Local regulation of [(3)H]-noradrenaline release from the isolated guinea-pig right atrium by P(2X)-receptors located on axon terminals

Abstract

In this study the regulation of cardiac sympathetic outflow by presynaptic P(2X) receptor-gated ion channels was examined. ATP (30 microM - 1 mM) and other P2-receptor agonists elicited [(3)H]-noradrenaline ([(3)H]-NA) outflow from the isolated guinea-pig right atrium with the potency order of ATP>2-methyl-thioATP>alpha,beta-methylene-ATP=ADP, whereas ss, gamma-methylene-L-ATP was inactive. Ca(2+)-free conditions abolished both electrical field stimulation (EFS)- and ATP-evoked release of tritium. Unlike from EFS-induced outflow, ATP-induced [(3)H]-NA outflow was not reduced by omega-Conotoxin-GVIA (100 nM), Cd(2+) (100 microM) and tetrodotoxin (1 microM). The rapid extracellular decomposition of ATP was revealed by HPLC analysis. However, the effect of ATP to promote [(3)H]-NA release was not prevented by 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 250 nM), 3, 7-dimethyl-1-propargylxanthine (DMPX, 250 nM), or by reactive blue 2 (RB2, 10 microM), antagonists of A(1)-, A(2)- and inhibitory P(2) receptors. Zn(2+) (50 microM), the P(2X)-receptor modulator potentiated, and P(2X) receptor antagonists, i.e. suramin (300 microM), pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 30 microM) and 2'-o-(trinitrophenyl)-adenosine 5'-triphosphate (TNP-ATP, 30 microM) antagonized the ATP (1 mM)-evoked response. RT - PCR study revealed the expression of P(2X2) and P(2X3) receptor mRNAs in guinea-pig superior cervical ganglion. PPADS (30 microM) significantly reduced the EFS-induced [(3)H]-NA outflow in the presence DPCPX (250 nM) and RB2 (10 microM). In summary a P(2X)-type purinoceptor regulates noradrenaline release from the isolated right atrium of the guinea-pig. The pharmacological profile of the receptor resemble to homo-oligomeric P(2X3) or hetero-oligomeric P(2X2)/P(2X3) complexes, and provide a new target to intervene on sympathetic neuroeffector transmission at the presynaptic site.

Figure 1

Electrical field stimulation- and ATP-induced release of [³H]-NA from the isolated guinea-pig right atrium. Tissues were superfused with Krebs' solution for 60 min and then subjected to electrical field stimulations (a,b) S₁, S₂, 25 V cm⁻¹, 2 Hz, 2.5 ms, 240 shocks) or to ATP (1 mM) application (b) as indicated. The tritium content in the perfusate samples was measured by liquid scintillation spectrometry and was expressed in fractional release (%, for calculation, see Methods), as a function of time. Data represent the means±s.e.mean of 6–8 identical experiments.

Figure 2

Effect of Ca²⁺ free medium, voltage dependent Ca²⁺ channel antagonists and tetrodotoxin on the electrical field stimulation-evoked and ATP-induced outflow of [³H]-NA in the isolated guinea-pig right atrium. The preparations were superfused and subjected to electrical field stimulation (S₁, 25 V cm⁻¹, 2 Hz, 2.5 ms, 240 shocks) or ATP (1 mM) application, according to the experimental protocol shown in Figure 1b. Ca²⁺ free solution (Ca²⁺ free), supplemented with 1 mM EGTA was applied from 60 min before the start of sample collection, and thereafter, ω-conotoxin GVIA (CGTX, 0.1 μM), Cd²⁺ (Cd²⁺, 100 μM) and tetrodotoxin (TTX, 1 μM) was perfused from 10 min before the start of the sample collection period, and thereafter. The net tritium release evoked by electrical field stimulation (EFS, open bars) or ATP (ATP, solid bars) were calculated by the area-under-the-curve method and expressed in fractional release (%). Data show the means±s.e.mean of 6–8 identical experiments. Asterisks indicate significant differences from respective controls, calculated by ANOVA followed by Dunnett test (^**P>0.01). n.d. not detectable.

Figure 3

Extracellular decomposition of ATP in the isolated guinea-pig right atrium. Preparations were incubated in 3 ml Krebs' solution in the presence of 60 nmol ATP. Aliquots (70 μl) were taken 2.5, 5, 10, 20, 25, and 30 min after the addition of ATP. The amount of nucleotides (ATP, ADP, AMP) and nucleosides (ADO, adenosine, INO, inosine) in the aliquots were determined by HPLC-UV method and expressed in μM. Data show the means±s.e.mean of three identical experiments.

Figure 4

Effect of different antagonists and Zn²⁺ on ATP-induced outflow of [³H]-NA. Isolated guinea-pig right atrium preparations were superfused and subjected to ATP (1 mM) application, according to the experimental protocol shown in Figure 1b. DPCPX (250 nM), DMPX (250 nM), reactive blue 2 (RB2, 10 μM), Zn²⁺ (50 μM) and PPADS (30 μM) were perfused 10 min before ATP application and thereafter. The net tritium release evoked by ATP was calculated by the area-under-the-curve method and expressed in fractional release (%). Data show the means±s.e.mean of 6–8 identical experiments. ^**P<0.01 indicates significant differences between ATP-evoked tritium outflow in the absence and presence of drugs or Zn²⁺, calculated by ANOVA followed by Dunnett test.

Figure 5

Concentration-response relationship of [³H]-NA outflow evoked by different P_2X receptor agonists. Isolated guinea-pig right atrium preparations were superfused and subjected to ATP (triangles), ADP (diamonds), 2-methyl-thioATP (thioATP, squares), α,β-methylene-ATP (α,β-mATP, circles) or β,γ-methylene-L-ATP (β,γ-L-mATP, crosses) application in different concentrations indicated on the abscissa, ranging from 30 μM to 1 mM according to the experimental protocol shown in Figure 1b. The net tritium release evoked by different agonists were calculated by the area-under-the-curve method and expressed in fractional release (%). Data show the means±s.e.mean of 6–8 identical experiments.

Figure 6

Effect of P_2X receptor antagonists on [³H]-NA outflow evoked by ATP. Concentration-response curves for ATP were made in the absence (triangles) or presence of PPADS (30 μM, squares), suramin (300 μM, circles) and TNP-ATP (30 μM, diamonds). The net tritium release evoked by different concentrations of ATP ranging from 30 μM to 1 mM were calculated by the area-under-the-curve method and expressed in fractional release (%). Data show the means±s.e.mean of 4–8 identical experiments.

Figure 7

RT–PCR analysis of P_2X2 and P_2X3 receptor subtype expression in the guinea-pig SCG and hippocampus. Total RNA samples from the SCG and hippocampus were reverse transcribed and amplified by PCR using primers specific to P_2X2 and P_2X3 transcripts. Amplification of β-actin was used as internal control. A 100 bp DNA ladder (Fermentas, Vilnius, Lithuania) was used to identify PCR fragment sizes. The gels shown are representative of at least three independent experiments.