ATP hydrolysis pathways and their contributions to pial arteriolar dilation in rats

Abstract

ATP is thought to be released to the extracellular compartment by neurons and astrocytes during neural activation. We examined whether ATP exerts its effect of promoting pial arteriolar dilation (PAD) directly or upon conversion (via ecto-nucleotidase action) to AMP and adenosine. Blockade of extracellular direct ATP to AMP conversion, with ARL-67156, significantly reduced sciatic nerve stimulation-evoked PADs by 68%. We then monitored PADs during suffusions of ATP, ADP, AMP, and adenosine in the presence and absence of the following: 1) the ecto-5'-nucleotidase inhibitor α,β-methylene adenosine 5'-diphosphate (AOPCP), 2) the A(2) receptor blocker ZM 241385, 3) the ADP P2Y(1) receptor antagonist MRS 2179, and 4) ARL-67156. Vasodilations induced by 1 and 10 μM, but not 100 μM, ATP were markedly attenuated by ZM 241385, AOPCP, and ARL-67156. Substantial loss of reactivity to 100 μM ATP required coapplications of ZM 241385 and MRS 2179. Dilations induced by ADP were blocked by MRS 2179 but were not affected by either ZM 241385 or AOPCP. AMP-elicited dilation was partially inhibited by AOPCP and completely abolished by ZM 241385. Collectively, these and previous results indicate that extracellular ATP-derived adenosine and AMP, via A(2) receptors, play key roles in neural activation-evoked PAD. However, at high extracellular ATP levels, some conversion to ADP may occur and contribute to PAD through P2Y(1) activation.

Fig. 1.

Sciatic nerve stimulation (SNS)-related data. A: representative time course of changes in pial arteriolar diameters associated with a 20-s SNS (gray bar). Each curve represents the average response measured in 2 animals, first in the absence (initial; ●) and, subsequently, in the presence of the ecto-pyrophosphatase/diphosphohydrolase blocker ARL-67156 (100 μM; ○). B: effect of ARL-67156 (100 μM) on SNS-evoked pial arteriolar dilations. Values are means ± SD. *P < 0.05 vs. initial; n = 5. Representative SNS-generated somatosensory-evoked potentials (SEPs) recorded from the contralateral cortical surface before (C) and after (D) ARL-67156 application are also shown. E: note that no differences in the SEP peak-to-peak amplitudes were observed, irrespective of whether the P1N1 or the P2N2 amplitude was measured. F: SNS-evoked pial arteriolar diameter percent increases from baseline measured before a CO₂ challenge (Pco₂, ∼70 mmHg for 3 min) and at 30-min intervals over the 2 h following CO₂ exposure. This time control group displayed a remarkably consistent pial arteriolar response to SNS when comparing SNS-evoked pial arteriolar reactivities measured pre- and posthypercapnia. Results in E and F are means ± SD; n = 5.

Fig. 2.

Pial arteriolar responses to the suffusion of 1, 10, and 100 μM ATP in the absence and presence of the ecto-5′-nucleotidase inhibitor α,β-methylene adenosine 5′-diphosphate (AOPCP; 300 μM; A), the A₂ receptor blocker ZM 241385 (10 μM; B), the P2Y₁ receptor blocker MRS 2179 (10 μM) and MRS 2179 plus the A₂ antagonist ZM 241385 (10 μM; C), and the ecto-diphosphohydrolase blocker ARL-67156 (100 μM; E). In D, the dose-related responses to ADP, in the presence or absence of ZM 241385 and ZM 241385 plus MRS 2179, are also shown. Pial arteriolar responses are expressed as the percent change from the baseline diameter value. Values are means ± SD. *P < 0.05 vs. initial; †P < 0.05 vs. AOPCP; n = 5 per group.

Fig. 3.

Effect of AOPCP (A) or ZM 241385 (B) on pial arteriolar responses to suffusions of 1, 10, and 100 μM AMP. C: depiction of responses to suffusions of AMP in the absence and presence of adenosine deaminase (ADA; 2 units/ml), ADA + AOPCP (300 μM), and ADA + AOPCP + ZM 241385 (10 μM). Values are means ± SD. *P < 0.05 vs. initial; †P < 0.05 vs. AOPCP; ‡P < 0.05 vs. ADA + AOPCP + ZM 241385; n = 5 per group.

Fig. 4.

Effects of AOPCP (300 μM; A) or ZM 241385 (10 μM; B) on pial arteriolar responses induced by suffusions of adenosine (Ado) at 10 and 100 μM. Values are means ± SD. *P < 0.05 vs. initial and AOPCP; n = 5 per group.