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1 Cell Biology & Anatomy, New York Medical College, 10595, New York, United States
2 Cell Biology and Anatomy, New York Medical College, Valhalla, New York, United States
* To whom correspondence should be addressed. E-mail: pravin_sehgal{at}nymc.edu.
We previously reported the disruption of caveolae/rafts, dysfunction of Golgi tethers, SNAREs and SNAPs, and inhibition of anterograde trafficking in endothelial cells in culture and in the rat lung when exposed to monocrotaline pyrrole (MCTP) as a prelude to the development of pulmonary hypertension. We have now investigated (a) whether this trafficking block affects eNOS subcellular localization and function, and (b) whether Golgi blockade and eNOS sequestration are observed after hypoxia and senescence. Immunofluorescence data revealed that MCTP-induced "megalocytosis" of pulmonary arterial endothelial cells (PAECs) was accompanied by a loss of eNOS from the plasma membrane with increased accumulation in the cytoplasm. This cytoplasmic eNOS was sequestered in heterogenous compartments which partially colocalized with Golgi and endoplasmic reticulum (ER) markers, caveolin-1, NOSTRIN and ER-tracker but not lysotracker. Hypoxia and senescence also produced enlarged PAECs with dysfunctional Golgi and loss of eNOS from the plasma membrane with sequestration in the cytoplasm. Live-cell imaging of caveolar and cytoplasmic NO using DAF-2DA as probe showed a marked loss of caveolar NO after MCTP, hypoxia and senescence. While ionomycin stimulated DAF-2DA fluorescence in control PAEC, this ionophore decreased that in MCTP-treated and senescent PAEC suggesting localization of eNOS in an aberrant cytoplasmic compartment which was readily discharged by Ca++-induced exocytosis. Thus monocrotaline, hypoxia and senescence all produce a Golgi blockade in PAEC leading to sequestration of eNOS away from its functional caveolar location providing a mechanism for the often-reported reduction in pulmonary arterial NO levels in experimental pulmonary hypertension despite sustained eNOS protein levels.
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