Bovine Vasopressin Research Articles

Comparative pharmacology of bovine, human and rat vasopressin receptor isoforms

In this study, we characterized the bovine vasopressin V 1a, V 1b, V 2 receptor isoforms and compared their pharmacological properties to those of corresponding rat and human vasopressin receptor subtypes. Specific binding sites of high affinity for vasopressin were found in all bovine tissues tested (kidney, liver and pituitary). Using a large series of recent peptidic and non-peptidic selective vasopressin agonists or antagonists, we demonstrated the presence of vasopressin V 2, V 1a or V 1b receptors in the kidney, liver and pituitary bovine tissues, respectively. This extensive characterization of bovine vasopressin receptor isoforms validates the pharmacological vasopressin receptor classification earlier established for the rat and human species. As expected, the bovine vasopressin receptors look much more like human receptors than rat ones. Interestingly, among the three vasopressin receptor isoforms studied, the vasopressin V 1b receptor subtype is the best conserved for the three species studied.

Pharmacological characterization of F-180: a selective human V(1a) vasopressin receptor agonist of high affinity.

1. The pharmacological properties of F-180, a vasopressin (VP) structural analogue, were determined on CHO cells expressing the different human vasopressin and oxytocin (OT) receptor subtypes. Binding experiments revealed that F-180 exhibited a high affinity for the human V(1a) receptor subtype (K(i)=11 nM) and was selective for this receptor subtype. 2. Functional studies performed on CHO cells expressing human V(1a) receptors indicate that similarly to AVP, F-180 can stimulate the accumulation of inositol phosphate. The activation constant (K(act)) for both F-180 and AVP was 1.7 nM. F-180 was also an agonist for the human V(2) and V(1b) receptor subtypes and an antagonist for the human OT receptor. 3. Since marked species pharmacological differences for vasopressin receptors have been described, we studied the properties of F-180 on various mammalian species. F-180 showed high affinity and good selectivity for human and bovine V(1a) receptors, but weak affinity and non selective properties for rat V(1a) receptors. 4. To assess the functional properties of F-180 on a native biological model, we performed studies on primary cultures of cells from bovine zona fasciculata (ZF). As AVP, F-180 stimulated inositol phosphate accumulation and cortisol secretion with similar efficiency. 5. In conclusion, we demonstrate that F-180 is the first selective V(1a) agonist described for human and bovine vasopressin receptors. Therefore F-180 can be used as a powerful pharmacological tool to characterize the actions of vasopressin that are mediated by V(1a) receptor subtypes.

Repression of vasopressin gene expression by glucocorticoids in transgenic mice: evidence of a direct mechanism mediated by proximal 5′ flanking sequence

Glucocorticoids are known to exert multiple effects upon neuronal systems and neuronal gene expression but the molecular mechanisms through which these effects are mediated are largely undefined. In this study, a transgenic mouse model that expresses a bovine vasopressin transgene was used to investigate the mechanisms by which this neuropeptide gene is repressed by glucocorticoids. Using both northern analysis and a reverse transcriptase–polymerase chain reaction assay, depletion of glucocorticoids with the 11,β-hydroxylase inhibitor metyrapone was shown to result in a dexamethasone-reversed increase in ectopic adrenal transgene messenger RNA levels. This result shows that sequences within the confines of the 3.5 kb transgene are sufficient to mediate repression by glucocorticoids, and indicates the involvement of a type II glucocorticoid receptor mechanism which is independent of cellular context. Evidence for the involvement of cis-acting repressive elements in the proximal 5′ flanking sequence was obtained in further studies in which bovine transgene constructs were shown to be negatively regulated by dexamethasone in 293 cells. The further demonstration that recombinant glucocorticoid receptor binds to a vasopressin promoter fragment in an in vitro electrophoretic mobility shift assay provided additional evidence of a direct mechanism of repression. Both in vitro studies were consistent with the presence of a glucocorticoid regulatory element within the region −300 to −155 of the transcription start site. The use of an in vivo transgenic system combined with in vitro analyses of gene promoter fragments enabled the characterization of the molecular mechanisms which effect physiological changes in vasopressin gene expression, and provided evidence of a direct mechanism of repression mediated by sequences within the vasopressin gene promoter.

