Cooling augments vasoconstriction mediated by 5-HT1 and α2-adrenoceptors in the isolated equine digital vein: involvement of Rho kinase

https://doi.org/10.1016/j.ejphar.2007.04.057Get rights and content

Abstract

The vasculature of the equine digit fulfils an important role in thermoregulation. In other species, it has been found that cooling may enhance the response of cutaneous vessels to 5-hydroxytryptamine (5-HT) and α2-adrenoceptor agonists. Translocation of α2-adrenoceptors to the smooth muscle cell membrane, mediated by Rho kinase, is thought to be involved in the cooling-enhanced response in mouse tail arteries. However, little is known about the effect of cooling on 5-HT receptor function. The present investigation compared the response of 5-bromo-6-(2-imidazolin-2-ylamino) quinoxaline (UK14304:1 nM to 30 μM), methoxamine (0.1 nM to 30 μM; in the presence of yohimbine 0.1 μM), 5-carboxamidotryptamine (5-CT; 0.1 nM to 10 μM) and α-methyl 5-HT (0.1 nM to 10 μM) in the isolated equine digital vein at 30 °C and 22 °C. The effect of the Rho kinase inhibitor, fasudil (1 μM), and the recovery of the response after the irreversible blockade of surface receptors with phenoxybenzamine (10 μM) or 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ;10 μM), was established. Moderate cooling significantly increased the maximum response to α-methyl 5-HT, 5-CT and UK14304 and shifted their response curves to the left. Cooling also augmented the phenoxybenzamine- and EEDQ-resistant response to UK14304 and 5-CT, respectively. Fasudil had no effect on the contractile response at 30 °C, but completely abrogated the effect of cooling on the response to 5-CT and UK14304. The response to methoxamine was not significantly affected by cooling. These results suggest that Rho kinase plays an important role in the cooling-enhanced response mediated by 5-HT1B/D receptors and α2-adrenoceptors. The exact mechanism by which Rho/Rho kinase enhances the functional responses mediated by these receptors in these vessels has yet to be determined.

Introduction

The equine digit is thought to have an important thermoregulatory function, by virtue of the counter-current arrangement between lamellar arteries and veins in the hoof and also the presence of a high density of arteriovenous anastomoses in the coronary band and lamellar corium (Pollitt, 1992). The temperature of the skin in the distal portion of the horse limbs has been shown to be lower than the core body temperature (Palmer, 1983, Bailey et al., 2004c). As the temperature of the skin is a sensitive indicator of the cutaneous blood flow in several species, including the horse (Dyson et al., 2001, Hood et al., 2001, Wright et al., 2006), the equine digital vascular bed and digit blood flow may be proportionally affected by changes in the environmental temperature. Reducing the blood flow to the appendages helps to maintain the core temperature in a cool environment while minimising the metabolic demands on the animal (Hill et al., 2004).

The integrated physiological system that regulates the cutaneous blood flow in response to environmental temperature changes involves both the autonomic nervous system and the local effect of temperature on the physiology of the cutaneous vessels (Kellogg, 2006). Cooling depresses the contractile response evoked by direct stimulation (depolarization) of the vascular smooth muscle by high potassium chloride solutions (Flavahan et al., 1985, Tsukada and Chiba, 2000a). Paradoxically however, cooling augments the contractile response mediated by α2-adrenoceptors and 5-hydroxytrypatamine (5-HT) receptors in the canine saphenous and human digital veins, respectively (Flavahan and Vanhoutte, 1986, Bodelsson et al., 1990b). The cooling-enhanced response seems to be an evolutionary specialization of some cutaneous vessels, since deeper vessels in the limbs (canine femoral vein) or the tail (rat tail artery) can display a reduced response to norepinephrine during cooling (Flavahan and Vanhoutte, 1986, Roberts et al., 2002). Furthermore, the behaviour of some cutaneous vessels during cooling could differ between species, such is the case of rabbit saphenous vein where the response to norepinephrine and UK14304 (a selective α2-adrenoceptor agonist) was not affected by cooling (Harker and Vanhoutte, 1989).

The enhanced response of cutaneous vessels to α-adrenoceptors agonists during cooling has been suggested to involve an apparent increase in the affinity of the α2-adrenoceptors (Flavahan and Vanhoutte, 1986). Moreover, Chotani et al. (2000) reported a cooling-enhanced response in the mouse tail artery specifically mediated by the α2c-adrenoceptor subtype, which was ‘silent’ at warm temperature. In a non-vascular cell line, it was demonstrated that cooling promotes the translocation of α2c-adrenoceptor from the Golgi compartment to the extracellular membrane (Jeyaraj et al., 2001). The same results were confirmed later, in the small mouse tail artery and human vascular smooth muscle cells, reporting the involvement of the cooling-induced production of reactive oxygen species and of the Rho/Rho kinase pathway in this physiological mechanism (Bailey et al., 2004a, Bailey et al., 2005). The activation of Rho kinase promotes the calcium sensitization of the vascular smooth muscle by inactivating the myosin light chain (MLC) phosphatase and maintaining the MLC phosphorylation state (Somlyo and Somlyo, 2000). It has been recognized that the activation of Rho kinase is implicated in the translocation of other membrane related proteins (Hayashi et al., 2004). Recently, it was shown that cold-induced cutaneous vasoconstriction in vivo, was blocked by a selective inhibitor of Rho kinase, fasudil, confirming the participation of this pathway in the thermoregulatory response to cooling (Thompson-Torgerson et al., in press).

