BackgroundSensory nerves innervating the airways play an important role in regulating various cardiopulmonary functions, maintaining homeostasis under healthy conditions and contributing to pathophysiology in disease states. Hypo-osmotic solutions elicit sensory reflexes, including cough, and are a potent stimulus for airway narrowing in asthmatic patients, but the mechanisms involved are not known. Transient receptor potential cation channel, subfamily V, member 4 (TRPV4) is widely expressed in the respiratory tract, but its role as a peripheral nociceptor has not been explored.ObjectiveWe hypothesized that TRPV4 is expressed on airway afferents and is a key osmosensor initiating reflex events in the lung.MethodsWe used guinea pig primary cells, tissue bioassay, in vivo electrophysiology, and a guinea pig conscious cough model to investigate a role for TRPV4 in mediating sensory nerve activation in vagal afferents and the possible downstream signaling mechanisms. Human vagus nerve was used to confirm key observations in animal tissues.ResultsHere we show TRPV4-induced activation of guinea pig airway–specific primary nodose ganglion cells. TRPV4 ligands and hypo-osmotic solutions caused depolarization of murine, guinea pig, and human vagus and firing of Aδ-fibers (not C-fibers), which was inhibited by TRPV4 and P2X3 receptor antagonists. Both antagonists blocked TRPV4-induced cough.ConclusionThis study identifies the TRPV4-ATP-P2X3 interaction as a key osmosensing pathway involved in airway sensory nerve reflexes. The absence of TRPV4-ATP–mediated effects on C-fibers indicates a distinct neurobiology for this ion channel and implicates TRPV4 as a novel therapeutic target for neuronal hyperresponsiveness in the airways and symptoms, such as cough.

Gold nanoparticles modified with anti-mouse IgG have been used to trace oligonucleotides carrying a c-myc peptide. Two strategies, a dot-blot format as well as inductively coupled plasma mass spectrometry (ICPMS) have been used to detect the nanoparticle tracer. For both cases, oligonucleotide-peptide conjugates were first applied to a nitrocellulose membrane using a manifold attached to a suction device. After immobilization of the oligonucleotide by UV radiation, the samples were incubated with an anti-c-myc monoclonal antibody. In the case of the dot-blot format strategy, it was followed by incubation with a secondary antibody conjugated to horseradish peroxidase and development with luminol as chemiluminescent substrate. In the case of ICPMS strategy, it was followed by incubation with the secondary antibody (anti-mouse IgG) conjugated to gold nanoparticles and their ICPMS detection after dissolving. The nonspecific adsorptions were found to be around zero. The limit of detection for peptide-modified DNA was 0.2 pmol. The method may have significant potential as an important ICPMS-based nonradioactive DNA detection method for the simultaneous determination of various sequences by labeling different kinds of inorganic nanoparticles.

Background and Purpose— Growing evidence supports that the immunomodulatory drug fingolimod is protective in stroke. Fingolimod binds to 4 of 5 sphingosine-1-phosphate (S1P) receptors and, among other actions, it induces lymphopenia. In this study, we investigated whether a selective S1P1 agonist is protective in experimental stroke. Methods— Drug selectivity was studied in vitro in cells overexpressing the human S1P receptors. Mice (n=54) received different doses of LASW1238 (3 or 10 mg/kg), fingolimod (1 mg/kg), or the vehicle intraperitoneal, and lymphopenia was studied at different time points. After intraluminal middle cerebral artery occlusion for 45 minutes and immediately after reperfusion, mice (n=56) received the drug treatment. At 24 hours, a neurological test was performed and infarct volume was measured. Treatment and all the analyses were performed in a blind fashion. Results— In vitro functional assays showed that LASW1238 is a selective agonist of the S1P1 receptor. At 10 mg/kg, this compound induced sustained lymphopenia in mice comparable with fingolimod, whereas at 3 mg/kg it induced short-lasting lymphopenia. After ischemia, both LASW1238 (10 mg/kg) and fingolimod reduced infarct volume, but only LASW1238 (10 mg/kg) showed statistically significant differences versus the vehicle. The neurological function and plasma cytokine levels were not different between groups. Conclusions— The selective S1P1 agonist LASW1238 reduces infarct volume after ischemia/reperfusion in mice, but only when lymphopenia is sustained for at least 24 hours. S1P1 and lymphocytes are potential targets for drug treatment in stroke. Defining the best drug dosing regimens to control the extent and duration of lymphopenia is critical to achieve the desired effects.