Molecular cloning and functional characterization of a vasotocin receptor subtype that is expressed in the shell gland and brain of the domestic chicken.

In chickens, oviposition is correlated with increased plasma levels of the neurohypophysial hormone vasotocin, and vasotocin stimulates contraction of uterine strips in vitro. A gene encoding a vasotocin receptor subtype that we have designated the VT1 receptor was cloned from the domestic chicken. The open reading frame encodes a 370-amino acid polypeptide that displays seven segments of hydrophobic amino acids, typical of guanine nucleotide-protein-coupled receptors. Other structural features of the VT1 receptor include two potential N-linked glycosylation sites in the extracellular N-terminal region, a conserved aspartic acid in transmembrane domain 2 that is found in nearly all guanine nucleotide-protein-coupled receptors, and two potential protein kinase C phosphorylation sites in the third intracellular loop and C-terminal tail. Expressed VT1 receptors in COS7 cells bind neurohypophysial hormones with the following rank order of potency: vasotocin congruent with vasopressin > oxytocin congruent with mesotocin > isotocin. In addition, the expressed VT1 receptor mediates vasotocin-induced phosphatidylinositol turnover and Ca(2+) mobilization. In the chicken, expression of VT1 receptor gene transcripts is limited to the shell gland (uterus) and the brain. Thus, the VT1 receptor that we have cloned may mediate contractions of the shell gland during oviposition and activate reproductive behaviors known to be stimulated by vasotocin in lower vertebrates.

Transcription of the beta2-adrenergic receptor gene in rat liver is regulated during early postnatal development by an upstream repressor element.

As early postnatal development of the male rat proceeds, there is a decline in transcription of the beta2-adrenergic receptor gene in liver which is associated with a decline in beta2-adrenergic receptor mediated glucose mobilization. In this study, primary cultures of rat hepatocytes transiently transfected with fusion genes containing various segments of beta2-adrenergic receptor gene 5'-flanking DNA fused to a promoterless luciferase reporter gene were used to identify genetic elements that might control beta2-adrenergic receptor gene expression during the first 10 days of postnatal life. We found that 261 bp of beta2-adrenergic receptor gene 5'-flanking region (-372 to -95, start of translation is +1) was sufficient to direct high luciferase expression in fetal day 18 hepatocytes and therefore included the beta2-adrenergic receptor gene promoter. Luciferase activities in fetal day 18 hepatocytes transfected with pbeta2AR(-372/-95), pbeta2AR(-1,335/-95) and pbeta2AR(-3,349/-95) were fourfold greater than that in either postnatal day 5 or postnatal day 10 hepatocytes transfected with the same fusion genes. By use of gel mobility shift assays, we observed increased protein binding to a 50 bp segment (-372 to -323) of the beta2-adrenergic receptor gene 5'-flanking region with nuclear extracts prepared from postnatal day 5 and postnatal day 10 hepatocytes compared to fetal day 18 hepatocytes. These findings suggest the presence of a regulatory element in the 5'-flanking region of the beta2-adrenergic receptor gene that appears to be involved in suppression of transcription of the beta2-adrenergic receptor gene in liver during early postnatal development.

Association of hepatic beta 2-adrenergic receptor gene transcript destabilization during postnatal development in the Sprague-Dawley rat with a M(r) 85,000 protein that binds selectively to the beta 2-adrenergic receptor mRNA 3'-untranslated region.

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.

Transcriptional and posttranscriptional regulation of hepatic beta 2-adrenergic receptor gene expression during development.

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.

Atrial natriuretic factor increases the magnitude of duodenal spontaneous phasic contractions.

