P2X3 Expression in DRG Neuron and Effects on Blood Pressure Response Following Femoral Artery Occlusion

Open Access
Author:
Li, Jialiu David
Area of Honors:
Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • James Harold Marden, Thesis Supervisor
  • James Harold Marden, Honors Advisor
  • Jianhua Li, Faculty Reader
Keywords:
  • P2X3 receptor
  • muscle afferents
  • peripheral artery disease
  • ATP
  • blood pressure
  • NGF
Abstract:
During exercise, muscle contraction increases sympathetic nerve activity and arterial blood pressure via activation of cardiovascular regulating nuclei in central nerve system. This neural mechanism is termed “Exercise Pressor Reflex”. Femoral artery occlusion augments the sympathetic nerve and pressor responses to static exercise (muscle contraction) and muscle metabolites (which produced in exercising muscles) injected into the arterial blood supply of the hindlimb muscles in rats. The underlying mechanism by which these reflex responses are enhanced after muscle vascular insufficiency is unclear. Purinergic P2X3 receptor has been reported to contribute to the metabolic component of the exercise pressor reflex. It is well known that muscle thin fibers afferent nerves (i.e., group III and IV, also named as A- and C- fibers) mediate the exercise pressor reflex. Additional study shows that femoral artery occlusion increases the levels of P2X3 receptor proteins in sensory neurons-dorsal root ganglion (DRG) neurons. Thus, the purpose of this study was to examine if chronic femoral occlusion would alter the expression of P2X3 in DRG neurons that project C- and/or A- fibers in control rats and in occluded rats. Also, P2X3-mediated blood pressure response was examined after femoral occlusion. In addition, the role played by nerve growth factor (NGF) in regulating blood pressure response to stimulation of P2X3 was examined. The dual immunofluorescence techniques were used to examine co-localization of fluorescent P2X3 and peripherin/NF200 immunoreactivity in the DRG neurons of control rats and occluded rats. Note that peripherin and NF200 are used to label neurons with C-fiber and A-fiber, respectively. The results of this experiment show that P2X3 staining appears in C-fibers of DRG neurons, but only few P2X3 staining appears in A-fibers of DRG neurons in both control and occlusion groups. Femoral artery occlusion increased expression of P2X3 in a large portion of DRG neurons with C-fiber compared with A-fiber of neurons. Furthermore, the results showed that responses of blood pressure to stimulation of P2X were greater in occluded rats than responses in control rats by injection of α, β-methylene ATP (α, β-me ATP, an agonist to P2X) into the arterial blood supply of the hindlimb muscle. In order to determine response of blood pressure to arterial injection of α, β-me ATP were mainly via P2X3, additional experiments were performed to give a specific antagonist to P2X3 (RO3) prior to α, β-me ATP and then the reflex muscle responses were observed. The data demonstrated that pretreatment of RO3 significantly attenuates blood pressure response evoked by α, β-me ATP in both control rats and occluded rats. Finally, infusion of NGF in the hindlimb muscles of healthy rats increased the pressor response to injection of α, β-me ATP. Likewise, blocking NGF attenuated exaggeration of the reflex response induced by α, β-me ATP in occluded rats. The findings of this study suggest that expression of P2X3 in primary afferent neurons projecting C-fibers are upregulated as the blood supply to the hindlimb is deficient under ischemic conditions, thereby leading to augmentation of the exercise pressor reflex. NGF is likely to be involved in increased reflex pressor response with P2X3 receptors activation observed in hindlimb muscle ischemia.