Nora Kovacs

Endothelial nitric oxide synthase gene transfer enhances dilation of newborn piglet pulmonary arteries

We determined the expression and functional correlate of in vitro transfection with a recombinant adenoviral vector encoding the gene for bovine endothelial nitric oxide synthase (AdCMVeNOS) or Escherichia coliβ-galactosidase (AdCMVLacZ) in pulmonary endothelial cells (EC), vascular smooth muscle cells (VSMC), and pulmonary arteries (PA) from newborn piglets. AdCMVeNOS and AdCMVeLacZ vectors, grown in 293-cell monolayers, were purified by double-cesium gradient ultracentrifugation. Cell cultures and PA were incubated with increasing vector titers for 30 or 60 min, followed by incubation in fresh medium for 18 h at 37°C. LacZ expression was assessed by histochemical staining; eNOS expression was evaluated by Western blot analysis. Functional eNOS expression was determined by measurement of cGMP and quantification of the relaxation response to bradykinin (BK). In PA, LacZ transgene expression was preferentially localized to the adventitia and endothelium. Increased eNOS protein expression was observed in EC and VSMC transfected with AdCMVeNOS. Functional studies revealed increased cGMP abundance in cultured cells and enhanced relaxation to BK in AdCMVeNOS-transfected PA. These studies demonstrate that gene transfer with AdCMVeNOS results in functional expression and altered vasoactive responses in the neonatal pulmonary vasculature. Gene transfer with replication-deficient adenovirus vectors is a useful tool for the study of targeted genes in vascular biology.

The Effects of 11,12-Epoxyeicosatrienoic Acid (11,12-EET) on Isolated Blood Vessels from the Newborn Pulmonary Circulation † 1651

The Effects of 11,12-Epoxyeicosatrienoic Acid (11,12-EET) on Isolated Blood Vessels from the Newborn Pulmonary Circulation † 1651

Effects of Acidosis and CO 2 on Vascular Tone in Newborn Piglet Pulmonary Resistance Vessels (PRV)

Effects of Acidosis and CO 2 on Vascular Tone in Newborn Piglet Pulmonary Resistance Vessels (PRV)

Vasoconstrictor Responses of Arteries, Veins and Microvessels from the Newborn Pulmonary Circulation |[dagger]| 1692

Background: There is considerable evidence that in the perinatal circulation, pulmonary veins (PV), as well as pulmonary arteries (PA), are an important site of action of vascular mediators. Furthermore, resistance to blood flow is controlled at the level of the pulmonary resistance vessels(PRV), which anatomically consist of the precapillary arterioles and the prearteriolar small arteries. Although segmental differences in the responses of the pulmonary arterial, venous and microcirculations have been documented in vivo, there are limited data comparing responses of isolated PRV to those of PA and PV from the newborn pulmonary circulation. Differential responses of PA, PRV and PV to endogenous and pharmacologic vasoconstrictors may influence pulmonary vascular resistance and blood flow, ventilation-perfusion matching and fluid exchange in the lung.Objective: Compare the concentration-dependent(10-11-10-6 M) responses of PA, PV and PRV isolated from newborn piglets to four vasoconstrictor agents: endothelin-1 (ET-1), U46619 (a thromboxane A2 analogue), PGF2α, and norepinephrine (NE).Methods: PA and PV rings were mounted in organ chambers for measurement of isometric tension; PRV were cannulated and pressurized to 15 mm Hg for measurement of lumen diameter. Results: ET-1, followed by U46619, induced the most potent constrictor responses in all vessel types. PV constricted more vigorously to ET-1, U46619 and PGF2α than did PA; constriction to NE was greater in PA than in PV. PRV were exquisitely sensitive to ET-1 and U46619, contracting to 70 ± 6 and 50 ± 3%, respectively, of baseline lumen diameter. PRV showed minimal reactivity to NE and PGF2α. In PRV, EC50 values were in the range of 1 nM, 2 nM, 10 nM and 100 nM for ET-1, U46619, NE and PGF2α, respectively. The EC50 values for U46619 were significantly lower for PV (3 nM) than for PA (10 nM). Conclusion: There are segmental differences in the vasoconstrictor responses of the newborn pulmonary vascular bed which can affect blood flow distribution and edema formation in the lung. The greater sensitivity of microvascular and venous vs. arterial segments to ET-1 and U46619 may play a role in lung liquid production in the fetus and in the pathogenesis of pulmonary hypertension and pulmonary edema in the post-natal period.

