-Bradykinin-induced activation of the pulmonary endothelium triggers nitric oxide production and other signals that cause vasorelaxation, including stimulation of largeconductance Ca 2ϩ -activated K ϩ (BKCa) channels in myocytes that hyperpolarize the plasma membrane and decrease intracellular Ca 2ϩ . Intrauterine chronic hypoxia (CH) may reduce vasorelaxation in the fetal-to-newborn transition and contribute to pulmonary hypertension of the newborn. Thus we examined the effects of maturation and CH on the role of BKCa channels during bradykinin-induced vasorelaxation by examining endothelial Ca 2ϩ signals, wire myography, and Western immunoblots on pulmonary arteries isolated from near-term fetal (ϳ140 days gestation) and newborn, 10-to 20-day-old, sheep that lived in normoxia at 700 m or in CH at high altitude (3,801 m) for Ͼ100 days. CH enhanced bradykinin-induced relaxation of fetal vessels but decreased relaxation in newborns. Endothelial Ca 2ϩ responses decreased with maturation but increased with CH. Bradykinin-dependent relaxation was sensitive to 100 M nitro-L-arginine methyl ester or 10 M 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, supporting roles for endothelial nitric oxide synthase and soluble guanylate cyclase activation. Indomethacin blocked relaxation in CH vessels, suggesting upregulation of PLA2 pathways. BKCa channel inhibition with 1 mM tetraethylammonium reduced bradykinin-induced vasorelaxation in the normoxic newborn and fetal CH vessels. Maturation reduced whole cell BKCa channel ␣1-subunit expression but increased ␤1-subunit expression. These results suggest that CH amplifies the contribution of BKCa channels to bradykinin-induced vasorelaxation in fetal sheep but stunts further development of this vasodilatory pathway in newborns. This involves complex changes in multiple components of the bradykinin-signaling axes. potassium channels; sheep; pulmonary artery; contractility; maturation; hypoxia REGULATION OF SMOOTH MUSCLE tone in pulmonary arteries during development is a delicate balance of vasoconstrictive and vasorelaxant pathways. Endothelial cells play a crucial role in determining the overall level of vasorelaxation (39, 67), and endothelium-dependent relaxation is partially mediated through bradykinin stimulation (31). Bradykinin is a potent vasodilator that is important in the fetal pulmonary circulation, as well as during inflammation, and its relationship to pulmonary hypertension has been explored (5, 31, 83).Endothelial bradykinin receptor activation induces vasorelaxation through modulation of several different intracellular signaling pathways that are largely dependent on a rise of endothelial intracellular Ca 2ϩ ([Ca 2ϩ ] i ) (67). The most widely studied pathway is bradykinin-induced activation of endothelial nitric oxide (NO) synthase (eNOS), an enzyme that generates NO (64). NO acts on nearby smooth muscle cells to cause downstream stimulation of soluble guanylate cyclase (sGC) pathways that leads to vasorelaxation (3,45). Previous studies have shown that regulatio...

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.

Endothelial activation is critical to pulmonary vasorelaxation during the fetal transition. Bradykinin (BK) relaxes vessels through endothelial‐induced nitric oxide (NO) and prostacyclin (PGI2) dependent signaling pathways. Intrauterine long term hypoxia (LTH) can stunt vasorelaxation in the fetal transition and cause pulmonary hypertension of the newborn. We therefore tested the hypothesis that LTH impairs maturation of BK‐mediated vasorelaxation by performing wire‐myography on pulmonary arteries (PA) isolated from fetal, newborn, or adult sheep that lived at low (720 m) or at high altitude (3,801 m) for >;100 days. Blocking eNOS‐dependent NO production with 100 μM LNAME and cGMP generation by soluble guanylate cyclase with 10 μM ODQ reduced 1 μM BK vasorelaxation in fetal and newborn but not adult vessels, effects largely independent of altitude. Inhibition of PGI2 production with 10 μM indomethacin also decreased BK‐vasorelaxation in fetal hypoxic and newborn normoxic PA. Interestingly, LTH enhanced BK‐mediated vasorelaxation in fetal vessels, suggesting a compensatory mechanism. Overall, the data imply that maturation has greater influence than altitude on eNOS ‐ cGMP mediated vasorelaxation. Moreover, cGMP as well as PGI2 pathways are important therapeutic avenues for pulmonary hypertension in newborns and adults. (NSF MRI923559, NIH R03HD69746, R01HD31226, R01HD3807, R01HL95973, P20MD6988)

