Alejandro D. Miguel-Martínez

Background/Objectives: Adenosine 5′-O-(β-thio)-diphosphate (ADPβS) is a stable analogue of adenosine diphosphate (ADP). In pithed rats, ADPβS produces (i) inhibition of both the cardioaccelerator sympathetic tone (via prejunctional P2Y₁₂/P2Y₁₃ receptors) and the vasodepressor sensory CGRPergic tone (via prejunctional P2Y₁ receptors) and (ii) vasodepressor/vasodilator responses via endothelial P2Y₁ receptors, and vasopressor/vasoconstrictor responses via P2Y₁/P2Y₁₂/P2Y₁₃ receptors. This study pharmacologically investigates whether P2Y₁, P2Y₁₂, and/or P2Y₁₃ receptors modulate the vasopressor sympathetic tone using ADPβS. Methods: A total of 126 male Wistar rats were pithed and classified into two groups to produce vasopressor responses by (i) spinal (T₇–T₉) electrical stimulation of the sympathetic vasopressor tone (0.03–3 Hz; n = 102) and (ii) intravenous (i.v.) boluses of noradrenaline (0.03–3 μg/kg; n = 24). Results: Continuous i.v. infusions of ADPβS (5.6 and 10 μg/kg·min) significantly inhibited the vasopressor responses to sympathetic electrical stimulation, without affecting those induced by exogenous noradrenaline. This sympathoinhibition by ADPβS (5.6 μg/kg·min) remained unchanged after vehicle (1 mL/kg, i.v.) or the antagonists PSB 0739 (P2Y_12; 300 μg/kg, i.v.) and MRS 2211 (P2Y_13; 1000 and 3000 μg/kg, i.v.). Conversely, it was abolished after MRS 2500 (P2Y_1; 300 μg/kg, i.v.) or glibenclamide (K_ATP channel blocker; 20 mg/kg, i.v.). Conclusions: ADPβS inhibits the vasopressor sympathetic tone via prejunctional P2Y₁ receptors through K_ATP channel-dependent mechanisms.

Vasoinhibin is a potent antiangiogenic protein that blocks the activation of endothelial nitric oxide synthase (eNOS) in response to vascular endothelial growth factor, bradykinin, and acetylcholine (ACh). In this regard, VIAN‐c4551, a new synthetic vasoinhibin analog, has therapeutic potential for targeting angiogenesis in oncology and ophthalmology. Given that cardiovascular actions are common complications of antiangiogenic drugs and eNOS inhibitors, this multidisciplinary study investigated the cardiovascular safety of VIAN‐c4551. Administered acutely, VIAN‐c4551 inhibited ACh‐induced eNOS phosphorylation/activation in cultured endothelial cells and in lung tissue treated in vivo, as well as the ACh‐induced relaxation of rat aortic segments. However, daily intravenous (i.v.) injections of VIAN‐c4551 (1 or 3 mg kg ⁻¹ ) for 5 days failed to significantly modify the vasodepressor responses to ACh and the baseline values of blood pressure in anesthetized rats (intact, vagotomized, or pithed). Furthermore, daily i.v. injections of 1 mg kg ⁻¹ VIAN‐c4551 (for 5 days) did not alter: (i) blood pressure or heart rate values in awake rats; (ii) cardiac autonomic and histological outcomes in anesthetized animals; or (iii) inflammatory (tumor necrosis factor‐α [TNF‐α] and interleukin‐6 [IL‐6]) and apoptotic (caspase‐3) markers. Although VIAN‐c4551 inhibited ACh‐induced eNOS activation in vitro and in vivo and vasorelaxation in ex vivo assays, in vivo experiments consistently showed no significant cardiovascular effects produced by this synthetic peptide. Thus, VIAN‐c4551 appears to be cardiovascularly safe for targeting angiogenesis‐related diseases.

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 P2X_2/3 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 K_ATP 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.