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cAMP-binding proteins in medullary tubules from rat kidney: effect of ADH
S M Gapstur1, S Homma, T P Dousa
1Department of Medicine, Mayo Clinic and Foundation, Rochester, Minnesota 55905.
Abstract:
Little is known of the regulatory steps in the cellular action of vasopressin (AVP) on the renal epithelium, subsequent to the cAMP generation. We studied cAMP-binding proteins in the medullary collecting tubule (MCT) and the thick ascending limb of Henle's loop (MTAL) microdissected from the rat kidney by use of photoaffinity labeling. Microdissected tubules were homogenized and photoaffinity labeled by incubation with 1 microM 32P-labeled 8-azido-adenosine 3',5'-cyclic monophosphate (N3-8-[32P]-cAMP); the incorporated 32P was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Both in MCT and MTAL preparations, the analyses showed incorporation of N3-8-[32P]cAMP into two bands (Mr = 49,000 and Mr = 55,000) that comigrated with standards of the cAMP-dependent protein kinase regulatory subunits RI and RII. In MCT, most of the 32P (80%) was incorporated into RI, whereas in MTAL the 32P incorporated into RI and RII was equivalent. When freshly dissected MCT segments were incubated with 10(-12)-10(-6) M AVP, the subsequent photoaffinity labeling of RI with N3-8-[32P]cAMP was markedly diminished in a dose-dependent manner compared with controls. Our results suggest that cAMP binds in MCT and MTAL to regulatory subunits RI and RII of cAMP-dependent protein kinase. However, in MCT the dominant type of cAMP-dependent protein kinase appears to be type I. The outlined procedure is suitable to indirectly measure the occupancy of RI by endogenous cAMP generated in MCT cells in response to physiological levels (10(-12) M) of AVP.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Researchers identified cyclic adenosine monophosphate (cAMP)-binding proteins in rat kidney tubules. Vasopressin (AVP) reduced cAMP binding to protein kinase type I in the medullary collecting tubule, indicating a regulatory mechanism.
Area of Science:
- Nephrology
- Molecular Biology
- Cellular Signaling
Background:
- The cellular mechanisms of vasopressin (AVP) action in the renal epithelium beyond cyclic adenosine monophosphate (cAMP) generation are not fully understood.
- Identifying cAMP-binding proteins is crucial for elucidating downstream signaling pathways in kidney tubules.
Purpose of the Study:
- To investigate the cAMP-binding proteins in the medullary collecting tubule (MCT) and thick ascending limb of Henle's loop (MTAL) of the rat kidney.
- To determine the effect of AVP on cAMP binding to these proteins in the MCT.
Main Methods:
- Microdissected rat kidney tubules (MCT and MTAL) were photoaffinity labeled with 32P-labeled 8-azido-adenosine 3',5'-cyclic monophosphate (N3-8-[32P]-cAMP).
- Incorporated 32P was analyzed using SDS-PAGE and autoradiography to identify cAMP-binding proteins.
- MCT segments were incubated with varying concentrations of AVP (10(-12) to 10(-6) M) before photoaffinity labeling to assess AVP's effect.
Main Results:
- Photoaffinity labeling identified two cAMP-binding proteins in both MCT and MTAL, corresponding to the regulatory subunits RI and RII of cAMP-dependent protein kinase.
- In MCT, 80% of cAMP binding was to RI, while MTAL showed equivalent binding to RI and RII.
- AVP treatment dose-dependently diminished cAMP binding to RI in MCT.
Conclusions:
- cAMP binds to RI and RII regulatory subunits of cAMP-dependent protein kinase in rat renal tubules (MCT and MTAL).
- The predominant cAMP-dependent protein kinase in MCT is type I.
- The described method allows indirect measurement of RI occupancy by endogenous cAMP in response to physiological AVP levels in MCT cells.