Detection of thromboxane A 2 receptor mRNA in rabbit nodose ganglion neurons

Abstract

Thromboxane A 2 (TXA 2) is an arachidonic acid metabolite that is released during tissue trauma and elicits platelet aggregation and vascular smooth muscle contraction. Previous research has shown that TXA 2 stimulates pulmonary and cardiac vagal afferent neurons. Therefore, we hypothesized that the presence of the TXA 2 receptor (TP) in vagal neurons would allow for stimulation or modulation of these neurons by TXA 2. To test this hypothesis, single cell RT-PCR was employed using neurons obtained from primary cell cultures of nodose ganglia excised from adult rabbits. Since the sequence for the rabbit TP gene was unknown, a portion of the rabbit TP cDNA was first amplified, cloned, and sequenced. Primer sets for TP were then designed based on this sequence and used in conjunction with a neuronal marker, medium weight neurofilament (NFM), in multiplex RT-PCR reactions. Ninety-three cells were isolated from culture and RT-PCR was carried out on individual cells. Using an aliquot from the initial RT-PCR reaction, a second round of PCR was then employed in which the NFM and TP primer sets were split up into separate reactions. Twenty-three of the 82 cells that were positive for NFM were also positive for TP. Therefore, we conclude that the presence of TP mRNA in a subset of cultured nodose ganglion neurons allows for the possibility that TXA 2 may directly stimulate or modulate vagal afferent neurons.