Elsevier

Vascular Pharmacology

Volume 47, Issues 5–6, November–December 2007, Pages 302-312
Vascular Pharmacology

Big ET-1 processing into vasoactive peptides in arteries and veins

https://doi.org/10.1016/j.vph.2007.08.006Get rights and content

Abstract

The endothelin (ET) peptides are more potent in contracting veins than arteries. The precursor big ET-1 is metabolized by endothelin converting enzyme [ECE; to ET-1 (1–21)], matrix metalloproteases [MMPs; to ET-1 (1–32)] and chymase [to ET-1(1–31)]. We hypothesized that arteries and veins were differently dependent in conversion of big ET-1 to vasoconstrictors. Immunohistochemical, western, zymographic and isometric contractile assays in rat aorta and vena cava were used. Big ET-1 contracted aorta [60 ± 17% phenylephrine contraction] but was more efficacious in vena cava [478 ± 61% norepinephrine contraction]. ECE and its product ET-1(1–21) were detected in aorta and vena cava, and the ECE inhibitors phosphoramidon and CGS-26393 reduced big ET-1-induced contraction. ET-1 (1–32) contracted aorta and vena cava but inhibition of MMPs with minocycline or GM6001 did not reduce big ET-1-induced contraction; zymography confirmed active tissue MMPs. Aorta and vena cava contracted to the product of chymase, ET-1 (1–31). Chymase was detected in aorta and only weakly in vena cava. Inhibition of chymase (chymostatin, 100 μM) reduced arterial (19% control) but not venous constriction to big ET-1. These results suggest at least one potential significant difference — the role of chymase — in in vitro enzymatic processing of big ET-1 in arteries and veins.

Introduction

Veins and arteries differ qualitatively and quantitatively in response to ET-1. ET-1 typically has a lower threshold of contraction, higher potency and greater relative efficacy in veins than arteries. Veins constrict faster to ET-1 than do arteries and veins do not desensitize to acute or chronic elevations in ET-1 as do arteries (Johnson et al., 2002, Thakali et al., 2004, Watts et al., 2002). These studies raise the idea that ET may be an important controller of endogenous venous tone. We have been interested in understanding the role of venomotor tone in control of blood pressure, specifically as it pertains to the ability of ET-1 to modify venous tone. The central hypothesis of this study is that veins and arteries differ in their enzymatic ability to metabolize big ET-1 and thus may be exposed to different profiles of vasoactive ET peptides.

Endothelin [ET-1 (1–21)] is one of the most potent vasoconstrictors to be isolated from blood vessels. Biosynthesis of this peptide begins with the actions of a furin-like protease on pro-ET-1 to form the peptide big ET-1 (1–38) (Gonzalez-Santiago et al., 2002, Kido et al., 1998b, Maguire et al., 1997, Schiffrin, 2001, Schweizer et al., 1997, Yanagisawa et al., 1988), with much of the proteolytic processing thought to take place in the vesicles of endothelial cells (Harrison et al., 1995). The most extensively studied processing pathway of big ET-1 is through the zinc-dependent endoprotease endothelin converting enzyme (ECE) to form ET-1 (1–21), commonly known as ET-1 (Harrison et al., 1995, Hisaki et al., 1991, Maguire et al., 1997, Schweizer et al., 1997, Struck et al., 2005). In recent years, other pathways for big ET-1 processing have been discovered.

