Mechanisms of ATP-mediated vasodilation in humans: modest role for nitric oxide and vasodilating prostaglandins

Abstract

ATP is an endothelium-dependent vasodilator, and findings regarding the underlying signaling mechanisms are equivocal. We sought to determine the independent and interactive roles of nitric oxide (NO) and vasodilating prostaglandins (PGs) in ATP-mediated vasodilation in young, healthy humans and determine whether any potential role was dependent on ATP dose or the timing of inhibition. In protocol 1 (n = 18), a dose-response curve to intrabrachial infusion of ATP was performed before and after both single and combined inhibition of NO synthase [N(G)-monomethyl-L-arginine (L-NMMA)] and cyclooxygenase (ketorolac). Forearm blood flow (FBF) was measured via venous occlusion plethysmography and forearm vascular conductance (FVC) was calculated. In this protocol, neither individual nor combined NO/PG inhibition had any effect on the vasodilatory response (P = 0.22-0.99). In protocol 2 (n = 16), we determined whether any possible contribution of both NO and PGs to ATP vasodilation was greater at low vs. high doses of ATP and whether inhibition during steady-state infusion of the respective dose of ATP impacted the dilation. FBF in this protocol was measured via Doppler ultrasound. In protocol 2, infusion of low (n = 8)- and high-dose (n = 8) ATP for 5 min evoked a significant increase in FVC above baseline (low = 198 ± 24%; high = 706 ± 79%). Infusion of L-NMMA and ketorolac together reduced steady-state FVC during both low- and high-dose ATP (P < 0.05), and in a subsequent trial with continuous NO/PG blockade, the vasodilator response from baseline to 5 min of steady-state infusion was similarly reduced for both low (ΔFVC = -31 ± 11%)- and high-dose ATP (ΔFVC -25 ± 11%; P = 0.70 low vs. high dose). Collectively, our findings indicate a potential modest role for NO and PGs in the vasodilatory response to exogenous ATP in the human forearm that does not appear to be dose or timing dependent; however, this is dependent on the method for assessing forearm vascular responses. Importantly, the majority of ATP-mediated vasodilation is independent of these putative endothelium-dependent pathways in humans.

Fig. 1.

Experimental timeline. Baseline measurements were made for 2 min before each experimental trial. A: protocol 1: ATP was infused at 3 progressive doses [1.25, 2.5, and 5 μg·dl forearm volume (FAV)⁻¹·min⁻¹] for 4 min at each dose. Dose response (D/R) occurred in control condition (trial 1) and following either single (trial 2) or combined (trial 3) infusion of N^G-monomethyl-l-arginine [l-NMMA; to acutely inhibit nitric oxide synthase (NOS) and thus NO synthesis] or ketorolac [KET; to acutely inhibit cyclooxygenase (COX) and thus prostaglandin (PG) synthesis]. Maintenance doses of each drug occurred for the remainder of the experiment, and 15 min of rest separated each trial. Drug order was reversed in half of the subjects (n = 9 both groups). B: protocol 2: ATP was infused at either a low (0.64 μg·dl FAV⁻¹·min⁻¹) or high dose (4.6 μg·dl FAV⁻¹·min⁻¹) in 2 different subject groups (n = 8 both groups) for 10 min as a time control (trial 1), 10 min with combined inhibition of NOS and COX (via l-NMMA and KET) during ATP infusion for the last 5 min (trial 2), or 5 min with prior NOS and COX inhibition (trial 3). Maintenance doses of each drug occurred for the remainder of the experiment and 15 min of rest separated each trial.

Fig. 2.

Protocol 1: forearm vasodilatory responses to ATP. A: control, independent PG inhibition (ketorolac) and combined PG/NO inhibition (ketorolac + l-NMMA) vasodilatory responses to 3 doses of intrabrachial ATP infusion. B: control, independent NO inhibition (l-NMMA) and combined NO/PG inhibition (l-NMMA + ketorolac) vasodilatory responses to 3 doses of intrabrachial ATP infusion. No significant differences were observed across all conditions in either group.

Fig. 3.

Protocol 2: effect of combined NO/PG inhibition during ATP infusion. Absolute forearm vascular conductance is presented for both doses [low dose (n = 8; 0.64 μg·dl FAV⁻¹·min⁻¹) and high dose (n = 8; 4.6 μg·dl FAV⁻¹·min⁻¹)] of ATP during baseline and 5 and 10 min of infusion during trial 2. Combined inhibition of NO/PG occurred from min 5 to 10. *P < 0.05 vs. control vasodilation (minute 5).

Fig. 4.

Protocol 2: effect of combined NO/PG inhibition before ATP infusion. Vasodilatory responses from baseline to steady-state vasodilation (minute 5) in control conditions (trial 2) and with prior NO/PG inhibition (trial 3) for low and high dose ATP. *P < 0.05 vs. control vasodilator response (trial 2).