Functional protease-activated receptors in the dorsal motor nucleus of the vagus

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Abstract
Background Protease‐activated receptors (PARs), a family member of G‐protein coupled receptors, are present and functionally active in a wide variety of cells. The object of this study was to demonstrate the presence and function of PAR‐1 and PAR‐2 in the dorsal motor nucleus of the vagus (DMV). Methods DMNV neurons were isolated from neonatal rat brainstems using micro‐dissection and enzymatic digestion. Neurons were cultured in Neurobasal medium A containing 2% B27 supplement. Intracellular calcium concentration ([Ca2 + ]i) was measured using fura‐2 based microspectrometry. Expression of PARs was detected by RT‐PCR and immunofluorescent staining. Key Result Thrombin and PAR‐1 agonist peptide activate PAR‐1 with a maximum change in [Ca2 + ]i expressed as ΔF/F0 of 229 ± 14% and 137 ± 7%, respectively. Trypsin and PAR‐2 agonist peptide activate PAR‐2 with a maximum ΔF/F0 change of 258 ± 12% and 242 ± 10%, respectively. Inhibition of phospholipase C (PLC) by U73312 (1 μm) decreased the maximal change in ΔF/F0 induced by PAR‐1 activation from 140 ± 17% to 21 ± 3%, while the PAR‐2‐mediated maximal change in ΔF/F0 decreased from 185 ± 21% to 19 ± 6%. Blockade of IP3 receptor with 2APB inhibited the maximal change in ΔF/F0 due to PAR‐1 and PAR‐2 activation by 72 ± 13% and 71 ± 20% respectively. PAR‐1 immnuoreactivity was present in DMV neurons. Increase in transcripts for PAR‐1 and PAR‐2 were detected in DMV tissues derived from IBD rats relative to control animals. Conclusions & Inferences Our results indicate that PAR‐1 and PAR‐2 are present in the DMV neurons, and their activation leads to increases in intracellular calcium via signal transduction mechanism that involves activation of PLC and the production of IP3.