Long-term hypoxia uncouples Ca²⁺and eNOS in bradykinin-mediated pulmonary arterial relaxation

Abstract

Bradykinin-induced activation of the pulmonary endothelium triggers a rise in intracellular Ca that activates nitric oxide (NO)-dependent vasorelaxation. Chronic hypoxia is commonly associated with increased pulmonary vascular tone, which can cause pulmonary hypertension in responsive individuals. In the present study, we tested the hypothesis that long-term high-altitude hypoxia (LTH) diminishes bradykinin-induced Ca signals and inhibits endothelial nitric oxide synthase (eNOS), prostacyclin (PGI), and large-conductance K (BK) channels in sheep, which are moderately responsive to LTH, resulting in decreased pulmonary arterial vasorelaxation. Pulmonary arteries were isolated from ewes kept near sea level (720 m) or at high altitude (3,801 m) for >100 days. Vessel force was measured with wire myography and endothelial intracellular Ca with confocal microscopy. eNOS was inhibited with 100 μM N-nitro-l-arginine methyl ester (l-NAME), PGI production was inhibited with 10 µM indomethacin that inhibits cyclooxygenase, and BK channels were blocked with 1 mM tetraethylammonium. Bradykinin-induced endothelial Ca signals increased following LTH, but bradykinin relaxation decreased. Furthermore, some vessels contracted in response to bradykinin after LTH. l-NAME sensitivity decreased, suggesting that eNOS dysfunction played a role in uncoupling Ca signals and bradykinin relaxation. The Ca ionophore A-23187 (10 µM) elicited an enhanced Ca response following LTH while relaxation was unchanged although l-NAME sensitivity increased. Additionally, BK function decreased during bradykinin relaxation following LTH. Western analysis showed that BK α-subunit expression was increased by LTH while that for the β subunit was unchanged. Overall, these results suggest that those even moderately responsive to LTH can have impaired endothelial function.

High Altitude Reduces NO-Dependent Myometrial Artery Vasodilator Response During Pregnancy

“…However, because the expression and activation of eNOS in myometrial vessels was measured under unstimulated conditions and the reduced maximal vasodilation to SNP seen at HA was not as marked as the decrease in ACh response, we cannot exclude the possibility that other mechanisms may also be contributing to the lack of ACh vasodilation at HA. Such mechanisms may include: a) attenuation of muscarinic acetylcholine receptor(s) expression or function, b) reduced Ca 2+dependent eNOS activation as has been shown in pulmonary arteries from chronicallyhypoxic sheep, 35 and c) the possibility that differences in eNOS activity might be present under stimulated conditions. Therefore, future studies assessing expression and function of muscarinic acetylcholine receptors, Ca 2+ -dependent eNOS activation and NO production under stimulated conditions are necessary to elucidate the mechanisms by which HA impairs ACh vasodilation.…”

Improved Workflow for Analysis of Vascular Myocyte Time-Series and Line-Scan Ca2+ Imaging Datasets

“…Data-driven and statistical-based screening techniques of spatial and temporal Ca 2+ signals based on LCPro-detected events were informative. LCPro performs well on rounded cells such as the rat pulmonary microvascular endothelial cell monolayers that were examined in Francis et al, 2012 [15], and in our more recent studies performed in recordings of pulmonary arterial endothelial cells in en face preparations [6,16]. Over the last decade, we have published extensively using manually curated Ca 2+ signaling datasets of vascular myocytes from LCPro.…”

“…We have examined highly deterministic G-protein receptor-coupled Ca 2+ signals and stochastic Ca 2+ events that occur in random locations and time intervals during the data recording. Examination of deterministic Ca 2+ signaling events due to agonist stimulation is relatively straightforward, as relevant data can be rapidly and easily identified based on the timing of cellular responses [5,6]. However, when performing quantitative analysis of stochastic events in complex tissues, our group estimates that we spend an order of magnitude more time performing image analysis than acquiring images at the microscope [7].…”

Piezo1 Channel Activation Reverses Pulmonary Artery Vasoconstriction in an Early Rat Model of Pulmonary Hypertension: The Role of Ca2+ Influx and Akt-eNOS Pathway

“…Several experimental models demonstrated the role of CH on eNOS regulation in vascular pulmonary beds from different species [ 35 ]. In addition, the eNOS expression was related to eNOS uncoupling during CH-induced PH [ 25 ].…”