Endothelial dependent relaxation of the pulmonary vasculature is important during the fetal transition to breathing air. Our studies show bradykinin‐mediated relaxation of the pulmonary vasculature is reduced before birth and that postnatal maturation of nitric oxide (NO) cGMP‐mediated signaling pathways is important to this process. Thus, we tested whether cGMP‐mediated vasorelaxation was blunted before birth by performing wire‐myography on pulmonary arteries (PA) isolated from term‐fetal, newborn, and adult sheep exposed to long term high altitude (3,801 m) hypoxia for >;100 days. The efficacy and potency for serotonin‐induced contraction were evaluated following YC1 activation of soluble guanylate cyclase (sGC) or when cGMP degradation was inhibited with the type 5 phosphodiesterase (PDE5) blocker sildenafil. YC1 and sildenafil decreased the potency for serotonin contraction more prominently in vessels from adults as compared to those from immature sheep. Yet, these drugs reduced the efficacy similarly in nearly all groups, with the exception of adult sheep where YC1 failed to alter the maximum contraction. Overall, sGC and PDE5 dependent cGMP signaling pathways are viable targets for reducing arterial tone and are relevant therapeutic avenues for pulmonary hypertension in newborns and adults. (NSF MRI 0923559, NIH R03HD069746, P01HD031226, R01HD003807, R01HL95973).

Bradykinin‐induced activation of the pulmonary endothelium triggers a rise in intracellular Ca2+, which activates a nitric oxide (NO)‐dependent signaling pathway. This pathway leads to vasodilation, thereby regulating pulmonary blood flow and 02 uptake. This vasodilation process includes stimulation of endothelial nitric oxide synthase (eNOS) and downstream activation of large‐conductance K+ (Maxi K+) channels. Chronic hypoxia (CH) is known to increase pulmonary pressures and restrict arterial relaxation, and can contribute to the development of pulmonary hypertension. We thus examined the effects of CH on bradykinin‐induced Ca2+ signals, bradykinin‐induced vasorelaxation, and the roles of eNOS and Maxi K+ channels in this relaxation. Wire‐myography and confocal microscopy studies were performed on pulmonary arteries (PA) from nonpregnant ewes that lived in a normoxic state at low altitude or a hypoxic state at high altitude (3,801 m) for >100 days. eNOS was inhibited with 100 μM NG‐nitro‐L‐arginine methyl ester (LNAME), and Maxi K+ channels were blocked with 1mM tetraethylammonium (TEA). The data show CH augmented endothelial Ca2+ signals, but restricted bradykinin relaxation. Further, CH caused bradykinin‐induced contraction. LNAME sensitivity was restricted, which suggests eNOS dysfunction is central to the uncoupling of Ca2+ signals and bradykinin relaxation. CH also abolished TEA‐sensitivity in bradykinin relaxation, suggesting there is loss of Maxi K+ function following CH. Overall, these results suggest that CH causes uncoupling of endothelial Ca2+ signaling and eNOS function and mediates major changes in the mechanisms of membrane hyperpolarization. It follows that CH‐induced Ca2+‐eNOS uncoupling and Maxi K+ channel dysfunction are important mechanisms to examine as future therapeutic avenues for pulmonary hypertension.Support or Funding InformationSupport from NIH, NSF and Loma Linda University School of Medicine