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Biochim Biophys Acta. 2016 Jul;1861(7):671-9. doi: 10.1016/j.bbalip.2016.04.014. Epub 2016 Apr 19.

Two functionally distinct pools of eNOS in endothelium are facilitated by myoendothelial junction lipid composition.

Author information

1
Department of Molecular Physiology and Biophysics, University of Virginia, USA; Robert M. Berne Cardiovascular Research Center, University of Virginia, USA.
2
Department of Pharmacology, University of Virginia, USA.
3
Department of Pharmacology, University of Virginia, USA; School of Biotechnology and Biomolecular Sciences, University of New South Wales, Australia.
4
Department of Pharmacology and Chemical Biology, University of Pittsburgh, USA; Heart, Lung, Blood and Vascular Medicine Institute; University of Pittsburgh, USA.
5
Department of Molecular Physiology and Biophysics, University of Virginia, USA; Robert M. Berne Cardiovascular Research Center, University of Virginia, USA. Electronic address: brant@virginia.edu.

Abstract

In resistance arteries, endothelial cells (EC) make contact with smooth muscle cells (SMC), forming myoendothelial junctions (MEJ). Endothelial nitric oxide synthase (eNOS) is present in the luminal side of the EC (apical EC) and the basal side of the EC (MEJ). To test if these eNOS pools acted in sync or separately, we co-cultured ECs and SMCs, then stimulated SMCs with phenylephrine (PE). Adrenergic activation causes inositol [1,4,5] triphosphate (IP3) to move from SMC to EC through gap junctions at the MEJ. PE increases MEJ eNOS phosphorylation (eNOS-P) at S1177, but not in EC. Conversely, we used bradykinin (BK) to increase EC calcium; this increased EC eNOS-P but did not affect MEJ eNOS-P. Inhibiting gap junctions abrogated the MEJ eNOS-P after PE, but had no effect on BK eNOS-P. Differential lipid composition between apical EC and MEJ may account for the compartmentalized eNOS-P response. Indeed, DAG and phosphatidylserine are both enriched in MEJ. These lipids are cofactors for PKC activity, which was significantly increased at the MEJ after PE. Because PKC activity also relies on endoplasmic reticulum (ER) calcium release, we used thapsigargin and xestospongin C, BAPTA, and PKC inhibitors, which caused significant decreases in MEJ eNOS-P after PE. Functionally, BK inhibited leukocyte adhesion and PE caused an increase in SMC cGMP. We hypothesize that local lipid composition of the MEJ primes PKC and eNOS-P for stimulation by PE, allowing for compartmentalized function of eNOS in the blood vessel wall.

KEYWORDS:

Diacylglycerol; Endoplasmic reticulum; Endothelial cell; Microcirculation; Myoendothelial junction; Nitric oxide synthase; Protein kinase C

PMID:
27106139
PMCID:
PMC4869716
[Available on 2017-07-01]
DOI:
10.1016/j.bbalip.2016.04.014
[Indexed for MEDLINE]

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