Spreading dilatation to luminal perfusion of ATP and UTP in rat isolated small mesenteric arteries

Abstract

Levels of ATP achieved within the lumen of vessels suggest a key autacoid role. P2Y receptors on the endothelium may represent the target for ATP, leading to hyperpolarization and associated relaxation of vascular smooth muscle through the endothelium-dependent hyperpolarizing factor (EDHF) pathway. EDHF signals radially from the endothelium to cause dilatation, and appears mechanistically distinct from the axial spread of dilatation, which we showed occurs independently of a change in endothelial cell Ca2+ in rat mesenteric arteries. Here we have investigated the potential of P2Y receptor stimulation to evoke spreading dilatation in rat resistance small arteries under physiological pressure and flow. Triple cannulation of isolated arteries enables focal application of purine and pyrimidine nucleotides to the endothelium, avoiding potential complicating actions of these agents on the smooth muscle. Nucleotides were locally infused through one branch of a bifurcation, causing near maximal local dilatation attributable to EDHF. Dilatation then spread rapidly into the adjacent feed artery and upstream against the direction of luminal flow, sufficient to increase flow into the feed artery. The rate of decay of this spreading dilatation was identical between nucleotides, and matched that to ACh, which acts only on the endothelium. In contrast, focal abluminal application of either ATP or UTP at the downstream end of cannulated arteries evoked constriction, which only in the case of ATP was also associated with modest spread of dilatation. The non-hydrolysable ADP analogue, ADPbetaS, acting at P2Y1 receptors, caused robust local and spreading dilatation responses whether applied to the luminal or abluminal surface of pressurized arteries. Dilatation to nucleotides was sensitive to inhibition with apamin and TRAM-34, selective blockers of small- and intermediate-conductance Ca2+-activated K+ channels, respectively. These data demonstrate that direct luminal stimulation of P2Y receptor on the endothelium of rat mesenteric arteries leads to marked spreading dilatation and thus suggests that circulating purines and pyrimidines may act as important regulators of blood flow.

Figure 1. Spreading dilatation responses to luminal perfusion of nucleotides in triple-cannulated arteries

Series 1 experiments. A, using a third pipette, one branch at an arterial bifurcation (Branch 1) was cannulated, through which perfusate containing agonists and carboxyfluorescein was infused. A typical fluorescence micrograph during the infusion period of an agonist shows the path of perfusate infusion. Note that the wall of the feed artery is visible due to autofluorescence. Bar = 500 μm. B, simultaneous traces of arterial dilatation (upper panel) and relative fluorescence (F/F_max in Branch 1, lower panel) in response to infusion of 3 μm ATP and 0.1 μm carboxyfluorescein into Branch 1. The small boxes in A (bottom panel) indicate the positions of fluorescence measurement, and relate to the positions at which diameter was measured from the simultaneous brightfield images, 2000 μm being the furthest upstream. The bar indicates the periods of infusion; l-NAME present in all experiments. See Supplemental Fig. 1 for animation of period between arrowheads.

Figure 2. Summary of dilatation responses to luminal perfusion of agonists in triple-cannulated arteries

Series 1 experiments. A, the dilatation at 0 μm in the feed artery (see Fig. 1A) was matched for ATP (1 or 3 μm, n= 6), ADPβS (1 or 3 μm, n= 6), UTP (3 or 10 μm, n= 6) and ADP (1 or 3 μm, n= 6), and responses simultaneously observed in Branch 1 and at upstream sites along the feed artery (0–2000 μm). B, the dilatation to 100 μm adenosine (n= 5) and 1 μm acetylcholine (ACh, n= 7) is shown for comparison to ATP (same data as A). To show relative rates of decay of spreading dilatation, the time points at which the dilatation response to 1 μm ACh reached 80% maximum dilatation at the 0 μm position is also shown. The bottom panels in A and B show the relative fluorescence at corresponding sites of diameter measurement (see Fig. 1), and indicate that in all experiments the solution infused into Branch 1 did not diffuse to the upstream sites in the feed artery. l-NAME present in all experiments.

