In this study, changes in plasma levels of calcitonin gene-related peptide (CGRP) and substance P (SP) during a spontaneous-like cluster headache attack provoked by nitroglycerin were evaluated. Peptide variations after spontaneous or sumatriptan-induced remission were also assessed. Blood was collected from the external jugular vein homolateral to the pain side of 30 male cluster headache patients; 18 men were in an active and 12 in a remission one. Plasma levels of CGRP and SP were determined using sensitive radioimmunoassays for each peptide. CGRP-like immunoreactivity (CGRP-LI) was found to be augmented in patients in an active period and became elevated further at the peak of the provoked attack. A complete reversal occurred both after spontaneous and sumatriptan-induced remission. On the contrary, nitroglycerin neither provoked a cluster headache attack nor altered CGRP-LI in the patients in a remission period. The augmented levels of CGRP-LI measured before and after nitroglycerin administration, when the provoked attack reached the maximum intensity, suggest an activation of the trigeminovascular system during the active period of cluster headache. Moreover, the clinical and biochemical actions showed by sumatriptan stress the involvement of serotonin in cluster headache mechanisms.

Neurogenic inf lammation is regulated by sensory nerves and characterized by extravasation of plasma proteins and infiltration of neutrophils from post-capillary venules and arteriolar vasodilatation. Although it is well established that substance P (SP) interacts with the neurokinin 1 receptor (NK1R) to initiate neurogenic inf lammation, the mechanisms that terminate inf lammation are unknown. We examined whether neutral endopeptidase (NEP), a cellsurface enzyme that degrades SP in the extracellular f luid, terminates neurogenic inf lammation in the colon. In NEP knockout mice, the SP concentration in the colon was Ϸ2.5-fold higher than in wild-type mice, suggesting increased bioavailability of SP. The extravasation of Evans blue-labeled plasma proteins in the colon of knockout mice under basal conditions was Ϸ4-fold higher than in wild-type mice. This elevated plasma leak was attenuated by recombinant NEP or the NK1R antagonist SR140333, and is thus caused by diminished degradation of SP. To determine whether deletion of NEP predisposes mice to uncontrolled inf lammation, we compared dinitrobenzene sulfonic acid-induced colitis in wild-type and knockout mice. The severity of colitis, determined by macroscopic and histologic scoring and by myeloperoxidase activity, was markedly worse in knockout than wild-type mice after 3 and 7 days. The exacerbated inf lammation in knockout mice was prevented by recombinant NEP and SR140333. Thus, NEP maintains low levels of SP in the extracellular f luid under basal conditions and terminates its proinf lammatory effects. Because we have previously shown that intestinal inf lammation results in down-regulation of NEP and diminished degradation of SP, our present results suggest that defects in NEP expression contribute to uncontrolled inf lammation.

Plasma extravasation from postcapillary venules is one of the earliest steps of inflammation. Substance P (SP) and bradykinin (BK) mediate extravasation and cause hypotension. The cell-surface enzyme neutral endopeptidase (NEP) inactivates both peptides. Thus, absence of NEP may predispose development of inflammation and hypotension. We examined these possibilities in mice in which the NEP gene was deleted by homologous recombination. There was widespread basal plasma extravasation in postcapillary venular endothelia in NEP-/- mice, which was reversed by recombinant NEP and antagonists of SP (NK1) and BK (B2) receptors. Mean arterial blood pressure was 20% lower in NEP-/- animals, but this was unaffected by reintroduction of recombinant NEP and the kinin receptor antagonists. The hypotension was also independent of nitric oxide (NO), because NEP-/- mice treated with a NO synthase inhibitor remained hypotensive relative to the wild type. Thus, NEP has important roles in regulating basal microvascular permeability by degrading SP and BK, and may regulate blood pressure set point through a mechanism that is independent of SP, BK and NO. The use of NEP antagonists as candidate drugs in cardiovascular disease is suggested by the blood pressure data reported herein.

Neurogenic inflammation is mediated by release of tachykinins from sensory nerves, which stimulate plasma extravasation from postcapillary venules. Because there are conflicting results regarding the importance of neurogenic inflammation in the gastrointestinal tract, we quantified plasma extravasation using Evans blue and identified sites of the leak using Monastral blue in the mouse. Substance P and bradykinin stimulated extravasation from postcapillary venules in the stomach, small and large intestine, pancreas, urinary bladder, trachea, and skin by two- to sevenfold by interacting with NK1 and B2 receptors, respectively. Stimulation of sensory nerves with capsaicin also induced extravasation. Capsaicin- and bradykinin-stimulated extravasation was attenuated by an NK1-receptor antagonist and is thus mediated by release of tachykinins and activation of the NK1 receptor. We conclude that 1) substance P stimulates extravasation in the gastrointestinal tract and pancreas of mice by interacting with the NK1 receptors, and 2) capsaicin and bradykinin induce plasma extravasation by stimulating tachykinin release from sensory nerves. Thus neurogenic mechanisms mediate inflammation in the gastrointestinal tract and pancreas of the mouse.

