This proposal represents a multi-PI collaboration between two complementary laboratories with unique expertise in the ductus arteriosus (Dr. Reese) and vascular chloride (Cl-) channels (Dr. Lamb). The goal of this proposal is to determine the mechanisms by which anions regulate the ductus arteriosus (DA). Vascular transition at birth requires rapid constriction of the DA. Persistent patency of the DA (PDA) is a serious problem affecting over 30% of preterm infants <1500g, yet current therapies have many shortcomings. The mechanisms that regulate fetal DA patency and postnatal closure are not fully resolved. We previously identified a number of ion channels that are promising targets for DA-specific therapies. Among them, Cl- channels are an intriguing prospect. Although cation channels have established roles in the DA, virtually no information exists regarding Cl- currents and DA contractility. In general, Cl- transport in vascular smooth muscle cells (VSMC) is dynamic and precisely tuned by specific channels and co-transporters. Alterations in vascular Cl- currents have been linked to hypertension and other vascular abnormalities. Moreover, it is known that clinical use of furosemide, a diuretic and Cl- transport inhibitor, is associated with PDA in preterm infants. These data suggest a role for Cl- balance in regulating DA tone. To probe the importance of Cl- flux in the DA, we first identified a subset of Cl--associated genes that were significantly enriched in the DA. We determined that the expression of specific Cl- import genes increased while Cl- export genes waned in the DA over the course of development, favoring Cl- export during fetal life, but switching to Cl- accumulation at birth. Patch-clamp recordings of isolated DA SMCs identified the presence of specific Cl- currents. Myography studies verified the functional significance of these currents. Inhibition of specific Cl- channels blocked O2- and agonist-induced constriction of the isolated mouse DA. Furosemide relaxed the ex vivo mouse DA, independent of NO or prostaglandin actions. Furthermore, treatment with a non-selective Cl- channel inhibitor produced a PDA phenotype in newborn mice. These data strongly implicate Cl- transport and Cl- currents as important, unexplored mediators of DA contractility. In this proposal, we will test the hypothesis that Cl- currents are critical mediators of DA tone, and that changes in anion transport at birth switch the DA VSMC transmembrane Cl- gradient from inhibitory to excitatory.
Aim 1 will identify the mechanisms by which Cl- currents contribute to in utero DA relaxation and postnatal DA closure.
Aim 2 will determine whether a developmental switch in Cl- transport modulates DA contractility and drives postnatal DA closure. Molecular, genetic, pharmacological, and electrophysiological approaches will define chloride-dependent mechanisms that regulate DA patency in vitro and in vivo. Completion of these aims will provide clinically relevant information on Cl- homeostasis in the DA and may identify new therapeutic targets for modulation of DA tone.
The ductus arteriosus (DA) is a fetal blood vessel whose patency is critical for survival in the womb, and whose closure is essential for vascular transition at the time of birth. Although chloride (Cl-) currents are tightly regulated in vascular smooth muscle cells, Cl- is a previously unrecognized mediator of the DA. This proposal is based on compelling preliminary data showing that Cl- channels play a key role in regulation of the DA, and will examine the hypothesis that abnormal Cl- balance contributes to pathological states like persistent patency of the DA (PDA) where the DA fails to constrict after birth.
