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Protein kinase A activity modulates natriuretic peptide-dependent cGMP accumulation in renal cells
S Ledoux1, J C Dussaule, C Chatziantoniou
1Institut National de la Santé et de la Recherche Médicale 64, Hôpital Tenon, Paris, France.
Abstract:
The purpose of this work was to examine whether the level of cAMP accumulation and protein kinase A (PKA) activity influence atrial natriuretic factor (ANF)-dependent guanosine 3',5'-cyclic monophosphate (cGMP) production in two renal cell types: rabbit cortical vascular smooth muscle cells (RCSMC) and SV-40-transformed human glomerular visceral epithelial cells (HGVEC-SV1). N-[2-(p-bromocinnamylamino)ethyl]- 5-isoquinolinesulfonamide (H-89), a PKA inhibitor, decreased ANF-stimulated cGMP production in RCSMC in a time- and concentration-dependent manner. ANF-stimulated cGMP production was markedly inhibited after prolonged 9- and 18-h incubations with 25 microM H-89 (52 and 65%, respectively) but was not altered after exposure of cells to this agent for 1 h. 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine and N-(2-[methylamino]ethyl)-5-isoquinolinesulfonamide, protein kinase inhibitors not selective for PKA, did not reproduce the effect of H-89, even at higher concentrations (50 and 100 microM). Cycloheximide (10 microM), a protein synthesis inhibitor, limited the inhibitory effect of H-89, although alone it did not modify the ANF-stimulated cGMP production. H-89 did not affect cGMP production when it was stimulated by SIN-1, a nitric oxide donor. Prolonged incubation (18 h) with 8-bromo cAMP or cholera toxin, an activator of Gs protein resulting in adenylate cyclase stimulation, enhanced ANF-dependent cGMP production by 225 and 176%, respectively. This stimulatory effect was blocked by 25 microM H-89. 125I-ANF binding to RCSMC at 4 degrees C was not affected by preincubation of the cells with H-89. There was a 44% decrease in the expression of ANF C receptors measured as the ANF-(4-23)-displaceable 125I-ANF binding at 37 degrees C, which could not, however, explain the inhibitory effect of H-89 on cGMP production. Modulation of ANF- and C-type natriuretic peptide-dependent cGMP production by H-89 and cholera toxin was also found in HGVEC-SV1 with the same characteristics as in RCSMC. Taken together, these results suggest that PKA activity controls the function of natriuretic peptide guanylate cyclase-coupled receptors in the two cell types studied. PKA-dependent inhibition of a negatively regulatory protein distinct from the receptor itself seems necessary for a full cGMP response.
Insights
Protein kinase A (PKA) activity influences atrial natriuretic factor (ANF)-dependent cyclic guanosine monophosphate (cGMP) production. PKA-dependent inhibition of a regulatory protein is necessary for a full cGMP response in renal cells.
Area of Science:
- Cellular and Molecular Physiology
- Renal Physiology
- Signal Transduction
Background:
- Atrial natriuretic factor (ANF) is a hormone that regulates blood pressure and fluid balance.
- Cyclic guanosine monophosphate (cGMP) is a second messenger involved in various cellular processes, including smooth muscle relaxation.
- The role of cAMP and PKA in ANF-stimulated cGMP production in renal cells is not fully understood.
Purpose of the Study:
- To investigate the influence of cAMP accumulation and PKA activity on ANF-dependent cGMP production in rabbit cortical vascular smooth muscle cells (RCSMC) and human glomerular visceral epithelial cells (HGVEC-SV1).
- To elucidate the specific mechanisms by which PKA modulates natriuretic peptide signaling.
Main Methods:
- Utilized N-[2-(p-bromocinnamylamino)ethyl]- 5-isoquinolinesulfonamide (H-89), a specific PKA inhibitor, to assess its effects on ANF-stimulated cGMP production.
- Employed protein synthesis inhibitors (cycloheximide) and nitric oxide donors (SIN-1) to differentiate signaling pathways.
- Investigated the impact of cAMP analogs (8-bromo cAMP) and Gs protein activators (cholera toxin) on cGMP production.
- Quantified ANF receptor binding at different temperatures to evaluate receptor expression and function.
Main Results:
- H-89 significantly inhibited ANF-stimulated cGMP production in RCSMC in a time- and concentration-dependent manner, particularly after prolonged incubations.
- Non-selective protein kinase inhibitors did not replicate H-89's effect, suggesting PKA specificity.
- Cycloheximide partially reversed H-89's inhibition, indicating a role for protein synthesis.
- H-89 did not affect cGMP production stimulated by SIN-1, ruling out nitric oxide pathway involvement.
- Elevated cAMP levels (via 8-bromo cAMP or cholera toxin) enhanced ANF-dependent cGMP production, an effect blocked by H-89.
- ANF receptor binding was not directly affected by H-89, but a decrease in ANF C receptor expression was observed.
- Similar modulatory effects of H-89 and cholera toxin on ANF- and C-type natriuretic peptide signaling were observed in HGVEC-SV1.
Conclusions:
- PKA activity plays a crucial role in controlling the function of natriuretic peptide guanylate cyclase-coupled receptors in renal cells.
- A PKA-dependent inhibition of a negatively regulatory protein, distinct from the receptor itself, is essential for optimal ANF-stimulated cGMP production.
- These findings provide insights into the complex regulation of renal natriuretic peptide signaling pathways.