Substance P (5-11)

Substance P at micromolar concentrations enhances the uptake of [14C]guanidinium in neuroblastoma X glioma hybrid cells, an effect which most likely indicates activation of Na+ permeability. The substance P receptor was characterized pharmacologically. Analogues of substance P with D-amino acids e.g. spantide, and substance P-methyl ester were similarly active. Substance P (free acid), fragments of the substance P precursor, and substance P-(1-9) displayed no activity. This indicates the importance of the hydrophobic C-terminal for stimulation of the hybrid cells. The potency was reduced with decreasing length the of C-terminal fragments. However, the substance P antagonists [D-Pro4,D-Trp7,9,Nle11]substance P-(4-11) and [D-Pro4,D-Trp7,9,10]substance P-(4-11) showed substantially greater activity than substance P-(4-11). Substance P-(6-11) (i.e. H-Arg-DTrp-MePhe-DTrp-Leu-Met-NH2) behaved as a mixed agonist-antagonist. At concentrations higher than 10 microM, it inhibited the stimulation exerted by substance P. No other peptides of the tachykinin family (neurokinins A and B, physalaemin, eledoisin, kassinin) nor the synthetic analogues with specificity for certain receptor subtypes ([pGlu6,Pro9]substance P-(6-11), DiMe-C7, i.e. [pGlu5,MePhe8,Sar9]substance P-(5-11) and senktide, i.e. N-succinyl-[Asp6,MePhe8]substance P-(6-11) had any effect on guanidinium uptake in the hybrid cells. Hence, the substance P site with low affinity on the hybrid cells does not fit into the usual classification of tachykinin receptors but resembles the site that modulates nicotinic acetylcholine receptors on chromaffin cells.

Using an antiserum directed at the COOH-terminus of tachykinins, we have examined postmortem tissue from two cases of metastatic ileal carcinoid for the presence of tachykinin-like immunoreactivity. The vast majority of the immunoreactive tachykinin-like material eluted from a Sephadex G-50 column as two peaks at positions corresponding to molecular weights of 1300 and 850. The 1300 dalton peak was resolved by reverse-phase-HPLC into two components which by Edman sequencing, amino acid analysis, and fast atom bombardment (FAB)-mass spectrometry criteria, were identified as substance P and substance K. The 850 dalton peak was also resolved on RP-HPLC into two peaks which were resistant to Edman degradation but from amino acid analysis and FAB-mass spectrometry criteria were identified as pyro-Glu-substance P 5-11 and oxidized pyro-Glu-substance P 5-11. In control experiments substance P 5-11 was converted to pyro-Glu-substance P 5-11 during the extraction procedure. Both tumors also contained a minor immunoreactive peak which eluted from a Sephadex G-50 sizing column at a position corresponding to a molecular weight of 4000 which probably represents neuropeptide K. These results suggest that beta-preprotachykinin is preferentially expressed in carcinoid tumors and that substance K may also play a role in the carcinoid syndrome.

Accumulating evidence shows that N- and C-terminal substance P fragments have significant biological activity. Substance P(1-9) and substance P(6-11) have been reported to be major substance P metabolites in rat striatum. We investigated the effects of these fragments on endogenous dopamine outflow in rat striatal slices. Substance P-(1-9) and substance P-(6-11) induced a significant increase in dopamine outflow at 0.1 and 1 nM. The effects of substance P-(6-11) (1 nM) were reversed by the tachykinin NK1 antagonist WIN 51,708 (17beta-hydroxy-17alpha-ethynyl-5alpha-androstano[3,2- b]pyrimido[1,2-a]benzimidazole) (2.5 nM), whereas the effects of substance P-(1-9) were not modified by the antagonist. Substance P-(1-9) and substance P-(6-11) (1 nM) did not increase the dopamine overflow induced by 25 mM KCI. The effects of the two fragments were reversed by the muscarinic antagonist atropine (1 microM) but not by nicotinic antagonists dihydro-beta-erythroidine (0.5 microM) and pempidine (10 microM). The co-incubation of tissue with substance P and each fragment in a 1/1 or 10/1 ratio of substance P to metabolite revealed a negative interaction between parent and fragments. A similar pattern was observed when substance P was co-administered with the active fragments substance P(1-4), substance P(1-7), substance P(5-11) and substance P(8-11). The data show that substance P-(1-9) and substance P-(6-11) have modulatory effects similar to substance P. However, the presence of active substance P metabolites does not appear to amplify the signal mediated by the parent peptide.