Increases in vanilloid TRPV1 receptor protein and CGRP content during endotoxemia in rats

doi:10.1016/j.ejphar.2007.03.032

European Journal of Pharmacology

Volume 566, Issues 1–3, 2 July 2007, Pages 145-152

https://doi.org/10.1016/j.ejphar.2007.03.032 Get rights and content

Abstract

The aim of the present study was to determine whether the transient receptor potential vanilloid (TRPV1) receptor protein as well as the calcitonin gene-related peptide (CGRP) content could be enhanced after the i.p. administration of 5 mg/kg lipopolysaccharide (LPS) to Sprague–Dawley rats. In tongue tissue, used as a representative model of TRPV1 receptors expression, there was a significant increase in the abundance of TRPV1 receptor protein 6 h after LPS administration. In mesenteric arteries, the density of the CGRP-positive nerves as well as the release of CGRP induced by 10 μM anandamide was also significantly increased 6 h after LPS administration. The relaxant responses induced by anandamide in mesenteric beds isolated from either untreated or LPS-treated rats were abolished after a 2 h exposure to 10 μM capsaicin. Moreover, anandamide-induced relaxations of untreated mesenteries were potentiated by the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 0.1 μM), but not by its inactive analogue 4α-phorbol (0.1 μM). The potentiation of anandamide effects caused by the PKC activator was accompanied by a significant increase in the overflow of CGRP induced by anandamide in the untreated rats. It is proposed that the overexpression of the TRPV1 receptors and the increased content of CGRP could contribute to the enhancement of anandamide effects during the endotoxemic shock. An eventual phosphorylation event linked to the overflow of CGRP could also participate in the enhanced relaxation caused by anandamide in endotoxemia.

Introduction

The induction of proinflammatory gene expression by bacterial lipopolysaccharide (LPS) plays a crucial role in triggering the cardiovascular collapse experienced by patients with systemic Gram negative bacterial infection, a major cause of morbidity and mortality (Karima et al., 1999). Increased circulating levels of anandamide, substance that has been proposed as a mediator in endotoxin-induced hypotension, were found under septic conditions (Varga et al., 1998, Kohro et al., 2004).

Enhancement of anandamide effects has been observed in several pathological situations, such as hypertensive states (Mendizabal et al., 2001, Li et al., 2003), cholestasis (Moezi et al., 2004) and cirrhosis (Domenicali et al., 2005). Potentiation of relaxant effects of anandamide in the mesenteric vasculature has also been reported for an early stage of septic shock, i.e. 6 h after intraperitoneal administration of 5 mg/kg of lipopolysaccharide (Orliac et al., 2003). Anandamide-induced relaxations were reported to be antagonized by the TRPV1 receptor antagonist capsazepine in mesenteric arteries isolated from either untreated (Mendizabal et al., 2001) or LPS-treated rats (Orliac et al., 2003). Relaxations to capsaicin were also enhanced in mesenteric beds of LPS-treated rats (Orliac et al., 2003).

The activation of transient receptor potential vanilloid 1 (TRPV1) receptors by anandamide has been proposed to have potential implications on inflammatory and cardiovascular disorders (Ross, 2003). Since vasorelaxant responses to anandamide in the rat vascular mesenteric bed are mainly mediated through the activation of TRPV1 receptors in perivascular sensory nerves and coupled to the release of calcitonine gene-related peptide (CGRP) (Zygmunt et al., 1999), the aim of the present work was to study whether TRPV1 receptor protein as well as CGRP content could be enhanced during septic shock. In addition, based on the evidence that sensitization of TRPV1 receptors is linked to an increased phosphorylation that results from the enhancement of protein kinase C (PKC) activity (Premkumar and Ahern, 2000), we also studied whether the direct activation of PKC through phorbol esters could potentiate the vasorelaxant effects of anandamide in the untreated rats.

Section snippets

Animal treatment

Male Sprague–Dawley rats weighting between 230–350 g were housed under 12:12-h light:dark cycle, at controlled room temperature with food and water ad libitum. Experiments were conducted in accordance with the Helsinki Declaration on research involving animals and human beings. Six hours prior to the beginning of the functional and biochemical studies, a single injection of LPS from Escherichia coli (5 mg/kg, i.p.) was administered in 0.25 ml saline/ 100 g body weight. The untreated rats

Changes in TRPV1 receptor protein levels

To test the possibility that changes in TRPV1 receptor protein expression could occur during endotoxemia, Western blot analysis of the TRPV1 receptor protein were performed in mesenteric bed as well as in tongue homogenates isolated from either untreated or LPS-treated rats. Fig. 1A shows a Western blot of mesenteric tissue where two bands of 120 kDa and 100 kDa of molecular weight were detected with the anti-TRPV1 antibody. These bands were obtained in a unique experiment carried out after

Discussion

The vanilloid TRPV1 receptor is a member of a superfamily of transient receptor potential ion channels that are gated by capsaicin, extracellular acidic pH and noxious heat (Caterina et al., 1997, Tominaga et al., 1998, Dedov and Roufogalis, 2000). The present observation that 6 h after i.p. administration of 5 mg/kg LPS to rats the abundance of vanilloid TRPV1 receptor protein as well as the CGRP content are increased, gives further support to the proposal that vanilloid TRPV1 receptors can

Acknowledgements

The technical assistance of Ms Fernanda De Fino and Ms Marina Galli is gratefully acknowledged.

Supported by grants FONCYT, PICT 5-14107 and CONICET, PIP 5695.

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^☆: This work is dedicated to the beloved memory of Maria Luz Orliac, who suddenly passed away on March 21, 2006.

^✠: Deceased.

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Increases in vanilloid TRPV1 receptor protein and CGRP content during endotoxemia in rats☆

Abstract

Introduction

Section snippets

Animal treatment

Changes in TRPV1 receptor protein levels

Discussion

Acknowledgements

Regul. Pept.

Eur. J. Pharmacol.

J. Biol. Chem.

Mol. Brain Res.

Mol. Med. Today

Cell Calcium

Eur. J. Pharmacol.

Eur. J. Pharmacol.

Eur. J. Pharmacol.

J. Pharmacol. Sci.

J. Biol. Chem.

Pharmacol. Ther.

Neuron

Brain Res. Mol. Brain Res.

Neuroscience

The capsaicin receptor: a heat-activated ion channel in the pain pathway

Nature

Mitochondrial calcium accumulation following activation of vanilloid (VR1) receptors by capsaicin in dorsal root ganglion neurons

Neuroscience

Increased anandamide induced relaxation in mesenteric arteries of cirrhotic rats: role of cannabinoid and vanilloid receptors

Gut

Increased iNOS activity is essential for intestinal epithelial tight junction dysfunction in endotoxemic mice

Shock