Behrendt Marc, Solinski Hans Jürgen, Schmelz Martin, Carr Richard
Department of Experimental Pain Research, MCTN, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany.
Front Cell Neurosci. 2022 Apr 13;16:843225. doi: 10.3389/fncel.2022.843225. eCollection 2022.
TRPM3 is a calcium-permeable cation channel expressed in a range of sensory neurons that can be activated by heat and the endogenous steroid pregnenolone sulfate (PS). During inflammation, the expression and function of TRPM3 are both augmented in somatosensory nociceptors. However, in isolated dorsal root ganglion (DRG) neurons application of inflammatory mediators like prostaglandins and bradykinin (BK) inhibit TRPM3. Therefore, the aim of this study was to examine the effect of preceding activation of cultured 1 day old mouse DRG neurons by the inflammatory mediator BK on TRPM3-mediated calcium responses. Calcium signals were recorded using the intensity-based dye Fluo-8. We found that TRPM3-mediated calcium responses to PS were enhanced by preceding application of BK in cells that responded to BK with a calcium signal, indicating BK receptor (BKR) expression. The majority of cells that co-expressed TRPM3 and BKRs also expressed TRPV1, however, only a small fraction co-expressed TRPA1, identified by calcium responses to capsaicin and supercinnamaldehyde, respectively. Signaling and trafficking pathways responsible for sensitization of TRPM3 following BK were characterized using inhibitors of second messenger signaling cascades and exocytosis. Pharmacological blockade of protein kinase C, calcium-calmodulin-dependent protein kinase II and diacylglycerol (DAG) lipase did not affect BK-induced sensitization, but inhibition of DAG kinase did. In addition, release of calcium from intracellular stores using thapsigargin also resulted in TRPM3 sensitization. Finally, BK did not sensitize TRPM3 in the presence of exocytosis inhibitors. Collectively, we show that preceding activation of DRG neurons by BK sensitized TRPM3-mediated calcium responses to PS. Our results indicate that BKR-mediated activation of intracellular signaling pathways comprising DAG kinase, calcium and exocytosis may contribute to TRPM3 sensitization during inflammation.
瞬时受体电位M型3（TRPM3）是一种钙通透性阳离子通道，表达于多种感觉神经元中，可被热和内源性类固醇硫酸孕烯醇酮（PS）激活。在炎症过程中，TRPM3在躯体感觉伤害感受器中的表达和功能均增强。然而，在分离的背根神经节（DRG）神经元中，应用前列腺素和缓激肽（BK）等炎症介质会抑制TRPM3。因此，本研究的目的是检测炎症介质BK对培养的1日龄小鼠DRG神经元的预先激活对TRPM3介导的钙反应的影响。使用基于强度的染料Fluo-8记录钙信号。我们发现，在对BK产生钙信号反应的细胞中，预先应用BK可增强TRPM3介导的对PS的钙反应，表明存在BK受体（BKR）表达。大多数共表达TRPM3和BKR的细胞也表达瞬时受体电位香草酸亚型1（TRPV1），然而，只有一小部分细胞共表达瞬时受体电位锚蛋白1（TRPA1），分别通过对辣椒素和超肉桂醛的钙反应来鉴定。使用第二信使信号级联和胞吐作用的抑制剂来表征BK后TRPM3致敏的信号传导和运输途径。蛋白激酶C、钙调蛋白依赖性蛋白激酶II和二酰甘油（DAG）脂肪酶的药理学阻断不影响BK诱导的致敏，但DAG激酶的抑制会影响。此外，使用毒胡萝卜素从细胞内储存释放钙也导致TRPM3致敏。最后，在存在胞吐作用抑制剂的情况下，BK不会使TRPM3致敏。总的来说，我们表明BK对DRG神经元的预先激活使TRPM3介导的对PS的钙反应致敏。我们的结果表明，BKR介导的包括DAG激酶、钙和胞吐作用的细胞内信号通路的激活可能有助于炎症期间TRPM3的致敏。
