NEUROTENSIN-MEDIATED INHIBITION OF CYCLIC-AMP FORMATION IN NEUROBLASTOMA N1E115-CELLS - INVOLVEMENT OF THE INHIBITORY GTP-BINDING COMPONENT OF ADENYLATE-CYCLASE

  • 1 May 1986
    • journal article
    • research article
    • Vol. 29  (5) , 489-496
Abstract
The tridecapeptide, neurotensin, inhibited prostaglandin E1-stimulated cyclic AMP production in intact plated neuroblastoma N1E115 cells. The peptide effect was concentration dependent (EC50=2 nM) and maximal inhibition reached 55% with 100 nM neurotensin. Acetyl neurotensin (8-13) was as active as neurotensin whereas neurotensins (1-8), (1-2), and (10-13) were barely active in inhibiting cyclic AMP production, thus showing the requirement of the carboxy terminal hexapeptide sequence of neurotensin for biological activity. The inhibitory effect of neurotensin on cyclic AMP production was largely prevented by pretreatment of N1E115 cells with islet-activating protein (pertassis toxin). In contrast, pertussis toxin did not inhibit neurotensin-stimulated cyclic GMP production in neuroblastoma cells. In cell membranes, the toxin promoted the selective ADP-ribosylation of a single protein having the same molecular weight (41,000) as the .alpha.-subunit of Ni, the inhibitory regulatory protein of adenylate cyclase. In membranes prepared from N1E115 cells, monoiodo[125I-Tyr3]neurotensin bound to a single population of receptors characterized, at 25.degree. and in the absence of monovalent cations and quanyl nucleotides, by a dissociation constant (Kd) of 56 pM and a maximal binding capacity (Bm) of 30 fmol/mg of protein. Na+ (10-100 mM) and GTP (0.1-100 .mu.M) inhibited neurotensin binding in a concentration-dependent manner. At 100 mM Na+ and 100 .mu.M GTP, receptor affinity was decreased by 5-and 2-fold, respectively. Li+ and K+ were less effective than Na+, and the effect of GTP was shared by GDP and guanyl-5''-yl-imidodiphosphate, but not by GMP, ATP, ADP, or adenyl-5''-yl-imidodiphosphate. It is concluded that in N1E115 cells, neurotensin attenuates cyclic AMP production by exerting an inhibitory effect on adenylate cyclase through an interaction of the peptide receptors with the regulatory GTP-binding protein Ni.