Simona Tolarova

Oxygen-induced constriction of rabbit ductus arteriosus occurs via inhibition of a 4-aminopyridine-, voltage-sensitive potassium channel.

The ductus arteriosus is a vital fetal structure allowing blood ejected from the right ventricle to bypass the pulmonary circulation in utero. Closure of the ductus arteriosus at birth, essential for postnatal adaptation, is initiated by an increase in oxygen (O2) tension. We recently demonstrated the presence of O2-sensitive potassium channels in the fetal and adult pulmonary circulation which regulate vascular tone in response to changes in O2 tension. In this study, we assessed the cellular mechanisms underlying O2-induced constriction of the ductus arteriosus in late-gestation fetal rabbits. We report that O2 reversibly inhibits a 58-pS voltage- and 4-aminopyridine-sensitive potassium channel, causing membrane depolarization, an increase in intracellular calcium through L-type voltage-gated calcium channels, and constriction of the ductus arteriosus. We conclude that the effector mechanism for O2 sensing in the ductus arteriosus involves the coordinated action of delayed rectifier potassium channels and voltage-gated calcium channels.

Redox control of oxygen sensing in the rabbit ductus arteriosus

How the ductus arteriosus (DA) closes at birth remains unclear. Inhibition of O2‐sensitive K+ channels may initiate the closure but the sensor mechanism is unknown. We hypothesized that changes in endogenous H2O2 could act as this sensor. Using chemiluminescence measurements with luminol (50 μm) or lucigenin (5 μm) we showed significantly higher levels of reactive O2 species in normoxic, compared to hypoxic DA. This increase in chemiluminescence was completely reversed by catalase (1200 U ml−1). Prolonged normoxia caused a significant decrease in K+ current density and depolarization of membrane potential in single fetal DA smooth muscle cells. Removal of endogenous H2O2 with intracellular catalase (200 U ml−1) increased normoxic whole‐cell K+ currents (IK) and hyperpolarized membrane potential while intracellular H2O2 (100 nm) and extracellular t‐butyl H2O2 (100 μm) decreased IK and depolarized membrane potential. More rapid metabolism of O2−· with superoxide dismutase (100 U ml−1) had no significant effect on normoxic K+ currents. N‐Mercaptopropionylglycine (NMPG), duroquinone and dithiothreitol all dilated normoxic‐constricted DA rings, while the oxidizing agent 5,5′‐dithiobis‐(2‐nitrobenzoic acid) constricted hypoxia‐dilated rings. NMPG also increased IK. We conclude that increased H2O2 levels, associated with a cytosolic redox shift at birth, signal K+ channel inhibition and DA constriction.

Mechanisms of Pulmonary Vasodilatation and Ductus Arteriosus Constriction by Normoxia

In the fetus, the lungs are not ventilated and arterial oxygen tension is approximately 22 mmHg. Under these conditions, the ductus arteriosus is open and the pulmonary vasculature is constricted. At birth, the ductus constricts and the pulmonary vessels relax. The resting membrane potential in vascular smooth muscle cells is largely determined by potassium current. It appears that oxygen increases the potassium current in the smooth muscle cells of small pulmonary arteries, thus causing membrane hyperpolarization and inhibition of the voltage-gated calcium channel, which in turn leads to a reduction in cytosolic calcium and to relaxation. In the adult, the oxygen-sensitive potassium channels are delayed rectifiers (Kv). In the ductus arteriosus the oxygen-sensitive potassium channel is also in the Kv group but oxygen inhibits, rather than activates, the potassium current and thus causes depolarization, calcium entry, and contraction. The mechanism by which the gating of potassium channels changes in response to oxygen is not clear. Transgenic mice that lack the 91-kDa subunit of NADPH oxidase still have normal hypoxic pulmonary vasoconstriction. Thus, while a role for NADPH oxidase is unlikely, it seems plausible that the oxygen sensor may be part of the channel or be coexpressed with it.

K+ Channels and the Normoxic Constriction of the Rabbit Ductus Arteriosus

The ductus arteriosus (DA) is a vital fetal structure that acts as a right-to-left shunt to divert blood flow away from the constricted pulmonary circulation in the developing fetus. At birth, the DA constricts as a direct result of the increase in oxygen (O2) tension that occurs. The mechanism for this O2-mediated constriction remains controversial. We have shown that the smooth muscle of the DA contains at least two types of potassium (K+) channel, a 4-aminopyridine-sensitive, delayed rectifier (Kv) channel and a tetraethlyammonium-sensitive, calcium- (Ca2+-) dependent K+ channel. Increased levels of O2 appear to selectively inhibit the activity of the Kv channel. Because this channel controls the resting membrane potential of DA smooth muscle cells, this inhibition results in the depolarization of the cell membrane, opening of voltage-gated L-type Ca2+ channels, influx of Ca2+, and hence constriction. We suggest that, under normal conditions, this mechanism may initiate the normoxic constriction of the DA.

AEROSOL DELIVERY OF DETANO, A NITRIC OXIDE ADDUCT, CAUSES SELECTIVE AND SUSTAINED PERINATAL PULMONARY VASODILATION † 2019

Postnatal adaptation of the pulmonary circulation is mediated by endothelium-derived nitric oxide (EDNO). Recent studies have demonstrated that inhaled nitric oxide (NO) causes selective and sustained vasodilation in infants with PPHN. The short half-life of NO limits its clinical application. We hypothesized that aerosol delivery of a NO-adduct, diethylenetriamine(DeTANO), can cause sustained and selective pulmonary vasodilation. To test this hypothesis, we studied the pulmonary vascular response of late-gestation fetal lambs (n=7; age=138 days; term = 147) to aerosolized DeTANO in the presence of an EDNO inhibitor, nitro-L-arginine (L-NA). At surgery, an ultrasonic flow transducer was placed on the left pulmonary artery (LPA) to measure flow (Q). Pressures were measured with catheters in the main pulmonary artery and aorta. After baseline measurements, L-NA (1 mg/min × 30 min) was infused into the LPA prior to ventilation (VENT) with 100% O2 for 30 minutes, followed by continued VENT with 10% O2 for 10 min. This represents the control period. VENT was continued with 100% O2 and DeTANO was given in doses of 0.1, 0.4, and 1.0 mg. 15 minutes following the last dose of DeTANO animals were ventilated with 10% O2.In the control period, after VENT with 100% total pulmonary resistance (TPR) was 0.724±.26 mmHg/cc/min. During VENT with 10% O2, TPR increased by 0.115±.068mmHg/cc/min. Aerosol delivery of DeTANO 0.1mg decreased TPR from 1.075±0.144 to 0.497±0.038 (p<0.05). After DeTANO, TPR did not change during VENT with 10% O2 (p<0.05, compared to control period. Aortic pressure did not change with DeTANO. We conclude that DeTANO can cause selective and sustained fetal pulmonary vasodilation. Aerosol delivery of DeTANO may increase the clinical applications of NO.