Corinne A.E. Martin

Influence of (+/-)-CP-96,345 and SR 48968 on electrical field stimulation of the isolated guinea-pig main bronchus.

The aim of this study was to investigate the effects of (+/-)-CP-96,345 and SR 48968, two new nonpeptide antagonists of neurokinin NK1 and NK2 receptors, respectively, on the response of isolated guinea pig main bronchi to electrical field stimulation (EFS). Bronchi were stimulated transmurally with biphasic pulses (16 Hz, 1 ms, 320 mA for 10 s) in the presence of indomethacin (10(-6) M) and propranolol (10(-6) M). Two successive contractile responses were observed. Both responses were abolished by tetrodotoxin (10(-6) M) whereas only the first rapid phase was abolished by atropine (10(-6) M). The late and prolonged second phase was strongly reduced by the neurokinin A (NK2) receptor antagonist SR 48968 (10(-11) to 10(-8) M) with an EC50 of 0.056 nM and a maximal inhibition of 83.3 +/- 10.8% (10(-8) M, n = 4). This second response was partially inhibited by the substance P (NK1) receptors antagonist (+/-)-CP-96,345 (10(-8) to 10(-6) M). An incubation of 2 h was necessary for SR 48968 to inhibit the EFS-evoked noncholinergic contraction. These results confirm that EFS of guinea-pig bronchi involves stimulation of cholinergic and noncholinergic excitatory nerves and demonstrate that the new developed tachykinin receptors nonpeptide antagonists (+/-)-CP-96,345 and especially SR 48968 are potent inhibitors of the noncholinergic contraction induced by EFS of the isolated guinea-pig main bronchus.

BETA 3 ADRENOCEPTOR AGONISTS, BRL 37344 AND SR 58611A DO NOT INDUCE RELAXATION OF HUMAN, SHEEP AND GUINEA – PIG AIRWAY SMOOTH MUSCLE IN VITRO

The existence of atypical- or beta 3-adrenoceptors has now been generally accepted. These receptors have been shown to be abundant in adipose tissue and in a number of gastrointestinal smooth muscle preparations. A recent study reported that beta 3-adrenoceptor stimulation mediated relaxation of isolated canine bronchial smooth muscle. The aim of the present study was to extend this observation to other species. We investigated the in vitro responses of guinea-pig, human and sheep bronchial smooth muscle to isoprenaline, salbutamol (a selective beta 2-adrenoceptor agonist), and BRL 37344 and SR 58611A (two presumably selective beta 3-adrenoceptor agonists). The preparations were precontracted to 60-70% of maximal tension with histamine 10(-6) M for guinea-pig and human bronchi, or acetylcholine 10(-6) M for sheep bronchi. In each species, SR 58611A produced a slight fall in tension of about 10% of the effects of theophylline (3 mM), but this decrease in tension was not significantly different from the spontaneous and weak relaxation observed with saline addition during the same duration of the experiment. These relaxations were not modified by either the nonselective beta-adrenoceptor antagonist propranolol or the selective beta 2-adrenoceptor antagonist ICI 118,551. In contrast, BRL 37344 induced a significant concentration-dependent fall in tension induced by both spasmogens. Its relaxant effects were inhibited both by propranolol and ICI 118,551 in human and guinea-pig airways, whereas on the isolated sheep bronchus BRL 37344-induced relaxations were only slightly, albeit significantly, reduced with either of the beta-adrenoceptor antagonists tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Rolipram, but not siguazodan or zaprinast, inhibits the excitatory noncholinergic neurotransmission in guinea-pig bronchi

