[Nle11]-Substance P

Four preparations, sensitive to tachykinins, the guinea-pig urinary bladder, the rat duodenum, the hamster and dog urinary bladders have been investigated and compared with four other preparations described before: the guinea-pig ileum and trachea, the dog carotid artery and the rabbit mesenteric vein. On the basis of the order of potency of agonists, evaluated with substance P, physalaemin, eledoisin, kassinin and neurokinin A, the preparations can be separated into three groups, the guinea-pig urinary bladder and the dog carotid artery, in which substance P is the most potent and neurokinin A the weakest tachykinin, the rabbit mesenteric vein, the guinea-pig trachea and the rat duodenum, in which the opposite order is observed and the hamster and dog urinary bladders, in which kassinin is the most potent agonist. The guinea-pig ileum shows similar sensitivity to the five tachykinins. C-terminal partial sequences appear to be weaker than SP-(1-11) in three of the four new preparations, SP-(6-11) being first in the rat duodenum and slightly weaker than SP-(1-11) in the hamster and dog urinary bladders. Studies performed with antagonists or inhibitors of endogenous agents suggest that substance P and neurokinin A act directly on specific receptors. The effects of the two peptides are reduced by antagonists analogues of the sequence SP-(4-11). One of the antagonists, [D-Pro4,Lys6,D-Trp7,9,10, Phe11]SP-(4-11) has been shown to be competitive against substance P and neurokinin A in the guinea-pig ileum, the guinea-pig urinary bladder and the rat duodenum. This compound, shows definitely higher activity against neurokinin A and kassinin, compared to substance P in various preparations. [D-Tyr4,D-Trp7,9,Nle11]SP-(4-11) is the most potent tachykinin antagonist in the hamster and dog urinary bladders. In these preparations, the antagonists act also against substance P, but with lower affinity. These findings with antagonists support the indication, emerged from the order of potency of agonists, that tachykinins may act on two and possibly three different receptor types.

The studies concerning the problem of whether an exogenous neuropeptide is able to enter the brain tissue were extended to the undecapeptide Substance P (SP). The amount of radioactivity 15 s after intracarotid injection of [3H] Nle11-SP or [14C] inulin was determined in 18 brain regions and the anterior pituitary of male rats. As compared to the reference [14C] inulin (mean +/- SD: 0.143 +/- 0.009% of the injected radioactivity per g tissue wet weight), the amount of radioactivity was higher after [3H]Nle11-SP injection (0.233 +/- 0.039%, p less than 0.001). Statistically significant differences could be found particularly in cortical and caudal areas as well as in the circumventricular organs studied. These observations do not refute the assumption that a low brain uptake of the labelled neuropeptide occurred due to an accumulation within structures of the blood-brain barrier and/or a penetration of the barrier system.

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.