The Effect of Alkalosis on Intracellular Calcium Concentrations[Ca++]i in Pulmonary Microvascular Endothelial Cells |[bull]| 1602

Background: The signaling mechanisms mediating alkalosis-induced pulmonary vasodilation are poorly understood. We have recently shown that an increase in pH from 7.4 to 7.7 stimulates cyclooxygenase (COX) and nitric oxide synthase (NOS) activity resulting in a 2-fold increase in cGMP and a 5-fold increase in prostacyclin production in pulmonary microvascular endothelial cells (EC) in culture. Objective: To test the hypothesis that alkalosis causes an increase in [Ca++]i by stimulating K+ channel activity resulting in membrane hyperpolarization. As EC do not have voltage-gated Ca++ channels, Ca++ entry in EC depends upon the electrochemical driving force. Methods: Bovine pulmonary microvascular EC were loaded for 1 hour at room temperature with 5 μM fura-2 AM and 0.01% Pluronic F127. Cultures were rinsed of free fura-2 and placed on the stage of an inverted microscope for measurement of[Ca++]i using a dual wavelength fluorescent imaging system(Intracellular Imaging, Inc.). Excitation wavelengths were altered between 340 and 380 nm and emission fluorescence was measured at 510 nm. Intracellular Ca++ concentrations were computed from Ca++ calibration curves generated from Ca++ standard solutions and the 340/380 ratios using the InCyt IM2 image acquisition and analysis software.Results: Basal [Ca++]i was 52 ± 22 nM. There was a pH-dependent increase in [Ca++]i: pH 9.0 caused an abrupt 7-fold increase, while pH 7.7 caused a 2.2-fold increase over basal levels. The pH-dependent increase in [Ca++]i was abolished in Ca++-free medium. TMB-8, an inhibitor of IP3-mediated Ca++ release, had no effect on the alkalosis-induced increase in[Ca++]i but abolished the Ca++ transient induced by bradykinin. Depolarization of membrane potential with an isotonic KCl buffer(50 mM) abolished the increase in [Ca++]i mediated by alkalosis. Pretreatment of EC for 15 min with tetraethylammonium (TEA) partially inhibited the alkalosis-induced increase in [Ca++]i.Conclusion: Alkalosis induces a pH-dependent increase in[Ca++]i in pulmonary microvascular EC, which is dependent upon the presence of extracellular Ca++. The alkalosis-induced increase in[Ca++]i is linked to K+ channel activation, as it is inhibited by membrane depolarization and partially blocked by TEA. These studies support the hypothesis that alkalosis mediates vasodilation by activating K+ channels, hyperpolarizing EC membrane potential, resulting in increased [Ca++]i, which ultimately activates Ca++- dependent enzymes, such as COX and NOS.

Adenoviral-mediated Gene Transfer in Newborn Piglet Pulmonary Conduit and Resistance Vessels Ex Vivo • 1601

Adenoviral-mediated Gene Transfer in Newborn Piglet Pulmonary Conduit and Resistance Vessels Ex Vivo • 1601

