Adrenomedullin (ADM) is a 52 amino acid peptide predominantly synthesized in endothelial, smooth muscle and mesangial cells. The antiproliferative effect of ADM on mesangial cells has gained much interest recently1,2. This effect is mediated by a decrease of extracellular signal-regulated kinase activity through an increase in protein phosphatase-2A activity1 and an increase in cAMP concentration after stimulation of the G-protein-coupled ADM receptor3,4. More recently, the antiproliferative effect of ADM on glomerular structures has been demonstrated in DOCA salt sensitive rats in vivo5. Particularly in the in vivo context, therefore, a cytoprotective function for glomerular cells might be attributed to ADM.
In contrast, a dual cytotoxic and cytoprotective potential has been attributed to nitric oxide (NO)6. This effect depends on the concentration of NO as well as on environmental conditions like the presence of oxygen radicals. Little attention, however, has been spent on the possibility that the quality of the NO effect might be modulated by downstream activation or inhibition of the regulatory peptide function. The principal possibility of this aspect has been demonstrated with regard to the interaction between NO and peptidergic mechanisms from a cardiovascular point of view, showing the impact of NO on the endothelin and neuropeptide Y system7,8,9,10. In addition, ADM has been demonstrated to increase the activity of the endothelial nitric oxide synthase11,12, suggesting cross talk between these systems.
Consequently, an activation of the mesangial ADM system might modulate the NO effect to a more protective than cytotoxic manner. To test for the impact of NO on ADM receptor function, we examined ADM ligand binding, signal transduction and ADM receptor gene expression under the influence of NO.
METHODS
Cell culture
After isolation rat mesangial cells were cultured up to fifteen passages in Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Eggenstein, Germany) containing 10% fetal calf serum (FCS), 5 mg/mL insulin, 100 U/mL penicillin, 100 mg/mL streptomycin, and 2 mmol/L L-glutamine (Sigma, Deisenhofen, Germany) in a 95% air/5% CO2 humidified atmosphere at 37°C. Details of the isolation process and characterization of mesangial cells are described elsewhere13.
Mesangial cells grown to confluence were washed twice with phosphate-buffered saline (PBS) and preincubated with the incubation medium again. The cells were pretreated with various concentrations of the NO-donors S-nitroso-N-acetyl-D,L-penicillamine (SNAP; Sigma, Munich, Germany) and S-nitrosoglutathione (GSNO; Sigma), with the stable cyclic 3',5'-guanosine monophosphate (cGMP) and cAMP analogs 8-bromo-cGMP and 8-bromo-cAMP and the inhibitor of the guanylate cyclase 1H-[1 2 4]oxadiazolo[4,3a]quinoxaline-1-one (ODQ). After washing the cells were incubated in PBS for an additional 30 minutes and used for the different experiments. Two chemically distinct NO donors were used for the experiments. The total number of cells was counted with an electronic cell counting system (Casy, Schärfe Inc., Reutlingen, Germany).
Competitive receptor binding assay
Competitive binding studies were performed as described elsewhere10,14,15. Primary rat mesangial cells were grown as monolayers in 24 well plates. After incubation with 100 
mol/L of the NO-donors GSNO and SNAP for 24 hours, the cells were washed with 1 mL of PBS and were then incubated in PBS for an additional 30 minutes. Subsequently, the cells were washed again with assay buffer containing HEPES (10 mmol/L), NaCl (150 mmol/L), KCl (5 mmol/L), CaCl2 (2.5 mmol/L), KH2PO4 (1.2 mmol/L), MgSO4 (1.2 mmol/L), NaHCO3 (25 mmol/L), bovine serum albumin (BSA) 10 mg/mL, 0.5 g/mL bacitracin, and 0.5 g/mL soybean trypsin inhibitor at pH 7.4. For radioligand binding studies 125I labeled ADM containing 103 counts per min/mL (Biotrend Chemikalien GmbH, Cologne, Germany) and various concentrations of unlabeled adrenomedullin (0.5 to 2000 nmol/L; Saxon Biochemicals GmbH, Hannover, Germany) were used. The tracer mixture was incubated for 90 minutes at 37°C. The incubation was stopped by removal of the assay buffer and subsequent washes with 1 mL of ice-cold fresh assay buffer and 1 mL of ice-cold saline. Maximal binding was calculated from specifically bound ADM. Radioligand binding curves and Scatchard blot were calculated.
