Michele Tognetto

An endogenous capsaicin-like substance with high potency at recombinant and native vanilloid VR1 receptors

The vanilloid receptor VR1 is a nonselective cation channel that is most abundant in peripheral sensory fibers but also is found in several brain nuclei. VR1 is gated by protons, heat, and the pungent ingredient of “hot” chili peppers, capsaicin. To date, no endogenous compound with potency at this receptor comparable to that of capsaicin has been identified. Here we examined the hypothesis, based on previous structure-activity relationship studies and the availability of biosynthetic precursors, that N-arachidonoyl-dopamine (NADA) is an endogenous “capsaicin-like” substance in mammalian nervous tissues. We found that NADA occurs in nervous tissues, with the highest concentrations being found in the striatum, hippocampus, and cerebellum and the lowest concentrations in the dorsal root ganglion. We also gained evidence for the existence of two possible routes for NADA biosynthesis and mechanisms for its inactivation in rat brain. NADA activates both human and rat VR1 overexpressed in human embryonic kidney (HEK)293 cells, with potency (EC50 ≈ 50 nM) and efficacy similar to those of capsaicin. Furthermore, NADA potently activates native vanilloid receptors in neurons from rat dorsal root ganglion and hippocampus, thereby inducing the release of substance P and calcitonin gene-related peptide (CGRP) from dorsal spinal cord slices and enhancing hippocampal paired-pulse depression, respectively. Intradermal NADA also induces VR1-mediated thermal hyperalgesia (EC50 = 1.5 ± 0.3 μg). Our data demonstrate the existence of a brain substance similar to capsaicin not only with respect to its chemical structure but also to its potency at VR1 receptors.

Agonists of proteinase-activated receptor 2 induce inflammation by a neurogenic mechanism.

Trypsin and mast cell tryptase cleave proteinase-activated receptor 2 and, by unknown mechanisms, induce widespread inflammation. We found that a large proportion of primary spinal afferent neurons, which express proteinase-activated receptor 2, also contain the proinflammatory neuropeptides calcitonin gene-related peptide and substance P. Trypsin and tryptase directly signal to neurons to stimulate release of these neuropeptides, which mediate inflammatory edema induced by agonists of proteinase-activated receptor 2. This new mechanism of protease-induced neurogenic inflammation may contribute to the proinflammatory effects of mast cells in human disease. Thus, tryptase inhibitors and antagonists of proteinase-activated receptor 2 may be useful anti-inflammatory agents.

The vanilloid receptor (VR1)-mediated effects of anandamide are potently enhanced by the cAMP-dependent protein kinase.

The endogenous cannabinoid receptor ligand, anandamide (AEA), is a full agonist of the vanilloid receptor type 1 (VR1) for capsaicin. Here, we demonstrate that the potency and efficacy of AEA at VR1 receptors can be significantly increased by the concomitant activation of protein kinase A (PKA). In human embryonic kidney (HEK) cells over‐expressing human VR1, AEA induces a rise in cytosolic Ca2+ concentration that is mediated by this receptor. The EC50 for this effect was decreased five‐fold in the presence of forskolin (FRSK, 1–5 µm) or the cAMP analogue, 8‐Br‐cAMP (10–100 µm). The effects of 8‐Br‐cAMP and FRSK were blocked by a selective PKA inhibitor. The FRSK (10 nm) also potently enhanced the sensory neurone‐ and VR1‐mediated constriction by AEA of isolated guinea‐pig bronchi, and this effect was abolished by a PKA inhibitor. In rat dorsal root ganglia slices, AEA‐induced release of substance P, an effect mediated by VR1 activation, was enhanced three‐fold by FRSK (10 nm). Thus, the ability of AEA to stimulate sensory VR1, with subsequent neuropeptide release, appears to be regulated by the state of activation of PKA. This observation supports the hypothesis that endogenous AEA might stimulate VR1 under certain pathophysiological conditions.