Matrix metalloproteases (MMPs) are serine proteases that can contribute to alteration in the extracellular matrix of blood vessels. Two intensively studied MMPs in the vasculature are MMP-2 and MMP-9, also referred to as gelatinase A and B. Their protease function allows dissolution of matrix and remodeling. Big ET-1 is acted upon by MMP-2 to form ET-1 (1–32), a product in which the terminal 6 amino acids of big ET-1 has been cleaved. ET-1 (1–32) functions as a vasoconstrictor in rat mesenteric arteries, and a potential vasodilator in rat renal arteries via activation of ETB receptors. Thus, the endogenous metabolism of big ET-1 via MMP leads to the possibility of production of an additional vasoactive ET peptide (Fernandez-Patron et al., 1999, Fernandez-Patron et al., 2000, Jeyabalan et al., 2003, Matsumura et al., 1991, Merchant and Davidge, 2004, Nagase et al., 2006, Roques, 1998). Similarly, the enzyme chymase can metabolize big ET-1 to ET-1 (1–31) (Guo et al., 2001, Ju et al., 2001, Kido et al., 1998a, Kido et al., 1998b, Kirimura et al., 2005, Mazzocchi et al., 2000, Nagata et al., 2000, Wypij et al., 1992). ET-1 (1–31) has recently received attention as an endogenous agonist of the ETA receptors, stimulation of which has been almost exclusively associated with vasoconstriction (Fecteau et al., 2005, Maguire and Davenport, 2004, Maguire et al., 1997, Maguire et al., 2001, Mazzocchi et al., 2000, Nagata et al., 2000).

We used an integrative set of techniques that allowed us to ask the questions of whether these three separate processing enzymes — ECE, MMPs and chymase—existed, whether the products of these 3 pathways produced substances that could influence smooth muscle tone in arteries and veins, and whether the pathways participated in the contraction of isolated vessels to big ET-1. Our model of vein and artery, the isolated thoracic vena cava and thoracic aorta, allowed us to carry out pharmacological, biochemical and immunohistochemical studies in concert.

Section snippets

Isometric contraction protocols

All animal procedures were approved by the Institutional Animal Care University Committee of Michigan State University. Male Sprague Dawley rats (Charles River) were deeply anesthetized with pentobarbital (50 mg kg− 1, i.p.) to the point of a loss of eyelid reflex and lack of withdrawal from painful stimuli. Thoracic aorta and vena cava were placed in physiologic salt solution consisting of (in mM) NaCl, 130; KCl, 4.7; KH2PO4, 1.18; MgSO4–7H2O, 1.17; CaCl2–2H2O, 1.6; NaHCO3, 14.9; dextrose,

ECE and big ET-1 in aorta and vena cava

The smooth muscle in the aorta is significantly more abundant than in the vena cava as demonstrated by the significantly darker and homogenous pink staining using a Modified Trichrome stain (Fig. 1A), consistent with the greater magnitude of force generated in the aorta observed to agonists (see keys in Fig. 1). Collagen staining (blue) was omitted from the Trichrome stain so the pink of the smooth muscle layer was more readily observed. The smooth muscle cell layer of the vena cava appears to

Discussion

This work was undertaken to understand how big ET-1 could be processed in an isolated artery and vein to form vasoactive peptide(s). ET-1(1–21), the product of ECE, was one of the peptides considered but other peptides were also investigated. Our main finding was a significant difference in the apparent expression and function of the enzyme chymase in arteries when compared to veins, with arteries possessing higher chymase function.

Since its discovery in 1988, ET-1 (1–21) has received intense

Limitations

There are several limitations to this study that need to be recognized. First, we have used large arteries and veins because of their ease of use in biochemical experiments, and thus this work may not necessarily apply to small arteries and veins. Our laboratories have, however, observed a similar relative response of small mesenteric veins and arteries with respect to ET-1 and adrenergic stimuli, where the ET peptides are amongst the most potent and efficacious venoconstrictors we have

Conclusions

  • These results suggest an overall similarity in the ability or large arteries and veins to convert big ET-1 into vasoactive peptides.

  • ET-1 (1–21) and likely ECE are present and functional in both arteries and veins.

  • The machinery for converting big ET-1 into two relatively understudied peptides — ET-1 (1–31) and ET-1 (1–32) — is present in both arteries and veins.

  • Arteries but not veins have the ability to utilize chymase as an ET processing enzyme.

  • Thus, there are differences in the in vitro

Acknowledgement

This study is supported by NIH PO1HL70687.

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