Calcium Influx Induced by Stimulation of ATP Receptors on Neurons Cultured from Rat Dorsal Root Ganglia

1. The electrophysiological actions of the P2-purinoceptor agonists, adenosine 5'-triphosphate (ATP), 2-methylthio ATP (2-meSATP) and alpha, beta-methyleneATP (alpha, beta-meATP) and of uridine 5'-triphosphate (UTP) were studied under concentration and voltage-clamp conditions in dissociated neurones of 1-6 day old rat dorsal root ganglia. 2. ATP (10 nM-100 microM) applied rapidly via a U-tube perfusion system (equilibration time < 10 ms) activated concentration-dependent inward currents with a latency to onset of a few ms, an EC50 of 719 nM and a Hill slope of 1.47. 3. 2-meSATP (10 nM- 100 microM) and alpha, beta-meATP (100 nM - 100 microM) also evoked transient inward currents. The EC50 and Hill slopes were 450 nM and 1.58 for 2-meSATP and 1.95 microM and 1.53 for alpha, beta-meATP respectively. There was no significant difference between the maximum currents evoked by the three agonists. 4. As the concentration of ATP increased so the rate of rise and decay of the currents also increased. At 100 and 300 nM ATP the decay of the current was best fitted by a single exponential, but at 1 microM and above two exponentials were required. Log-log plots of the rise time or time constants of decay versus concentration were linear. Currents evoked by 2-meSATP and alpha, beta-meATP showed a similar concentration-dependence in their kinetics. 5. Inward currents evoked by ATP, 2-meSATP and alpha, beta-meATP (300 nM) were abolished by the P2-purinoceptor antagonist, suramin (100 microM). 6. UTP (10 microM) evoked similar transient inward currents, which were sensitive to suramin (100 microM). ATP (10 microM), applied 2 min beforehand, reduced the response to UTP (10 microM) by 80 +/- 10%. 7. This study shows that ATP, 2-meSATP and alpha, beta-meATP act via a suramin-sensitive P2x-purinoceptor to evoke rapid, transient inward currents in dissociated neurones of rat dorsal root ganglia. The pyrimidine nucleotide, UTP, was also active. It is likely that the agonists were acting at the P2x3-subtype to produce these effects.

Section: Discussioncontrasting

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Characterization of a P_2X‐purinoceptor in cultured neurones of the rat dorsal root ganglia

Robertson

Rae

Rowan

et al. 1996

“…A relatively high permeability of ATP‐activated cation channels to Ca 2+ was found in rat pheochromocytoma cells (PC12) (Nakazawa et al , 1990), nucleus tractus solitarii neurons (Ueno et al , 1992) and human embryonic kidney (HEK 293) cells with exogenously transfected P2X receptors under the voltage‐clamp condition (Evans et al , 1996). Fura‐2 fluorescence recording also showed that ATP causes a rise in intracellular Ca 2+ ([Ca 2+ ] i ) associated with catecholamine release in PC12 cells (Nakazawa & Inoue, 1992) and [Ca 2+ ] i influx into rat cultured DRG neurons through a P2X receptor channel complex (Bouvier et al , 1991). There is increasing evidence that Ca 2+ influx through the P2X receptor plays an important role in the modulation of this receptor type (Burnstock & Wood, 1996; Khiroug et al , 1997).…”

Section: Introductionmentioning

confidence: 99%

Characterization of Ca²⁺ influx through recombinant P2X receptor in C6BU‐1 cells

Ueno

Koizumi

Inoue

1998

1 The e ects of exogenous adenosine 5'-triphosphate (ATP) and a,b-methylene ATP (a,bmeATP) on C6BU-1 cells transfected with P2X 2 and P2X 3 subtypes, separately or together (P2X 2+3 ), were investigated using fura-2¯uorescence recording and whole-cell patch clamp recording methods. 2 Untransfected C6BU-1 cells showed no intracellular Ca 2+ ([Ca 2+ ] i ) increase in response to depolarizing stimulation with high K + or stimulation with ATP. There was no current induced by ATP under voltage clamp conditions in untransfected C6BU-1 cells. ATP caused Ca 2+ in¯ux only from extracellular sources in C6BU-1 cells transfected with the P2X subtypes, suggesting that the C6BU-1 cell line is suitable for the characterization of Ca 2+ in¯ux through the P2X subtypes. 3 In C6BU-1 cells transfected with the P2X 2 subtype, ATP (more than 10 mM) but not a,bmeATP (up to 100 mM) evoked a rise in [Ca 2+ ] i . 4 In the cells transfected with the P2X 3 subtype, current responses under voltage clamp conditions were observed at ATP concentrations higher than 0.1 mM of a,bmeATP were required. This discrepancy in the concentration dependence of the agonist responses with respect to the [Ca 2+ ] i rise and the current response was seen only with the P2X 3 subtype. In addition, the agonist-induced rise in [Ca 2+ ] i was observed only after the ®rst application because of desensitization of this subtype. 5 In C6BU-1 cells co-transfected with P2X 2 and P2X 3 , ATP at 1 mM evoked a [Ca 2+ ] i rise. This responsiveness was higher than that of the other subtype combinations tested. The e ciency of expression was improved by co-transfection with P2X 2 and P2X 3 , when compared to transfection with the P2X 3 subtype alone. The desensitization of the P2X 2+3 was apparently slower than that of the P2X 3 subtype alone. Therefore, this combination could respond to the repeated application of agonists each time with a [Ca 2+ ] i rise. 6 These results suggest that the P2X 2 and P2X 3 subtypes assemble a heteromultimer and that this heterogeneous expression acquires more e ective Ca 2+ dynamics than that by homogenously expressed P2X 2 or P2X 3

“…The cell body diameters ranged between 10 and 25 μm and ∼90% of the cells had two or more, short‐ or medium‐sized processes. We then examined their responsiveness to bath applications of capsaicin which acts selectively on sensory neurones (Bevan & Szolcsanyi, 1990) and α,β‐methylene adenosine‐triphosphate, an agonist at purinergic receptors (Bouvier et al ., 1991).…”

Section: Resultsmentioning

confidence: 99%

Analysis of GABA_A‐ and GABA_B‐receptor mediated effects on intracellular Ca²⁺ in DRG hybrid neurones

Yokogawa

Kim

Krieger

et al. 2001