RNAs encoded by a 3.5-kb bovine vasopressin gene construct are targeted to the neurohypophysis of transgenic mice

Recent studies have established that the RNA coding for the neuropeptide arginine-vasopressin (AVP) is expressed in the neurohypophyseal compartment of the hypothalamo-neurohypophyseal system. In order to determine the molecular mechanisms that direct this novel expression pattern we have now investigated whether an AVP transgene is similarly regulated. Using a reverse transcriptase-polymerase chain reaction (RT-PCR) approach that permits simultaneous analysis of both endogenous and transgene RNA levels, we have demonstrated that RNA derived from a 3.5-kb bovine vasopressin transgene is expressed in the neurohypophysis of transgenic mice, and is up-regulated by a physiological stimulus (salt-loading) in a similar manner to mouse AVP RNA. Sequences conserved between this region of the murine and bovine AVP genes are therefore sufficient to mediate neurohypophyseal expression. These lines of transgenic mice will serve as a model for the delineation of sequences that target expression beyond the neuronal perikaryon.

Bovine Oxytocin Transgenes in Mice: HYPOTHALAMIC EXPRESSION, PHYSIOLOGICAL REGULATION, AND INTERACTIONS WITH THE VASOPRESSIN GENE

To gain insights into the molecular mechanisms that restrict the expression of the oxytocin gene to anatomically defined groups of neurons in the hypothalamus, we generated transgenic mice bearing bovine oxytocin genomic fragments. Appropriate neuron-specific and physiological regulation was observed in mice bearing transgene bOT3.5, which consists of the oxytocin structural gene flanked by 0.6 kilobase pair (kbp) of upstream and 1.9 kbp of downstream sequences. bOT3.5 is expressed in oxytocin magnocellular neurons in the mouse supraoptic nucleus and paraventricular nucleus, but transgene RNAs are excluded from vasopressin neurons. Replacement of the drinking diet of the transgenic mice with 2% (w/v) NaCl for 7 days significantly increased the abundance of bovine oxytocin transcripts in the supraoptic nucleus, but not in the paraventricular nucleus, in parallel with the endogenous mouse oxytocin RNA. Surprisingly, mimicry of the endogenous oxytocin gene expression pattern was lost with larger transgenes. Addition of 0.7 kbp of contiguous downstream sequences (transgene bOT) or linkage to the bovine vasopressin gene (transgene VP-B/bOT3.5) repressed hypothalamic expression. No mice were derived bearing transgene bOT6.4, which consists of the oxytocin structural gene flanked by 3 kbp of upstream and 2.6 kbp of downstream sequences, suggesting that the presence of this DNA is detrimental to normal embryonic development. These data suggest that while bOT3.5 contains sufficient cis-acting sequences to mediate expression to particular subsets of hypothalamic neurons, the overall regulation of the oxytocin gene is governed by multiple interacting enhancers and repressors.

Neuron-specific expression and physiological regulation of bovine vasopressin transgenes in mice.

We have used transgenic mice to analyse the regulation of the bovine vasopressin (BVP) gene. We find that the restriction of BVP gene expression to anatomically and functionally distinct hypothalamic neuronal groups is achieved, in part, by selective repression. The expression of a 1.25 kb BVP proximal promoter, which on its own confers general expression of a reporter to most peripheral and brain tissues, was limited by sequences in the BVP structural gene to neural cells in the adrenal medulla and brain. Transgene expression in the hypothalamus was shown to be regulated by the physiological stimulus of dehydration in parallel with the endogenous gene. The expression of a larger 13.4 kb BVP transgene, containing 9 kb of 5' upstream sequence, the VP structural gene and 1.5 kb 3' of the transcription unit, was even more restricted and resembles that of the endogenous mouse gene. Hypothalamic expression of the 13.4 kb BVP transgene was regulated appropriately in response to an osmotic challenge.