The present investigation was designed to determine if atrial natriuretic factor relaxes non-vascular smooth muscle. Rather than cause a relaxation, atrial natriuretic factor induced a two-to-four fold enhancement in the amplitude of the spontaneous phasic contractions of duodenal longitudinal muscle. Dose-response curves revealed that ANF enhanced these contractions over a concentration range of 10 picomoles to 100 nanomoles with the ED50 at 1 nanomolar. The increased amplitude of contraction began within 30 seconds and was calcium-dependent. The increased force of contraction was associated with a three-fold increase in cyclic GMP levels and activation of particulate guanylate cyclase [E.C.4.5.1.2.]. Atrial natriuretic factor had its half-maximal [ED50] activation of guanylate cyclase at its 1 nM concentration while maximal enhancement was at its 100 nM concentration in duodenum, jejunum, and ileum. Atrial natriuretic factor did not stimulate adenylate cyclase [E.C.4.6.1.1.]. Thus, atrial natriuretic factor increases the force of the spontaneous phasic contractions of the small intestine which are calcium-dependent and associated with activation of the guanylate cyclase-cyclic GMP system.

Atrial natriuretic prohormone peptides 1-30, 31-67, and 79-98 vasodilate the aorta.

Human prohormone atrial natriuretic peptides 1-30, 31-67, and 79-98 caused vasodilation of porcine aortas which began in 30 seconds and was maximal at 10 minutes. These three peptides were found to be equally potent to atrial natriuretic factor in their vasodilatory activity which was found with or without endothelium present. This vasodilation was associated with a 4 to 5 fold increase in cyclic GMP in the aorta secondary to activation of particulate guanylate cyclase [E.C. 4.6.12]. These data demonstrate that three N-terminal peptide segments of the atrial natriuretic factor prohormone cause vasodilation.

The kinetic properties and sensitivity to pyruvate inhibition of tissue lactate dehydrogenase in diving and nondiving reptiles.

The kinetic properties and susceptibility to pyruvate inhibition of lactate dehydrogenase (LDH) were examined in three tissues of the freshwater turtle, Pseudemys scripta, and two snake species, Elaphe obsoleta and Nerodia rhombifera. Brain and heart LDH activity of the snakes surpassed that of Pseudemys at pyruvate concentrations between 0.03 and 0.50 mM. The snakes also had lower apparent Km and higher Vmax values than Pseudemys. Liver LDH activity, apparent Km, and Vmax were highest in Elaphe, and there were no differences between Km or Vmax values of Nerodia and Pseudemys. In each tissue, snake LDH was less susceptible to pyruvate inhibition than was Pseudemys LDH. The results indicate that Pseudemys LDH is no better adapted to withstand anaerobic conditions than is that of Nerodia or Elaphe.

Effects of hyperbaric oxygen and glutathione on mammalian liver metabolism.

The effects of hyperbaric oxygen tensions on oxygen consumption and succinate dehydrogenase (SDH) activity of mouse liver were investigated. Liver homogenates exposed to a PO2 of 3837 mm Hg for 30 min showed a 50.6% reduction in oxygen consumption compared to controls exposed to nitrogen at ambient pressure. The SDH activity was significantly reduced during a 3-h exposure to a PO2 of 3796 mm Hg. The effects of glutathione as a protective agent against oxygen toxicity were also examined. Mouse liver pretreated with reduced glutathione and exposed to hyperbaric oxygen tensions showed higher rates of oxygen consumption than untreated controls. Oxidized and reduced glutathione protected SDH against hyperbaric oxygen inactivation. It is concluded that glutathione can stimulate oxygen consumption and maintain SDH activity after exposure to hyperbaric oxygen by increasing succinate formation through the glutathione-succinate shunt.

Oxygen-induced inhibition of mouse brain lactate dehydrogenase.

The lactate dehydrogenase (LDH) activity of mouse brain homogenates was examined after exposure to hyperbaric oxygen (5763.8 mm Hg Po2) and compared to room air controls (158.8 mm Hg Po2). The effect of reduced glutathione on LDH activity after hyperbaric oxygen exposure was also examined. The activity of LDH after treatment with hyperbaric oxygen was significantly diminished when compared with controls. In the presence of reduced glutathione, homogenates exposed to hyperbaric oxygen demonstrated higher activity than did homogenates incubated without glutathione. It is concluded that oxygen-induced inhibition occurs through the oxidation of essential free sulfhydryl groups and that this oxidation can either be prevented by reduced glutathione or the disulfide bridges may be reduced to free sulfhydryl groups by the glutathione after oxidation.