cGMP Formation In Cells Research Articles

Ca2+/calmodulin-dependent cyclic nucleotide phosphodiesterase in cGMP metabolism in rabbit parotid acinar cells

Muscarinic cholinergic receptor activation provokes cGMP formation in parotid acinar cells. We investigated the involvement of Ca(2+)/calmodulin-dependent cyclic nucleotide phosphodiesterase (PDE1) in cGMP breakdown in rabbit parotid acinar cells. The muscarinic agonist carbachol stimulated cGMP formation in the cells. The carbachol-induced cGMP formation was enhanced in the presence of 8-methoxymethyl-3-isobutyl-1-methylxanthine (MM-IBMX), a PDE1 inhibitor. cGMPPDE activity in rabbit parotid acinar cells was reduced by about 25% in the absence of Ca(2+)/ calmodulin or in the presence of MM-IBMX. Ca(2+)/calmodulin-dependent cGMP-PDE in rabbit parotid acinar cells was purified using Calmodulin-Sepharose 4B and Mono Q ion-exchange column chromatography. Two dominant fractions with cGMP-PDE activity, referred to as the P-1 and P-2 fractions, were eluted from the Mono Q ion-exchange column. The Km values for cGMP of PDE in the P-1 and P-2 fractions were 0.82 microM and 0.40 microM, respectively, which were much lower than that for cAMP. The EC(50) for Ca(2+) and calmodulin of PDEs in the P-1 and P-2 fractions were 458 nM and 426 nM, respectively, and 32 nM and 137 nM, respectively. Protein bands that crossreacted with anti-PDE1A antibody were detected. These results suggest that Ca(2+)/calmodulin-dependent PDE, PDE1A, is involved in cGMP breakdown in rabbit parotid acinar cells.

Activation of Constitutive Nitric-oxide Synthase Activity Is an Early Signaling Event Induced by Ionizing Radiation

Ionizing radiation at clinical dose levels activates both pro- and anti-proliferative signal transduction pathways, the balance of which determines cell fate. The initiating and amplifying mechanisms involved in the activation are poorly understood. We demonstrate that one mechanism involves stimulation of constitutive nitric-oxide synthase (NOS) activity. NOS activity of Chinese hamster ovary cells was measured by the arginine --> citrulline conversion assay. Irradiation stimulated a transient activation of NOS with maximal activity at 5 min of post-irradiation. Western blot analysis and genetic manipulation by overexpression of wild type or dominant negative NOS mutant identify the radiation-induced isoform as NOS-1. Further evidence that NOS-1 is activated by radiation was the demonstration of radiation-induced cGMP formation in cells transiently transfected with the NO-dependent soluble guanylate cyclase. Protein Tyr nitration, a footprint of peroxynitrite formation, followed radiation exposure and was inhibited by expression of a dominant negative NOS-1 mutant. Radiation-induced ERK1/2 kinase activity, a cytoprotective response to radiation, was also blocked by inhibiting NOS activity. These experiments establish NO-dependent signal transduction pathways as being radio-responsive. Given the lipophilic and relatively stable properties of NO, these results also suggest a possible mechanism by which ionization events in one cell may activate signaling processes in adjacent cells.