Figure 5. Contribution of NO to spreading dilatation responses in triple-cannulated arteries

Series 1 experiments. Data from Fig. 2 in the presence of l-NAME are compared to separate experiments performed in the absence of l-NAME (No LN, A) and endothelial cell damage in the feed artery and Branch 2 (EC Damage, B). Responses were simultaneously observed in Branch 1 and at upstream sites along the feed artery (0–2000 μm). A, In the absence of l-NAME, the dilatation at 0 μm in the feed artery (see Fig. 1A) was matched for ATP (1 or 3 μm, n= 5), and time points were the dilatation response to 1 μm ACh reached 80% maximum dilatation (n= 4). B, following selective endothelial cell damage in the feed artery, and in the presence of l-NAME, the dilatation in Branch 1 was matched as closely as possible for ATP (3 μm, n= 4) and ACh (1 μm, n= 4). Note the constriction to ATP at 0 μm. The bottom panels in A and B show the relative fluorescence at corresponding sites of diameter measurement (see Fig. 1), and indicate that in all experiments the solution infused into Branch 1 did not diffuse to the upstream sites in the feed artery.

Figure 3. Effect of spreading dilatation on feed artery flow in triple-cannulated arteries

Series 2 experiments. A, simultaneous traces of diameter (upper panel) and flow rate (lower panel) in response to infusion of 1 μm ACh and 0.1 μm carboxyfluorescein at 2 μl min⁻¹ into Branch 1. Diameter was simultaneously measured at positions 0–2000 μm upstream from the bifurcation. The bar indicates the period of infusion. B, summary of 7 paired diameter (upper panel) and flow (lower panel) responses to luminal infusion of 1 μm ACh into Branch 1. Upon switching on the syringe pump, after a short delay, a rise in fluorescence intensity was observed in Branch 1, but not at any position in the feed artery (not shown). In addition to the raw values for flow, data are expressed as a percentage of the maximum diameter or flow, and should be compared to Supplemental Fig. 2B. l-NAME present in all experiments.

Figure 4. Effect of inhibitors of K⁺ channels on dilatation to luminal perfusion of nucleotides in unbranched arteries

Series 3 experiments. Agonist responses were fully inhibited by the combination of TRAM-34 and apamin, indicating an EDHF-type response. A, concentration-dependent responses to ATP (n= 3–14), and B, comparison of responses to 1 μm ATP (n= 3–14), 1 μm ADPβS (n= 3–12) and 3 μm UTP (n= 3–12). C, effect of MRS2179 on responses to luminal perfusion of purinoceptor agonists. MRS2179 (1 μm) fully inhibited the response to 1 μm ATP (n= 3) and 1 μm ADPβS (n= 7), but had no effect on the dilatation to 3 μm UTP (n= 3) or 1 μm acetylcholine (ACh, n= 6). l-NAME present in all experiments, except that in A one set of experiments was performed in the absence of l-NAME (No LN). *Significantly different from control.

Figure 6. Spreading dilatation response to abluminal application of nucleotides in unbranched arteries

Series 4 experiments. A, using a pipette positioned at the downstream end of the artery, agonists were pressure-pulse ejected as bolus doses of agonist (local response, 0 μm), and spreading dilatation responses observed upstream from the direction of superfusion flow (500–2000 μm). Representative responses to ATP (B, 1 mm, 100 ms), ADPβS (C, 1 mm, 30 ms), and UTP (D, 1 mm, 300 ms). l-NAME present in all experiments.

Figure 7. Summary of spreading dilatation responses to abluminal application of agonists in unbranched arteries

Series 4 experiments. The biphasic nature of responses to purinoceptor agonists is shown by peak dilatation (continuous lines) and constriction (dotted lines) responses to each agonist. ATP (1 mm, 30–300 ms, n= 4), ADPβS (1 mm, 30 ms, n= 4), UTP (1 mm, 30–1000 ms, n= 4) and acetylcholine (ACh, 1 mm, 80–180 ms, n= 6). l-NAME present in all experiments.

Figure 8. Effect of TRAM-34 and apamin on spreading dilatation to abluminal application of nucleotides in unbranched arteries

Series 4 experiments. The biphasic nature of responses to agonists is shown by peak dilatation (continuous lines) and constriction (dotted lines) responses to each agonist. A, ATP (1 mm, 100 ms, n= 3), and B, ADPβS (1 mm, 10–30 ms, n= 3). l-NAME present in all experiments.