Calcium requirement for cGMP production during muscarinic activation of N1E-115 neuroblastoma cells

Muscarinic agonists elicit large increases in intracellular Ca2+ and guanosine 3',5'-cyclic monophosphate (cGMP) in N1E-115 neuroblastoma cells. Both signals are blocked in cells loaded with the Ca2+ buffer 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid showing that the increase in intracellular Ca2+ concentration ([Ca2+]i) is necessary to stimulate cGMP accumulation. Inhibition of nitric oxide synthase (NOS) blocks the cGMP response without affecting the peak amplitude of the intracellular Ca2+ signal, and it is concluded that Ca(2+)-dependent activation of NOS is required for cGMP production. cGMP accumulation is reduced by 60% when cells are bathed in Ca(2+)-free saline, but the peak change in [Ca2+]i is not affected. This suggests that Ca2+ influx is strongly coupled to the activation of cGMP production, even though it makes a smaller contribution to the intracellular Ca2+ signal than does Ca2+ release. Thapsigargin, which releases Ca2+ from intracellular stores, activates Ca2+ influx and increases cGMP. The cGMP increase is transient and follows approximately the same time course as Ca2+ store depletion. Ca2+ influx remains activated after store depletion, however, which indicates that influx alone cannot sustain cGMP production. It is concluded that summation of Ca2+ influx and Ca2+ release is necessary to reach a threshold Ca2+ level needed to stimulate cGMP accumulation. Because of the large contribution from Ca2+ influx, we suggest that NOS or a cofactor necessary for its activation may be located close to Ca2+ channels in the membrane.