Shigemi Yoshihara

Role of kinins in anaphylactic-induced bronchoconstriction mediated by tachykinins in guinea-pigs.

1 In the present study, we have investigated the role of kinins in allergen‐induced bronchoconstriction. 2 Anaesthetized guinea‐pigs were sensitized to ovalbumin, ventilated artificially, pretreated with atropine (1.4 μmol kg−1, i.v.) and total pulmonary resistance (RL) measured. In preliminary studies in the presence of the neutral endopeptidase inhibitor, phosphoramidon (4.5 μmol kg−1, i.v.), the bradykinin B2 receptor antagonist Hoe 140 (0.1 μmol kg−1, i.v.) completely abolished the increase in RL following aerosolized bradykinin (1 mm, 40 breaths), but had no effect on the increase in RL following aerosolized neurokinin A (NKA, 10μm, 40 breaths). On the other hand, a combination of the NK1 (CP‐96,345, 2 μmol kg‐1, i.v.) and NK2 (SR 48968, 0.3 μmol kg−1, i.v.) tachykinin receptor antagonists abolished completely the increase in RL produced by NKA and partially inhibited the increase in RL produced by bradykinin. These results confirm previous studies that suggest that bradykinin induces the release of tachykinins from sensory nerves in guinea‐pig airways. 3 Aerosolized ovalbumin (0.5%, 5 breaths) increased RL in sensitized guinea‐pigs pretreated with atropine (1.4mmol kg−1, i.v.), an effect that began within 2 min and reached a maximum within 5 min; RL remained above baseline at 20 min. Pretreatment with the bradykinin B2 receptor antagonist, Hoe 140, decreased the bronchoconstrictor effect of ovalbumin markedly at 10 to 20 min. In the presence of phosphoramidon (4.5 μmol kg−1, i.v.) the inhibition induced by Hoe 140 was apparent earlier and remained over the 20 min period of study. 4 Pretreatment with a combination of NK1 (CP‐96,345) and NK2 (SR 48968) tachykinin receptor antagonists also markedly inhibited ovalbumin‐induced bronchoconstriction; addition of the bradykinin B2 receptor antagonist to the NK1 and NK2 tachykinin receptor antagonists had no additional inhibitory effect on antigen‐induced bronchoconstriction. 5 These findings confirm that activation of sensory nerves to release tachykinins in guinea‐pig airways contribute to antigen‐induced bronchoconstriction, and provide evidence that tachykinin release is due to kinins generated during the allergic response.

Cold air-induced bronchoconstriction is mediated by tachykinin and kinin release in guinea pigs

In the present study, we investigated the role of acetylcholine, tachykinins and kinins in the bronchoconstriction induced by cold air inhalation. Cold air was delivered to anaesthetised, artificially ventilated guinea pigs through a tracheal cannula. Inhalation of cold air increased the maximum total pulmonary resistance (RL) in a time-dependent manner, reaching a maximum after 15 min of exposure. The increase in RL induced by exposure to cold air for 10 min was not affected by pretreatment with atropine (1.4 mu mol/kg, i.v.); it was abolished by the tachykinin NK2 receptor antagonist, SR 48968 (0.3 mu mol/kg, i.v.) and was reduced by 58% by the kinin B2 receptor antagonist, HOE 140 (0.1 mu mol/kg, i.v.). These findings suggest that cold air induces bronchoconstriction in guinea pigs via a cascade that involves the release of kinins and tachykinins.

Inhibition of bronchoconstriction by pituitary adenylate cyclase activating polypeptide (PACAP 1–27) in guinea‐pigs in vivo

1 We studied the inhibitory effect of pituitary adenylate cyclase activating polypeptide (PACAP 1–27) on the increase in total pulmonary resistance (RL) caused either by allergen or histamine in anaesthetized, ventilated guinea‐pigs. 2 PACAP 1–27 given via i.v. infusion (0.045‐4.5 nmol kg−1 min−1) dose‐dependently reduced the increase in RL caused by inhaled ovalbumin and histamine. At the highest dose, PACAP 1–27 prevented the increase in RL caused by ovalbumin and histamine completely. Infusion of PACAP 1–27 and the β2‐adrenoceptor agonist, salbutamol (0.045‐4.5 nmol kg‐1 min−1) inhibited the increase in RL similarly, but salbutamol increased the heart rate more than PACAP 1–27. 3 PACAP 1–27 and salbutamol given via inhaled aerosol (0.1 mM, 20 breaths) significantly reduced the increase in RL caused by histamine infused i.v., whereas aerosolised sterile saline did not. Both PACAP 1–27 and salbutamol caused bronchodilator effects within 1 min of drug inhalation and these effects remained throughout the 20 min of study. 4 Because PACAP 1–27 produced significant bronchodilatation and rapid onset of sustained action in vivo and without pronounced cardiovascular side effects, we conclude that this peptide may have therapeutic potential as a bronchodilator.