Neurogenic responses mediated by vanilloid receptor‐1 (TRPV1) are blocked by the high affinity antagonist, iodo‐resiniferatoxin

Stimulation of the vanilloid receptor‐1 (TRPV1) results in the activation of nociceptive and neurogenic inflammatory responses. Poor specificity and potency of TRPV1 antagonists has, however, limited the clarification of the physiological role of TRPV1. Recently, iodo‐resiniferatoxin (I‐RTX) has been reported to bind as a high affinity antagonist at the native and heterologously expressed rat TRPV1. Here we have studied the ability of I‐RTX to block a series of TRPV1 mediated nociceptive and neurogenic inflammatory responses in different species (including transfected human TRPV1). We have demonstrated that I‐RTX inhibited capsaicin‐induced mobilization of intracellular Ca2+ in rat trigeminal neurons (IC50 0.87 nM) and in HEK293 cells transfected with the human TRPV1 (IC50 0.071 nM). Furthermore, I‐RTX significantly inhibited both capsaicin‐induced CGRP release from slices of rat dorsal spinal cord (IC50 0.27 nM) and contraction of isolated guinea‐pig and rat urinary bladder (pKB of 10.68 and 9.63, respectively), whilst I‐RTX failed to alter the response to high KCl or SP. Finally, in vivo I‐RTX significantly inhibited acetic acid‐induced writhing in mice (ED50 0.42 μmol kg−1) and plasma extravasation in mouse urinary bladder (ED50 0.41 μmol kg−1). In in vitro and in vivo TRPV1 activated responses I‐RTX was ∼3 log units and ∼20 times more potent than capsazepine, respectively. This high affinity antagonist, I‐RTX, may be an important tool for future studies in pain and neurogenic inflammatory models.

NOCICEPTIN RECEPTOR ACTIVATION INHIBITS TACHYKINERGIC NON ADRENERGIC NON CHOLINERGIC CONTRACTION OF GUINEA PIG ISOLATED BRONCHUS

We studied the action of nociceptin (NC) on the atropine-resistant contractions of the guinea pig isolated bronchus evoked by the electrical field stimulation (EFS), an effect that is mediated by the activation of excitatory non adrenergic-non cholinergic (eNANC) nerves and the subsequent release of tachykinins. The functional site by which NC acts in this preparation was investigated using few different NC receptor agonists and the newly discovered NC receptor antagonist, [Phe1psi(CH2-NH)Gly2]NC(1-13)NH2 ([F/G]NC(1-13)NH2). NC inhibited in a concentration dependent manner (pEC50 7.14; Em - 87 +/- 3% of control values) EFS induced contractions. NC effect was mimicked by the NC analogues, NCNH2 and NC(1-13)NH2, but not by NC(1-9)NH2. NC (1 microM) did not affect the contractile effects of exogenously applied neurokinin A (1 microM). [F/G]NC(1-13)NH2 (10 microM) completely prevented the inhibition induced by NC (1 microM), whereas naloxone (1 microM) was found inactive. Both naloxone and ([F/G]NC(1-13)NH2 were per se inactive on basal resting tone as well as on the electrically induced contractions. The present findings show that NC inhibits the atropine-resistant EFS-induced contraction in the guinea pig bronchus by inhibiting eNANC nerves, and suggest the presence of NC receptors, distinct from opioid receptors, on the nerves of the guinea pig bronchus.

Evidence that PAR-1 and PAR-2 mediate prostanoid-dependent contraction in isolated guinea-pig gallbladder

