Cardiovascular pharmacology
Extracellular ATP signaling in equine digital blood vessels

https://doi.org/10.1016/j.ejphar.2013.01.018Get rights and content

Abstract

The functional distribution of ATP-activated P2 receptors is well characterized for many blood vessels, but not in the equine digital vasculature, which is a superficial vascular bed that displays thermoregulatory functions and has been implicated in ischemia-reperfusion injuries of the hoof. Isolated equine digital arteries (EDA) and veins (EDV) were submitted to isometric tension studies, whereby electric field stimulation (EFS) and concentration–response curves to exogenously applied agonists were constructed under low tone conditions. Additionally, immunofluorescent localization of P2X and P2Y receptor subtypes was performed. EFS-induced constriction was abolished by tetrodotoxin (1 μM, n=4). Endothelium denudation did not modify the EFS-induced constriction (n=3). The EFS-induced constriction in EDA was inhibited by phentolamine (67.7±1.8%, n=6; 10 μM), and by the non-selective P2 receptor antagonist suramin (46.2±1.3%, n=6; 10 μM). EFS-induced constriction in EDV was reduced by suramin (48.2±2.4%, n=6; 10 μM), the P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (58.3±4.5%, n=6; 10 μM), and phentolamine (23.2±2.5%, n=6; 10 μM). Exogenous methoxamine and ATP mimicked EFS-induced constriction in EDA and EDV. Immunostaining for P2X1, P2X2 and P2X3, and, for P2X1 and P2X7 receptor subunits were observed in EDA and EDV smooth muscle and adventitia, respectively. ATP and noradrenaline are co-transmitters in sympathetic nerves supplying the equine digital vasculature, noradrenaline being the dominant agonist in EDA, and ATP in EDV. In conclusion, P2X receptors mediate vasoconstriction in EDA and EDV, although different P2X subunits are involved in these vessels. The physiological significance of this finding in relation to thermoregulatory functions and equine laminitis is discussed.

Introduction

Adenosine 5′-triphosphate (ATP) contributes to the maintenance of vascular tone. It is released from perivascular nerves as a cotransmitter with noradrenaline and from endothelial cells in response to changes in blood flow (shear stress) and hypoxia (Ralevic, 2009). The P2X receptor family consists of seven ion channel subunits (P2X1–7) that can combine to form either trimeric homomers (P2X1–7) or heteromers (P2X1/2, 2/3, 2/6, 1/4, 1/5, 4/6, 4/7), all of which are ligand-gated ion channels. There are eight G protein-coupled P2Y receptor subtypes (P2Y1, 2, 4, 6, 11–14), which may also form heteromultimeric complexes (Burnstock, 2007).

The P2X1 receptor subtype is the principal P2 receptor expressed on vascular smooth muscle (Bo and Burnstock, 1993, Hansen et al., 1999, Wallace et al., 2006). P2X2 receptor subunits are also expressed on smooth muscle cells of rat mesenteric, renal and pulmonary arteries (Hansen et al., 1999). P2X4 receptor subunits are expressed in rat aorta, vena cava, coronary, pulmonary, renal and femoral arteries (Soto et al., 1996, Nori et al., 1998). It is generally accepted that P2X receptors play a role in the maintenance of vascular tone, and depending on their location and expression, can mediate both vasoconstriction (e.g. P2X1), and/or vasodilation (e.g. P2X2 and/or P2X4) (Burnstock, 2010).

P2Y receptors are involved in mediating vasodilatation (Wallace et al., 2006). P2Y1, 2, 4, 6 receptors have been shown to mediate endothelium-dependent vasodilatation (Knight et al., 2003). Interestingly, ATP-mediated vasodilatation (in the rat mesenteric bed) has been shown to be mediated via endothelial nitric oxide or endothelium-derived hyperpolarizing factor (Stanford et al., 2001). That the application of uridine 5′-triphosphate (UTP) elicits smooth muscle constriction suggests that P2Y2, 4 and/or P2Y6 are also players mediating vasoconstriction in some vessels (Erlinge and Burnstock, 2008, Gitterman and Evans, 2001).

The contribution of ATP, when released from perivascular nerves as a co-transmitter with noradrenaline, varies greatly between vascular bed, vessel type and species (Knight et al., 2003, Wallace et al., 2006, Erlinge and Burnstock, 2008). For example, the rabbit mesenteric artery is predominantly purinergic, whereas the rabbit pulmonary artery is mainly noradrenergic (Knight et al., 2003). A change in the ratio of ATP to noradrenaline release from sympathetic nerves has also been linked to animal models of hypertension and in the postsynaptic vascular response to temperature changes (Pelleg and Burnstock, 1990, Kluess et al., 2005, Ralevic, 2009).

The anatomical arrangement of the equine digital vasculature bed is complex and is involved in thermoregulatory function (Zerpa et al., 2010). Furthermore, it has been proposed that increased post-capillary resistance, due to venoconstriction, could be one of the pathophysiological mechanisms involved in the development of laminitis in horses (Moore et al., 2004). The aim of this study was to describe the extracellular ATP signaling in equine digital vessels.

Section snippets

Animals and tissues

Equine digital arteries (EDA) and veins (EDV) were collected in a local abattoir. The hind limbs of healthy mixed breed adult horses of either gender were removed within 10 min of death. The digital artery was cannulated and 120 ml of ice-cold modified Krebs Henseleit (Krebs solution) was infused through the catheter. The skin was then reflected from above the coronet band to reveal the digital coronet venous plexus and the plantar digital artery. The digital coronet venous plexus and the distal

Frequency–response curves from equine digital arteries and veins.

EFS of autonomic nerves in rings of EDA and EDV caused frequency-dependent constriction, with peak responses obtained at 32 Hz. The EFS-induced constriction was abolished by pre-treatment with TTX (1 μM) in EDA (96.8±0.5% inhibition, n=4, P<0.05, Fig. 1A), and in EDV (97.8±0.9% inhibition, n=4, P<0.05, Fig. 1C). The presence of the endothelium did not modify the EFS-induced constriction in either EDA or EDV (Fig. 1B and D, respectively).

EFS, in the presence of the non-selective α-adrenoceptor

Discussion

The effect of P2 receptor antagonists on EFS-mediated constriction and the contractile response to natural and synthetic P2 receptor agonists in conjunction with the immunoreactivity for P2X receptors in the smooth muscle of EDA and EDV demonstrate the presence of P2X receptors on vascular smooth muscle mediating constriction of EDA and EDV. Furthermore, and not surprisingly given that ATP and noradrenaline are cotransmitters in sympathetic nerves, we also report dual control of both EDA and

Acknowledgments

Acknowledgments go to Dr Yoel Berhane for his assistance and advice with organ bath experiments.

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