Transglutaminase-synthesized spermine derivative of substance P recognizes rat portal vein neurokinin-3 receptors

The effects of the transglutaminase-sinthesized polyamine derivatives of Substance P (SP) have been further characterized by their ability to contract in vitro the rat portal vein strip (RPV), a pharmacological preparation particularly rich in NK-3 receptors. The effects of selective agonists of NK-1, NK-2 and NK-3 receptors [Sar(9),Met(O-2)(11)]SP beta-Ala(8) NKA(4-10),and senktide respectively, were also evaluated by measuring RPV concentration-response curves. Peptide [GR-82334 (NK-1) and MEN-10,376 (NK-2)] and nonpeptide [WIN 51,708 (NK-1) and SR 142801 (NK-3)] NK receptor antagonists were used to confirm the participation of the different NK receptors to contractile response. Our results demonstrated that the spermine derivative of SP (Spm-SP), previously shown to be unable to recognize NK-1 and NK-2 receptors in some bioassays, contracts RPV (EC(50) = 588 nM) better than the native neuropeptide (EC(50) = 1120 nM). A pretreatment with thiorphan, an inhibitor of neutral endopeptidases, significantly reduced such a difference. While this inhibitor shifts the SP concentration-response curves to the left (EC(50) = 720 nM) the action of Spm-SP and [Sar(9),Met(O-2)11]SP were completely thiorphan-resistant. In the absence of thiorphan we found the following rank order of potency: senktide>>beta-Ala(8) NKA(4-1O)>[Sar9,Met(O-2)(11)]SP=Spm-SP>SP. Among the mentioned NK receptor antagonists, only the selective NK-3 receptor antagonist, SR 142801, shifted to the right Spm-SP and [Sar(9),Met(O-2)(11)]SP concentration-response curve, showing pK(B) values of 5.84 and 5.88, respectively. Therefore, the reported results suggest that the introduction of a Spm moiety into the SP alters the parent peptide molecule by increasing its affinity for NK-3 receptors and/or by preventing its degradation by some proteolytic enzymes.