells at this early stage of preparation. The decrease in l-isoproterenol responsive adenylate cyclase following cell rupture is different from other tissues and suggests a difference in the actions of beta-agonist in smooth muscle compared with other tissues.

Hamster spermatozoa, preincubated for 3 h in a culture medium containing bovine serum albumin (BSA) and a protein-free ultrafiltrate of a bovine adrenal cortex preparation, were unable to fertilize cumulus-free eggs. In contrast, if the culture medium also contained a protein-free ultrafiltrate of a bovine adrenal medulla preparation, 92% of the inseminated eggs were fertilized within 2 h. Replacement of the adrenal medulla preparation with 20.0 pM (-)-epinephrine (BSA and the adrenal cortex preparation also present) resultedin 85% of the inseminated eggs being fertilized. If 20.0 pM (-)-phenylephrine, an 3-adrenergic agonist or 20.0 pM (-)-isoproterenol, a j3-adrenergic agonist, was used in place of epinephrine, the percentage of fertilized eggs was 11% and 50%, respectively. With both phenylephrine and isoproterenol present. 90% of the eggs were fertilized. Furthermore, fertilization in the presence of 20.0 pM (-)-epinephrine could be inhibited by equimolar concentrations of either phentolamine, an n-adrenergic antagonist, (0% fertilization) or (-)-propranolol, a 13-adrenergic antagonist, (18% fertilization). These results, together with our previous work, suggest the involvement of adrenergic receptors in the development of the fertilizing ability of hamster spermatozoa in vitro.

In this study, we have used an alpha 1-adrenergic receptor photoaffinity ligand, 2-[4-(4-azido-3-iodo-benzoyl)-piperazin-1-yl]-4-amino-6, 7-dimethoxyquinazoline (125I-APD), to label covalently the alpha 1-adrenergic receptor in a smooth muscle cell line. Our results indicate that in the absence of light, (125I)APD binds reversibly to a site in the DDT1 MF-2 cell membranes having pharmacological characteristics of an alpha 1-adrenergic receptor. Following incorporation of (125I)APD into partially purified membranes a single labeled band of protein with a Mr of 81 000 was visualized by autoradiography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incorporation of (125I)-APD into this band was affected by adrenergic agonists and antagonists in a manner consistent with an alpha 1-adrenergic interaction. Prazosin (alpha 1-selective) blocked incorporation of the label into the Mr = 81 000 protein while yohimbine (alpha 2-selective) did not. Of the adrenergic agonists, (-)-epinephrine and (-)-norepinephrine but not (-)-isoproterenol blocked labeling of the Mr = 81 000 protein. We conclude that the ligand binding site of the DDT1 MF-2 cell alpha 1-adrenergic receptor resides in a Mr = 81 000 protein.

Putative receptors for arginine-vasopressin (AVP) in rat brain were localized and characterized using autoradiographic and radioligand- binding techniques. When brain slices were incubated with tritium- labeled vasopressin, an intense concentration of binding sites was identified which included the medial aspects of the amygdala. Binding of labeled AVP to membrane preparations from this brain region was saturable, specific, reversible, and of high affinity. An antagonist of the peripheral pressor activity of vasopressin was found to be a potent competitor for binding to this site.

The liver undergoes a biochemical and morphological circadian transformation. In this paper, we document circadian variation in the binding parameters of the hepatic epidermal growth factor receptor (EGFR). Liver membranes were prepared from ad libitum fed or fasted male CD2F1 mice killed at different circadian phases at 4 h intervals. Bmax (maximum binding) and Kd (dissociation constant) varied in a rhythmic fashion. The range of change for Bmax along the 24 h time scale was 423%. For Kd, it was 162%. Both peaked late in the dark span, and decreased late in the light span. Fasting and EGF treatment reduced Bmax and the amplitude of circadian variation.