Cultures of Airway Parasympathetic Nerves Express Functional M₂ Muscarinic Receptors

To study the control of acetylcholine release from airway parasympathetic neurons, primary cultures of these cells were established. Guinea pig tracheas were disaggregated with collagenase and plated onto matrigel-coated plates in medium that contained cytosine arabinoside to inhibit growth of dividing cells. Over 7 to 10 days neurites grow from the cell bodies, reaching a length of 2 mm. The vast majority of the cells in these cultures were neurons, as identified by morphology and staining with Neurotag® and with antibody to neuron-specific antigen protein gene product 9.5. Cultured neurons contained acetylcholine, which was released by electrical field stimulation. Thus these were parasympathetic neurons. Staining with antibodies to M₁, M₂, and M₄ muscarinic receptors revealed the presence of only M₂ receptors. Likewise, reverse transcription-polymerase chain reaction using primers for M₁, M₂, and M₄ muscarinic receptors revealed mRNA only for M₂ receptors. Blocking these M₂ receptors using atropine potentiated the stimulated release of acetylcholine, demonstrating that the M₂ receptors inhibit acetylcholine release, as they have been shown to do in vivo. Thus airway parasympathetic neurons can be grown in culture, they retain the ability to synthesize and release acetylcholine, and they express functional inhibitory M₂ muscarinic receptors.