Extracellular Histones Activate Plasma Membrane Toll-Like Receptor 9 to Trigger Calcium Oscillations in Rat Pancreatic Acinar Tumor Cell AR4-2J

Abstract

In acute pancreatitis, histones are released by infiltrating neutrophils, but how histones modulate pancreatic acinar cell function has not been investigated. We have examined histone modulation of rat pancreatic acini and pancreatic acinar tumor cell AR4-2J by calcium imaging. Histones were found to have no effect on calcium in pancreatic acini but blocked calcium oscillations induced by cholecystokinin or acetylcholine. Both mixed (Hx) and individual (H1, H2A, H2B, H3, H4) histones induced calcium oscillations in AR4-2J. RT-PCR and Western blot verified the expression of histone-targeted Toll-like receptor (TLR) 2, 4 and 9. Immunocytochemistry identified TLR2/TLR4 on apical plasma membrane and TLR9 in zymogen granule regions in pancreatic acini. TLR2 was found on neighboring and TLR9 on peripheral plasma membranes, but TLR4 was in the nucleus in AR4-2J clusters. Neither TLR2 agonist zymosan-A nor TLR4 agonist lipopolysaccharide had any effect on calcium, but TLR9 agonist ODN1826 induced calcium oscillations; TLR9 antagonist ODN2088 blocked H4-induced calcium oscillations in AR4-2J, which also disappeared after treatment of AR4-2J with glucocorticoid dexamethasone, with concurrent TLR9 migration from plasma membrane to cell interiors. TLR9 down regulation with siRNA suppressed H4-induced calcium oscillations. These data together suggest that extracellular histones activate plasma membrane TLR9 to trigger calcium oscillations in AR4-2J cells.

Photodynamic Activation of Cholecystokinin 1 Receptor with Different Genetically Encoded Protein Photosensitizers and from Varied Subcellular Sites

“…AR4-2J was bought from The American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in DMEM/F12 supplemented with 20% fetal bovine serum in a CO 2 incubator under humidified atmosphere (5% CO 2 /95% air) at 37 °C, as reported previously [ 32 , 62 , 63 , 64 ].…”

“…Cytosolic calcium was measured in an inverted fluorescent microscope (Nikon TE-2000U) (Shanghai, China) coupled to a Photon Technology International (PTI Inc., now HORIBA, Edison, NJ, USA) calcium measurement system with alternating excitations at 340 nm/380 nm (DeltaRam X); emitted Fura-2 fluorescence (dichroic mirror 400DCLP, emitter 510 ± 40 nm) was detected with a charge-coupled device (CCD) camera (NEO-5.5-CL-3, Andor/Oxford Instruments, Belfast, Northern Ireland, UK). Calcium concentration was expressed as Fura-2 fluorescence ratios F 340 /F 380 and plotted against time with SigmaPlot (Palo Alto, CA, USA), as reported before [ 29 , 30 , 32 , 62 , 63 , 64 , 66 ]. In figures shown in Results, original colored calcium tracings were each from individual cells, all from 1 out of N (as indicated in figures, N ≥ 3) identical experiments.…”

NanoLuc Bioluminescence-Driven Photodynamic Activation of Cholecystokinin 1 Receptor with Genetically-Encoded Protein Photosensitizer MiniSOG

“…AR4-2J was bought from ATCC (Rockville, MD, USA) and cultured in DMEM/F12 supplemented with 20% fetal bovine serum in a CO 2 incubator under humidified atmosphere (5% CO 2 /95% air) at 37 • C, as reported before [1,2,28,33,52].…”

“…The rhodopsin-type or A class calcium-mobilizing G protein-coupled receptor (GPCR) cholecystokinin type 1 (CCK1 or CCK1R) [1,2] is widely expressed both in the central nervous system (CNS) [3][4][5][6] and in peripheral tissues [7][8][9][10][11][12]. CCK1R in the CNS regulates neurotransmitter release [13][14][15], anxiety [16,17], appetite [18], neurogenesis [19], brain development [6], learning, and memory formation [20].…”

“…Owing to the wide distribution and participation in important physiological functions, CCK1R and coupled signaling pathways are rather well characterized [27], including coupling to calcium signaling [1,2,28]. In contrast to reversible activation by agonists, one strikingly conspicuous feature of CCK1 receptor pharmacology is that CCK1R such as those present at the basal plasma membrane in rat pancreatic acini are permanently activated by the lowest lying excited state of molecular oxygen, the delta singlet oxygen ( 1 O 2 ) [29][30][31][32].…”