Regulation of Adrenomedullin Signaling in Kidney Interstitial Fibroblasts

Adrenomedullin (AM), a potent vasodilatory peptide has beneficial effects in the kidney in vivo. The major aim of the present study was to determine the presence of AM receptor and the biological outcomes of AM on kidney interstitial fibroblasts in culture. Utilizing RT-PCR we found that NRK-49F cells express calcitonin receptor like receptor (CRLR) and receptor activity modifying protein 2 (RAMP2) but not RAMP3. Treatment of these cells with AM resulted in a concentration-dependent increase in cAMP activation. The activation of adenylate cyclase system was enhanced by over-expression of CRLR, RAMP2 and RAMP3. Furthermore, AM-stimulated adenylate cyclase activity was inhibited by AM-[22-52] the AM receptor antagonist. AM also caused a PKA-dependent increase in CRE-luciferase activity. To test the biological consequences of AM treatment and the signaling pathways mediating them, we examined the effect of AM on proliferation of NRK-49F cells and the desensitization of AM receptor. AM caused a significant decrease in proliferation that was AM-receptor mediated but was PKA independent. In addition, AM also caused desensitization of cAMP response within a few minutes of treatment. This effect of AM was also not mediated via cAMP pathway as forskolin failed to desensitize AM receptor, and a PKA-inhibitor did not inhibit the desensitization. Taken together these results demonstrate that NRK-49F cells express functional AM receptor that when activated by AM results in a significant reduction of cell proliferation. Although cAMP activation by AM, as in other systems, is also observed in NRK-49F cells, PKA-independent pathways lead to some of the biological responses observed in these cells.