We have investigated the ability of protease‐activated receptor‐1 (PAR‐1), PAR‐2, PAR‐3 and PAR‐4 agonists to induce contractile responses in isolated guinea‐pig gallbladder. Thrombin, trypsin, mouse PAR‐1 activating (SFLLRN‐NH2) peptide, and mouse PAR‐2 activating (SLIGRL‐NH2) and human PAR‐2 activating (SLIGKV‐NH2) peptides produced a concentration‐dependent contractile response. Mouse PAR‐4 activating (GYPGKF‐NH2) peptide, the mouse PAR‐1 reverse (NRLLFS‐NH2) peptide, the mouse PAR‐2 reverse (LRGILS‐NH2) and human PAR‐2 reverse (VKGILS‐NH2) peptides caused negligible contractile responses at the highest concentrations tested. An additive effect was observed following the contractile response induced by either trypsin or thrombin, with the addition of a different PAR agonist (SFLLRN‐NH2 and SLIGRL‐NH2, respectively). Desensitization to PAR‐2 activating peptide attenuated the response to trypsin but failed to attenuate the response to PAR‐1 agonists, and conversely desensitization to PAR‐1 attenuated the response to thrombin but failed to alter contractile responses to PAR‐2 agonists. The contractile responses produced by thrombin, trypsin, SFLLRN‐NH2 and SLIGRL‐NH2 were markedly reduced in the presence of the cyclo‐oxygenase inhibitor, indomethacin, whilst the small contractile response produced by NRLLFS‐NH2 and LRGILS‐NH2 were insensitive to indomethacin. The contractile responses to thrombin, trypsin, SFLLRN‐NH2 and SLIGRL‐NH2 were unaffected by the presence of: the non‐selective muscarinic antagonist, atropine; the nitric oxide synthase inhibitor, L‐NAME; the sodium channel blocker, tetrodotoxin; the combination of selective tachykinin NK1 and NK2 receptor antagonists, (S)‐1‐[2‐[3‐(3,4‐dichlorphenyl)‐1 (3‐isopropoxyphenylacetyl) piperidin‐3‐yl] ethyl]‐4‐phenyl‐1 azaniabicyclo [2.2.2] octane chloride (SR140333) and (S)‐N‐methyl‐N‐[4‐acetylamino‐4‐phenylpiperidino‐2‐(3,4‐dichlorophenyl)‐butyl] benzamide (SR48968), respectively. The results indicate that PAR‐1 and PAR‐2 activation causes contractile responses in the guinea‐pig gallbladder, an effect that is mediated principally by prostanoid release, and is independent of neural mechanisms.

Evidence for in vitro expression of B1 receptor in the mouse trachea and urinary bladder

Motor responses to des‐Arg9‐bradykinin and bradykinin were studied in the isolated mouse trachea (precontracted with carbachol, 10 μM) and the urinary bladder of either Swiss, C57Bl/6J or bradykinin B2 receptor knockout (Bk2r−/−) mice after 1–6 h in vitro. The expression of mRNA for the mouse B1 receptor in tracheal and urinary bladder tissues was also studied by using Northern blot analysis. In isolated tracheae, des‐Arg9‐bradykinin produced a relaxant response that increased over time: no response was observed after 1 h of incubation, whereas after 6 h the maximum response (1 μM) was 68–84% of the relaxation produced by isoproterenol (1 μM) in the three mouse strains. The relaxant response to bradykinin (1 μM) observed at 1 h (38–51% of isoproterenol) was increased (62–65% of isoproterenol) after 6 h in Swiss and C57Bl/6J mice, but was absent in Bk2r−/− mice. In the presence of cycloheximide, des‐Arg9‐bradykinin did not cause any response at 6 h. Similar findings were obtained in the urinary bladder: at 1 h des‐Arg9‐bradykinin (1 μM) did not cause any motor effect, whereas at 6 h it caused a contraction that was 28–59% of that produced by carbachol (1 μM) in the three mouse strains. Cycloheximide blocked the response to des‐Arg9‐bradykinin. Bradykinin (1 μM) contracted urinary bladders at 1 h (34–35% of carbachol), as well as at 6 h (66–77% of carbachol) in Swiss and C57Bl/6J strains, but was without effect in Bk2r−/− mice. Northern blot hybridization with a specific cDNA probe against mouse B1 receptor mRNA using total RNA extracted from tracheae and urinary bladders freshly removed from Swiss and Bk2r−/− mice revealed minimal expression. However, marked hybridization was detected 150 min after in vitro exposure in both tissues. Evidence is provided that in vitro exposure of mouse trachea and urinary bladder causes a time‐dependent induction of B1 receptors that cause relaxation and contraction, respectively.

