Search

Prostaglandin E1 (PGE1)-mediated transmembrane signal control systems were investigated in intact murine neuroblastoma cells (clone N1E-115). PGE1 increased intracellular levels of total inositol phosphates (IP), cyclic GMP, cyclic AMP, and calcium ([Ca2+]i). PGE1 transiently increased inositol 1,4,5-trisphosphate formation, peaking at 20 s. There was more than a 10-fold difference between the ED50 for PGE1 at cyclic AMP formation (70 nM) and its ED50 values at IP accumulation (1 microM), cyclic GMP formation (2 microM), and [Ca2+]i increase (5 microM). PGE1-mediated IP accumulation, cyclic GMP formation, and [Ca2+]i increase depended on both the concentration of PGE1 and extracellular calcium ions. PGE1 had more potent intrinsic activity in cyclic AMP formation, IP accumulation, and cyclic GMP formation than did PGE2, PGF2 alpha, or PGD2. A protein kinase C activator, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate, had opposite effects on PGE1-mediated IP release and cyclic GMP formation (inhibitory) and cyclic AMP formation (stimulatory). These data suggest that there may be subtypes of the PGE1 receptor in this clone: a high-affinity receptor mediating cyclic AMP formation, and a low-affinity receptor mediating IP accumulation, cyclic GMP formation, and intracellular calcium mobilization.

Acute desensitization of M1 muscarinic receptor-mediated responses (cyclic GMP formation and inositol phosphate release) was studied in murine neuroblastoma cells (N1E-115 clone). After a 45-min incubation at 37 degrees of N1E-115 cells either in monolayer or in suspension, with the muscarinic agonist carbachol (1 mM), the receptor-mediated cyclic GMP response to carbachol was nearly completely lost. This loss was associated with greater than 80% loss of carbachol-mediated inositol phosphate release. The protein kinase C activator phorbol 12-myristate 13-acetate (PMA) inhibited both responses with similar potencies. Carbachol or PMA reduced by 30-40% the number of muscarinic receptor sites for antagonist and agonist on intact cells (determined in binding assays using [3H]N-methylscopolamine) only for cells in monolayer and not for those in suspension. PMA but not carbachol pretreatment of cells in monolayer or in suspension caused a translocation of [3H]phorbol 12,13-dibutyrate binding and protein kinase C activity. In addition, desensitization to carbachol occurred in cells largely depleted of protein kinase C by chronic exposure to PMA. Thus, agonist-mediated down-regulation is not needed for muscarinic M1 receptor desensitization, which may be a result of the activation of a receptor-activated kinase different from protein kinase C.

Neurotensin, some of its analogs, and neuromedin N were examined for comparison of their potencies at stimulating inositol phospholipid hydrolysis and cyclic GMP synthesis in intact murine neuroblastoma cells (clone N1E-115). Neurotensin(8-13) and acetylneurotensin(8-13) had the highest potencies for the stimulation of the hydrolysis of inositol phospholipid, which were about three times as potent as neurotensin (EC50 = 0.9 nM). On the other hand, fragments of the amino-terminal portion of neurotensin, such as neurotensin(1-6), neurotensin(1-8) and neurotensin(1-11), showed no ability to stimulate this hydrolysis. Neuromedin N, which is similar in structure to neurotensin(8-13) and which has been demonstrated to stimulate cyclic GMP formation [J.A. Gilbert and E. Richelson, Eur. J. Pharmac. 129, 379 (1986)], had EC50 values of 2.5 and 4.5 nM for release of [3H]inositol phosphates and stimulation of cyclic [3H]GMP respectively. A strong correlation was obtained between the EC50 values for neurotensin and several analogs in the stimulation of the release of inositol phosphates and the EC50 values for these peptides in the stimulation of cyclic GMP formation in neuroblastoma clone N1E-115 cells under similar experimental conditions. Thus, these two different biochemical effects of neurotensin and its analogs appear to be mediated by the same receptor site, which may also have been the site of action of neuromedin N in these cells.