Theophylline has been reported to inhibit excitatory noncholinergic but not cholinergic-neurotransmission in guinea-pig bronchi. As theophylline might exert this effect through an inhibition of phosphodiesterases (PDE), and since many types of PDE have now been described, the aim of this study was to investigate the effects of three specific inhibitors of PDE on the electrical field stimulation (EFS) of the guinea-pig isolated main bronchus in vitro. The drugs used were siguazodan, rolipram and zaprinast, which specifically inhibit PDE types, III, IV and V, respectively. Guinea-pig bronchi were stimulated transmurally with biphasic pulses (16 Hz, 1 ms, 320 mA for 10 s) in the presence of indomethacin 10(-6) M and propranolol 10(-6) M. Two successive contractile responses were observed: a rapid cholinergic contraction, followed by a long-lasting contraction due to a local release of neuropeptides from C-fibre endings. Rolipram (10(-9) to 10(-6) M) but not siguazodan or zaprinast, inhibited the peptidergic contraction in a concentration-dependent manner. Conversely, the cholinergic response was unaffected. Contractile responses induced by exogenous acetylcholine (10(-8) to 10(-3) M) or [Nle10]NKA(4-10) (10(-10) to 10(-6) M) were also unaffected by rolipram, siguazodan and zaprinast (10(-7) M). These results demonstrate that concentrations of rolipram, similar to those which inhibit PDE, reduce the release of sensory neuropeptides from C-fibre endings, and suggest that the cyclic adenosine monophosphate (AMP) PDE type IV is specifically involved in this effect, as in other anti-inflammatory effects.

1-Benzazepine derivatives acting as ATP-dependent potassium-channels antagonists

Summary In order to select between various pharmacological activities related to the 1-benzazepine nucleus, a set of 1-benzazepine derivatives bearing different N-1 or C-2 substituents were synthesized and tested in vitro as ATP-dependent K + -channel antagonists on isolated guinea-pig trachea. Cumulative concentration curves to cromakalim were constructed in the absence or presence of the various compounds tested in comparison with glibenclamide. Products bearing N-1 substituents, especially (2-imidazolinyl)methyl showed a non-competitive antagonism towards ATP-dependent K + channels.

In vitro effects of HOE 140 in human bronchial and vascular tissue

Bradykinin is a potent inflammatory mediator which may be involved in various airway diseases. A selective and potent antagonist of the bradykinin B2 receptor has recently been discovered (HOE 140: D-Arg-[Hyp3,Thi5,D-Tic7,Oic8]bradykinin). The purpose of this study was to evaluate the potency of this compound in isolated human tissue (bronchus, pulmonary artery endothelium, umbilical artery and vein smooth muscle). Bradykinin induced contractions of the isolated human bronchus and umbilical artery and vein (the umbilical vessels were pretreated with indomethacin and L-nitro-arginine to inhibit prostaglandin and nitric oxide synthesis). It provoked an endothelium-dependent relaxation in the human pulmonary artery. HOE 140 was a non-competitive antagonist in human bronchial tissue (pKB: 8.19 +/- 0.30) and a competitive one in vascular tissue (pA2: 7.97 +/- 0.12, 8.16 +/- 0.16 and 8.00 +/- 0.11 in human pulmonary artery, umbilical artery and vein respectively). The effect of HOE 140 was selective as it did not influence the umbilical vein contractile response to serotonin and histamine. HOE 140 up to 3 x 10(-6) M was devoid of residual agonistic activity in the various human preparations studied. Furthermore, although the effects of HOE 140 were fully reversible, in isolated bronchial airways and umbilical veins, HOE 140 (10(-6) M) still possessed activity 1 h after being washed out in both tissues. Our results indicate that HOE 140 is a potent and potentially long-acting antagonist of the human bradykinin B2 receptor.

