Functional evidence for the expression of P2X1, P2X4 and P2X7 receptors in human lung mast cells

Abstract

Background and purpose: P2X receptors are widely expressed in cells of the immune system with varying functions. This study sought to characterize P2X receptor expression in the LAD2 human mast cell line and human lung mast cells (HLMCs). Experimental approach: Reverse transcriptase polymerase chain reaction (RT-PCR) and patch clamp studies were used to characterize P2X expression in mast cells using a range of pharmacological tools. Key results: RT-PCR revealed P2X1, P2X4 and P2X7 transcripts in both cell types; mRNA for P2X6 was also detected in LAD2 cells. Under whole-cell patch clamp conditions, rapid application of ATP (1-1000 mM) to cells clamped at -60 mV consistently evoked inward currents in both types of cells. Brief application of ATP (1 s) evoked a rapidly desensitizing P2X1-like current in both cell types. This current was also elicited by abmethylene ATP (10 mM, 94% cells, n = 31) and was antagonized in LAD2 cells by NF 449 (1 mM) and pyridoxal phosphate-6-azo(benzene-2,4-disulphonic acid) (1-10 mM). A P2X7-like nondesensitizing current in response to high concentrations of ATP (1-5 mM) was also seen in both cell types (96% LAD2, n = 24; 54% HLMCs, n = 24) which was antagonized by AZ11645373 (1 mM). P2X7-like responses were also evoked in LAD2 cells by 2′(3′)-0-(4-benzoylbenzoyl)ATP (300 mM). A P2X4-like current was evoked by 100 mM ATP (80% LAD2, n = 10; 21% HLMCs, n = 29), the amplitude and duration of which was potentiated by ivermectin (3 mM).

Purinergic signaling in inflammatory cells: P2 receptor expression, functional effects, and modulation of inflammatory responses

“…Human lung mast cells (HLMC) were reported to express mRNA transcripts for P2X1 and P2X4 but not for P2X2, P2X3, and P2X5-P2X7 [213] in a study that did not investigate the P2Y receptor family, while others found that of the 3 P2 receptor transcripts assessed in the study, P2Y 1 and P2Y 2 but not P2X7 were expressed [200]. In contrast to the absence of the P2X7 receptor in above mentioned studies, Wareham et al reported its presence in HLMC next to P2X1 and P2X4 [214] (Table 1). Also, the human mast cell line HMC-1 was shown to highly express the P2X7 receptor being further upregulated upon PMA (phorbol 12-myristate 13-acetate) or ionomycin stimulation [200], while Wareham et al also confirmed the presence of P2X7 receptors next to P2X1, P2X4, and P2X6 in the LAD2 human cell line [214].…”

“…In contrast to the absence of the P2X7 receptor in above mentioned studies, Wareham et al reported its presence in HLMC next to P2X1 and P2X4 [214] (Table 1). Also, the human mast cell line HMC-1 was shown to highly express the P2X7 receptor being further upregulated upon PMA (phorbol 12-myristate 13-acetate) or ionomycin stimulation [200], while Wareham et al also confirmed the presence of P2X7 receptors next to P2X1, P2X4, and P2X6 in the LAD2 human cell line [214]. Among the P2Y receptors, the presence of P2Y 1 , P2Y 2 , P2Y 11 , P2Y 12 , and P2Y 13 subtypes were shown by RT-PCR on cord blood-derived human mast cells (hMCs) [23].…”

“…The application of ATP is known to evoke inward currents in the mast cells [214], yielding an increase in intracellular Ca 2+ which is necessary for IgE-mediated and/or ATP-mediated/potentiated mediator release from mast cells [23,195,200,202,213,[217][218][219][220]. The first paper functionally linking P2 receptors to Ca 2+ -dependent mediator release by mast cells was the work from Schulman et al who reported that adenine nucleotides consistently enhanced anti-IgE-induced histamine release with a rank order of potency of ATP > 2mSATP > αβmATP > βγmATP [200].…”

P2X ion channel receptors and inflammation

“…P2X receptors have been implicated in the participation of the immune system in inflammatory pain [88,89]. P2X1, P2X4, P2X7, and perhaps P2X3 receptors are expressed by mast cells [90,91]. Mast cells were the cell type in which the properties of the P2X7 receptor were initially observed and characterized by Cockcroft and Gomperts [92].…”

The role of P2Y14 and other P2Y receptors in degranulation of human LAD2 mast cells

“…The roles of P2 receptors in mast cell biology have recently been reviewed [7]. The expression and some functions of human P2X receptors have recently been characterized using both LAD2 mast cells and human lung mast cells [8]. Nucleotides, especially adenosine 5′-diphosphate (ADP) and adenosine 5′-triphosphate (ATP), have been studied previously in the context of mast cell degranulation [9,10], but specific receptor subtypes mediating these functions have not been systematically explored.…”