The identification of a cis-acting element involved in cyclic 3‘,5‘-adenosine monophosphate regulation of bovine vasopressin gene expression.

Cyclic adenosine 3',5'-monophosphate (cAMP) has been implicated as an intracellular messenger mediating osmotic regulation of expression of the gene encoding the neuropeptide vasopressin (VP) in the hypothalamus. We have used a heterologous transient transfection system to demonstrate that cAMP regulates the bovine VP gene promoter following transfection into CV1 cells. Mutational analysis identified a bovine VP cAMP-responsive element (BVP-CRE) 120-112 base-pairs upstream of the start of transcription. DNase I footprint analysis using nuclear protein extract from CV1 cells showed protection at the site of the BVP-CRE. Protection of the BVP-CRE was also observed using purified AP1 protein, while there was a weak interaction with the BVP-CRE using purified rat CREB protein. Nuclear proteins purified from the rat supraoptic nucleus bind to the BVP-CRE. As transgenic mouse studies have shown that the bovine VP gene is subject to appropriate physiological regulation in the mouse hypothalamus (Ang, H. L., Funkhouser, J., Carter, D. A., Ho, M. Y., and Murphy, D. (1991) Soc. Neurosci. Abstr. 513, 12), these data indicate a role for the BVP-CRE element in mediating VP gene expression in vivo. These data demonstrate that cAMP regulates bovine VP gene expression in vitro via a cis-acting element within the VP promoter, and this activation may be mediated by members of the AP1/ATF/CREB family of transcription factors.

Morphology of adenohypophysial tumors in mice transgenic for vasopressin-SV40 hybrid oncogene.

Transgenic mice for the promoter sequence of bovine arginine vasopressin (AVP) gene fused to large SV40 T-antigen coding sequence develop pituitary tumors and insulin-producing pancreatic tumors. In order to establish the cellular composition of the pituitary tumors, histological, immunocytochemical, in situ hybridization, and electron microscopic technics were applied. Pituitary anterior lobe tumors were identified in 10 out of 14 glands examined. In 2 of these cases, intermediate lobe tumors were also found. The anterior lobe tumors contained a variable number of GH immunoreactive cells. In situ hybridization performed in 7 cases revealed a diffuse distribution of GH messenger RNA over all tumor cells. Ultrastructurally, the tumors contained undifferentiated cells with very small secretory granules and rare cells showing some resemblance to somatotrophs. The results indicate that these pituitary tumors are composed of undifferentiated somatotrophs. The presence of a few PRL immunoreactive cells in four tumors and scattered TSH immunoreactive cells in two tumors supports the view that somatotrophs have the potential to produce PRL and TSH. The intermediate lobe tumors were immunoreactive for ACTH and intensely positive for POMC mRNA. In the nontumorous adenohypophyses, no hyperplasia of any cell type was noted. Several GH immunoreactive cells exhibited pleomorphic, giant nuclei and mitoses. In conclusion, the majority of transgenic mice for AVP/large T-antigen develop pituitary tumors originating in and composed of somatotrophs. Less frequently, intermediary lobe tumors were present as well. AVP/SV40 transgenic mice provide a unique experimental model for somatotroph tumors that are neither preceded by, nor associated with somatotroph hyperplasia.

