New agonist and antagonist analogues of bradykinin.

Three new analogues of bradykinin (BK) have been tested for their agonistic and antagonistic actions on the rabbit jugular vein and the guinea pig ileum (B2 receptors), and six were studied on rabbit aorta strips (B1 receptors). Substitution of Gly4, Phe5, and Phe8 in BK with D-Trp gives analogues with a relative affinity lower than 1.0% as compared with BK. These analogues have no antagonistic properties on the rabbit jugular vein and on guinea pig ileum (B2 receptors). Substitution of Pro7 in des-Arg9-BK by Gly and by D-Ala give compounds that antagonise the effects of kinins on the rabbit aorta strips (B1-receptor system). These new antagonists are fairly potent with a pA2 value of 6.03 to 7.29 and seem competitive because the pA2--pA10 values approximate 0.95. These results suggest that the orientation of Phe8 is critical for the activation of B1 receptors by kinins.

Effects of bradykinin and some of its fragments on smooth muscles and chemotaxis.

Bradykinin (BK) and two of its C-terminal fragments, namely H-Phe-Ser-Pro-Phe-Arg-OH and H-Ser-Pro-Phe-Arg-OH were found to be potent inhibitors of the chemotaxis of rat polymorphonuclear neutrophils (PMN). The activity of the three peptides was significantly enhanced by SQ 14225, a rather specific inhibitor of kininase II. A shorter C-terminal sequence of BK (Phe-Arg-OH) was inactive. The whole peptide (BK) exerted potent actions on blood pressure and on isolated organs, e.g. the rabbit mesenteric vein or the guinea pig ileum, but none of its fragments showed similar effects. The present findings suggest that rat PMN possess membrane receptors which cannot be considered identical to B1- and B2-receptors, previously characterized on isolated smooth muscles.

Effects of kinins on isolated blood vessels. Role of endothelium.

Bradykinin (BK) and des-Arg9-BK were used to determine whether the stimulatory and inhibitory actions of the kinins in various isolated vessels require the presence of endothelium and may be mediated by arachidonic acid metabolites. It was found that the presence of intact endothelium is required only for the relaxation of the dog common carotid artery in response to bradykinin. Stimulatory actions of both BK and des-Arg9-BK in arterial (rabbit aorta) and venous (rabbit jugular and mesenteric vein) smooth muscle do not require the presence of endothelium. Inhibition of the arachidonic acid cascade at various levels affects the relaxing action of acetylcholine (rabbit aorta and dog common carotid artery) while being inactive against both the relaxing (dog common carotid artery) and contractile actions (rabbit aorta, rabbit jugular and mesenteric veins) of bradykinin and des-Arg9-BK. Inhibitors of the arachidonic acid cascade also do not affect the inhibitory action of isopropylnoradrenaline on the rabbit aorta. The present results indicate that stimulant actions of kinins in isolated vascular smooth muscles do not require the presence of endothelium. Endothelium is required for the inhibitory actions of acetylcholine and bradykinin but not for that of isopropylnoradrenaline on the dog carotid artery. Moreover, the inhibition of arachidonic acid metabolism only affects the response of isolated vessels to acetylcholine. The present results suggest that several mechanisms may be involved in the inhibition of vascular tone by vasodilators.

Synthesis and biological activities of substance P antagonists.

Several substance P analogues containing various D-amino acid modifications have been synthesized by the solid-phase procedure, detached from the solid support by ammonolysis, and purified by gel filtration combined with reversed-phase chromatography. Three compounds were fair to very potent competitive antagonists of substance P on three bioassays, i.e., guinea pig ileum, rabbit mesenteric vein, and guinea pig trachea. [Arg6,D-Trp10]SP(6-11) is a reasonable antagonist in all three bioassays and [D-Pro4,D-Trp7,9]SP(4-11) is a very potent competitive antagonist with pA2 values ranging around 6.0.

Substance P antagonists active in vitro and in vivo.

[Pro4, Trp7,9]SP-(4-11) and a newly developed compound, [Pro4, Trp7,9,10]SP-(4-11) were shown to be potent antagonists of substance P (SP). They inhibited the hypotensive effect of SP in the anesthetised rats, the vasodilator effect in the rabbit perfused heart and the relaxation induced by SP in dog carotid arteries contracted with noradrenaline. Stimulatory actions of SP in the guinea pig ileum and the guinea pig trachea were also inhibited by the two antagonists, which were shown to be specific for substance P and related peptides in all the pharmacological preparations utilised in the present study.

Effects of peptides and amines on isolated guinea pig tracheae as influenced by inhibitors of the metabolism of arachidonic acid.

Stimulants (histamine, acetylcholine and substance P) and relaxants (isoprenaline and bradykinin) of the guinea pig trachea were tested in the absence and presence of mepacrine, eicosatetraynoic acid, BW 755C and indomethacin in order to evaluate the involvement of prostaglandins and congeners in the effects of amines and peptides. While histamine appeared to stimulate tracheal smooth muscle directly, acetylcholine acted in part by promoting the release of contractile leukotrienes. Substance P was unable to express its full stimulating effect because of a likely release of an inhibitory prostaglandin. Isoprenaline inhibited tracheal smooth muscles by a direct action, while the relaxation of the trachea in response to bradykinin appeared to depend on the release of prostaglandins, since it was reduced in the presence of inhibitors of cyclo- and lipooxygenase. It is concluded that enzymes involved in the metabolism of arachidonic acid are present in the guinea pig trachea and are activated both by relaxant and stimulant agents.

Pharmacological effects of peptides on tracheal smooth muscle.

Peptide and non-peptide agents were tested for their stimulatory or inhibitory effects on circular strips of guinea pig isolated tracheae. Substance P, eledoisin, physalaemin, neurotensin, angiotensin, histamine and carbachol were found to contract, while noradrenaline, dopamine, bradykinin, nucleotides (AMP, ADP, ATP) and prostaglandins (PGE1, PGE2, PGA2) induced concentration-dependent relaxations of tracheae contracted with substance P or carbachol. Indomethacin (2.8 X 10(-6) M) significantly potentiated the effect of substance P and blocked that of bradykinin. The contractions to substance P of tissues treated with indomethacin were not modified by atropine, methysergide, diphenhydramine, cimetidine, propranolol, phentolamine, [Leu8]-ATII, [Leu8]-des-Arg9-bradykinin, naloxone and baclofen. The order of potency of C-terminal fragments of substance P was: hexa(6-11) greater than hepta(5-11) greater than substance P greater than = octa(4-11). It is concluded that the guinea pig isolated trachea is a pharmacological preparation sensitive to numerous agents and useful for studying structure-activity relationship and the mechanism of cellular action of several peptides, particularly substance P.