Pharmacological Profile of the Purinergic P2Y Receptors That Modulate, in Response to ADPβS, the Vasodepressor Sensory CGRPergic Outflow in Pithed Rats

Abstract

Calcitonin gene-related peptide (CGRP), an endogenous neuropeptide released from perivascular sensory nerves, exerts a powerful vasodilatation. Interestingly, adenosine triphosphate (ATP) stimulates the release of CGRP by activation of prejunctional P2X2/3 receptors, and adenosine 5′-O-2-thiodiphosphate (ADPβS), a stable adenosine diphosphate (ADP) analogue, produces vasodilator/vasodepressor responses by endothelial P2Y1 receptors. Since the role of ADP in the prejunctional modulation of the vasodepressor sensory CGRPergic drive and the receptors involved remain unknown, this study investigated whether ADPβS inhibits this CGRPergic drive. Accordingly, 132 male Wistar rats were pithed and subsequently divided into two sets. In set 1, ADPβS (5.6 and 10 µg/kg·min) inhibited the vasodepressor CGRPergic responses by electrical stimulation of the spinal T9–T12 segment. This inhibition by ADPβS (5.6 µg/kg·min) was reverted after i.v. administration of the purinergic antagonists MRS2500 (300 µg/kg; P2Y1) or MRS2211 (3000 µg/kg; P2Y13), but not by PSB0739 (300 µg/kg; P2Y12), MRS2211 (1000 µg/kg; P2Y13) or the KATP blocker glibenclamide (20 mg/kg). In set 2, ADPβS (5.6 µg/kg·min) failed to modify the vasodepressor responses to exogenous α-CGRP. These results suggest that ADPβS inhibits CGRP release in perivascular sensory nerves. This inhibition, apparently unrelated to activation of ATP-sensitive K+ channels, involves P2Y1 and probably P2Y13, but not P2Y12 receptors.

Pharmacological Nature of the Purinergic P2Y Receptor Subtypes That Participate in the Blood Pressure Changes Produced by ADPβS in Rats

“…The fact that the dose-dependent vasodepressor responses caused by ADPβS, but not by the vehicle, were immediate in the four groups of rats (Figure 2) suggests the possible role of peripheral (mainly endothelial) mechanisms [20] rather than direct central mechanisms. This suggestion is consistent with: (i) our results demonstrating that the vasodepressor responses to ADPβS were also elicited in pithed rats (without or with a methoxamine infusion; Figure 2), in which the CNS is not operative [30,37,38]; and (ii) the hydrosolubility of ADPβS (and its stability, so it is unlikely to be converted to adenosine, unlike ADP), which implies that it does not readily penetrate the blood-brain barrier. It is noteworthy that these ADPβS-induced vasodepressor responses were highly reproducible in all four groups, as they remained unchanged (p > 0.05) when the D-R curves were repeated two more times (Figure 2).…”

“…Clearly, none of these treatments produced a significant effect (p > 0.05) on DBP compared with that of: (i) the control animals (p > 0.05); or (ii) its respective baseline value (before any treatment; not presented for greater clarity), but the baseline values in each group are shown above in Section 2.1). It is to be noted that the baseline values of heart rate have previously been reported to remain without significant effects (p > 0.05) after administration of these compounds in pithed rats [21,30]. For simplicity, we decided not to show these heart rate data.…”

“…In an attempt to gain in-depth insight into the pharmacological characteristics of the receptors that participate in these responses (with selective antagonists at P2Y 1 , P2Y 12 , and P2Y 13 receptors), in the present study a wide range of ADPβS doses (i.e., 0.3, 0.56, 1, 1.8, 3.1, 5.6, and 10 µg/kg, i.v.) was used (see Section 4.3 of methods), in: (i) anaesthetised rats without (Group 1) and with bilateral vagotomy (Group 2) to explore the potential role of vagal and other baroreflex compensatory mechanisms of central origin, triggered by blood pressure changes [17,[34][35][36], as the CNS is intact; and (ii) pithed rats without (Group 3) and with a methoxamine infusion (Group 4) for systemic vascular tone restoration to confirm peripheral mechanisms, as the CNS is not functional [30,37,38].…”

“…Interestingly, in Group 4, where peripheral vascular resistance was restored at the postjunctional level by using pithed rats continuously infused with the α 1 -adrenergic agonist methoxamine [30,37,38], the profile of the ADPβS responses was different; namely, the vasodepressor responses to ADPβS were followed by secondary vasopressor responses consisting of predominant increases in systolic blood pressure. This latter finding: (i) leads us to infer that ADPβS may also increase cardiac contractility, as systolic blood pressure is considered an index cardiac (left ventricle) contractility [1,28,29]; and (ii) may have triggered baroreflex compensatory mechanisms of central origin in anaesthetised rats, which could have counteracted the increases in systolic blood pressure [1,[3][4][5][6].…”

“…The doses selected for these antagonists in the present study: (i) have previously been justified in terms of their binding/affinity values and other properties by our group [21,30]; and (ii) have been demonstrated to cause a complete blockade of their respective receptor subtypes that elicit cardiovascular responses in pithed rats [21,30].…”