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2011). The function of CaSR in the central nervous system has been investigated extensively. While its roles are still not fully characterized, CaSR has been implicated in a variety of functions, including neurotransmission and neuronal growth, differentiation, migration and injury (Bouschet & Henley, 2005; Phillips et al. 2008; Vizard et al. 2008; Vyleta & Smith, 2011). For example, it has recently been reported that stimulation of CaSR inhibits the activity of a non-selective cation channel (Chen et al. 2010) and synapse transmission (Phillips et al. 2008) in neocortical nerve terminals. Although CaSR has been cloned from rat dorsal root ganglion, which indeed shows Ca2+ sensitivity when stably expressed in HEK 293 cells (Wang et al. 2003b; Awumey et al. Alectinib nmr 2007), the function of CaSR in peripheral sensory transmission has not been well studied. The present study aimed to investigate whether CaSR is expressed in vagal bronchopulmonary sensory neurons and, if so, whether this G protein-coupled receptor can modulate the function of TRPV1, an important polymodal transducer in bronchopulmonary C fibres. The procedures described click here were performed in compliance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals (Office of Laboratory Animal Welfare, Amended August 2002) and US Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research and Training. These procedures were also approved by the Mercer University Institutional Animal GPX4 Care and Use Committee. Sensory neurons innervating the airway and lungs were identified by retrograde labelling of lungs using the fluorescent tracer 1,1��-dioctadecyl-3,3,3��,3��-tetramethylindocarbocyanine perchlorate (DiI), as described in our recent studies (Gu & Lee, 2009; Gu & Lin, 2010). Briefly, young Sprague�CDawley rats were anaesthetized by continuous inhalation of isoflurane administered via a nose-cone connected to a vaporizing machine (Smiths Medical, Dublin, OH, USA). A small mid-line incision was made on the ventral neck skin to expose the trachea. The DiI (0.2 mg ml?1 for cell culture and 1 mg ml?1 for immunohistochemistry; 50 ��l in volume) was instilled into the lungs via a 30 gauge needle inserted into the lumen of the trachea; the skin incision was then closed. Animals were kept undisturbed for 7�C10 days until they were used for the cell culture or immunohistochemistry. Rats (150�C250 g body weight; n= 4) were anaesthetized by inhalation of isoflurane, killed and transcardially perfused with 0.01 m PBS (pH 7.4), followed by freshly prepared 0.01 m phosphate buffer (pH 7.4) containing 4% paraformaldehyde. The nodose and jugular ganglia, trachea and lungs were dissected and fixed in 4% paraformaldehyde for 4 h.