Testicular Oxytocin Gene Expression in Seminiferous Tubules of Cattle and Transgenic Mice*

We are using transgenic mice to study the regulation of the bovine vasopressin (VP) and oxytocin (OT) genes. Prompted by the observation that mice bearing a bovine OT transgene express bovine OT RNA in their testes, we investigated the expression of the VP-OT locus in normal mice and cattle. Normal wild-type mice do not have detectable levels of either VP or OT RNA in their testes. Normal cattle are also devoid of detectable VP transcripts, but have relatively high levels of testicular OT RNA. Additionally, OT, but not VP, peptide is detectable by HPLC. In situ hybridization to RNA in bovine testicular tissue sections localized OT transcripts to seminiferous tubules, with a distribution similar to that of alpha-inhibin, suggesting expression in Sertoli cells. Interestingly, the bovine OT RNAs in the transgenic mouse testes were also shown by in situ hybridization to have the same distribution. These data suggest that the cis-acting regulatory sequences responsible for expression of the OT gene in bovine Sertoli testis reside within the limits of the transgene used in this study. Further, the trans-acting factors present in murine testicular cells are able to recognize these elements, although they do not express the endogenous mouse OT gene in this tissue.

A morphological analysis endocrine tumour genesis in pancreas and anterior pituitary of AVP/SV40 transgenic mice.

Insertion into the mouse genome of the hybrid oncogene made up of bovine vasopressin gene derived 5' upstream sequences and the coding sequences of SV40 large T-antigen promoted tumours in anterior pituitary and endocrine pancreas of mice bearing this transgene. In order to investigate the morphology of the steps in the neoplastic process, we used light and electron microscopy to study these organs in 42 animals belonging to the 3rd, 4th and 5th generations, subdivided into 4 age groups from 20 days to 100 days of life. Antibodies to large T-antigen were used to identify sites of expression of the hybrid oncogene, thus monitoring the steps in neoplastic transformation. Large T-antigen immunoreactivity was identified in dysplastic lesions of younger animals and in both dysplastic lesions and tumours of older mice. Insulin (100% of cases) and pancreatic polypeptide (25% of cases) immunoreactivities were revealed in pancreatic lesions but no hormonal immunoreactivity was detected in the pituitary lesions. The ultrastructural study confirmed that the majority cell population of the pancreatic neoplasms was B-type and that the anterior pituitary tumours were poorly granulated. The subcellular localization of large T-antigen immunoreactivity was investigated by the immunogold method and was confined to the heterochromatin of tumour cell nuclei. These findings provide evidence for the dysplasia-neoplasia sequence in the genesis of endocrine tumours of pituitary and pancreas of transgenic mice. The vasopressin-SV40 large T-antigen transgenic mice may therefore be an useful model for the study of endocrine cell oncogenesis.

Chromosomal assignment of human sequences encoding arginine vasopressin-neurophysin II and growth hormone releasing factor.

Complementary DNA clones encoding bovine vasopressin and human pancreas growth hormone releasing factor have been used to map homologous sequences in the human genome. Assignment of both cloned sequences to human chromosome 20 was accomplished by hybridization of insert DNAs to a panel of human-rodent somatic cell hybrids. Both these probes have been used to examine the structure of their respective genes in DNA from various individuals. No restriction fragment variants for growth hormone releasing factor have yet been found. Analysis of populations for restriction fragment length polymorphisms associated with disease states involving arginine vasopressin is underway.

Recent gene conversion involving bovine vasopressin and oxytocin precursor genes suggested by nucleotide sequence.

The nonapeptide hormones arginine vasopressin (AVP) and oxytocin (OT) are synthesized in the hypothalamus together with their carrier proteins, the neurophysins, as common polypeptide precursors. The organization of these precursors has been established by sequence determination of cloned bovine cDNAs encoding prepro-arginine vasopressin-neurophysin II (prepro-AVP-NPII) and prepro-oxytocin-neurophysin I (prepro-OT-NPI). When the mRNA sequences coding for the conserved middle part of the neurophysins were compared, we found that these sequences are not merely similar but identical. The primary structure of the chromosomal genes now determined shows that both genes, which appear to have arisen by a gene duplication, are split into three exons, each encoding a functional domain of the precursor polypeptide. Sequence comparison reveals that the stretch of sequence identity within the two mRNAs is probably the result of a gene conversion encompassing exon B, which encodes the conserved part of the neurophysins, and part of the preceding intron.