Beyond the physiological thermoregulatory role of the cutaneous digital circulation, haemodynamic alterations in digital blood flow may have pathological consequences in some species. Equine laminitis is an ischaemic condition of the equine digit that compromises the integrity of the anatomical relationship between the third phalanx and the hoof. It has been proposed that the development of an increase in the postcapillary resistance, due to venoconstriction, could be one of the pathophysiological mechanisms involved (Bailey et al., 2004b). This condition in the horse, has been compared with Raynaud's phenomenon in humans, a similar condition that is characterized by digital ischaemia-reperfusion injury, linked to a cooling-induced enhancement of α2-adrenoceptor-mediated vasoconstriction (Hood et al., 1990, Flavahan et al., 2003). Considering the superficial cutaneous location of the equine digital veins (Fig. 1) and their hypothetical role in the pathophysiology of equine laminitis, the aim of this study was to investigate the cooling-enhanced response mediated by 5-HT1 receptors and α2-adrenoceptors in the equine digital vein. The effect of fasudil, a selective antagonist of the Rho/Rho kinase pathway, was used to determine the role of this pathway in this thermoregulatory mechanism. Part of these results has been presented in abstract form elsewhere (Zerpa et al., 2006).

Section snippets

Animals and tissues

Equine digital veins were collected in a local abattoir. The hindlimbs of healthy mix breeds horses were removed within 10 min of death. The digital artery was cannulated at the level of the fetlock and 120 ml of ice-cold modified Krebs-Henseleit (Krebs solution) was infused through the catheter. The skin was then reflected from above the coronary band to reveal the coronary venous plexus. The coronary venous plexus was dissected and removed, placed in ice-cold Krebs solution and transported to

Effect of moderate cooling on the response of equine digital veins to agonists of 5-HT receptors and α-adrenoceptors

All vessels contracted in response to DKS 118 mM. The response to DKS 118 mM (n = 34) was similar at 30 °C and 22 °C (0.43 ± 0.03 g/mg of tension and 0.39 ± 0.03 g/mg of tension, respectively).

Discussion

Moderate cooling induced an increase in the first phase responses of the concentration response curve evoked by UK14304 and 5-CT in the equine digital veins. Our laboratory has previously described that these initial phases are mediated by the activation of α2-adrenoceptors and 5-HT1B/D receptors respectively (Elliott, 1997, Bailey and Elliott, 1998). The results presented here also show that fasudil (1 μM) selectively inhibited the augmented response mediated by these receptors at 22 °C, but

Acknowledgements

HZ was supported by CDCH-UCV and the Programme Alβan, the European Union Programme of Scholarships for Latin America, scholarship no. E05D054410VE. The authors are also grateful to Helen Woodcock for her technical support.

References (53)

  • M. Roberts et al.

    Adrenoceptor and local modulator control of cutaneous blood flow in thermal stress

    Comp. Biochem. Physiol., A

    (2002)
  • J.M. Van Nueten et al.

    Ketanserine and vascular contractions in response to cooling

    Eur. J. Pharmacol.

    (1984)
  • C.I. Wright et al.

    Non-invasive methods and stimuli for evaluating the skin's microcirculation

    J. Pharmacol. Toxicol. Methods.

    (2006)
  • D. Zhao et al.

    Non-adrenergic inhibition at prejunctional sites by agmatine of purinergic vasoconstriction in rabbit saphenous artery

    Neuropharmacology

    (2005)
  • S.R. Bailey et al.

    Rho kinase mediates cold-induced constriction of cutaneous arteries. Role of α2c-adrenoceptor translocation

    Circ. Res.

    (2004)
  • S.R. Bailey et al.

    The effects of vasoactive amines found in the equine hindgut on digital blood flow in the normal horse

    Equine Vet. J.

    (2004)
  • S.R. Bailey et al.

    Reactive oxygen species from smooth muscle mitochondria initiate cold-induced constriction of cutaneous arteries

    Am. J. Physiol., Heart Circ. Physiol.

    (2005)
  • M. Bodelsson et al.

    Cooling enhances α2-adrenoceptor mediated vasoconstriction in human hands veins

    Acta. Physiol. Scand.

    (1990)
  • M. Bodelsson et al.

    Effect of cooling on smooth muscle response to 5-hydroxytryptamine in human hand veins

    Acta. Physiol. Scand.

    (1990)
  • M.A. Chotani et al.

    Silent α2c-adrenergic receptors enable cold-induced vasoconstriction in cutaneous arteries

    Am. J. Physiol., Heart Circ. Physiol.

    (2000)
  • S. Davies et al.

    Specificity and mechanism of action of some commonly used protein kinase inhibitors

    Biochem. J.

    (2000)
  • S. Dyson et al.

    Factors influencing blood flow in the equine digit and their effect on uptake of 99 m technetium methylene diphosphonate into bone

    Equine Vet. J.

    (2001)
  • J. Elliott

    α-adrenoceptors in equine digital veins: evidence for the presence of both α1 and α2-receptors mediating vasoconstriction

    J. Vet. Pharmacol. Ther.

    (1997)
  • J. Elliott et al.

    Gastrointestinal derived factors are potential triggers for the development of acute equine laminitis

    J. Nutr.

    (2006)
  • J. Elliott et al.

    The role of nitric oxide in the responses of equine digital veins to vasodilator and vasoconstrictor agents

    Equine Vet. J.

    (1994)
  • N.A. Flavahan et al.

    Cooling and α1-and α2-adrenergic responses in cutaneous veins: role of receptor reserve

    Am. J. Physiol., Heart Circ. Physiol.

    (1985)
  • Cited by (10)

    View all citing articles on Scopus
    View full text