Activation of Adenosine A3 Receptors Suppresses Airway Hypersensitivity

Airway hypersensitivity, characterized by exaggerated sensory and reflexogenic responses to various stimuli, is a common pathophysiological feature in patients with airway inflammatory diseases (e.g., asthma, bronchitis). Increasing and compelling evidence indicates that sensitized pulmonary C-fiber afferents play a vital role in the pathogenesis of airway hypersensitivity. Many mediators are known to sensitize these sensory fibers, whereas only very few have been identified to suppress the sensory hypersensitivity. Agonists of adenosine A3 receptor (AA3R) are recognized as protective agents in clinical studies in some fields of anti-inflammation. Recently, AA3R agonists have been shown to suppress the chronic inflammation-induced hyperalgesia and its associated hyper-excitability of spinal sensory neurons, whose suppression is believed as an indirect effect via its anti-inflammatory actions. A recent report has suggested a direct suppressive effect of AA3R agonist on neuronal excitability, in which, in isolated retinal ganglion cells, AA3R agonist reduces glutamate-induced rise of intracellular [Ca2+]. This [Ca2+] rise is a key step to elevate neuronal excitability. However, whether the AA3R agonist exerts a direct suppressive effect on neuronal excitability still remains unknown. This proposal will study the novel effect of AA3R agonist on suppressing hypersensitivity of the pulmonary neuronal excitability and airway reflexes. The proposed study of the 1st-year aims to determine 1) whether activation of the AA3R suppresses the airway hypersensitivity, 2) whether the AA3R activation suppresses the hypersensitivity of pulmonary C-fiber afferents and 3) whether the AA3R located in the brain and phrenic motor nerves is important to the suppressive effect on airway hypersensitivity. The proposed study of 2nd-year aims to determine 1) whether the AA3R agonist acts at the level of overall neuronal excitability of pulmonary C-fibers, but not at specific sensory transduction, 2) whether the AA3R-induced suppression is a direct action on pulmonary C-neurons and 3) whether the AA3R is expressed on C-fiber sensory nerves in lungs. The proposed study of 3rd-year aims to study 1) the cellular signal transduction involved in the AA3R activation-induced suppression on pulmonary C-neurons and 2) whether the AA3R-induced suppressive effect is still existed while applied as post-treatment. The possible signaling pathways will include Gi protein, adenylyl cyclase, protein kinase A (PKA), PKC, ATP-sensitive K+ channels. Hypersensitivity is induced by prostaglandin E2 (PGE2), a mediator released during airway inflammation. In the in vivo study of 1st- and 2nd-years, hypersensitivity of airway reflex will be determined by elevation of cough and apnea reflexes measured in conscious guinea pigs and spontaneously breathing anesthetized rats, respectively. The afferent activity of the pulmonary C fibers will be measured using “single-fiber” recording technique in artificially ventilated anesthetized rats. In the ex vivo study of 1styear, the hypersensitivity of phrenic neuromuscular transmission will be determined using a rat isolated phrenic nerve-diaphragm preparation. In the in vitro study of 2nd and 3rd-years, neuronal responses will be determined using perforated whole-cell patch-clamp in rat cultured primary pulmonary sensory neurons. Our preliminary results reveal that 2-Cl-IB-MECA (a selective agonist of AA3R) suppresses the PGE2-induced (1) hypersensitivity of apnea reflexes, (2) hyper-excitability of pulmonary C-fibers in anesthetized rats and (3) hypersensitivity of inward current in isolated pulmonary C-neurons. Furthermore, (4) the 2-Cl-IB-MECA-induced suppressive effect of the neuronal hypersensitivity is, in part, mediated by PKA. The results thus suggest the feasibility of this proposed study. The findings of this proposal may provide a new insight of AA3R agonist into potential therapies of airway hypersensitive diseases.