Calcium requirement for cGMP production during muscarinic activation of N1E-115 neuroblastoma cells
Thompson, S.H.; Mathes, C.; Alousi, A.A.
American Journal of Physiology 269(4 Pt 1): C979-C985
1995
ISSN/ISBN: 0002-9513 PMID: 7485468 Document Number: 439345
Muscarinic agonists elicit large increases in intracellular Ca-2+ and guanosine 3',5'-cyclic monophosphate (cGMP) in N1E-115 neuroblastoma cells. Both signals are blocked in cells loaded with the Ca-2+ buffer 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid showing that the increase in intracellular Ca-2+ concentration ((Ca-2+)-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 Ca-2+ 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 (Ca-2+)-i is not affected. This suggests that Ca-2+ influx is strongly coupled to the activation of cGMP production, even though it makes a smaller contribution to the intracellular Ca-2+ signal than does Ca-2+ release. Thapsigargin, which releases Ca-2+ from intracellular stores, activates Ca-2+ influx and increases cGMP. The cGMP increase is transient and follows approximately the same time course as Ca-2+ store depletion. Ca-2+ influx remains activated after store depletion, however, which indicates that influx alone cannot sustain cGMP production. It is concluded that summation of Ca-2+ influx and Ca-2+ release is necessary to reach a threshold Ca-2+ level needed to stimulate cGMP accumulation. Because of the large contribution from Ca-2+ influx, we suggest that NOS or a cofactor necessary for its activation may be located close to Ca-2+ channels in the membrane.