Effects of two β3-adrenoceptor agonists, SR 58611A and BRL 37344, and of salbutamol on cholinergic and NANC neural contraction in guinea-pig main bronchi in vitro

1 The aim of the present study was to investigate the type of adrenoceptor which modulates constriction of the guinea‐pig isolated main bronchus in response to electrical field stimulation (EFS). Drugs used were salbutamol and two agonists reportedly selective for the putative β3‐adrenoceptor: BRL 37344 and SR 58611A. 2 At basal tone, all three drugs induced relaxation, however, SR 58611A and BRL 37344 (10−9 to 10−6 m) relaxed guinea‐pig isolated main bronchus more weakly than salbutamol (10−9 to 10−6 m). The effects observed at 10−6 m were 43% ± 9%, 63% ± 4% and 98% ± 1% of the maximal effect induced by theophylline (3 × 10−3 m) for SR 58611A, BRL 37344 and salbutamol, respectively. 3 SR 58611A and BRL 37344 (10−8 to 10−6 m) did not significantly modify the cholinergic component of the response to EFS, but caused a concentration‐dependent reduction of the nonadrenergic non‐cholinergic (NANC) excitatory component (41.8% ± 10.1% and 56.8% ± 7.4% respectively at 10−6 m, n = 6–7). Salbutamol (10−9 to 10−7 m) strongly inhibited both components, with 91.1% ± 4.2% of inhibition for the NANC contraction and 62.0% ± 5.2% of inhibition for the cholinergic contraction (10−7 m, n = 7). 4 Whereas the inhibitory effects of salbutamol were strongly inhibited by both propranolol (10−6 m) and ICI 118,551 (10−6 m), those of BRL 37344 were only slightly, albeit significantly reduced by both propranolol and ICI 118,551, and those of SR 58611A were unaffected by treatment with either β‐adrenoceptor antagonist. An α2‐adrenoceptor antagonist, yohimbine, did not influence the inhibitory effects of any of the β‐adrenoceptor agonists tested. 5 Concentration‐response curves to acetylcholine (10−8 to 10−3 m), [Nle10]NKA(4–10) (10−10 to 10−6 m) and substance P (10−10 to 3 × 10−6 m) were also significantly shifted to the right by salbutamol (10−6 m), whereas SR 58611A and BRL 37344 (10−6 m) had no effect. 6 These results suggest that the stimulation of putative β3‐adrenoceptors exerts a specific prejunctional inhibitory action on NANC excitatory response induced by EFS of the isolated main bronchus of the guinea‐pig. They also suggest that a β2‐adrenoceptor agonistic component may be involved in the effects of BRL 37344.

Inhibition of cholinergic neurotransmission in isolated guinea-pig main bronchi by SR 48968

SR 48968 (10(-6) to 10(-5) M) inhibited the cholinergic response of the isolated guinea-pig main bronchus to electrical field stimulation. Since this effect was reversed by naloxone 10(-5) M and since SR 48968 had no effect on the contractile response to exogenous acetylcholine, we conclude that SR 48968 acts at a prejunctional level and that opioid receptors are involved. This effect was observed at concentrations approximately 75,000 times higher than those needed for blockade of tachykinin NK2 receptors.

Effects of cromakalim on bradykinin-, histamine- and substance P-induced airway microvascular leakage in the guinea-pig

The effects of cromakalim on the increase in microvascular permeability induced by histamine, substance P or bradykinin in guinea-pig airways were studied in vivo. Extravasation of i.v. injected Evans blue dye was used as an index of permeability. We also studied the effects of cromakalim on the contractile effect of substance P, histamine or bradykinin on the isolated guinea-pig main bronchus and on the contractile response of isolated guinea-pig main bronchi to electrical field stimulation. Cromakalim (30 to 300 micrograms.kg-1) did not inhibit the increase in microvascular permeability induced by histamine (30 micrograms.kg-1) in guinea-pig airways and potentiated (30 and 100 micrograms.kg-1) the effects of substance P (0.3 microgram.kg-1) in trachea, main bronchi and proximal intrapulmonary airways. In contrast, cromakalim (30 and 300 micrograms.kg-1) reduced the increase in microvascular permeability induced by bradykinin (0.3 microgram.kg-1). However, a significant potentiation of the effects of bradykinin was observed with cromakalim (100 micrograms.kg-1) in main bronchi and intrapulmonary airways. In the isolated guinea-pig main bronchus, the contractile effects of bradykinin, histamine and substance P were not modified by cromakalim (10(-5) M). Conversely, cromakalim (10(-5) M) significantly reduced both cholinergic and noncholinergic contractile responses induced by electrical field stimulation of the isolated guinea-pig main bronchus. In conclusion, cromakalim can partially inhibit the increase in microvascular permeability induced by i.v. bradykinin. It is suggested that this effect might occur through inhibition of the nonadrenergic noncholinergic excitatory (NANC) nerves preventing release by bradykinin of inflammatory neuropeptides such as substance P.