Measurements of cAMP
After washing twice with PBS, cells were incubated with SNAP, GSNO, and ODQ as described above. The cells were washed again three times with the assay buffer. After adding 0.5 mmol/L 3-isobutyl-1-methylxanthine to the assay buffer, the cells were incubated for 20 minutes with increasing concentrations of ADM. Controls were incubated with ADM only. The time course was examined after pretreatment of the cells with 100 mol/L GSNO for 6, 24, and 48 hours. The incubation was stopped by aspiration of the buffer and immediate addition of 1 mL 65% (vol/vol) ethanol. The accumulated cAMP was measured by radioimmunoassay using a commercial kit (Amersham, Braunschweig, Germany). Details of the procedure have been described previously15.
RNA extraction and TaqMan real time PCR
RNA was extracted using a commercial RNA isolation kit (RNAzol-B isolation kit; WAK-Chemie Medical GmbH, Bad Homburg, Germany). In order to monitor gene expression, we used quantitative real time, RT-PCR analysis. The use of the TaqMan reaction has been described in a number of original and review articles16,17. This approach makes use of the 5' exonuclease activity of the DNA polymerase (AmpliTaq Gold). Briefly, within the amplicon defined by a gene specific PCR primer pair an oligonucleotide probe labeled with two fluorescent dyes is created, designated as TaqMan probe. As long as the probe is intact, the emission of the reporter dye (that is, 6-carboxy-fluorescein, FAM) at the 5'-end is quenched by the second fluorescence dye (6-carboxy-tetramethyl-rhodamine, TAMRA) at the 3'-end. During the extension phase of PCR, the polymerase cleaves the TaqMan probe resulting in a release of reporter dye. The increasing amount of reporter dye emission is detected by an automated sequence detector combined with a special software (ABI Prism 7700 Sequence Detection System; Perkin-Elmer, Foster City, CA, USA). The oligonucleotides of each target of interest were designed by the Primer Express software (Perkin-Elmer) using uniform selection parameters that allowed for the application of standard cycle conditions18. The following primers and TaqMan probes were used:
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH): forward 5'-CCCATGTTCGTCATGGGTGT-3', reverse 5'-TGGTCATGAGTCCTTCCACGATA-3', TaqMan probe 5'(FAM)-CTGCACCACCAACTGCTTAGCACCC-(TAMRA)3'.
-actin: forward 5'-GCGAGAAGATGACCCAGGATC-3', reverse 5'-CCAGTGGTACGGCCAGAGG-3', TaqMan probe 5'(FAM)-CCAGCCATGTACGTTGCTATCCAGGC-(TAMRA)3'.
ADM-receptor: forward 5'-TGCCTCAGCATTGACCGCTACGTC-3', reverse 5'-CTGGAGTACACCTGGCTCTGG-3', TaqMan probe 5'(FAM)-CCAGGAGGGAGAGGTATTGGT-(TAMRA)3'.
ADM: forward 5'-ACCGCCAGAGCATGAACCAGGG-3', reverse 5'-CTCAGAGCACAGCCCACATTC-3', TaqMan probe 5'(FAM)-GCATTGTGCAGGTCCCAAAG-(TAMRA)3'.
Determination of 3H-thymidine uptake
Mesangial cells were subcultured in 96-well plates in medium supplemented with 10% FCS until subconfluence, and growth arrested for 72 hours in medium supplemented with 0.4% FCS. Quiescent mesangial cells were then exposed to fresh medium containing 0.4% FCS with or without GSNO for six hours before stimulation with FCS in a final concentration of 5%. Cells were pulsed with 1 mCi/mL [3H-methyl]-thymidine (specific activity, 5 mCi/mmol/L; ICN, Costa Mesa, CA, USA) from 0 to 24 hours after the addition of 5% FCS. The cells were washed twice with PBS, lysed with distilled water, and harvested onto filters by an automated cell harvester (Dunn, Asbach, Germany). Incorporated counts were measured by a liquid scintillation counter (Beckmann, Fullerton, CA, USA).
