Physalaemin

Abstract: Substance P (6.25-25 p-mole) produced dose-dependent flare and wheal responses when injected intradermally into the volar surface of the human forearm. The maximum flare response was obtained within the first 3 min of injection and declined thereafter. The wheal response reached a maximum after 12 min following the injection. Only those peptides having one or more basic residues in the N-terminal region were effective in producing a flare reaction. Eledoisin-related peptide and SP1-9 were 17 and 7 times less active than substance P respectively, whilst [D-pro2, D-phe7, D-trp9]SP1-11 was twice as active. The N-terminal tetrapeptide, SP1-4 and eledoisin were inactive in the dose range tested. Wheal-producing activity was not dependent on the presence of basic residues and the rank order of relative potencies was: physalaemin (2.0): [D-pro2, D-phe7, D-trp9]SP1-11 (1.1): SP1-11 (1.0): SP4-11 (0.4): SP1-9 (0.15): eledoisin-related peptide (0.08): eledoisin (0.06). The N-terminal tetrapeptide failed to produce a wheal response in the dose range tested. Substance P was approximately equi-active with poly-L-arginine in the production of wheal and flare and both of these agents were about 10 times more potent than histamine. Adenosine triphosphate (25-400 n-mole) produced dose-dependent wheal and flare responses and was 10,000 times less potent than substance P. Pre-treatment of the subjects with the H1 histamine antagonist, chlorpheniramine, (20 mg I.V.) reduced the wheal and flare responses to substance P. Local anaesthetic injection into the skin reduced the spread of the flare response but did not affect the development of the wheal response. Pre-treatment of the skin with capsaicin reduced the flare but not the wheal response to intradermal injection of histamine. The results are discussed in relation to the mechanism of the ‘axon reflex’ vasodilatation in skin. This is thought to involve mast cells in addition to substance P-containing primary afferent neurones.

Abstract: 1. Human skin mast cells, unlike other human mast cells so far studied, released histamine in a concentration-related manner in response to substance P, vasoactive intestinal peptide (VIP) and somatostatin (1 microM to 30 microM). In contrast, eledoisin, physalaemin, neurokinin A, neurokinin B, calcitonin gene-related peptide (CGRP), neurotensin, bradykinin and Lys-bradykinin induced negligible histamine release. 2. The low histamine releasing activity of physalaemin, eledoisin, neurokinin A and neurokinin B relative to substance P suggests that the human skin mast cell activation site is distinct from the tachykinin NK-1, NK-2 or NK-3 receptors described in smooth muscle. 3. The relative potencies of substance P and its fragments SP2-11, SP3-11, SP4-11 and SP1-4 in releasing histamine from human skin mast cells suggests that both the basic N-terminal amino acids and the lipophilic C-terminal portion of substance P are essential for activity. 4. Peptide-induced histamine release, like that induced by compound 48/80, morphine and poly-L-lysine, is rapid, reaching completion in 10-20 s, is largely independent of extracellular calcium but requires intact glycolysis and oxidative phosphorylation. 5. The substance P analogue, [D-Pro4,D-Trp7,9,10] SP4-11 (SPA), not only reduced substance P-induced histamine release in a concentration-related manner but also inhibited that induced by VIP, somatostatin, compound 48/80, poly-L-lysine and morphine but not anti-IgE. 6. The similar characteristics of histamine release induced by substance P, VIP, somatostatin, compound 48/80, poly-L-lysine and morphine suggest that they share a common pathway of activation-secretion coupling distinct from that of IgE-dependent activation. Furthermore, the ability of human skin mast cells to respond to basic non-immunological stimuli including neuropeptides may reflect a specialised function for these cells.

Abstract: The most widely used smooth muscle preparations for neurokinin bioassays have been critically analyzed in order to determine whether neurokinins act directly or by the intermediary of other natural agents. Indeed, part of the contraction of the GPI in response to neurokinins appears to be mediated by acetylcholine and possibly prostaglandins. Active metabolites of the arachidonic acid cascade also intervene in the response of the HUB. Neurokinins produce relaxation of the DCA by stimulating the release of a vascular smooth muscle relaxing factor from the endothelium. In the other preparations (the RD, the RPA without endothelium and the RPV) neurokinins may act directly on the smooth muscle fibers. Neurokinins produce their biological effects by activating specific receptors. Three different receptor types, one for each mammalian neurokinin, have been identified by using four groups of natural peptide sequences and some selective agonists. The receptor for SP is particularly sensitive to SP and physalaemin and shows higher affinity for the whole natural peptides (SP, NKA) than for their C-terminal fragments. The receptor for neurokinin A is highly sensitive to NKA and eledoisin: it shows high affinity for heptapeptide fragments such as NKA4-10 and SP5-11. The receptor for NKB is sensitive to NKB and kassinin more than to the other natural peptides and their fragments. The natural peptides show however little selectivity. Synthetic analogues active on a single receptor type (selective agonists) have been used to find out whether the responses of the isolated organs are due to the activation of one or more than one receptor. It has been found that the GPI, the RD and the HUB contain all three or at least two receptors, while the DCA has only the NK1, the RPA has only the NK2 and the RPV only the NK3 type. Binding sites specific for each neurokinin have been identified in brain and peripheral organs with accurate biochemical assays, using labeled neurokinins. Competitive displacement assays have been performed with a variety of neurokinin-related peptides, and their Ki have been determined. By plotting Ki values against the ED50, estimated from biological assays, positive significant correlations have been found for the monoreceptor (DCA, RPA, RPV) but not for the multiple receptor systems (GPI, RD, HUB). This suggests that pharmacological receptors may be identical with the recognition sites which bind the labeled neurokinins. The availability of monoreceptor systems and of selective agonists opens the way for the identification of potential antagonists and accurate estimation of their affinities.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: 1. The algesic effect of several substances following intraarterial injection into the rabbit ear was measured: by recording the action potentials of the auricular nerve in the isolated ear (method A) and by recording the reflex fall in systemic blood pressure elicited by intraarterial injection into the isolated perfused ear which was still connected to the body by the nerve (method B). 2. The order of activity of the algesic substances was: bradykinin, substance P>ACh>ATP, histamine, serotonin, KCl. A good correlation of the actions of all substances tested was found in both preparations. Physalaemin, eledoisin, adenosine, adrenaline, angiotensin, vasopressin, oxytocin and prostaglandin E1 were found to be inactive. 3. Infusion of prostaglandin E1 into the ear, however, enhanced the effect of all substances. 4. The vasoconstriction in the ear observed with most of the substances was not correlated with their activity in eliciting afferent impulses.