Work described in this application will involve the use of amnion cells to elucidate principal mechanisms of up and down regulation of the OTR. The investigator will: (1) isolate the rabbit OTR gene from rabbit genomic libraries, knowledge of the 5-prime flanking and coding sequences will allow the completion of aims 2 to 5; (2) determine the effects of regulatory agents on OTR gene expression. These studies involve measurement of OTR mRNA levels by RNAase protection and transcription initiation rates by nuclear runoff assays. The investigator will also study OTR mRNA stability; (3) describe the promoter region of the rabbit OTR gene and use transient tranfection of cultured cells with deletion and replacement mutants to characterize regulatory elements; (4) determine the effects of regulatory agents on foot printing sites in the OTR promoter and 5' flanking sequence. These studies will define which of the potential regulatory elements (deduced from consensus sequence analysis and transfection studies) are functional when the OTR gene is transcriptionally active and elucidate essential regulators of OTR gene expression; and (5) determine the effects of OTR regulatory agents on the synthesis of OTR protein by immunoabsorption after labelling amnion proteins with (35S)methionine. Antisera will be produced to three separate domains of the OTR.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD026168-08
Application #
2609078
Study Section
Human Embryology and Development Subcommittee 1 (HED)
Project Start
1989-08-01
Project End
2000-11-30
Budget Start
1997-12-01
Budget End
1998-11-30
Support Year
8
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Texas Medical Br Galveston
Department
Obstetrics & Gynecology
Type
Schools of Medicine
DUNS #
041367053
City
Galveston
State
TX
Country
United States
Zip Code
77555
Copland, John A; Zlatnik, Marya G; Ives, Kirk L et al. (2002) Oxytocin receptor regulation and action in a human granulosa-lutein cell line. Biol Reprod 66:1230-6
Jeng, Y J; Liebenthal, D; Strakova, Z et al. (2000) Complementary mechanisms of enhanced oxytocin-stimulated prostaglandin E2 synthesis in rabbit amnion at the end of gestation. Endocrinology 141:4136-45
Soloff, M S; Jeng, Y J; Copland, J A et al. (2000) Signal pathways mediating oxytocin stimulation of prostaglandin synthesis in select target cells. Exp Physiol 85 Spec No:51S-58S
Hoare, S; Copland, J A; Strakova, Z et al. (1999) The proximal portion of the COOH terminus of the oxytocin receptor is required for coupling to g(q), but not g(i). Independent mechanisms for elevating intracellular calcium concentrations from intracellular stores. J Biol Chem 274:28682-9
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Strakova, Z; Copland, J A; Lolait, S J et al. (1998) ERK2 mediates oxytocin-stimulated PGE2 synthesis. Am J Physiol 274:E634-41
Strakova, Z; Soloff, M S (1997) Coupling of oxytocin receptor to G proteins in rat myometrium during labor: Gi receptor interaction. Am J Physiol 272:E870-6
Jeng, Y J; Hinko, A; Soloff, M S (1995) Effectors of cyclic adenosine 5'-monophosphate up-regulating-oxytocin receptors in rabbit amnion cells: isoproterenol, parathyroid hormone-related protein, and potentiation by cortisol. Biol Reprod 53:1051-6
Czaja, M; Kruszynski, M; Hinko, A et al. (1994) Arginine vasopressin antagonism of oxytocin-stimulated PGE2 release from rabbit amnion cells and the activities of thioanalogs of oxytocin and arginine vasopressin. Pol J Pharmacol 46:429-37
Hinko, A; Soloff, M S (1993) Up-regulation of oxytocin receptors in rabbit amnion by adenosine 3',5'-monophosphate. Endocrinology 132:126-32

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