Central GPR109A Activation Mediates Glutamate-Dependent Pressor Response in Conscious Rats

Abstract

G protein-coupled receptor 109A (GPR109A) activation by its ligand nicotinic acid (NA) in immune cells increases Ca 21 levels, and Ca 21 induces glutamate release and oxidative stress in central blood pressure (BP)-regulating nuclei, for example, the rostral ventrolateral medulla (RVLM), leading to sympathoexcitation. Despite NA's ability to reach the brain, the expression and function of its receptor GPR109A in the RVLM remain unknown. We hypothesized that NA activation of RVLM GPR109A causes Ca 21-dependent L-glutamate release and subsequently increases neuronal oxidative stress, sympathetic activity, and BP. To test this hypothesis, we adopted a multilevel approach, which included pharmacologic in vivo studies along with ex vivo and in vitro molecular studies in rat pheochromocytoma cell line (PC12) cells (which exhibit neuronal phenotype). We present the first evidence for GPR109A expression in the RVLM and in PC12 cells. Next, we showed that RVLM GPR109A activation (NA) caused pressor and bradycardic responses in conscious rats. The resemblance of these responses to those caused by intra-RVLM glutamate and their attenuation by NMDA receptor (NMDAR) blockade (2-amino-5-phosphonopentanoic acid) and enhancement by L-glutamate uptake inhibition (L-trans-pyrrolidine-2,4-dicarboxylic acid, PDC) supported our hypothesis. NA increased Ca 21 , glutamate, nitric oxide and reactive oxygen species (ROS) levels in PC12 cells and increased RVLM ROS levels. The inactive NA analog isonicotinic acid failed to replicate the cardiovascular and biochemical effects of NA. Further, GPR109A knockdown (siRNA) abrogated the biochemical effects of NA in PC12 cells. These novel findings yield new insight into the role of RVLM GPR109A in central BP control.

Rostral Ventrolateral Medulla EP3 Receptor Mediates the Sympathoexcitatory and Pressor Effects of Prostaglandin E2 in Conscious Rats

“…Femoral artery catheterization and stereotaxic implantation of an RVLM guide cannula were performed under anesthesia (9 mg/100 g ketamine and 1 mg/100 g xylazine, i.p.) and appropriate analgesia (0.03 mg/kg buprenorphine) as detailed in our recent study (Rezq and Abdel-Rahman, 2016). Histologic verification of the RVLM position was done via intra-RVLM injection of fast green dye (EM Sciences, Cherry Hill, NJ).…”

“…The protocol used in our recent report (Rezq and Abdel-Rahman, 2016) was used for c-Fos-ir neuron studies in coronal sections containing the RVLM rostrally from 212.8 to 211.8 mm relative to the bregma (Paxinos et al, 1980). Frozen sections from brains of treated and control rats (n 5 5-7) were incubated for 48 hours at 4°C in a rabbit polyclonal anti-c-Fos antibody (1:200; Santa Cruz Biotechnology, Santa Cruz, CA).…”

“…DCF was used to generate the standard curve. ROS level was expressed in terms of relative fluorescence units of produced DCF, as detailed in our recent study (Rezq and Abdel-Rahman, 2016).…”

“…A rat pheochromocytoma cell line (PC12 cells) purchased from American Type Culture Collection (Manassas, VA) was used according to the protocol detailed in our recent study (Rezq and Abdel-Rahman, 2016).…”

“…L-Glutamate release in cultured PC12 cells was measured using the Amplex Red kit (Molecular Probes, Invitrogen) following the manufacturer's instructions and as detailed in our recent study (Rezq and Abdel-Rahman, 2016).…”