Novel tool for the study of cholecystokinin-stimulated pancreatic enzyme secretion.

The molecular events that mediate cholecystokinin (CCK)-stimulated pancreatic secretion are not well defined because of the complex receptor-binding and concentration-response characteristics of this hormone. Functional models of receptor occupancy initiating the cascade leading to secretion have been complicated by the inhibition of secretion effected by supramaximal concentrations of CCK. Recent report of a CCK analogue that does not exhibit supramaximal inhibition led us to synthesize a similar analogue that could also be radiolabeled for studies of receptor binding and affinity labeling, and for studies of second messenger activity. This probe, D-Tyr-Gly-[(Nle28,31)CCK-26-32]-phenethyl ester, was a fully efficacious secretagogue with no supramaximal inhibition, and, unlike native hormone, bound to a single class of sites present on both acini and membranes. Occupation of this site correlated well with stimulation of secretion. Evidence that this was indeed a CCK-binding site were the abilities of CCK and the antagonist L-364, 718 to inhibit binding of this analogue. Affinity labeling confirmed the identity of the site mediating secretory stimulation as a Mr = 85,000-95,000 protein. Whereas the nonhydrolyzable guanosine triphosphate analogue, 5'-guanylyl-imidodiphosphate, was a potent inhibitor of CCK binding, it had no effect on binding of this secretagogue, suggesting that a novel cascade not involving a guanine nucleotide-binding protein mediates CCK stimulation of pancreatic secretion.

Neurotensin and its analogs--correlation of specific binding with stimulation of cyclic GMP formation in neuroblastoma clone N1E-115.

The receptors which mediate neurotensin-stimulated intracellular cyclic GMP formation in murine neuroblastoma clone N1E-115 [J. A. Gilbert and E. Richelson, Eur. J. Pharmac. 99, 245 (1984)] were further characterized. The binding of [3H]neurotensin to intact N1E-115 cells at 0 degree displayed specificity, saturability, reversibility, and tissue linearity. A single class of neurotensin receptors was demonstrated with an apparent KD of 9-11 nM and a Bmax of 180-250 fmoles/10(6) cells, determined by the type of serum employed in the cellular culture medium. A number of neurotensin analogs and fragments were compared for their ability to inhibit [3H]neurotensin binding and stimulate intracellular cyclic GMP formation with intact N1E-115 cells. A direct correlation was found to exist between the KD and EC50 for each peptide. The carboxyl-terminal portion of neurotensin proved to be responsible for the binding and biochemical activities of this peptide with clone N1E-115. Neurotensin(8-13) was, in fact, fifty times more potent than native neurotensin in stimulating intracellular cyclic GMP formation and had an 18-fold higher affinity for the neurotensin receptor on this neuronal cell type.

Neurotensin(8-13): comparison of novel analogs for stimulation of cyclic GMP formation in neuroblastoma clone N1E-115 and receptor binding to human brain and intact N1E-115 cells.

Neurotensin(8-13), the carboxyl-terminal portion of neurotensin, is 4-50 times more potent than native neurotensin in binding to intact neuroblastoma N1E-115 cells and human brain tissue and in stimulation of intracellular cyclic GMP production and inositol phospholipid hydrolysis in clone N1E-115 (Gilbert JA and Richelson E, Eur J Pharmacol 99: 245-246, 1984; Gilbert JA et al., Biochem Pharmacol 35: 391-397, 1986; Kanba KS et al., J Neurochem 46: 946-952, 1986; and Kanba KS and Richelson E, Biochem Pharmacol 36: 869-874, 1987). A series of novel analogs of neurotensin (8-13) was synthesized, and a structure-activity study was done comparing the abilities of these peptides to stimulate intracellular cyclic GMP production in intact neuroblastoma clone N1E-115 and to inhibit the binding of [3H]neurotensin to these cells and to membranal preparations from human brain. A direct correlation was found for each analog between its EC50 for biochemical activity and its KD for binding ability in studies with clone N1E-115. Furthermore, a strong correlation existed for each peptide between its KD for binding to neurotensin receptors on these cells and its KD for binding to neurotensin receptors in human brain tissue. In this study, the residues that were important to the biochemical and binding activities of neurotensin (8-13) proved to be identical to the amino acids that are necessary for the functional integrity of native neurotensin (Gilbert JA et al., Biochem Pharmacol 35: 391-397, 1986.

Selectivity of antimuscarinic compounds for muscarinic receptors of human brain and heart.

Seven antimuscarinic compounds, used mostly for the treatment of extrapyramidal problems, were tested in vitro in radioligand binding assays for evidence of selectivity for two different pharmacological subtypes of the human muscarinic receptor, M1, a predominant form in brain, and M2, a predominant form in heart. Although biperiden, scopolamine, procyclidine, and benztropine showed significant selectivity in the in vitro assays, it is likely that in clinical practice biperiden would be the drug of choice to avoid any antimuscarinic effects on the heart.

Structure-antinociceptive activity of neurotensin and some novel analogues in the periaqueductal gray region of the brainstem.

Neurotensin, an endogenous tridecapeptide, produces a potent, naloxone-insensitive antinociceptive response when it is microinjected into the periaqueductal gray region of the rat brainstem. In the present study, the ED50 for neurotensin in inducing antinociception was 1.5 nmol, two times more potent than morphine. We sought to find whether neurotensin's antinociceptive effects were mediated by the same receptor that mediates its other functions. We found that the structure-activity relationship of neurotensin-induced antinociception was different from that required for the stimulation of intracellular cyclic GMP production in neuroblastoma clone N1E-115 and the binding to N1E-115 cells, human brain tissue, or rat periaqueductal gray. These data suggest there exists a subtype of neurotensin receptors in neural tissue that mediates its antinociceptive actions.