Abstract
Norepinephrine (NE) controls many vital body functions by activating adrenergic receptors (ARs). Average core body temperature (CBT) in mice is 37°C. Of note, CBT fluctuates between 36 and 38°C within 24 hours, but little is known about the effects of CBT changes on the pharmacodynamics of NE. Here, we used Peltier element-controlled incubators and challenged murine hypothalamic mHypoA -2/10 cells with temperature changes of ±1°C. We observed enhanced NE-induced activation of a cAMP-dependent luciferase reporter at 36 compared with 38°C. mRNA analysis and subtype specific antagonists revealed that NE activates β 2- and β 3-AR in mHypoA-2/10 cells. Agonist binding to the β 2-AR was temperature insensitive, but measurements of cytosolic cAMP accumulation revealed an increase in efficacy of 45% ± 27% for NE and of 62% ± 33% for the β 2-AR-selective agonist salmeterol at 36°C. When monitoring NE-promoted cAMP efflux, we observed an increase in the absolute efflux at 36°C. However, the ratio of exported to cytosolic accumulated cAMP is higher at 38°C. We also stimulated cells with NE at 37°C and measured cAMP degradation at 36 and 38°C afterward. We observed increased cAMP degradation at 38°C, indicating enhanced phosphodiesterase activity at higher temperatures. In line with these data, NE-induced activation of the thyreoliberin promoter was found to be enhanced at 36°C. Overall, we show that physiologic temperature changes fine-tune NE-induced cAMP signaling in hypothalamic cells via β 2-AR by modulating cAMP degradation and the ratio of intra- and extracellular cAMP. SIGNIFICANCE STATEMENT: Increasing cytosolic cAMP levels by activation of G protein-coupled receptors (GPCR) such as the β 2-adrenergic receptor (AR) is essential for many body functions. Changes in core body temperature are fundamental and universal factors of mammalian life. This study provides the first data linking physiologically relevant temperature fluctuations to β 2-AR-induced cAMP signaling, highlighting a so far unappreciated role of body temperature as a modulator of the prototypic class A GPCR.
Highlights
Norepinephrine (NE) together with epinephrine belongs to the neurotransmitter family of catecholamines
We provide the first evidence that physiologic temperature changes affect signaling pathways regulated by transmembrane receptors in hypothalamic cells and that the cAMP response elements binding protein (CREB) and signal transducer and activator of transcription (STAT) pathways are significantly affected
CREB activity induced by the direct adenylyl cyclase (AC) activator FSK (10 mM) was enhanced at 36C
Summary
Norepinephrine (NE) together with epinephrine belongs to the neurotransmitter family of catecholamines. Depending on the species, CBT in mammals fluctuates by 1–4C within 24 hours, with a nadir in temperature at the end of the resting state and ABBREVIATIONS: ABCC, ATP-binding cassette subfamily C; AC, adenylyl cyclase; AR, adrenergic receptor; BAY60-6583, 2-[[6-amino-3,5dicyano-4-[4-(cyclopropylmethoxy)phenyl]-2-pyridinyl]thio]-acetamide; BIM-X, bisindolylmaleimide X; BK, bradykinin; BMAL1, brain and muscle ARNT-like protein-1; CBT, core body temperature; CREB, cAMP response elements binding protein; CYP, cyanopindolol; DMEM, Dulbecco’s modified Eagle’s medium; ESI-09, a-[(2-(3-Chlorophenyl)hydrazinylidene]-5-(1,1-dimethylethyl)-b-oxo-3-isoxazolepropanenitrile; FOXO, forkhead-box-protein-O; FSK, forskolin; GPCR, G protein–coupled receptor; Gpp(NH)p, guanosine 50-[b,c-imido]triphosphate; HBSMC, human bronchial smooth muscle cell; 3H-cAMP, adenosine 30,50-cyclic monophosphate [50,8-3H]; IBMX, 3-isobutyl-1-methylxantine; ICI118,551, (2R,3R)-rel-1-[(2,3-dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol; IFN, interferon; ISO, isoproterenol; KT-5720, 2,3,9,10,11,12-hexahydro-10S-hydroxy-9-methyl-1-oxo-9R,12S-epoxy-1H-diindolo[1,2,3-fg:30,20,10-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid, hexylester; mHypoA-2/10, murine hypothalamic A 2/10 cells; mHypoA-2/12, murine hypothalamic A 2/12 cells; NE, norepinephrine; PDE, phosphodiesterase; PVN, paraventricular nucleus; R, active; R*, inactive; rEC-cAMP, ratio of extracellular and cytosolic cAMP; RLU, random light unit; Sal, salmeterol; signal transducer and activator of transcription; SR59230A, 1-(2-Ethylphenoxy)-3-[[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]-(2S)-2-propanol hydrochloride
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