PACAP 1-38 as an Inhaled Bronchodilator in Guinea Pigs In Vivo

The effect on total pulmonary resistance (R1) was examined for inhaled PACAP 1-38, PACAP 1-27 and VIP in anesthetized, ventilated guinea pigs. Two minutes after inhalation, PACAP 1-38 (36 +/- 6%), PACAP 1-27 (42 +/- 9%) and VIP (48 +/- 19%) inhibited the increase in R1 (% inhibition of histamine-induced R1 prior to inhalation) caused by histamine i.v., whereas the vehicle (-1 +/- 10%) did not. This inhibitory effect lasted five times longer for PACAP 1-38 (> 50 min) than for PACAP 1-27 and VIP (< 10 min). The inhaled peptides caused no sustained effects on heart rate or blood pressure. Infusion of PACAP 1-38 i.v. dose-dependently inhibited the increase in R1 caused by inhaled histamine and by carbachol i.v..

Corticotropin-releasing factor inhibits antigen-induced plasma extravasation in airways

We investigated the potential of corticotropin-releasing factor (CRF) to reduce neurogenic plasma extravasation in sensitised guinea pig airways evoked by antigen challenge. Inhalation of 5% ovalbumin for 2 min in the presence of phosphoramidon (2.5 mg/kg, i.v.) increased extravasation of Evans blue dye in the trachea and main bronchi. The increase in plasma extravasation induced by antigen challenge was significantly reduced by pretreatment with CRF (30 nmol/kg, i.v.) (73% in the trachea and 42% in the main bronchi). The inhibition of plasma extravasation by CRF (30 nmol/kg, i.v.) alone was not different from the inhibition induced by the combination of CRF and the tachykinin NK1 receptor antagonist, CP-99,994 (4 mg/kg, i.v.) (73% in the trachea and 38% in the main bronchi). CRF (30 nmol/kg, i.v.) inhibited by 32% in the trachea and by 43% in the main bronchi plasma extravasation induced by aerosolised bradykinin but did not reduce the plasma extravasation caused by aerosolised substance P in the presence of phosphoramidon. These findings suggest that CRF reduces ovalbumin-induced plasma extravasation in guinea pig airways by inhibiting the release of tachykinins from primary sensory nerves.

Tachykinins mediate the potentiation of antigen-induced bronchoconstriction by cold air in guinea pigs

The role of tachykinins in the potentiation of antigen-evoked bronchoconstriction induced by inhalation of cold air was studied in guinea pigs. Cold air was delivered through a tracheal cannula to anesthetized, artificially ventilated guinea pigs sensitized with ovalbumin and pretreated with atropine (1.4 micromol/kg). Inhalation of cold air increased total pulmonary resistance (RL) in a time-dependent manner; inhalation of cold air for 10 or 15 minutes, but not for 5 minutes, produced a significant increase in RL. Aerosolized ovalbumin (5 breaths) increased RL in a dose-dependent manner (0.5% to 5%). Inhalation of cold air for 5 minutes significantly enhanced both the peak and the duration of the increase in RL induced by 0.5% ovalbumin. The tachykinin neurokinin 2-receptor antagonist, SR 48968 (0.3 micromol/kg intravenously) inhibited both the peak and the duration of the bronchoconstriction induced by 5-minute inhalation of cold air and ovalbumin (0.5%), whereas it did not affect the response to ovalbumin (0.5%) alone. These findings suggest that exposure to cold air potentiates the bronchoconstriction response to antigen and that this potentiation is mediated by tachykinin release from sensory nerves.

