FUNCTIONAL-STUDIES WITH SUBSTANCE-P ANALOGS - EFFECTS OF N-TERMINAL, C-TERMINAL, AND C-TERMINUS-EXTENDED ANALOGS OF SUBSTANCE-P ON NICOTINE-INDUCED SECRETION AND DESENSITIZATION IN CULTURED BOVINE ADRENAL CHROMAFFIN CELLS

Substance P (SP) and SP analogues, including C-terminal, N-terminal, a nd C-terminus-extended analogues, have been investigated for their abi lity to modulate nicotine-induced secretion from bovine adrenal chroma ffin cells in culture. Secretion was monitored by measuring the releas e of endogenous catecholamines by electrochemical detection following separation on HPLC and the release of endogenous ATP with an on-line l uciferin-luciferase bioluminescence technique. SP is known to have the following two effects on nicotine-induced secretion of catecholamines (see Livett and Zhou, 1991): inhibition of the nicotinic response and protection against nicotinic desensitization. Secretion induced by 10 (-5) M nicotine was inhibited 70-80% by SP, SP-methyl ester, and the C -terminus-extended analogue SP-Tyr(12)-NH2, 65% by (Ala(3))SP-NH2, 45% by the C-terminal analogue SP(4-11), and 20 and 5% by the N-terminal analogues SP(1-7) and SP(1-5), respectively, when these peptides were present at 3 X 10(-5) M concentrations. The order of potency was SP = SP-methyl ester = SP-Tyr(12)-NH2 > (Ala(3))SP-NH2 > SP(4-11) > SP(1-7) > SP(1-5). SP, SP-methyl ester, and (Ala(3))SP-NH2 protected against nicotinic desensitization by 40-55%, and SP(4-11) protected by 20% (al l at 3 x 10(-5) M). In contrast, the N-terminal analogues SP(1-7) and SP(1-5) and the C-terminus-extended analogue SP-Tyr(12)-NH2 at 3 X 10( -5) M did not protect against nicotinic desensitization. Cyclo-SP(3-9) , Ac-SP(3-9)-NH2, SP(3-9), and SP(3-6) had neither inhibitory nor faci litatory effects on secretion. Of the 20 SP analogues extended at the C terminus by one amino acid, there were only three that protected aga inst nicotinic desensitization, whereas the majority inhibited nicotin e-evoked catecholamine secretion. The present work indicates that for inhibition of nicotine-evoked secretion, both the C terminus and N ter minus of SP are necessary. For the protection against nicotine-induced desensitization, the C terminus of SP is important. This suggests tha t the two mechanisms, inhibition of nicotine-evoked secretion and prot ection against nicotinic desensitization, are regulated independently.