Proteinase‐activated receptor‐1 (PAR‐1) activation contracts the isolated human renal artery in vitro

The in vitro motor function of protease‐activated recepter‐1 (PAR‐1), PAR‐2 and PAR‐4 and the presence by immunohistochemistry of PAR‐1 in the human renal artery have been investigated. Thrombin and the human PAR‐1 (SFLLRN‐NH2) activating peptide, but not the PAR‐1 reverse peptide (NRLLFS‐NH2), contracted both endothelial‐intact and endothelial‐denuded human renal artery strips, whereas no relaxation was observed either in strips non‐precontracted or precontracted with phenylephrine. Maximum contraction by thrombin or SFLLRN‐NH2 was about 60% of phenylephrine. However, thrombin was approximately 1000‐fold more potent than SFLLRN‐NH2. PAR‐1 desensitisation, using repeated applications of SFLLRN‐NH2, almost completely blocked the response to thrombin. The contractile effect produced by thrombin and SFLLRN‐NH2 was not affected by nitric oxide synthase inhibition, but was significantly reduced by cyclooxygenase blockade. Trypsin, the PAR‐2 (SLIGKV‐NH2 and SLIGRL‐NH2) and PAR‐4 (GYPGQV‐NH2 and AYPGKF‐NH2) activating peptides did not produce any significant contraction or relaxation. In agreement with the motor function data immunohistochemistry showed specific staining patterns for PAR‐1 in the human renal artery. Combined, these studies would suggest a possible role for PAR‐1 in renal vascular homeostasis.

Tachykinins and kinins in airway allergy.

Using models of airway diseases, our understanding of the role of tachykinins and kinins in airway pathophysiology has been greatly enhanced by the recent development of a large series of peptide, peptoid and non-peptide antagonists for tachykinin and kinin receptors. This article reviews the experimental findings of the contribution of kinins and tachykinins and their respective receptors, in models of airway inflammation in response to agents known to trigger or worsen asthma attacks, such as antigen and cold air. Some new antagonists, mostly of a non-peptide nature, exhibit excellent pharmacodynamic and pharmacokinetic profiles; a brief account of early clinical studies in which they have been used is also given.

Bradykinin B2 receptors mediate contraction in the normal and inflamed human gallbladder in vitro

BACKGROUND & AIMS The components of the kinin system, including kinongens, kininogenases, and B(2) and B(1) receptors, are expressed and activated during inflammation. Here, we investigated the expression of the kinin B(2) receptor messenger RNA, kininogen and kallikrein immunoreactivity, and the ability of kinins to contract control and inflamed gallbladders in vitro. METHODS Human gallbladders, obtained from patients undergoing cholecystectomy either for acute cholecystitis secondary to gallstone disease or during elective gastro-entero-pancreatic surgery (controls), were processed for reverse-transcription polymerase chain reaction analysis, kallikrein and kininogen immunohistochemistry, binding studies, and in vitro contractility studies. RESULTS Tissue expression of B(2) receptor messenger RNA and specific binding of [(3)H]-bradykinin increased significantly in acute cholecystitis compared to controls. Kallikrein immunoreactivity was detected in the epithelium and infiltrating leukocytes, whereas kininogen immunoreactivity in the lumen of blood vessels and interstitial space. Bradykinin contracted isolated strips of control and acute cholecystitis gallbladders. In acute cholecystitis tissue, efficacy of bradykinin was higher than that of control gallbladders and similar to that of cholecystokinin. The contraction induced by bradykinin was significantly attenuated by B(2) receptor antagonism but not by cyclooxygenase inhibition and B(1), muscarinic, or tachykinin receptor antagonism. CONCLUSIONS All the components of the kinin system are expressed in the human gallbladder. Bradykinin is a powerful spasmogen via B(2) receptor activation in the normal and, especially, in the inflamed human gallbladder.