Hypoxia Augments TRPM3‐mediated Calcium Influx in Vagal Sensory Neurons

Abstract

Exposure to low systemic oxygen (hypoxia, Hx) elevates blood pressure, heart rate and respiration via reflex sensory and central nervous system activation. We have shown transient receptor potential (TRP) vanilloid channels contribute to afferent‐evoked activation of the brainstem nucleus tractus solitarii (nTS) following chronic Hx. Short sustained Hx exposure (10% O2, 24hr) in rats also enhances nTS activity, although the mechanisms are not completely understood. Recent evidence from others shows TRP melastatin 3 (TRPM3) channels contribute to afferent activity and nTS glutamate signaling, but their role in afferent signaling in Hx is unknown. We hypothesize that TRPM3 increases afferent neuronal activity in nTS‐projecting nodose ganglia (NG) neurons, and its contribution is elevated following Hx. Rats were exposed to either in vivo normal room air (normoxia) or 24‐hr of 10% O2 (Hx). A subset of NG neurons from normoxic rats were exposed to in vitro incubation for 24‐hr in 21% or 1% O2, 5% CO2. Intracellular calcium (Ca2+) of dissociated NG neurons was monitored via Fura‐2 fluorescence imaging. TRPM3 was activated via 1‐min bath application of Pregnenolone sulfate (Preg, 30 and 100 µM) or CIM0216 (5 ‐ 20 µM). The increase in cellular fluorescence to Preg or CIM0216 was normalized to that of its prior baseline fluorescence (ΔF/F%). NG neurons were also exposed to vehicle (Veh) alone and their Ca2+ ΔF/F% responses were used as the threshold for TRPM3 responders under comparable timeframes. RNAScope identified TRPM3 transcripts in the normoxic NG. Veh alone did not alter Ca2+ from their initial baseline (ΔF/F%, less than 102.5% increase across all preparations). Activation of TRPM3 via Preg (30 and 100 µM) elevated Ca2+ in 51% and 55% of NG neurons by a similar magnitude [120.4 ± 3.8% and 116.6 ± 3.0% (mean ± SEM), respectively]. CIM0216 also augmented Ca2+ with the greatest response occurring during 20 µM (~56% of NG neurons, 115.6 ± 3.1% elevation). Preg responses were significantly reduced by pre‐application of the TRPM3 antagonist ononetin (20 µM; Preg, 125.9 ± 7.7% vs. ononetin + Preg, 101.7 ± 0.6%), confirming drug specificity. Elevated Ca2+ by 30 µM Preg was also eliminated by bathing cells in 0 mM Ca2+ (reduced to 100.9 ± 0.4%) suggesting Ca2+ elevation occurs via opening of membrane‐bound TRPM3. In NG neurons isolated from Hx‐exposed rats, the Preg‐induced elevation of Ca2+ was significantly greater than their paired normoxic‐exposed rats (134.1 ± 4.2% vs 118.1 ± 3.9%), although the number of responders were comparable (40% vs 43%). To determine if the increase in TRPM3 Ca2+ responses to in vivo Hx was due to the reduction of oxygen or the reflex‐induced change in neuronal activity, cells were isolated then incubated in either 21% or 1% O2 for 24 hrs. Following 24 hr in vitro Hx, 30 µM Preg enhanced Ca2+ more than in 24 hr normoxic controls (115.3 ± 2.2% vs 108.1 ± 0.1%). The proportion of Preg responders was similar between groups (58% vs 61%). Altogether, these results suggest a Hx specific increase in TRPM3 function, driven by the influx of extracellular calcium.