Quantitation of DNA fragmentation
DNA fragmentation was assayed as reported19,20. Briefly, following the incubations, cells (2.5 
105 cells/assay) were centrifuged, resuspended in 250 L TE buffer [10 mmol/L Tris-HCl, 1 mmol/L ethylenediaminetetraacetic acid (EDTA), pH 8.0] and lysed by adding 250 L cold lysis buffer [2 mmol/L EDTA, 0.5% Triton X-100 (vol/vol), 5 mmol/L Tris-HCl, pH 8.0]. After 30 minutes at 4°C, disintegrated cells were centrifuged (14000 g, 15 min) to separate intact chromatin (pellet) from DNA fragments (supernatant). Pellets were resuspended in 500 L TE buffer and the DNA content of pellets versus supernatants were measured using the diphenylamine reagent. Diphenylamine was purchased from Sigma.
Determination of lactate dehydrogenase release
Following incubations, the medium of approximately 2.5 105 mesangial cells was collected, and cells were supplemented with 0.2% (vol/vol) Triton-X 100 in PBS. Cells were lysed for four hours at 4°C. A total of 500 L of reaction mix containing 50 mmol/L triethanolamine dissolved in 5 mmol/L EDTA, pH 7.6, 127 mmol/L pyruvate, and 14 mmol/L NADH in 1% NaHCO3 was added to 300 L cell medium or lysed cells. Lactate dehydrogenase (LDH) activity was monitored by the oxidation of NADH following the decrease in absorbance at 334 nm. The percentage of LDH released was defined as the ratio of LDH activity in the supernatant to the sum of the LDH amount released plus the activity measured in the cell lysate.
Statistical analysis
All results are expressed as the mean 
SEM. Differences in ADM receptor binding, cAMP concentration and mRNA expression were compared using ANOVA and post-hoc t tests. In case of multiple tests, P values were corrected according to Bonferroni. Concentration-response curves for ADM binding were analyzed by fitting the experimental data to two side binding curves. The bottoms of the curves were fixed at 0 and their Hill slopes at 100%. Scatchard plot analysis was used for estimating the number of receptors expressed on the cell surface and the ligand affinity21. All calculations were performed using iterative non-linear regression analysis (GraphPAD Prism; GraphPAD Software, San Diego, CA, USA). A P value less than 0.05 was considered statistically significant.
RESULTS
ADM radioligand binding
There was a significant increase in ADM binding after 24 hours of incubation with 100 mol/L each of the two NO donors GSNO and SNAP (P < 0.001 for both NO donors; Figure 1). Consequently, maximum ADM radioligand binding to the ADM receptor increased from 52% 4% to 100% 4% (P < 0.001) and 81% 2% (P < 0.001) after 24 hours of incubation with GSNO and SNAP, respectively (N = 6).
Figure 1.
Adrenomedullin (ADM) radioligand binding assay showing the increase in ADM binding after 24 hours of incubation with 100 
mol/L of each of the two NO donors GSNO and SNAP (P < 0.001 for both NO donors). Symbols are: (
) control; (
) GSNO 100 mol/L; (
) SNAP 100 mol/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.
To calculate the expression levels of the ADM receptor in primary rat mesangial cells Scatchard blot analysis was used. In the cells not preincubated with NO donors the mean number of receptors per cell was 7.9 10-17 1.6 10-17 mol/cell. The level of expression increased to 14.3 10-17 1.8 10-17 mol/cell (P = 0.02) and 11.1 10-17 1.1 10-17 mol/cell (P = 0.05) in the cells pretreated with GSNO and SNAP, respectively. The affinity of the ADM radioligand to the ADM receptor increased three- to sixfold in the cells pretreated with the NO donors (Kd was 0.17 nmol/L for control cells, 0.51 nmol/L for GSNO pretreated cells, and 0.96 nmol/L for cells incubated with SNAP).
Cyclic AMP concentration
After a 24 hour pretreatment with both NO donors GSNO (100 mol/L) and SNAP (100 mol/L), ADM stimulation of the mesangial cell ADM receptor led to a significant increase in intracellular cAMP concentration (P < 0.001 for both NO donors; N = 6). This increase was seen in comparison to the cells stimulated with ADM without pretreatment with NO donors. The effect could be imitated by the pretreatment of the cells with 50 mol/L 8-bromo-cGMP. Combined pretreatment of the mesangial cells with the inhibitor of the guanylate cyclase ODQ (100 mol/L) and GSNO (100 mol/L) reduced ADM-induced cAMP activation to the levels seen in control cells Figure 2. Subsequently, the logarithm of the EC50 was -7.0 0.2 log (nmol/L) for the control cells and dropped to -9.2 0.4 log (nmol/L) for GSNO 50 mol/L, -9.3 0.3 log (nmol/L) for 100 mol/L GSNO, and -8.9 0.1 log (nmol/L) for 50 mol/L 8-bromo-cGMP. Combined treatment with ODQ (100 mol/L) and GSNO (100 mol/L) reversed the GSNO effect on log EC50 to -7.6 0.2 log (nmol/L), a value similar to the control group.