Long lasting smooth muscle relaxation by a novel PACAP analogue in guinea-pig and primate airways in vitro

1 We compared the relaxant effect of pituitary adenylate cyclase activating peptide (PACAP) 1–27 with that of a newly developed PACAP 1–27 analogue, [Arg15,20,21Leu17]‐PACAP‐Gly‐Lys‐Arg‐NH2, in the guinea‐pig trachea and primate bronchi in vitro (n=4–5). 2 In the guinea‐pig trachea precontracted by a submaximally effective carbachol concentration (0.1u2003μM), cumulative administration of PACAP 1–27 and the β2‐adrenoceptor agonist salbutamol (3u2003nM–3u2003μM) caused significant and concentration‐dependent smooth muscle relaxation, with salbutamol being approximately one log‐step more potent in this model. However, in primate bronchi precontracted by carbachol (0.1u2003μM), cumulative administration of PACAP 1–27 and salbutamol caused concentration‐dependent smooth muscle relaxation with very similar potencies and maximum relaxant effects. 3 In the guinea‐pig trachea, non‐cumulative administration of the PACAP 1–27 analogue and the original PACAP 1–27 (0.3–3u2003μM) caused concentration‐dependent relaxation with a very similar maximum relaxant effect and potency. However, the onset and offset of action was markedly slower for the PACAP 1–27 analogue than for the original PACAP 1–27 (>90% versus <10% of peak relaxation remaining 6u2003h after administration). Separate experiments confirmed that the PACAP 1–27 analogue also caused significant relaxation with slower onset and offset of action than did the original PACAP 1–27 in primate bronchi. 4 Peptidase inhibition by captopril (10u2003μM) and phosphoramidon (1u2003μM) significantly increased the maximum relaxant effect and duration of action of PACAP 1–27 but not of the PACAP 1–27 analogue, during the 3u2003h of observation in the guinea‐pig trachea. 5 We conclude that [Arg15,20,21Leu17]‐PACAP‐Gly‐Lys‐Arg‐NH2 produces significant, concentration‐dependent and sustained airway smooth muscle relaxation in vitro. The sustained relaxant effect is due, at least in part, to the PACAP 1–27 analogue being less susceptible to cleavage by peptidases than the original peptide PACAP 1–27.

FUNCTIONAL TYPE II VIP-PACAP RECEPTORS IN HUMAN AIRWAY EPITHELIAL-LIKE CELLS

VIP-PACAP receptors were characterized in a human airway epithelial-like cell line (Calu-3), Pituitary adenylate cyclase activating polypeptide (PACAP) 1-27, PACAP 1-38, vasoactive intestinal polypeptide (VIP) and the beta 1- and beta 2-adrenoceptor agonist isoproterenol (3 nM-1 microM) increased cAMP concentration dependently. The peptides and isoproterenol displayed similar potencies (range of means pEC50[M]: 6.5-7.1). The maximum increase in cAMP (Emax in % of basal cAMP level) was similar for the peptides (range of means Emax: 2500-5100%). Pretreatment with the peptidase inhibitors captopril (10 microM) and phosphoramidon (1 microM) significantly increased the cAMP response to PACAP 1-38 (to 480% of control) only.

A Novel PACAP 1‐27 Analogue Causes Sustained Smooth Muscle Relaxation in Guinea‐Pig Tracheaa

Two endogenous derivatives of the pituitary adenylate cyclase-activating peptide (PACAP) have been identified, PACAP 1-27 and PACAP 1-38. Of these two peptides, it has been demonstrated that PACAP 1-38 alone produces smooth muscle relaxation in rodent tracheas in vitro,2,3 whereas PACAP 1-27 was recently shown to be a potent bronchodilator in guinea pigs in V ~ V O . ~ The physiological role of the PACAPs is not yet certain, but because immunoreactivity for the PACAPs is present in the airways of various species including it can be speculated that the PACAPs, in addition to catecholamines, vasoactive intestinal polypeptide (VIP), and nitric oxide, may function as endogenous bronchodilators. We hypothesized that the activation of airway VIP-PACAP receptors may constitute a useful pathway not only for endogenous but also for exogenous bronchodilators. Because recent data on rats and dogs in vivo suggest that PACAP 1-27 has fewer cardiovascular effects than PACAP 1-38 and we chose PACAP 1-27 as a basis for molecular modeling of a novel bronchodilator. We thus examined the airway smooth muscle relaxation in vitro induced an Nand C-terminally modified PACAP 1-27 molecule, [Arg15~2*2Le~17]-PACAP-Gly-Lys-Arg-NHZ and compared it with the original PACAP 1-27. We also evaluated the effect of peptidase inhibition on smooth muscle relaxation caused by these two peptides.