The neurophysin domain of human vasopressin precursor

Bovine vasopressin precursor is composed of three domains, namely vasopressin, neurophysin II (MSEL-neurophysin) and a glycopeptide, which are cut out by intraneuronal processing [ 11. In most placental mammals investigated, on the one hand two neurohypophysial hormones, arginine vasopressin and oxytocin have been identified [2,3], on the other hand two neurophysins, termed MSELand VLDV-neurophysins according to the nature of amino acids in positions 2,3,6 and 7, have been characterized ]2,4]. In man, the two hormones have been isolated [5] using the neurophysin complex procedure [6] and the two neurophysins have been purified by polyacrylamide gel electrophoresis [7,8]. As a response to nicotine stimulation, arginine vasopressin and MSEL-neurophysin are selectively secreted in blood [7,8] and this co-secretion agrees with the presence of a common precursor similar to the one identified in ox. Furthermore a human neurohypophysial glycopeptide, homologous to the bovine glycopeptide [9], has recently been identified [IO]. We report here the complete amino acid sequence of human MSEL-neurophysin, homologous to the neurophysin component of the bovine vasopressin precursor. pressor activity, the fourth 0.34 U/mg of oxytocic activity and 0.36 U/mg of pressor activity. Extraction, carried out on l-g samples with 0.1 M HCl (50 ml/g), was followed by molecular sieving on Sephadex G-75 in 0.1 M formic acid and the crude neurophysin fraction was subjected to ion-exchange chromatography on DEAE-Sephadex A-50 using a stepwise ionic-strength gradient made with pyridine acetate (pH 5.9) under the conditions described for bovine neurophysins [ 111. MSEL-neurophysin was recovered in the second peak of the fraction eluted over 0.15-0.30 M and VLDV-neurophysin in the third peak. A C-terminal truncated MSEL-neurophysin was found in the peak eluted over 0.4-0.6 M. In a typical experiment, 1 g acetonic posterior pituitary powder gave 33.5 mg crude neurophysins from which 2.7 mg (300 nmol) of intact MSEL-neurophysin were isolated.

Differential effects of dithiothreitol and iodoacetamide on corticotropin-releasing factor (CRF) activity of bovine hypothalamic CRFs and vasopressin.

Various fractions were tested in vivo for corticotropin-releasing factor (CRF) activity after Sephadex G-100 fractionation of 0.1-N HCl extracts of bovine hypophyseal stalk or cerebral cortex. Female rats pretreated with chlorpromazine, morphine, and Nembutal were used for CRF assay. CRF-A (void volume fractions; big CRF), CRF-B (Kav = 0.583), and CRF-C (salt volume fractions) of bovine hypophyseal stalk and lysine or arginine vasopressin all induced clear-cut stimulation of ACTH and corticosterone in the assay rat, whereas they were ineffective in acutely hypophysectomized rats. Control fractions purified from bovine cerebral cortex had no CRF activity. Treatment of arginine and lysine vasopressin and CRF-C with dithiothreitol and iodoacetamide completely abolished their CRF activity, whereas the CRF activities of CRF-A and CRF-B were unaltered by these treatments. Treatment with iodoacetamide alone had no effect on the CRF activity of any of these substances. Fractionation of either CRF-C or arginine vasopressin on Sephadex G-15 yielded a CRF-active peak at a Kav of 0.35. We conclude that 1) three different forms of CRF exist in bovine hypophyseal stalk; 2) CRF-A and CRF-B are unrelated to vasopressin and require neither a disulfide bond(s) nor a sulfhydryl group(s) for their CRF activity; 3) reduction of the disulfide bond of vasopressin destroys both CRF and antidiuretic activities; 4) CRF-C requires an intact disulfide bond(s) for its CRF activity and is likely to be either vasopressin itself or a substance closely related to vasopressin; and 5) CRF-B is likely to be the physiologically important form of bovine CRF.