Gz- and not Gi-proteins are coupled to pre-junctional µ-opioid receptors in bovine airways.

We investigated the signal transmission pathway by which activation of µ-opioid receptors attenuates acetylcholine (ACh) release in bovine trachealis. Electrical stimulation (ES)-induced [(3)H]-ACh release was determined in bovine tracheal smooth muscle strips pre-incubated with either the Gi-protein inhibitor pertussis toxin (PTX, 500 ng/ml and 1 µg/ml) or the Gz-protein specific inhibitor arachidonic acid (AA, 10(-6)M and 10(-5)M) and then treated with DAMGO (D-Ala(2),N-MePhe(4),Gly-ol(5)-enkephalin) 10(-5)M. Indomethacin 10(-5)M was used to block AA cascade. The inhibitory effect of DAMGO on ES-induced [(3)H]-ACh release was PTX-insensitive, but, by contrast, ablated by AA in a concentration-dependent manner. AA 10(-5)M alone reduced [(3)H]-ACh release, an effect that was prevented by iberiotoxin 10(-7)M, suggesting an involvement of Ca(2+)-activated K(+)-channels. Western blot analysis consistently showed immunoreactive bands against a specific antibody anti-Gz-α subunit at ∼40 kDa, consistent with the presence of Gz-protein. The present findings suggest that in isolated bovine trachealis, activation of µ-opioid receptors inhibits ACh-release through a signal transmission pathway involving Gz-protein rather than Gi-protein.

Effects of κ- and µ-opioid agonists on cholinergic neurotransmission and contraction in isolated bovine trachealis.

The effects of the selective µ-opioid agonist DAMGO and the selective κ-opioid agonist U-50488H on tritiated acetylcholine release ([(3)H]-ACh) and contractile responses to electrical stimulation (ES) were simultaneously determined in isolated bovine trachealis. The inhibitory effect of DAMGO 10(-5)M on [(3)H]-ACh release was not significantly different from the effect of the non-selective muscarinic agonist pilocarpine 10(-5)M, whereas the effect of U-50488H 10(-5)M was significantly greater. The effects of both opioids were not significantly different when muscles were pre- or co-incubated with the unselective muscarinic antagonist atropine 10(-7)M. Both DAMGO and U-50488H attenuated ES-induced contraction and this effect was significantly correlated with the inhibition of [(3)H]ACh-release (r(2)=0.8552). These data suggest that (1) opioids are important modulators of airway smooth muscle tone, (2) their effect is not altered by the activity of muscarinic autoregulation, and (3) their inhibitory effect of airway smooth muscle contraction can be almost totally explained by inhibition of ACh release.

Special article: T. H. Seldon (1905-1991).

Dr. Seldon was Editor of Anesthesia & Analgesia from 1954 to 1977. We examined how he led the effort to transform this journal into a clinically relevant and vital publication for the specialty.

Pre-junctional muscarinic autoreceptors in bovine airways.

UNLABELLED: We searched for pre-junctional inhibitory muscarinic receptors in isolated bovine trachealis strips and bronchial rings. Electric stimulation (ES)-induced tritiated acetylcholine ([(3)H]-ACh)-release and isometric contractions were determined in muscles incubated with the non-selective muscarinic agonist pilocarpine, the non-selective muscarinic antagonist atropine, the selective M(2)-receptor antagonists methoctramine and gallamine, or the selective M(4)-receptor antagonist PD102807. Electric field stimulation (EFS)-induced isometric contractile responses were assessed in trachealis strips and bronchial rings treated with 10(-9)-10(-5)M methoctramine, gallamine or PD102807. Pilocarpine (10(-6) and 10(-5)M) and atropine (10(-7)M) significantly decreased and increased ES-evoked [(3)H]-ACh-release, respectively. The enhancing effect of atropine on [(3)H]-ACh-release prevailed over the inhibitory effect of pilocarpine. M(2)- and M(4)-receptor antagonists did not increase EFS-induced contraction or ES-induced [(3)H]-ACh-release. However, 10(-7)M methoctramine, gallamine or PD102807 significantly attenuated the inhibitory effects of pilocarpine 10(-5)M on ES-induced [(3)H]-ACh-release. CONCLUSIONS: Muscarinic autoregulation is present in bovine airways but is not fully accounted for by M(2)- and M(4)-receptor subtypes.

