CALCIUM REQUIREMENT FOR CGMP PRODUCTION DURING MUSCARINIC ACTIVATION OF N1E-115 NEUROBLASTOMA-CELLS

Muscarinic agonists elicit large increases in intracellular Ca2+ and g uanosine 3',5'-cyclic monophosphate (cGMP) in N1E-115 neuroblastoma ce lls. Both signals are blocked in cells loaded with the Ca2+ buffer ,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 (N OS) blocks the cGMP response without affecting the peak amplitude of t he intracellular Ca2+ signal, and it is concluded that Ca2+-dependent activation of NOS is required for cGMP production. cGMP accumulation i s reduced by 60% when cells are bathed in Ca2+-free saline, but the pe ak 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 intracellul ar stores, activates Ca2+ influx and increases cGMP. The cGMP increase is transient and follows approximately the same time course as Ca2+ s tore depletion. Ca2+ influx remains activated after store depletion, h owever, which indicates that influx alone cannot sustain cGMP producti on. It is concluded that summation of Ca2+ influx and Ca2+ release is necessary to reach a threshold Ca2+ level needed to stimulate cGMP acc umulation. Because of the large contribution from Ca2+ influx, we sugg est that NOS or a cofactor necessary for its activation may be located close to Ca2+ channels in the membrane.