Volume 61, Issue 5, Pages (May 2002)

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Volume 61, Issue 5, Pages 1707-1713 (May 2002) Nitric oxide increases adrenomedullin receptor function in rat mesangial cells Jörg Dötsch, M.D., Ellen Schoof, Harald O. Schöcklmann, Bernhard Brüne, Ina Knerr, Reinald Repp, Wolfgang Rascher Kidney International Volume 61, Issue 5, Pages 1707-1713 (May 2002) DOI: 10.1046/j.1523-1755.2002.00330.x Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 1 Adrenomedullin (ADM) radioligand binding assay showing the increase in ADM binding after 24 hours of incubation with 100 μmol/L of each of the two NO donors GSNO and SNAP (P < 0.001 for both NO donors). Symbols are: (○) control; (▴) GSNO 100 μmol/L; (▪) SNAP 100 μmol/L. Values are presented as the mean ± SEM. The maximal binding of the GSNO pretreated cells was normalized to 100%. The results are based on 6 experiments. Kidney International 2002 61, 1707-1713DOI: (10.1046/j.1523-1755.2002.00330.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 2 Cyclic AMP concentration following ADM receptor stimulation in rat mesangial cells after 24 hour pretreatment with the NO donors S-nitrosoglutathione (GSNO 100 μmol/L) and S-nitroso-N-acetyl-d,l-penicillamine (SNAP 100 μmol/L), the cyclic GMP analog 8-bromo-cGMP (50 μmol/L), and combined pretreatment with GSNO (100 μmol/L) and the guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3a]quinoxaline-1-one (ODQ 100 μmol/L). The control cells were only stimulated with ADM without any pretreatment. The maximal cAMP concentration was normalized to 100%. The values represent the mean ± SEM of 6 experiments. Symbols are: (□) control; (▴) GSNO 50 μmol/L (P < 0.001); (•) GSNO 100 μmol/L (P < 0.001); (▵) ODQ 100 μmol/L and GSNO 100 μmol/L (P < 0.05); (♦) 8-Br-cGMP 50 μmol/L (P < 0.01). Kidney International 2002 61, 1707-1713DOI: (10.1046/j.1523-1755.2002.00330.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 3 ADM receptor mRNA expression related to GAPDH gene expression in rat mesangial cells as quantified by TaqMan real time polymerase chain reaction (PCR). ADM receptor gene expression decreases after incubation with different concentrations of the NO donors GSNO and SNAP. Values are shown as the mean ± SEM of 6 experiments. Symbols are: (□) control cells; () 100 μmol/L SNAP; **P < 0.01. (A) ADM receptor mRNA expression as a function of incubation time with 100 μmol/L SNAP. (B) ADM receptor mRNA expression as a function of the concentration of GSNO (after 24 hours of preincubation). Kidney International 2002 61, 1707-1713DOI: (10.1046/j.1523-1755.2002.00330.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 4 3H-thymidine incorporation after pretreatment with ADM for 48 hours. There was a significant dose dependent inhibition of the 5% FCS-induced 3H-thymidine incorporation as compared to the controls (P = 0.002). Kidney International 2002 61, 1707-1713DOI: (10.1046/j.1523-1755.2002.00330.x) Copyright © 2002 International Society of Nephrology Terms and Conditions

Figure 5 DNA fragmentation is slightly but significantly increased after incubation of primary rat mesangial cells with 100 nmol/L ADM for 24 hours (P < 0.001). Kidney International 2002 61, 1707-1713DOI: (10.1046/j.1523-1755.2002.00330.x) Copyright © 2002 International Society of Nephrology Terms and Conditions