In this study, we highlight a role for the nitric oxide-cGMPdependent protein kinase (NO-G-kinase) signaling pathway in glial intercellular Ca 2ϩ wave initiation and propagation. Addition of the NO donor molsidomine (100-500 M) or puffing aqueous NO onto primary glial cell cultures evoked an increase in [Ca 2ϩ ] i in individual cells and also local intercellular Ca 2ϩ waves, which persisted after removal of extracellular Ca 2ϩ . High concentrations of ryanodine (100-200 M) and antagonists of the NO-G-kinase signaling pathway essentially abrogated the NO-induced increase in [Ca 2ϩ ] i , indicating that NO mobilizes Ca 2ϩ from a ryanodine receptor-linked store, via the NO-G-kinase signaling pathway. Addition of 10 M nicardipine to cells resulted in a slowing of the molsidomine-induced rise in [Ca 2ϩ ] i , and inhibition of Mn 2ϩ quench of cytosolic fura-2 fluorescence mediated by a bolus application of 2 M aqueous NO to cells, indicating that NO also induces Ca 2ϩ influx in glia. Mechanical stress of individual glial cells resulted in an increase in intracellular NO in target and neighboring cells and intercellular Ca 2ϩ waves, which were NO, cGMP, and G-kinase dependent, because incubating cells with nitric oxide synthase, guanylate cyclase, and G-kinase inhibitors, or NO scavengers, reduced ⌬[Ca 2ϩ ] i and the rate of Ca 2ϩ wave propagation in these cultures. Results from this study suggest that NO-Gkinase signaling is coupled to Ca 2ϩ mobilization and influx in glial cells and that this pathway plays a fundamental role in the generation and propagation of intercellular Ca 2ϩ waves in glia.
Key words: nitric oxide; glia; calcium waves; mobilization; influx; ryanodine receptors; nitric oxide synthase; DAF-2; phospholipase C; astrocytesIt is becoming increasingly apparent that intercellular Ca 2ϩ waves might be the result of combined contributions of intracellular and extracellular Ca 2ϩ signaling pathways in glial cells. One generally accepted mode of intercellular Ca 2ϩ wave propagation involves the diffusion of Ca 2ϩ mobilizing second messengers, including inositol-1,4,5-trisphosphate (IP 3 ) and Ca 2ϩ , across gap junctions (Nedergaard, 1994;Charles, 1998). Extracellular, gap junction-independent modes of Ca 2ϩ signaling, involving the release of a diffusible messengers, also appear to operate in this particular system however, with ATP release from cells and P 2 receptor-coupled elevation of [Ca 2ϩ ] i having recently been suggested as a likely candidate (Cotrina et al., 1998;Guthrie et al., 1999). There are however other species that may participate as extracellular messengers in glial intercellular Ca 2ϩ waves. There is increasing evidence that the highly diffusible messenger nitric oxide (NO) can induce Ca 2ϩ mobilization in several cell types (Publicover et al., 1993; Willmott et al., 1995a,b,c;Clementi et al., 1996) either via the cGMP-dependent protein kinase (G-kinase)-coupled activation of ADP-ribosyl cyclase, resulting in an increased synthesis of the potent Ca 2ϩ mobilizing agent cyclic ADP-rib...