Listeria monocytogenes multidrug resistance transporters activate a cytosolic surveillance pathway of innate immunity.

To gain insight into the interaction of intracellular pathogens with host innate immune pathways, we performed an unbiased genetic screen of Listeria monocytogenes mutants that induced an enhanced or diminished host innate immune response. Here, we show that the major facilitator superfamily of bacterial multidrug resistance transporters (MDRs) controlled the magnitude of a host cytosolic surveillance pathway, leading to the production of several cytokines, including type I IFN. Mutations mapping to repressors of MDRs resulted in ectopic expression of their cognate transporters, leading to host responses that were increased up to 20-fold over wild-type bacteria, and a 20-fold decrease in bacterial growth in vivo. Mutation of one of the MDRs, MdrM, led to a 3-fold reduction in the IFN-beta response to L. monocytogenes infection, indicating a pivotal role for MdrM in activation of the host cytosolic surveillance system. Bacterial MDRs had previously been associated with resistance to antibiotics and other toxic compounds. This report links bacterial MDRs and host immunity. Understanding the mechanisms through which live pathogens activate innate immune signaling pathways should lead to the discovery of adjuvants, vaccines, and perhaps new classes of therapeutics. Indeed, we show that the mutants identified in this screen induced vastly altered type I IFN response in vivo as well.

Modulation of cholinergic responsiveness through the [beta]-adrenoceptor signal transmission pathway in bovine trachealis.

The effects of pharmacological stimulation at different levels of the beta-adrenoceptor (AR) pathway, including the receptor, the receptor-coupled Gs protein, and adenylyl cyclase, were studied by simultaneous measurements of acetylcholine (ACh) release and isometric force evoked by electric stimulation in isolated bovine trachealis. The beta-AR agonists isoproterenol (10-6 and 10-5 M) and salbutamol (10-7 to 10-5 M) significantly attenuated both ACh release and contractile force. Forskolin, at 10-6 M, significantly increased ACh release without effect on contractile force, whereas at 10-5 M it increased ACh release but significantly decreased force. Activation of Gs protein by cholera toxin (10 microg/ml) significantly attenuated both ACh release and contractile force, but its effect on ACh release was abolished by calcium-activated potassium (KCa)-channel blocker iberiotoxin (10-7 M). The KCa-channel opener NS-1619 (10-4 M) attenuated significantly both ACh release and contractile force. It is concluded that beta-AR agonists attenuate cholinergic neurotransmission in isolated bovine trachealis model by a mechanism not involving cAMP but KCa channels.

Beclomethasone rapidly ablates allergen-induced beta 2-adrenoceptor pathway dysfunction in human isolated bronchi.

Bronchial rings from nonatopic humans were passively sensitized with serum from allergic subjects. Allergen challenge significantly reduced the relaxant effect of salbutamol on carbachol-induced contractions, suggesting beta(2)-adrenoceptor (beta(2)-AR) pathway dysfunction. Incubation of challenged rings for 3 h with 3 x 10(-6) M beclomethasone dipropionate (BDP) restored the relaxant effect, suggesting reversal of beta(2)-AR pathway dysfunction. Incubation with the G(s)alpha protein-stimulating cholera toxin attenuated contractile responses to carbachol significantly less in challenged than in unchallenged rings. Treatment of challenged rings with BDP resulted in an inhibitory effect of cholera toxin that was similar to the effect in unchallenged rings. G(s)alpha protein expression was not significantly altered by BDP, suggesting that the activity of G(s)alpha protein was increased. Relaxation of challenged rings by forskolin was not significantly affected by BDP, suggesting that beta(2)-AR pathway dysfunction was proximal to the adenylyl cyclase. In conclusion, short-term (3-h) treatment with BDP after allergen challenge ablated beta(2)-AR pathway dysfunction by increasing the activity of the G(s)alpha protein in human isolated bronchi.