We have identified three different, GTP-sensitive PGE receptors in canine renal outer medulla, papilla and Madin-Darby canine kidney (MDCK) cells, respectively. We propose that each receptor mediates a unique response to PGE2 either in the thick ascending limb or the collecting tubule. The goals of the proposed research are to determine the properties. location and function of these three PGE receptors. One PGE receptor is concentrated in the renal outer medulla, but is undetectable in papilla. This receptor is associated with the inhibitory guanine nucleotide regulatory protein, N/1, and is designated a PGE-N/1 receptor. In the first part of our studies, we propose to isolate cortical and medullary thick limb cells from canine kidney and to test the concepts (a) that the PGE-N/1 receptor is present in thick limb cells and (b) that PGE2 acts through the PGE-N/1 receptor to inhibit adenylate cyclase in the thick limb. We have solubilized and partially purified PGE-N/1, and we also propose to continue efforts to purify this receptor. A second PGE receptor, designated PGE-N/x, comprises more than 80% of the PGE binding activity of MDCK cells. Based on preliminary, indirect evidence from studies with a PGE receptor antagonist, we suggest that PGE-N/x mediates intracellular Ca++ mobilization. We propose to test the concepts (a) that PGE-N/x is present in collecting tubule cells, (b) that this receptor mediates stimulation of a Ca++-dependent cAMP phosphodiesterase, and (c) that PGE2 acts via the PGE-N/x receptor to inhibit the hydroosmotic effect of arginine vasopressin. We also will attempt to find a more abundant source of the PGE-N/x receptor, perhaps fundic mucosa, to facilitate biochemical characterization of the protein. A third GTP-sensitive PGE receptor, designated PGE-N/s, is the most abundant PGE receptor in renal papilla. We think that this receptor mediates stimulation of adenylate cyclase. In the final part of our study, we propose to determine if the papillary PGE receptor is actually couped in N/s. We will use papillary membranes determine (a) if cholera toxin specifically eliminates PGE2-induced GTPase activity or to the inhibitory effect of GTP on PGE2 binding and (b) if there is a correlation between the ability to PGE analogs to affect PGE2 binding and to stimulate adenylate cyclase in papillary membranes. We will also detemine if there is a papillary-like PGE-N/s receptor in collecting tubule cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37DK022042-14
Application #
3483419
Study Section
General Medicine B Study Section (GMB)
Project Start
1978-08-01
Project End
1992-07-31
Budget Start
1991-08-01
Budget End
1992-07-31
Support Year
14
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Michigan State University
Department
Type
Schools of Medicine
DUNS #
193247145
City
East Lansing
State
MI
Country
United States
Zip Code
48824
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Kang, Yeon-Joo; Wingerd, Byron A; Arakawa, Toshi et al. (2006) Cyclooxygenase-2 gene transcription in a macrophage model of inflammation. J Immunol 177:8111-22
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Kuklev, Dmitry V; Smith, William L (2004) Synthesis of long chain n-3 and n-6 fatty acids having a photoactive conjugated tetraene group. Chem Phys Lipids 130:145-58
Kuklev, Dmitry V; Smith, William L (2003) A procedure for preparing oxazolines of highly unsaturated fatty acids to determine double bond positions by mass spectrometry. J Lipid Res 44:1060-6
Smith, William L; Song, Inseok (2002) The enzymology of prostaglandin endoperoxide H synthases-1 and -2. Prostaglandins Other Lipid Mediat 68-69:115-28
Smith, W L; Langenbach, R (2001) Why there are two cyclooxygenase isozymes. J Clin Invest 107:1491-5
Smith, W L; DeWitt, D L; Garavito, R M (2000) Cyclooxygenases: structural, cellular, and molecular biology. Annu Rev Biochem 69:145-82

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