Stimulation of Rb+ influx by bradykinin through Na+/K+/Cl− cotransport and Na+/K+-atpase in NIH-3T3 fibroblasts

Bradykinin receptor stimulation results in G-protein-coupled phospholipase activation, initiating protein kinase C (PKC) stimulation and cytosolic free Ca2+ concentration ([Ca2+]i) rises as signalling pathways. Using Rb+ as a tracer for K+, we have studied the mechanisms involved in bradykinin-stimulated Rb+ influx in NIH-3T3 fibroblasts. The furosemide-sensitive Na+/K+/Cl- cotransport and the ouabain-sensitive Na+/K(+)-ATPase were both involved in Rb+ influx under resting conditions with a ratio Na+/K+/Cl- cotransport/Na+/K(+)-ATPase (r) = 0.73. Bradykinin stimulated Rb+ influx (+82.6%) through both systems without changing their ratio (r = 0.72). PKC stimulation by a 15-min-treatment with phorbol 12-myristate 13-acetate (PMA) (2x10(-7) M) increased Rb+ influx in resting cells by 75.7% without affecting r (0.75). PKC inhibition by H-7, and PKC down-regulation by 24-h PMA (10(-6) M) treatment decreased the bradykinin-induced stimulation of Rb+ influx (+31% and +14.9% above control, respectively). Both down-regulation and inhibition of PKC dramatically reduced the furosemide-sensitive Na+/K+/Cl- cotransport, as r fell to 0.239 and 0.032 in bradykinin-stimulated cells after H-7 and 24-h PMA treatments, respectively. BAPTA/AM pretreatment (10(-4) M, 60 min), which complexed with [Ca2+]i, not only prevented the bradykinin-induced [Ca2+]i raise, but also partially inhibited bradykinin-induced Rb+ influx stimulation (+39% above control), without modifying r (0.76). We conclude that stimulation of PKC is a major pathway involved in bradykinin stimulation of Rb+ influx in NIH-3T3 fibroblasts, and that rises in [Ca2+]i participate in bradykinin signalling, possibly through PKC activation. Our data also suggest that active PKC is required for basal and bradykinin-stimulated Na+/K+/Cl- cotransport activity in these cells.

Neurotensin modulates cholinergic and noncholinergic neurotransmission in guinea-pig main bronchi in vitro

Guinea-pig main bronchi were stimulated transmurally in vitro by electrical field stimulation in the presence of indomethacin 10(-6) M, propranolol 10(-6) M and phosphoramidon 10(-5) M. Two contractile neurogenic responses were successively observed. The second noncholinergic contraction was concentration dependently inhibited or abolished by neurotensin whereas the first cholinergic contraction was only partially inhibited. SR 48692, a novel antagonist of neurotensin receptors, reduced the inhibition induced by neurotensin (pKB = 9.75) whereas levocabastine, an antagonist of low-affinity neurotensin receptors, did not significantly modify the inhibitory effects of neurotensin on both neurally-mediated contractions. These results demonstrate that neurotensin exerts an inhibitory effect on neurotransmission in guinea-pig airways. Furthermore, the present study shows that the newly developed neurotensin receptors antagonist, SR 48692, is a potent inhibitor of the neurotensin inhibitory effects on cholinergic and noncholinergic contractions induced by electrical field stimulation of the guinea-pig isolated main bronchus.