Abstract
Some major pathobiologic processes in renal mesangial cells, elicited in response to immunoinflammatory stimuli, are modulated via cAMP-protein kinase A (PKA) signaling pathways; namely, generation of reactive oxygen metabolites (ROM) and accelerated proliferation of mesangial cells. We investigated the role of cAMP phosphodiesterase (PDE) isozymes in these regulatory mechanisms. Generation of ROM in cultured rat mesangial cells was inhibited by selective inhibitors of PDE4, rolipram and denbufylline, whereas PDE3 inhibitors, cilostamide and lixazinone, had no effect. Conversely, cilostamide or lixazinone suppressed mitogenic synthesis of DNA in mesangial cells, but 1 microM rolipram or 1 microM denbufylline showed no inhibitory effect. The efficacy of PDE isozyme inhibitors (IC50) to suppress [3H]thymidine incorporation or ROM generation paralleled IC50 values for inhibition of cAMP PDE. Incubation of mesangial cells with either rolipram alone or with cilostamide alone increased significantly in situ activity of PKA in mesangial cells, assessed by (-cAMP/+cAMP) PKA activity ratio, and the stimulatory effects were additive. Results indicate that in mesangial cells a cAMP pool that is metabolized by PDE4 activates PKA and thereby inhibits ROM generation; another cAMP pool that is metabolized by PDE3 activates another PKA (isozyme or pool) which suppresses proliferation of mesangial cells. We propose that in mesangial cells, a cAMP-PKA pathway that regulates mitogenesis is determined by activity of PDE3, whereas another cAMP-PKA pathway is directed by activity of PDE4 and controls ROM generation. Therefore, two PDE isozymes within one cell type compartmentalize distinct cAMP signaling pathways.
Highlights
Reactive oxygen metabolites (ROM)1 [3, 4]
We observed that generation of reactive oxygen metabolites (ROM) in freshly isolated rat glomeruli is suppressed when the cAMP-protein kinase A (PKA) pathway is activated by incubating whole glomeruli with rolipram, a selective inhibitor of PDE isozyme type-4 (PDE4), and to a lesser degree with cilostamide, a selective inhibitor of PDE3 [10]
All of these cell types can contribute to ROM production by the whole glomeruli; it remained unknown in which cells and to what extent inhibitors of PDE3 and/or PDE4s exert its inhibitory effects upon ROM generation [10]
Summary
Reactive oxygen metabolites (ROM)1 [3, 4]. According to the current view, mesangial cells have a key role in maintaining the integrity of renal glomerular structure and function mainly by regulating capillary blood flow, uptake of macromolecules by phagocytosis, and synthesis of the extracellular matrix [1, 3, 4]. We observed that generation of ROM in freshly isolated rat glomeruli is suppressed when the cAMP-PKA pathway is activated by incubating whole glomeruli with rolipram, a selective inhibitor of PDE4, and to a lesser degree with cilostamide, a selective inhibitor of PDE3 [10]. Interpretation of these results was limited since all three cell types populating renal glomeruli, i.e. mesangial, epithelial, and endothelial cells, are endowed with NADPH oxidase, an enzyme complex that catalyzes ROM generation [11, 12]. The results provide evidence for the intracellular compartmentalization of cAMP signaling pathways effected by PDE isozymes within mesangial cells
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