Different role of TTX-sensitive voltage-gated sodium channel (NaV 1) subtypes in action potential initiation and conduction in vagal airway nociceptors.

KEY POINTS: The action potential initiation in the nerve terminals and its subsequent conduction along the axons of afferent nerves are not necessarily dependent on the same voltage-gated sodium channel (NaV 1) subunits. The action potential initiation in jugular C-fibres within airway tissues is not blocked by TTX; nonetheless, conduction of action potentials along the vagal axons of these nerves is often dependent on TTX-sensitive channels. This is not the case for nodose airway Aδ-fibres and C-fibres, where both action potential initiation and conduction is abolished by TTX or selective NaV 1.7 blockers. The difference between the initiation of action potentials within the airways vs. conduction along the axons should be considered when developing NaV 1 blocking drugs for topical application to the respiratory tract. ABSTRACT: The action potential (AP) initiation in the nerve terminals and its subsequent AP conduction along the axons do not necessarily depend on the same subtypes of voltage-gated sodium channels (NaV 1s). We evaluated the role of TTX-sensitive and TTX-resistant NaV 1s in vagal afferent nociceptor nerves derived from jugular and nodose ganglia innervating the respiratory system. Single cell RT-PCR was performed on vagal afferent neurons retrogradely labelled from the guinea pig trachea. Almost all of the jugular neurons expressed the TTX-sensitive channel NaV 1.7 along with TTX-resistant NaV 1.8 and NaV 1.9. Tracheal nodose neurons also expressed NaV 1.7 but, less frequently, NaV 1.8 and NaV 1.9. NaV 1.6 were expressed in ∼40% of the jugular and 25% of nodose tracheal neurons. Other NaV 1 α subunits were only rarely expressed. Single fibre recordings were made from the vagal nodose and jugular nerve fibres innervating the trachea or lung in the isolated perfused vagally-innervated preparations that allowed for selective drug delivery to the nerve terminal compartment (AP initiation) or to the desheathed vagus nerve (AP conduction). AP initiation in jugular C-fibres was unaffected by TTX, although it was inhibited by NaV 1.8 blocker (PF-01247324) and abolished by combination of TTX and PF-01247324. However, AP conduction in the majority of jugular C-fibres was abolished by TTX. By contrast, both AP initiation and conduction in nodose nociceptors was abolished by TTX or selective NaV 1.7 blockers. Distinction between the effect of a drug with respect to inhibiting AP in the nerve terminals within the airways vs. at conduction sites along the vagus nerve is relevant to therapeutic strategies involving inhaled NaV 1 blocking drugs.

Pharmacological examination of the neurokinin-1 receptor mediating relaxation of human intralobar pulmonary artery.

The effect of selective tachykinin receptor agonists and antagonists on human isolated intralobar pulmonary arterial rings was investigated. Neither Substance P (SP) nor neurokinin A (NKA) contracted the arteries. Both of these agonists, however, were potent and efficacious at relaxing the arteries that were precontracted with phenylephrine. The negative log (M) EC(50) values for SP and NKA were 9.0 and 8.3, respectively. The neurokinin (NK)-3 selective agonist, senktide-analog, and the NK-2 receptor selective agonist, [beta-Ala(8)]NKA(4-10), caused neither contractions nor relaxations of the arteries, whereas the NK-1 receptor agonist Ac-[Arg6, Sar9, Met(O2)11]SP(6-11) (ASM-SP) relaxed the tissue with a potency similar to SP. The relaxations to SP, NKA, and ASM-SP were competitively antagonized by the selective NK-1 receptor antagonist CP 99994, with a pK(b) in the nanomolar range. Antagonism of the ASM-SP-induced relaxations was also noted with FK 888, RP 67580, and L 732,138, although these antagonists were much less potent than CP 99994 in this regard. Another NK-1 receptor selective antagonist, SR 140333, caused an insurmountable antagonism of the SP-induced relaxations. The NK-1 receptor-mediated relaxations could be blocked by removing the endothelium, or by a combination of N-nitro-L-arginine and indomethacin. Measurement of prostanoid generation revealed that in endothelial-intact but not endothelial-denuded tissue, ASM-SP caused a selective increase in the production of 6-keto-PGF1alpha, the stable metabolite of prostacyclin. The results indicate that stimulation of NK-1 receptors leads to relaxation of human intralobar pulmonary arteries, which is mediated largely by nitric oxide and prostacyclin released from the endothelium of these vessels.

Characterization of vagal afferent subtypes stimulated by bradykinin in guinea pig trachea.

