The Pathways Of ATP-induced Intracellular Calcium Signal Transduction In The Rat Nociceptive Trigeminal Ganglion Neurons

Objective: Adenosine 5′-triphosphate (ATP), in addition to its function as an intracellular energy donor, is now recognized as an important neurotransmitter or cotransmitter in both the central and peripheral nervous systems, which may mediate a variety of function activities in the body. According to the previous study, ATP plays a prominent role in nociception when ATP increased or injected, which could cause facial and skin pain. However, the mechanism of ATP modulating pain is not clearly till now. Particularly, the origins of the cytoplasmic Ca2+ rise induced by ATP remain unknown in the trigeminal ganglion. In this experiment, we used Fura-2-based microfluorimetry techniques to measure [Ca2+]i and pharmacological tools to determine the nature of Ca2+ stores in order to clarify the intracellular calcium signal transduction pathway in nociceptive TG neurons.Methods: 1. Sprague-Dawley rats (80-120 g) were anesthetized with ether and rapidly decapitated, in accordance with the Animal Care and Use Committee of Anhui Medical University. The TGs were dissected out and transferred immediately into Dulbecco's modified Eagle's medium (DMEM) at pH 7.4. After the removal of the surrounding connective tissues, the TGs were minced with fine spring scissors and the ganglion fragments were placed in a flask containing 5 mL of DMEM supplemented with trypsin (0.5 mg/mL, type II-S) and collagenase (1.0 mg/mL, type I-A) and incubated at 37°C in a shaking water bath for 35 min. While incubating, TGs were triturated by fine fire-polished Pasteur pipettes three times (10 min each). After incubation, TG neurons were separated by centrifugation (1000 rpm, 5 min). Enzymatic digestion was stopped by addition of phosphate buffered saline with 10% normal cattle serum. Cell suspensions were then plated onto culture dishes and studied within 10 hrs.2. Calcium imaging: [Ca2+]i was measured using TILLvision Imaging System with a Ca2+-sensitive dye Fura-2 acetoxymethyl ester (Fura-2/AM). The Fura-2/AM was prepared as a stock solution and diluted with extracellular solution to the final concentration (2μmol/L) immediately before use. Isolated TG neurons were incubated for 30 min in a bath solution containing Fura-2/AM at 37°C, then washout Fura-2/AM with extracellular solution. The well Fura-2/AM-loaded small neurons were selected to do calcium image experiments. The Fura-2/AM fluorescence, which can go through cell membrane, was measured with a 10 Hz alternating wavelength time scanning with excitation wavelengths of 340 nm and 380 nm, and an emission wavelength of 510 nm. F340 and F380 representing the fluorescence intensity elicited by 340 nm and 380 nm excitation light, respectively. The results were expressed as the ratio of F340/F380 intensity to estimate the change in [Ca2+]i by TILL vision software. All experiments were performed at room temperature.3. Statistical analysis: Data were analyzed with Igor Pro software (Wavemetrics, Inc. Oregon, USA). Statistical data are presented as means±SEM. Comparisons between means were performed using Student's unpaired t-test. Differences were considered to be significant when p < 0.05.Results1.In small TG neurons, the application of thapsigargin (1μmol/L), caffeine (20 mmol/L) or ATP (100μmol/L) induced a transient [Ca2+]i increase under the condition of normal extracellular solution or Ca2+-free condition. Those results indicate that there exist both IP3- and ryanodine-sensitive Ca2+ stores in rat nociceptive TG neurons.2.In the extracellular Ca2+-free condition, the ATP-induced [Ca2+]i transient rise was reversibly inhibited by thapsigargin (n = 8,p < 0.001), but were not affected by caffeine in extracellular Ca2+-free condition (n = 6, p > 0.05). Those results indicate that ATP-induced [Ca2+]i rise in Ca2+-free conditions occurs is the calcium released from IP3-sensitive Ca2+ stores and not from ryanodine-sensitive Ca2+ stores.3.In the extracellular Ca2+-free condition, ATP-induced [Ca2+]i transient rise was reversibly inhibited by suramin (100μmol/ L, 60 s), an antagonist against P2 receptors, indicating that ATP induced the [Ca2+]i rise via acting on P2Y purinoreceptors. 4.In normal extracellular solution, ATP (100μM) could still induced a transient [Ca2+]i rise after pretreated with thapsigargin (1μM) which had induced [Ca2+]i rise to a high platform in small TG neurons. These results indicate that besides P2YRs, P2XRs activation may cause extracellular calcium to flow into intracellular and induce [Ca2+]i rise.ConclusionThere exist inositol-1,4,5-triphosphate (IP3) - and ryanodine-sensitive Ca2+ stores in rat nociceptive TG neurons. Two pathways are involved in the purinoreceptor-mediated [Ca2+]i rise in nociceptive TG neurons. One pathway involved the metabotropic P2Y receptors, associated with the IP3 Ca2+ sensitive store; the second being coupled to the ionotropic P2X receptors inducing Ca2+ influx.