RESPONSES OF ISOLATED, PRESSURIZED NEWBORN PULMONARY RESISTANCE VESSELS TO CHANGES IN EXTRACELLULAR pH (æ pH) † 1459

Isolated vessel segments have been useful for studying control of vascular tone. For technical reasons, most studies of pulmonary vascular responses have utilized large conductance vessels. However, the principal site of increased pulmonary vascular resistance is the small precapillary arteriole (<300μm diameter). Indeed, the unique pressor response of the intact pulmonary circulation to hypoxia is observed only in isolated resistance vessels. We have similarly found that large conductance vessels do not demonstrate consistent responses to changes in pH (Δ pH). We hypothesized that isolated, pressurized pulmonary resistance vessels would mimic the unique responses of the intact lung to ▵ pH. Pulmonary resistance vessels(<300 μm) from newborn piglets were isolated, cannulated at both ends, pressurized to 15 mm Hg and superfused with NaHCO3-containing buffer equilibrated with gas mixtures containing room air (RA) plus 14, 5 or 2% CO2 to achieve a buffer pH of 6.8, 7.4 or 7.7, respectively. The vessel image was projected on a monitor for continuous measurement of the lumen diameter (LD) with a video dimension analysis system. Spontaneous responses to▵ pH were studied in 9 pulmonary resistance vessels from 5 piglets (mean LD 206; range 115-265 μm). All vessels constricted to 50 mM KCl (21± 13% decrease in LD) and demonstrated intact endothelial-dependent dilation to bradykinin (1 μM). After one hour equilibration with stable LD in RA/5% CO2 (pH 7.4) the superfusion gas was changed to RA/14% CO2 (pH 6.8). Acidosis caused a 9 ± 4.6% decrease in LD, which was immediately reversed by changing to RA/2% CO2 (pH 7.7). Continued exposure to alkalosis caused a 15.2 ± 6.6% dilation to a LD consistently larger than that observed at pH 7.4. We conclude that isolated, pressurized pulmonary resistance vessels can be used as a model to study signaling mechanisms mediating the unique responses of the pulmonary circulation to ▵pH. These findings emphasize the importance of viewing the pulmonary circulation as a non-homogeneous unit and of focusing on small precapillary arterioles in investigations of pulmonary vascular responses to hypoxia and pH. (Supported by March of Dimes #6-FY96-0703)

EFFECTS OF CHANGES IN EXTRACELLULAR pH ( ▵ pH) ON ISOLATED, PRESSURIZED NEWBORN RESISTANCE VESSELS

EFFECTS OF CHANGES IN EXTRACELLULAR pH ( ▵ pH) ON ISOLATED, PRESSURIZED NEWBORN RESISTANCE VESSELS

DIFFERENT SIGNALING MECHANISMS MEDIATE BRADYKININ (BK)-INDUCED VASORELAXATION IN ISOLATED, NEWBORN PULMONARY RESISTANCE VESSELS (PRV) VS. CONDUCTANCE VESSELS. • 1460

DIFFERENT SIGNALING MECHANISMS MEDIATE BRADYKININ (BK)-INDUCED VASORELAXATION IN ISOLATED, NEWBORN PULMONARY RESISTANCE VESSELS (PRV) VS. CONDUCTANCE VESSELS. • 1460

Alkalosis Stimulates Release of Nitric Oxide (No) and Prostacyclin(Pgi2) From Pulmonary Microvascular Endothelial Cells

Alkalosis is a widely practiced therapeutic modality for persistent pulmonary hypertension of the newborn. Yet, little is known about the cellular mechanisms mediating alkalosis-induced pulmonary vasodilation. To determine if alkalosis stimulates release of endothelial-dependent vasodilators, NO and PGI2 production were measured at pH 7.4 and pH 7.7 by enzyme immunoassay for cGMP and 6-keto PGF1α, respectively. Compared with pH 7.4, alkalosis (pH 7.7) caused a 2-fold increase in both basal and bradykinin (BK, 0.1 μM)-stimulated cGMP production in mixed cultures of pulmonary microvascular endothelial cells (EC) and vascular smooth muscle cells. This was inhibited by 0.1 mM nitro-L-arginine (L-NA). In EC, alkalosis also caused a 5-fold increase in basal release and a 50% increase in BK-stimulated release of PGI2, which was inhibited by 0.01 mM indomethacin. Interestingly, L-NA caused partial inhibition of PGI2 release at pH 7.4, but not at pH 7.7. We conclude that alkalosis stimulates production of both NO and PGI2 from pulmonary EC. Furthermore, at pH 7.4, there is a link between the nitric oxide synthase (NOS) and cyclooxygenase (COX) pathways, suggesting a stimulatory role for NO in eicosanoid production. Alkalosis appears to uncouple the interaction between the NOS and COX pathways.