In vitro electrophysiological techniques were used to examine the effect of bradykinin on guinea pig trachea and bronchus afferent nerve endings arising from the nodose or jugular ganglia. The data reveal that bradykinin activates nerve terminals of jugular C and Adelta fibers. Although the fibers were too few in number to study rigorously, bradykinin also stimulated nodose C fibers innervating the trachea and bronchus. In contrast, Adelta fibers arising from the nodose ganglion were unresponsive to bradykinin challenge. The responses in both jugular C and Adelta fiber types were blocked by a selective bradykinin B2 receptor antagonist and were not dependent on the efferent release of sensory neuropeptides. These data indicate that the sensitivity of guinea pig airway afferent fibers to bradykinin is dependent more on the ganglionic origin of the cell body than on the conduction velocity of its axon.

Selective stimulation of jugular ganglion afferent neurons in guinea pig airways by hypertonic saline.

We evaluated the ability of hyperosmolar stimuli to activate afferent nerves in the guinea pig trachea and main bronchi and investigated the neural pathways involved. By using electrophysiological techniques, studies in vitro examined the effect of hyperosmolar solutions of sodium chloride (hypertonic saline) on guinea pig airway afferent nerve endings arising from either vagal nodose or jugular ganglia. The data reveal a differential sensitivity of airway afferent neurons to activation with hypertonic saline. Afferent fibers (both A delta and C fibers) with cell bodies located in jugular ganglia were much more sensitive to stimulation with hypertonic saline, compared with afferent neurons with cell bodies located in nodose ganglia. Additional studies in vivo demonstrated that inhalation of aerosols of hypertonic saline induced plasma extravasation in guinea pig trachea that was mediated via tachykinin NK1 receptors. Identification of a differential sensitivity of guinea pig airway afferent nerves to hypertonic saline leads to the speculation that airway responses to hyperosmolar stimuli may result from activation of afferent neurons originating predominantly from the jugular ganglion.

Inhibition of neurally mediated nonadrenergic, noncholinergic contractions of guinea pig bronchus by isozyme-selective phosphodiesterase inhibitors.

We studied the effect of inhibiting phosphodiesterase (PDE) isozyme types III, IV and V on the cholinergic and noncholinergic (tachykinergic) contractile responses to electrical field stimulation (EFS) in the guinea pig isolated bronchus. SKF 94836, a PDE III inhibitor, had a slight (approximately 30%) but significant inhibitory effect on the noncholinergic contractions. Rolipram, an inhibitor of PDE IV isozymes, dramatically inhibited the noncholinergic contractions by nearly 70%. The EC50 for rolipram was approximately 20 nM. Rolipram (1 microM) had no effect on contractions elicited by either capsaicin or neurokinin A. EFS, but not direct vagus nerve stimulation, elicits small nonadrenergic, noncholinergic relaxations of the bronchus that were potentiated by rolipram. Rolipram had the same inhibitory effect on EFS- and vagus nerve stimulation-induced noncholinergic contractions. The effect of rolipram was mimicked by another PDE IV inhibitor, RO-201724. Inhibition of PDE V with zaprinast (3 microM) had no effect on the tachykinergic contractile response. None of the PDE inhibitors affected the EFS-induced cholinergic contractions. The data suggest that the contraction due to stimulation of tachykinergic fibers is significantly reduced by selective inhibition of PDE IV and, to a lesser extent, PDE III isozymes. This is unlikely to be due to functional antagonism at the level of the smooth muscle. It is also unlikely to be due to potentiation of the nonadrenergic relaxant response to nerve stimulation. Rather, the data are in agreement with the hypothesis that selective inhibition of PDE isozymes leads to inhibition of electrically evoked tachykinin release from capsaicin-sensitive fibers in the guinea pig bronchus.

Induction of vascular smooth muscle bradykinin B1 receptors in vivo during antigen arthritis.

Antigen arthritis in rabbits was associated with induction of bradykinin B1 receptors in isolated aorta smooth muscle 24 h following intra-articular injection of antigen in sensitized animals. Control tissues developed responsiveness to desArg9-bradykinin or bradykinin during 3 h incubation, but failed to respond to either kinin at the beginning of experiments. Aorta from rabbits 24 h after induction of arthritis not only developed responsiveness to kinins more rapidly than controls, but also responded at the outset of experiments. Antigen arthritis was characterized by acute phase protein synthesis and joint swelling. This is the first demonstration of induction of smooth muscle responsiveness to desArg9-bradykinin during an immune complex disease.

D-Arg[Hyp3-Thi5-D-Tic7-Tic8]-bradykinin, a potent antagonist of smooth muscle BK2 receptors and BK3 receptors.

D-Arg[Hyp3-Thi5-D-Tic7-Tic8]-bradykinin (NPC 16731) inhibited bradykinin (BK) binding and BK-induced contraction in guinea-pig ileum, being markedly more potent than D-Phe7-BK analogues as a BK2 receptor antagonist. In isolated trachea NPC 16731, unlike other BK2 antagonists, inhibited BK binding and BK-induced contraction, and 45Ca2+ efflux in tracheal smooth muscle cells. That NPC 16731 potently inhibits BK effects in trachea provides further evidence for the existence of the airway BK3 receptor.