Ca2+/calmodulin-dependent cyclic GMP phosphodiesterase activity in granule neurons and astrocytes from rat cerebellum

Cyclic GMP (cGMP) formation induced by agonist stimulation of Ca2+/calmodulin-dependent nitric oxide (NO) synthase type I is known to occur in both granule cell and astrocyte cultures from rat cerebellum. Here we show that in these same cells cGMP is predominantly hydrolyzed by a Ca2+/calmodulin-dependent phosphodiesterase. At 10 mu M cGMP, Ca2+ (25 mu M) stimulated basal (Ca2+-independent) phosphodiesterase activity about 6 times in granular neurons and 15 times in astrocytes. The calmodulin antagonist calmidazolium blocked the Ca2+-dependent phosphodiesterase activity and exogenous calmodulin increased 3-4-fold the stimulatory potency of Ca2+ in both cell types (EC,, values 1.26 +/- 0.20 and 1.50 +/- 0.42 mu M in the absence and 0.38 +/- 0.11 and 0.39 +/- 0.14 mu M in the presence of 1 mu M calmodulin, for neurons and astrocytes, respectively). In both cell types K-m values for cGMP at 25 mu M Ca2+ were similar (1.72 +/- 0.20 and 1.92 +/- 0.09 mu M) and phosphodiesterase activities were inhibited by isozyme-selective phosphodiesterase inhibitors with potencies analogous to tho se de scribed for Ca2+ /calmodulin-phosphodiesterases or phosphodiesterase type 1 isoforms in other preparations. The nonselective phosphodiesterase inhibitor 3-isobutyl-l-methylxantine (IBMX) effectively blocked the Ca2+/calmodulin-phosphodiesterase activity in granule cell and astrocyte extracts (IC50 values at 1 mu M cGMP: 31 +/- 10 mu M and 46 +/- 6 mu M, respectively), in contrast to the apparent inability of this compound to inhibit the Ca2+-dependent activity reported in whole brain extracts. These results demonstrate that comparable phosphodiesterase type 1 activities are found in the cytosols of cerebellar granule cells and astrocytes and suggest that these activities may play an important role in controlling cGMP levels in cells where the Ca2+-dependent NO synthase type I is stimulated. (C) 1997 Elsevier Science B.V.