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

Abstract

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).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 ([Ca 2 + ] i ) was measured using fura-2 based microspectrometry. Expression of PARs was detected by RT-PCR and immunofluorescent staining.Thrombin and PAR-1 agonist peptide activate PAR-1 with a maximum change in [Ca 2 + ] 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. 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.