Two functionally distinct pools of eNOS in endothelium are facilitated by myoendothelial junction lipid composition.
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.
Department of Molecular Physiology and Biophysics, University of Virginia, USA; Robert M. Berne Cardiovascular Research Center, University of Virginia, USA.
Lauren A
LA
Biwer
Department of Pharmacology, University of Virginia, USA.
Evan P
EP
Taddeo
Department of Pharmacology, University of Virginia, USA.
Brandon M
BM
Kenwood
Department of Pharmacology, University of Virginia, USA; School of Biotechnology and Biomolecular Sciences, University of New South Wales, Australia.
Kyle L
KL
Hoehn
Department of Pharmacology and Chemical Biology, University of Pittsburgh, USA; Heart, Lung, Blood and Vascular Medicine Institute; University of Pittsburgh, USA.
Adam C
AC
Straub
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.
Brant E
BE
Isakson
D001692:Biological Transport; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002450:Cell Communication / Q000187:drug effects; D018920:Coculture Techniques; D006152:Cyclic GMP / Q000378:metabolism; D004075:Diglycerides / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D017629:Gap Junctions / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate; D047028:Macrocyclic Compounds / Q000494:pharmacology; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D052250:Nitric Oxide Synthase Type III / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D010080:Oxazoles / Q000494:pharmacology; D010656:Phenylephrine / Q000494:pharmacology; D010718:Phosphatidylserines / Q000378:metabolism; D010766:Phosphorylation; D061251:Primary Cell Culture; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology
D004075:Diglycerides; D047028:Macrocyclic Compounds; D010080:Oxazoles; D010718:Phosphatidylserines; D047428:Protein Kinase Inhibitors; C511704:xestospongin C; D010656:Phenylephrine; D004533:Egtazic Acid; D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D011493:Protein Kinase C; D006152:Cyclic GMP; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D001920:Bradykinin; D002118:Calcium
Diacylglycerol; Endoplasmic reticulum; Endothelial cell; Microcirculation; Myoendothelial junction; Nitric oxide synthase; Protein kinase C