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 mol/L) and S-nitroso-N-acetyl-D,L-penicillamine (SNAP 100 mol/L), the cyclic GMP analog 8-bromo-cGMP (50 mol/L), and combined pretreatment with GSNO (100 mol/L) and the guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3a]quinoxaline-1-one (ODQ 100 mol/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 mol/L (P < 0.001); (
) GSNO 100 mol/L (P < 0.001); (
) ODQ 100 mol/L and GSNO 100 mol/L (P < 0.05); (
) 8-Br-cGMP 50 mol/L (P < 0.01).
The effect of preincubation of mesangial cells with 10 mol/L GSNO was not time dependent. The logarithm of the EC50 was -9.2 0.2 log (nmol/L) after six hours of GSNO incubation and subsequent ADM stimulation, -9.3 0.3 log (nmol/L) after 24 hours of GSNO incubation and -9.1 0.2 log (nmol/L) after 48 hours of preincubation.
ADM receptor mRNA expression
In contrast to radioligand binding and cAMP concentration, there was no stimulatory effect of the NO donors GSNO and SNAP on ADM receptor gene expression related to GAPDH gene expression. In fact, concentrations as low as 1 mol/L of GSNO led to a significant reduction in ADM receptor/GAPDH gene expression Figure 3a. Similar results were seen after incubation of the cells with SNAP. The negative impact on ADM receptor/GAPDH gene expression was similar after 6, 24, and 48 hours Figure 3b. Incubation of mesangial cells with 8-bromo-cGMP (50 mol/L) led to a significant decrease in ADM receptor/GAPDH mRNA expression from 0.025 0.0005 relative units to 0.013 0.002 relative units (P < 0.003; N = 5). In contrast, there was no effect of 8-bromo-cAMP on the ADM receptor/GAPDH gene expression in rat mesangial cells.
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 mol/L SNAP; **P < 0.01. (A) ADM receptor mRNA expression as a function of incubation time with 100 mol/L SNAP. (B) ADM receptor mRNA expression as a function of the concentration of GSNO (after 24 hours of preincubation).
Similar results with respect to relative differences and P values were obtained when ADM mRNA expression was normalized to the gene expression of the housekeeping gene -actin (data not shown).
Determination of 3H-thymidine uptake
There was a significant dose dependent inhibition of the 5% FCS-induced 3H-thymidine incorporation after incubation with ADM for 48 hours as compared to the controls (P = 0.002; Figure 4).
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).
Full figure and legend (21K)Quantitation of DNA fragmentation and determination of lactate dehydrogenase release
DNA fragmentation was slightly but significantly increased after incubation of primary rat mesangial cells with 100 nmol/L ADM for 24 hours (P < 0.001; Figure 5). Similarly, lactate dehydrogenase release increased slightly from 15.6 0.2% to 22.0 1.0% (P = 0.01).
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).
Full figure and legend (33K)ADM mRNA expression
Incubation of primary rat mesangial cells with various concentrations (10 to 100 mol/L) of GSNO and SNAP for time intervals of 1 to 48 hours had no influence on ADM gene expression related to GAPDH gene expression. For instance, the mRNA expression of ADM/GAPDH incubation of cells with 100 mol/L GSNO and SNAP were 0.0060 0.0009 relative units and 0.0054 0.0004 relative units, respectively (N = 5). There was no difference to the values obtained for ADM/GAPDH gene expression in the control group (0.0053 0.0010 relative units; N = 5; P> 0.1). Similar values were obtained for normalization of gene expression to the housekeeping gene -actin.
DISCUSSION
The results of the present study suggest an activation of ADM receptor function by NO. This effect becomes most evident on the level of the ADM second messenger cAMP, which is one important mediator of the antiproliferative effects of ADM on mesangial cells3. The effect of NO can be completely reversed by the addition of ODQ, which inhibits the generation of the NO second messenger cGMP. Therefore, these results suggest a specific NO-mediated activation of ADM signal transduction.