Bradykinin, applied locally to the airways, is a weak bronchoconstrictor agent in guinea pigs in vivo and it may cause constriction or dilatation of guinea pig airways smooth muscle in vitro. We examined the motor effect of bradykinin perfused through the lumen of isolated guinea pig tracheal tubes with or without nitric oxide (NO) synthase inhibitors. In the presence of NG-nitro-D-arginine methyl ester (D-NAME) or NG-monomethyl-D-arginine (D-NMMA) intraluminal bradykinin caused a moderate concentration-dependent relaxation. In contrast, in the presence of NG-nitro-L-arginine methyl ester (L-NAME) or NG-monomethyl-L-arginine (L-NMMA) tracheas developed a sustained increase in tone, and bradykinin caused a marked, concentration-dependent contraction, both effects being reversible by pretreatment with L-arginine, but not with D-arginine. The ability of bradykinin to relax (in the presence of D-NAME) or contract (in the presence of L-NAME) guinea pig tracheal tubes was not affected by indomethacin. Bradykinin contracted epithelium-denuded tracheas in the presence of either L-NAME or D-NAME. Both contraction and relaxation by bradykinin were blocked by the kinin B2 receptor antagonist, HOE 140. Baseline production of guanosine 3',5'-cyclic monophosphate (cyclic GMP) in strips of guinea pig trachealis in vitro was markedly reduced by L-NAME, but not by D-NAME. Bradykinin increased baseline cyclic GMP concentration. These results indicate that bradykinin releases NO or a NO-related molecule, which, possibly by increasing cyclic GMP concentrations, mediates relaxation and opposes contraction induced by bradykinin itself, and further, that bradykinin releases NO from the tracheal epithelium.

Abstract-The use of angiotensin-converting enzyme (ACE) has been associated with the occurrence of adverse effects, including cough and angioneurotic edema. Accumulation of kinins has been suggested to play a major role in these adverse effects of ACE inhibitor, although conclusive evidence for such a role is lacking. We investigated whether ACE inhibition increases plasma extravasation in mice (Swiss, C57Bl/6J, and J129Sv/Ev strains) via inhibition of bradykinin metabolism and stimulation of neurogenic inflammatory mechanisms. Intravenous captopril and enalapril increased the extravasation of Evans blue dye in all tissues examined (trachea, stomach, duodenum, and pancreas

1 This study investigated the possibility that tachykinins relax the guinea-pig isolated trachea by releasing nitric oxide (NO) from the epithelium. The types of tachykinin receptor mediating both relaxation and contraction of the trachea were also studied. Isometric tension was recorded in isolated tracheal tube preparations precontracted with acetylcholine (10 gM) in which compounds were administered intraluminally in the presence of phosphoramidon and indomethacin (both gUM) and 5 These results demonstrate that tachykinins relax guinea-pig tracheal tube preparations by releasing NO via the stimulation of epithelial NK, receptors by a mechanism independent of histamine release.The NK, receptor type involved is sensitive to SP, NKA, NKB and [Sar9, Met(O2)"]-SP but not to septide, and is pharmacologically distinct from the NKI receptor that mediates contraction, which is stimulated by all the agonists, including septide.

Inhalation of cold air in guinea pigs increases total pulmonary resistance (RL), an effect that is mediated by kinins and tachykinins. Bronchoconstriction induced by bradykinin (BK) inhalation in guinea pigs is markedly inhibited by nitric oxide (NO) release from the airway epithelium. We investigated whether endogenous NO modulates the increase in RL induced by inhalation of cold air. In anesthetized and artificially ventilated guinea pigs pretreated with atropine, cold-air inhalation (13 degrees C in the trachea) for 5 min did not increase RL. Pretreatment with intravenous N(G)-nitro-L-arginine methyl ester (L-NAME) (but not with its inactive enantiomer, D-NAME) increased RL, an effect reversed by L-Arg. The increase in RL induced by cold air after L-NAME was abolished by the tachykinin NK2-receptor antagonist SR 48968 or the kinin B2-receptor antagonist, HOE 140. After administration of SR 48968, inhalation of cold air reduced baseline airway tone. However, after HOE 140, cold-air inhalation did not affect baseline airway tone. L-NAME exaggerated the bronchoconstriction induced by BK. However, L-NAME did not affect capsaicin-induced bronchoconstriction. BK increased cyclic guanosine monophosphate (cGMP) levels in strips of guinea pig trachealis muscle in vitro, whereas the selective tachykinin NK2-receptor agonist [betaAla8]neurokinin A (4-10) was without effect. The present data suggest that bronchoconstriction induced by cold-air inhalation and mediated by kinin and tachykinin release is inhibited by endogenous NO, and that kinins, but not tachykinins or cold air alone, release bronchorelaxant NO.