Lawrence E. Cornett

Characterization and localization of 3H-arginine8-vasopressin binding to rat kidney and brain tissue

Anatomic, behavioral and pharmacologic evidence suggests that arginine8-vasopressin (AVP) serves as a CNS neurotransmitter or neuromodulator. We have characterized AVP binding to membrane and tissue slice preparations from brain and kidney, and examined the anatomical distribution of these binding sites. Conditions for the binding assay were optimized using kidney medullary tissue. Binding of 3H-AVP (S.A. = 30-51 Ci/mmol, NEN) to brain and kidney membranes and tissue slices was saturable, temperature dependent, linearly related to protein concentration (or number of tissue slices), reversible, and specific since the ability of cold AVP to displace 3H-AVP from binding was greater than oxytocin and other related peptide fragments. Autoradiographic localization of 3H-AVP binding was restricted to kidney medullary tissue. In brain tissue, 3H-AVP binding was found to occur in concentrated foci. Brainstem areas such as the nucleus tractus solitarius (NTS) showed a high density of AVP binding sites. Since local injections of AVP into the NTS have been shown to influence blood pressure, the present study presents the first anatomical evidence for the presence of AVP specific binding sites which might mediate this effect.

Plasma levels of immunoreactive mesotocin and vasotocin during oviposition in chickens: Relationship to oxytocic action of the peptides in vitro and peptide interaction with myometrial membrane binding sites

Plasma concentrations of immunoreactive vasotocin (AVT) and mesotocin (MT) were measured periodically before and subsequent to spontaneous oviposition in conscious chickens. The concentrations of AVT and MT approximately an hour prior to oviposition were 5.2 +/- 1.1 microU/ml and 14.7 +/- 5.1 pg/ml, respectively. Plasma AVT levels increased abruptly at oviposition (25.1 +/- 3.3 microU/ml) and decreased to 5.0 +/- 0.6 microU/ml within 30 min postoviposition. Significant changes in MT were not observed. The data indicate that AVT is selectively released during oviposition. The uterus was removed immediately after oviposition and the oxytocic potencies of several peptides were tested on muscle strips in vitro. The order of oxytocic potencies was AVT greater than or equal to arginine vasopressin (AVP) much greater than MT = pressinoic acid. Partially purified membranes were prepared from separate portions of the uteri used in the oxytocic assay. [3H]arginine8 vasopressin, [3H]AVP, bound to membranes saturably (Bmax = 17 fmol/mg protein) and with high affinity (Kd = 0.7 nM). The rank order of potency of the peptides in displacing [3H]AVP from the binding sites was the same as in the oxytocic assay which suggests that the [3H]AVP binding sites in uterine membranes represent physiological receptors that interact with AVT during oviposition.

Molecular Cloning and Functional Characterization of a Vasotocin Receptor Subtype That Is Expressed in the Shell Gland and Brain of the Domestic Chicken

Abstract 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 ≅ vasopressin > oxytocin ≅ mesotocin > isotocin. In addition, the expressed VT1 receptor mediates vasotocin-induced phosphatidylinositol turnover and Ca2+ 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.

Molecular cloning and functional characterization of a vasotocin receptor subtype expressed in the pituitary gland of the domestic chicken (Gallus domesticus): avian homolog of the mammalian V1b-vasopressin receptor.

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.

An in situ hybridization and immunohistochemical study of vasotocin neurons in the hypothalamus of water-deprived chickens

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.

Aging: changes in cardiac α1-adrenoceptor responsiveness and

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)

Characteristics of an Adenylate Cyclase Coupled β-Adrenergic Receptor in a Smooth Muscle Tumor Cell Line

AbstractWe have shown that binding of 3H-dihydroalprenolol ([3H] DHA) to DDT1 MF-2 cells and cell membranes was of high affinity, saturable, stereoselective and reversible. The [3H]DHA dissociation constants were 0.63 ± 0.15 nM (n=6) and 0.83 ± 0.04 nM (n=5) for intact cells and cell membranes, respectively, with a binding site concentration for cells of 27,300 ± 5,200 sites/ cell (n=6) and for membranes 468 ± 24 fmoles/mg protein (n=5). The order of agonist competition for the [3H]-DHA binding site of DDT1 cell membranes was isoproterenol (Ki = 0.20 ± 0.07 μM) > epinephrine (Ki = 0.4 ± 0.2 μM) > norepinephrine (Ki = 66.5 ± 5.15 μM) consistent with a β2-selective receptor interaction. Zinterol, a β2-selective antagonist, (Ki = 0.05 ± 0.01 μM) was 18x more effective than metoprolol, a β1-selective antagonist (Ki = 0.9 ± 0.1 μM), in competing for the DHA binding site. A nonlinear iterative curve fitting analysis of zinterol and metoprolol binding isotherms indicated that (p>0.05) DDT1 cells possess a pure p...

Regulation of the β2-Adrenergic Receptor and its Mrna in the Rat Lung by Dexamethasone

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.

Glucocorticoid induction of β-adrenergic receptors in the DDT1 MF-2 smooth muscle cell line involves synthesis of new receptor

We have shown that glucocorticoids induce the appearance of β2-adrenergic receptors in membranes of the ductus deferens smooth muscle cell line (DDT1 MF-2). A concomitant increase in isoproterenol stimulated adenylate cyclase activity in the absence of exogenously applied GTP was observed as was a significantly increased (p < 0.05) sensitivity of the adenylate cyclase system to exogenously applied GTP. However, no significant difference in the maximal velocity of adenylate cyclase between control and steroid treatment was measurable in the presence of sodium fluoride. Induction of β2-adrenergic receptors in DDT1 MF-2 cells is correlated with the presence of steroid receptors (androgen and glucocorticoid) in the cells since estrogens and progesterones had no effect on receptor levels. Finally, utilizing dense amino acid labeling of cells to measure old versus newly synthesized receptor sites by a density shift method, we have documented that glucocorticoid induction of β2-adrenergic receptors involves synthesis of new receptor protein.

Vasopressin receptor subtypes in dorsal hindbrain and renal medulla

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.