Co-expression and modulation of neuronal and endothelial nitric oxide synthase in human endothelial cells

doi:10.1016/j.yjmcc.2004.07.006

Journal of Molecular and Cellular Cardiology

Volume 37, Issue 5, November 2004, Pages 939-945

https://doi.org/10.1016/j.yjmcc.2004.07.006 Get rights and content

Abstract

Despite originally identified in neurones, the neuronal type of nitric oxide synthase (nNOS) is present also in cardiac and skeletal myocytes. Whether nNOS is functionally expressed in human endothelial cells—as the endothelial enzyme (eNOS)—is unknown. Human umbilical vein endothelial cells (HUVEC) were studied under control culture conditions and after 48 h treatment with cytomix (human tumour necrosis factor-α, interferon-γ and E. coli endotoxin). We tested: (i) localisation and expression of nNOS and eNOS proteins by immunostaining and immunoblotting; (ii) activity of nNOS and eNOS by measuring l-arginine to l-citrulline conversion with 1-(2-trifluoromethylphenyl)imidazole (TRIM), a specific nNOS antagonist, in sub-cellular fractions; (iii) intracellular cGMP levels, as a marker for nitric oxide production, after TRIM pre-treatment, by radioimmunoassay. nNOS protein was expressed in the cytosolic fraction and immunolocalised in cultured HUVEC, and co-localised with the eNOS protein in frozen sections of the human umbilical cord. nNOS protein contributed to total l-citrulline production as TRIM selectively and dose-dependently reduced l-citrulline synthesis in the cytosolic but not particulate fraction of HUVEC. Similarly, TRIM reduced intracellular cGMP content both at baseline and after stimulation with a calcium ionophore. Cytomix down-regulated the expression and function of both nNOS and eNOS while no inducible NOS (iNOS) was detected. In conclusion, a functional neuronal type of NOS is co-expressed with the endothelial NOS type in HUVEC, suggesting a possible role for nNOS in regulation of blood flow.

Introduction

Nitric oxide synthase (NOS) is the key enzyme in the synthesis of nitric oxide (NO), a short-lived radical with important biological activities. Up-to-date, three structurally distinct isoforms of NOS have been identified and cloned: the neuronal (nNOS), the inducible (iNOS) and the endothelial (eNOS) one [1], [2], [3]. The three NOS isoforms were first identified in the brain, macrophages and endothelium, but later have been found widely distributed in other cell types [4]. The nNOS and eNOS are expressed constitutively under physiological conditions whilst iNOS may be induced only in pathological conditions, e.g. inflammation [4].

Human endothelial cells, whose products importantly contribute to the regulation of vascular tone and blood homeostasis, clearly express eNOS. Controversial data are available on iNOS induction while scanty data are available on nNOS in human endothelial cells [5], [6], [7], [8]. Recently, nNOS has been found also in non-neuronal cells such as cardiac and skeletal myocytes of rabbits and humans, these findings being limited to positive nNOS immunoreactivity in humans [9], [10]. Hence, the aims of this study were to test whether: (i) nNOS is present in the human endothelium; (ii) the endothelial nNOS is functional; (iii) the endothelial nNOS is susceptible to modulation by inflammatory agents.

Section snippets

Cell culture and treatment

Human umbilical vein endothelial cells (HUVEC) were isolated from umbilical cords according to the method described by Jaffe et al. [11], re-suspended in a culture medium (M199-RPMI 1640 1:1 containing 20% pooled human serum, glutammine 2 mmol/l, Hepes 10 mmol/l, and 1% penicillin-streptomycin) and seeded on gelatine-coated culture wells. HUVEC showed the typical cobblestone morphology and reacted positive to von Willebrand factor (vWF).

For each experiment, pooled cells derived from two to five

Nitric oxide synthases immunostaining

All cells from cytology HUVEC samples were positive for vWF (Fig. 1a). The whole cytoplasm showed either a diffuse or a granular staining pattern. The vast majority of HUVEC displayed a positivity for eNOS at the level of the cell membrane and a finely granular positivity in the cytoplasm (Fig. 1b). A large proportion of HUVEC was immunoreactive for nNOS. The positivity appeared in the cytoplasm, mostly in the perinuclear area, in the form of fine granules (Fig. 1c).

The immunostaining for

Discussion

Expression of nNOS protein has been found in endothelial cells of the bovine brain pial arteries but not of the parenchymal vessels [18]. Loesch and Burnstock [19] confirmed this finding in endothelial cells of the rat cerebral basilar artery. Moreover, nNOS was also detected in skeletal muscle capillaries of the hamster [20]. In humans, nNOS immunoreactivity was found in endothelial cells of small testicular arteries and in capillary endothelial cells of the lung [7], [8].

The results from our

Acknowledgements

We would like to thank the staff of the Delivery Room and the Transfusion Centre of the Spedali Civili of Brescia for providing human umbilical cords and serum. We also thank Professor Pier Giovanni Grigolato for von Willebrand factor immunostaining and Dr. Alessandro Bettini for editing the text. This study was supported by a grant from MURST (ex 40%) entitled “Relationship among cytokines, apoptosis and hypertrophy in ventricular and vascular remodelling: experimental and clinical aspects”.

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Original ArticleCo-expression and modulation of neuronal and endothelial nitric oxide synthase in human endothelial cells

Abstract

Introduction

Section snippets

Cell culture and treatment

Nitric oxide synthases immunostaining

Discussion

Acknowledgements

Adv. Pharmacol.

Adv. Pharmacol.

Am. J. Hypertens.

Respir. Physiol.

Biochem. Biophys. Res. Commun.

Neurosci. Res.

Brain Res.

Lancet

Enzymatic function of nitric oxide synthases

Cardiovasc. Res.

Nitric oxide synthases: which, where, how, and why?

J. Clin. Invest.

Regulation of nitric oxide synthesis by proinflammatory cytokines in human umbilical vein endothelial cells

J. Clin. Invest.

Evidence for production and functional activity of nitric oxide in seminiferous tubules and blood vessels of the human testis

J. Clin. Endocrinol. Metab.

Nitric oxide synthase in cardiac sarcoplasmic reticulum

Proc. Natl. Acad. Sci. USA

Culture of human endothelial cells derived from umbilical veins

J. Clin. Invest.

Original Article
Co-expression and modulation of neuronal and endothelial nitric oxide synthase in human endothelial cells