Acute Normoxia Increases Fetal Pulmonary Artery Endothelial Cell Cytosolic Ca2+ Via Ca2+-Induced Ca2+ Release

Abstract

To test the hypothesis that an acute increase in O 2 tension increases cytosolic calcium ([Ca 2ϩ ] i ) in fetal pulmonary artery endothelial cells (PAECs) via entry of extracellular calcium and subsequent calcium-induced calcium release (CICR) and nitric oxide release, low-passage PAECs (Ͻ10 passages) were isolated from the intralobar pulmonary artery (PA) of fetal sheep and maintained under hypoxic conditions (PO 2 , 25 Torr). Using the calciumsensitive dye fura-2, we demonstrated that acute normoxia (PO 2 ϭ 120 Torr) increased PAECs [Ca 2ϩ ] i by increasing the rate of entry of extracellular calcium. In the presence of either ryanodine or 2-aminoethoxy-diphenylborate (2APB), normoxia did not lead to a sustained increase in PAECs [Ca 2ϩ ] i Whole-cell patch clamp studies demonstrated that acute normoxia causes PAEC membrane depolarization. When loaded with the nitric oxide (NO)-sensitive dye, DAF -FM, acute normoxia increased PAEC fluorescence. In PAECs derived from fetal lambs with pulmonary hypertension, an acute increase in O 2 tension had no effect on either [Ca 2ϩ ] i or NO production. Hypoxia increases loading of acetylcholine-sensitive calcium stores, as hypoxia potentiated the response to acetylcholine We conclude that acute normoxia increases [Ca 2ϩ ] i and NO production in normotensive but not hypertensive fetal PAECs via extracellular calcium entry and calcium release from calcium-sensitive intracellular stores. I n the fetus, pulmonary blood flow is limited. At birth, pulmonary blood flow increases eight-to 10-fold (1) and pulmonary arterial pressure decreases to 50% of systemic levels within 24 h after birth (2). The critical physiologic stimuli that account for perinatal pulmonary vasodilation include rhythmic distention of the lung (3), an increase in both shear stress (4), and oxygen tension (5). In response to physiologic stimuli, the pulmonary endothelium elaborates vasoactive mediators such as NO (6), endothelin (7), bradykinin, and prostaglandins. PAEC NO production is necessary (8), although not always sufficient, for the transition of the pulmonary circulation from a high to low resistance circuit.Vasoactive products produced by the PAECs simultaneously preserve and constrain fetal pulmonary blood flow. During fetal life, inhibition of endothelin, a vasoconstrictive protein produced by the endothelium (7), causes marked pulmonary vasodilation (9). Conversely, inhibition of NO increases pulmonary vascular resistance (8). Among the most important functions of the PAECs is the ability to sense and respond to an increase in oxygen tension at birth. Interestingly, the capacity of the pulmonary circulation to sense and respond to an acute increase in oxygen tension is developmentally regulated. Not until gestation is approximately 85% complete is the fetal pulmonary circulation able to respond to an increase in oxygen tension (10). Vasodilator agents that act through endothelial cell-mediated mechanisms produce only transient pulmonary vasodilation (5,11). Perhaps the relativ...

Rho kinase modulates postnatal adaptation of the pulmonary circulation through separate effects on pulmonary artery endothelial and smooth muscle cells

“…Whereas the oxygen-induced increase in fetal PAEC NO production has been previously described (30), ROCK inhibition augmented NO production in response to normoxia and had no effect on PAEC NO production under hypoxic conditions (Fig. 1).…”

“…Cell culture. Techniques used for cell isolation and culture have been previously described (14,20,30). Late-gestation fetal sheep (139 days; term ϭ 147 days) from ewes with time-dated pregnancies were used in this study.…”

Developmental regulation of hypoxia-inducible factor 1 and prolyl-hydroxylases in pulmonary vascular smooth muscle cells

“…The dispersed cell suspension was aliquoted onto 25-mm 2 glass coverslips coated with poly-L-lysine (Sigma) and into 25-cm 2 tissue culture flasks (Falcon Plastics, Oxnard, CA) at a density of 5 Ϫ10 ϫ 10 3 cells/cm 2 . The cells were incubated at 37°C in either a humidified 5% oxygen, 5% CO 2 balance nitrogen atmosphere or a humidified air, 5% CO 2 atmosphere (27,28). After 18-24 h the cultures were washed once with Hanks' balanced salt solution to remove nonadherent cells and debris and refed with fresh medium.…”

“…For Western blotting and ELISAs, fetal and PA SMC were incubated under either normoxic or hypoxic conditions as described (27,28). Eight micrograms of nuclear extract was used in ELISAs (Active Motif, Carlsbad CA), and results were standardized to COS7 nuclear protein.…”

Voltage-Dependent Anion Channel-2 Interaction with Nitric Oxide Synthase Enhances Pulmonary Artery Endothelial Cell Nitric Oxide Production

“…Distal (>4th generation) pulmonary arteries were excised and FPAECs were isolated from late-gestation fetal sheep (z141 d), as previously described (28). hPAECs (Lonza, Basel, Switzerland) were cultured in EGM-2 media (Lonza).…”