The potential of metabolomic analysis techniques for the characterisation of α1-adrenergic receptors in cultured N1E-115 mouse neuroblastoma cells

Abstract

Several studies of neuropathic pain have linked abnormal adrenergic signalling to the development and maintenance of pain, although the mechanisms underlying this are not yet fully understood. Metabolomic analysis is a technique that can be used to give a snapshot of biochemical status, and can aid in the identification of the mechanisms behind pathological changes identified in cells, tissues and biological fluids. This study aimed to use gas chromatographymass spectrometry-based metabolomic profiling in combination with reverse transcriptase-polymerase chain reaction and immunocytochemistry to identify functional a 1 -adrenergic receptors on cultured N1E-115 mouse neuroblastoma cells. The study was able to confirm the presence of mRNA for the a 1D subtype, as well as protein expression of the a 1 -adrenergic receptor. Furthermore, metabolomic data revealed changes to the metabolite profile of cells when exposed to adrenergic pharmacological intervention. Agonist treatment with phenylephrine hydrochloride (10 lM) resulted in altered levels of several metabolites including myo-inositol, glucose, fructose, alanine, leucine, phenylalanine, valine, and n-acetylglutamic acid. Many of the changes observed in N1E-115 cells by agonist treatment were modulated by additional antagonist treatment (prazosin hydrochloride, 100 lM). A number of these changes reflected what is known about the biochemistry of a 1 -adrenergic receptor activation. This preliminary study therefore demonstrates the potential of metabolomic profiling to confirm the presence of functional receptors on cultured cells.

Noradrenergic Mechanisms in Fentanyl-Mediated Rapid Death Explain Failure of Naloxone in the Opioid Crisis

“…Subsequent studies with similar models of human SH-SY5Y neuroblastoma cells have demonstrated the presence of endogenous muscarinic (M) 3 receptor sites on these cells, where fentanyl might act as a potential antagonist (Yamanoue et al, 1993;Bundey and Nahorski, 2001). Animal studies have also demonstrated evidence of both a 2 -and a 1 -adrenergic receptors (specifically, the a 1D subtype) on neuroblastoma cells where fentanyl has a 1 -subtype antagonist effects (Baron and Siegel, 1989;Wenner et al, 2016). This study was performed to better understand the relationship between neuronal amine uptake and analgesia and was not analyzed or performed in the context of FIMR or WCS.…”

Current Developments on the Role of α1-Adrenergic Receptors in Cognition, Cardioprotection, and Metabolism

“…When fatty acid oxidation was suppressed centrally in the brain, α 1 -ARs stimulated the counter-regulatory increases in plasma glucose levels (Sajapitak et al, 2008). A metabolomic analysis in a neuronal cell culture also showed that α 1 -AR stimulation results in lower levels of carbohydrates (Wenner et al, 2016).…”

“…When fatty acid oxidation was suppressed centrally in the brain, α 1 -ARs stimulated the counter-regulatory increases in plasma glucose levels ( Sajapitak et al, 2008 ). A metabolomic analysis in a neuronal cell culture also showed that α 1 -AR stimulation results in lower levels of carbohydrates ( Wenner et al, 2016 ). These results are consistent with other studies in the α 1B -AR KO mice which displayed insulin resistance and dysfunctional glucose homeostasis ( Burcelin et al, 2004 ) and the use of prazosin treatment, an α 1 -AR antagonist, which increases risk of metabolic syndrome and high fasting plasma glucose levels in patients with benign prostatic hyperplasia ( Lee et al, 2013 ).…”

Untargeted metabolomics of neuronal cell culture: A model system for the toxicity testing of insecticide chemical exposure

“…For measurement of metabolites by GC-MS, dried metabolite extracts were derivatized by methoximation and silylation to increase thermal stability and volatility of metabolite compounds. The derivatization process had been previously optimized for a range of different biological samples (Abbiss, Rawlinson, Maker, & Trengove, 2015;Abbiss et al, 2012;Ng, Ryan, Trengove, & Maker, 2012;Wenner, Maker, Dawson, Drummond, & Mullaney, 2016). Briefly, 40 μL of 20 mg ml −1 methoxyamine hydrochloride in pyridine was added to a dried extract and incubated at 30°C for 90 min with agitation in a Thermomixer Comfort (Eppendorf) at 1200 rpm, followed by addition of 20 μl of N-methyl-trimethylsilyltrifluoroacetamide and incubation at 37°C for 30 min with agitation at 300 rpm.…”