Abstract
The ductus arteriosus (DA), an essential vascular shunt for fetal circulation, begins to close immediately after birth. Although Ca(2+) influx through several membrane Ca(2+) channels is known to regulate vasoconstriction of the DA, the role of the T-type voltage-dependent Ca(2+) channel (VDCC) in DA closure remains unclear. Here we found that the expression of alpha1G, a T-type isoform that is known to exhibit a tissue-restricted expression pattern in the rat neonatal DA, was significantly up-regulated in oxygenated rat DA tissues and smooth muscle cells (SMCs). Immunohistological analysis revealed that alpha1G was localized predominantly in the central core of neonatal DA at birth. DA SMC migration was significantly increased by alpha1G overexpression. Moreover, it was decreased by adding alpha1G-specific small interfering RNAs or using R(-)-efonidipine, a highly selective T-type VDCC blocker. Furthermore, an oxygenation-mediated increase in an intracellular Ca(2+) concentration of DA SMCs was significantly decreased by adding alpha1G-specific siRNAs or using R(-)-efonidipine. Although a prostaglandin E receptor EP4 agonist potently promoted intimal thickening of the DA explants, R(-)-efonidipine (10(-6) m) significantly inhibited EP4-promoted intimal thickening by 40% using DA tissues at preterm in organ culture. Moreover, R(-)-efonidipine (10(-6) m) significantly attenuated oxygenation-induced vasoconstriction by approximately 27% using a vascular ring of fetal DA at term. Finally, R(-)-efonidipine significantly delayed the closure of in vivo DA in neonatal rats. These results indicate that T-type VDCC, especially alpha1G, which is predominantly expressed in neonatal DA, plays a unique role in DA closure, implying that T-type VDCC is an alternative therapeutic target to regulate the patency of DA.
Highlights
The ductus arteriosus (DA), an essential vascular shunt for fetal circulation, begins to close immediately after birth
R(Ϫ)-Efonidipine Delayed Closure of the Rat Neonatal DA— The present study demonstrated that Ca2ϩ influx via T-type voltage-dependent Ca2؉ channel (VDCC), especially ␣1G, stimulated smooth muscle cells (SMCs) migration, intimal cushion formation, and vasoconstriction in the cultured DA SMCs or DA explants
In terms of functional DA closure, we found that blockade of T-type
Summary
Animals—Timed pregnant Wistar rats were purchased from Japan SLC, Inc. (Shizuoka, Japan). Quantitative Reverse Transcription-PCR Analysis—Both isolation of total RNA from pooled tissues or cultured SMCs and generation of cDNA and reverse transcription-PCR analysis for ␣1G subunit were carried out as described previously [5]. Intracellular Ca2ϩ Concentration in DA SMCs—DA SMCs were loaded with fura-2/AM (Dojindo, Kumamoto, Japan) in a Tyrode solution (137 mM NaCl, 2.7 mM KCl, 1.4 mM CaCl2, 5.6 mM glucose, 0.5 mM MgCl2, 0.3 mM NaH2PO4, 12 mM NaHCO3, pH7.4). Intracellular Ca2ϩ concentration of DA SMCs was performed using the ARVOTMMX fluorescence microplate reader (PerkinElmer Life Sciences) from 5 min at room air after the hypoxic incubation. Fetal arteries including the DA on the 19th day of gestation were stimulated for 2 days by R(Ϫ)-efonidipine under the following conditions: a humidified chamber, a concentration of 10Ϫ6 M in 0.5% FCS containing Dulbecco’s modified Eagle’s medium, 5% CO2, and 95% ambient mixed air at 37 °C. Comparisons between data from multiple groups were performed by unpaired analysis of variance followed by the Student’s-Newmann-Keuls test. p values Ͻ0.05 were considered statistically significant
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