One mechanism of this effect may be seen in an increase in receptor availability and affinity for ADM by NO was evident by the increase in ADM radioligand binding and receptor affinity after the incubation with different NO donors. The molecular processes leading to the increased number of receptor binding sites, as assessed by Scatchard plot analysis, are not quite clear. NO might stabilize receptor proteins either by protecting them from external destruction, for example, by other radicals or might prevent internalization of the receptor by interacting with the process of internalization. Alternatively, NO might interact with the configuration of the so-called receptor activity-modifying proteins (RAMPS) that determine whether the receptor works as ADM or calcitonin gene-like receptor22.
However, two observations suggest that the more important mechanism of ADM receptor activation might operate on the second messenger level of the peptide: First, the extent of increase in ADM induced cAMP activation by both NO donors exceeds by far the rise in available ADM receptors. Second, there is a complete reversal of cAMP activation to levels seen in the control cells by ODQ that blocks NO signal transduction. The exact mechanism of the NO effect on ADM receptor function remains speculative. Since the effect is not dose dependent and already present after six hours of preincubation with NO donors, a relatively fast mechanism may be suspected. It appears possible that the elevated intracellular cGMP levels following pretreatment with NO lead to an inhibition of the phosphodiesterase PDE3A, that has been demonstrated previously23. As a consequence, cAMP inactivation would be reduced leading to elevated cAMP concentrations as seen in mesangial cells after NO donor pretreatment.
Of note is the reduced ADM receptor gene expression following the treatment of mesangial cells with the NO donors GSNO and SNAP. This effect was already evident at low concentrations in the NO donors; it is mediated via the NO second messenger cGMP but not by a stable analog of the ADM second messenger cAMP. These data suggest that NO down-regulates ADM receptor gene expression in a negative feedback manner. Similar effects of NO on the gene expression of peptide receptors have been observed previously10. From a methods point of view, the use of TaqMan real time PCR for the quantitation of gene expression has been shown to be at least as reliable as the application of other quantitative PCR techniques like competitive PCR24.
It is of importance that there are clear indicators of a biologically-active ADM receptor in the primary rat mesangial cells used in our experiments: The effect of ADM on 3H-thymidine incorporation in the rat mesangial cells used in our experiments reflects antiproliferative capabilities of the peptide that have been demonstrated previously1,2. In contrast, NO, applied to the same cells using the same assay, has ambiguous effects that range between antiproliferative and neutral effects25,26. Moreover, ADM exhibits moderate apoptotic effects in our mesangial cell model. This again suggests the presence of a functional ADM receptor in these cells. In contrast, the impact of NO on mesangium cells is ambiguous19,20. Future studies will have to address whether there is a synergetic effect between ADM and NO on the proliferation of mesangial cells. This question is most relevant in vivo in respect to mesangioproliferative glomerulonephritis, such as in IgA nephropathy.
In mesangial cells, NO promotes the activation of soluble guanylate cyclase in a cytoprotective manner on one hand, whereas it may lead to cytotoxicity on the other. The cytoprotective action is characterized by cGMP formation and signal propagation toward myosin light chain kinase, the effector system that regulates F-actin assembly, thereby affecting reversible relaxation/contraction of mesangial cells. The cytotoxic effect is characterized by the initiation of morphological and biochemical alterations that are reminiscent of apoptosis such as condensation, p53 or Bax accumulation as well as caspase-3 activation27.
Our data suggest one additional mechanism for the modulation of the effect of NO on mesangial cells. In case of stimulation of the ADM receptor, NO, at least in the concentrations applied in this study, is capable of increasing the ADM effect considerably. Ongoing studies will have to demonstrate whether this modulation leads to a reduction in mesangial cell proliferation and a modulation of apoptosis.
Unlike ADM receptor function, there appears to be no impact of NO donors on ADM mRNA synthesis in rat mesangial cells. This is different from human umbilical vein cells (HUVEC) where NO leads to an increase in both gene expression and peptide synthesis of ADM28.
In summary, our data show an activation of ADM receptor function in rat mesangial cells by NO via the cGMP pathway. The effect is partially counteracted on the level of ADM gene expression.
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Acknowledgments
The excellent technical assistance of Ms. Ida Allabauer, Ms. Ulla Jacobs, and Ms. Andrea Lüdke is very much appreciated. We thank our colleagues from the SFB 423 for stimulating discussions of the results.
