We would thus argue that activation of FFA3 should suppress catecholamine release and thus sympathetic function. to produce voltage-dependent inhibition of N-type Ca2+channels (Cav2.2) in sympathetic neurons. In addition to acetate and propionate, we show that -hydroxybutyrate (BHB), a metabolite produced during ketogenic conditions, is also an FFA3 agonist. This contrasts with previous interpretations of BHB as an antagonist at FFA3. Together, these results indicate that endogenous BHB levels, especially when elevated under certain conditions, such as starvation, diabetic ketoacidosis, and ketogenic diets, play a potentially important role in regulating the activity of the SNS through Lacidipine FFA3. == Introduction == Free fatty acid receptor 3 (FFA3) and 2 (FFA2), originally termed GPR41 and GPR43, respectively, are paralogs in a G-protein-coupled receptor (GPCR) family tandemly encoded at a single chromosomal locus. Short-chain fatty acids (SCFAs), most notably, acetate (C2), propionate (C3), and butyrate (C4), are potential endogenous agonists for FFA2 and FFA3 (Brown et al., 2003;Le Poul et al., 2003;Nilsson et al., 2003). Anerobic bacterial fermentation of undigested carbohydrate in the lower gut is the major source for SCFAs (Sellin, 1999;Topping and Clifton, 2001). Lacidipine FFA3 and FFA2 are expressed in the intestine where SCFAs are produced (Karaki et al., 2008;Samuel et al., 2008;Tazoe et al., 2009) as well as at other sites involved with metabolism, including pancreatic islets, blood cells (monocytes and neutrophils), and white adipose tissue (Le Poul et al., 2003;Nilsson et al., 2003;Xiong et al., 2004;Brown et al., 2005;Kebede et al., 2009). Recently, SCFAs and a ketone body, -hydroxybutyrate (BHB), were reported to regulate the sympathetic nervous system (SNS) directly through FFA3 expressed in sympathetic superior cervical ganglionic (SCG) neurons (Kimura et al., 2011). In this study, SCFAs promoted sympathetic outflow by serving as agonists for FFA3, whereas BHB, a metabolite produced during ketogenic conditions, antagonized FFA3, thereby suppressing the SNS. Under conditions, such as fasting, diabetes, and ethanol consumption, endogenous ligands for FFA3 attain plasma concentrations (low millimolar) sufficient to activate the receptor, thereby potentially influencing body energy expenditure and metabolic homeostasis. However, thus far, physiological signaling pathways for FFA3 in sympathetic neurons are unknown. Moreover, the source of the endogenous ligands for activating FFA3 in sympathetic ganglia remains unclear. The majority of FFA3 studies have used biochemical assays in cell lines heterologously expressing the receptor. To better understand the details of native FFA3 signaling in tissue, we used dissociated rat sympathetic neurons as a model system for electrophysiological studies. In this well-explored system, natively and heterologously expressed GPCRs efficiently couple to endogenous G-proteins to modulate natively expressed N-type voltage-gated Ca2+channels after external application of cognate agonists (Ikeda and Schofield, 1989;Zhu and Ikeda, 1993a,b;Ikeda, 1996). Using whole-cell patch-clamp, we examined the pharmacology and signaling pathways of both native and heterologous FFA3 expressed in sympathetic neurons. We found that native FFA3 responses were asymmetric within the SNS with an increasing cranial to caudal gradient. FFA3 transcript levels, as decided with qRT-PCR andin situhybridization, correlated well with the magnitude of physiological responses. Finally, in contrast to previous findings, we identify BHB as an agonist rather than an antagonist at both heterologously and natively expressed FFA3. We conclude that BHB is likely the physiologically relevant endogenous ligand for FFA3 in sympathetic neurons during epochs of increased plasma BHB levels that occur because of starvation, diabetes, or ketogenic diets. == Materials and Methods == == == == == == Cloning. == The open reading frames for FFAs and GPR109A (accession no. for mFFA3:NM_001033316, mFFA2:BC019570, rFFA3:NM_001108912, rFFA2:NM_001005877, rGPR109A:NM_181476) were amplified by PCR from genomic DNA (gDNA) Tbp (mouse, Promega; rat, Lacidipine Clontech) with Phusion polymerase (New England Biolabs). PCR primers incorporated 5.