Long-term high-altitude hypoxia influences pulmonary arterial L-type calcium channel-mediated Ca²⁺ signals and contraction in fetal and adult sheep

Abstract

Long-term hypoxia (LTH) has a profound effect on pulmonary arterial vasoconstriction in the fetus and adult. Dysregulation in Ca signaling is important during the development of LTH-induced pulmonary hypertension. In the present study, we tested the hypothesis that L-type Ca channels (Ca), which are voltage dependent and found in smooth, skeletal, and cardiac muscle, are important in the adaptation of pulmonary arterial contractions in postnatal maturation and in response to LTH. Pulmonary arteries were isolated from fetal or adult sheep maintained at low or high altitude (3,801 m) for >100 days. The effects were measured using an L-type Ca channel opener FPL 64176 (FPL) in the presence or absence of an inhibitor, Nifedipine (NIF) on arterial contractions, intracellular Ca oscillations, and ryanodine receptor-driven Ca sparks. FPL induced pulmonary arterial contractions in all groups were sensitive to NIF. However, when compared with 125 mM K, FPL contractions were greater in fetuses than in adults. FPL reduced Ca oscillations in myocytes of adult but not fetal arteries, independently of altitude. The FPL effects on Ca oscillations were reversed by NIF in myocytes of hypoxic but not normoxic adults. FPL failed to enhance Ca spark frequency and had little impact on spatiotemporal firing characteristics. These data suggest that Ca-dependent contractions are largely uncoupled from intracellular Ca oscillations and the development of Ca sparks. This raises questions regarding the coupling of pulmonary arterial contractility to membrane depolarization, attendant Ca facilitation, and the related associations with the activation of Ca oscillations and Ca sparks.

Long-Term High-Altitude Hypoxia and Alpha Adrenoceptor-Dependent Pulmonary Arterial Contractions in Fetal and Adult Sheep

“…Following tissue collection, sheep were sacrificed by intravenous injection of the proprietary euthanasia solution, Euthasol (2 ml/kg; Virbac, Ft. Worth, TX, USA). All tissue bath and calcium experimental procedures were performed as previously described (Goyal et al, 2011; Papamatheakis et al, 2011, 2012; Blum-Johnston et al, 2016; Shen et al, 2018) within the regulations of the Animal Welfare Act, the National Institutes of Health Guide for the Care and Use of Laboratory Animals, “The Guiding Principles in the Care and Use of Animals” approved by the Council of the American Physiological Society, and the Animal Care and Use Committee of the Loma Linda University. Although the animals were housed in a rarified environment at the White Mountain Research Station and then the low PaO 2 was maintained at the Loma Linda University by breathing hypoxic gasses, all experimental studies were performed under normoxic conditions at the Loma Linda University.…”

Long-Term Hypoxia Negatively Influences Ca2+ Signaling in Basilar Arterial Myocytes of Fetal and Adult Sheep

“…Intracellular Ca 2+ of basilar arterial myocytes was measured in situ with a Ca 2+ sensitive fluorescent dye (Fluo-4 AM, Cat No F14201, Invitrogen, Carlsbad, CA, United States) using a Zeiss 710 NLO laser scanning confocal imaging work station (Thornwood, NY, United States) with an inverted microscope (ZEISS Axio Observer), using procedures based on previous studies ( Hadley et al, 2012 ; Harraz et al, 2014 ; Shen et al, 2018 ). Fluo-4 AM was dissolved in DMSO creating a 1 mM stock solution.…”

“…A time series of 300 full frame Fluo-4 fluorescence images of 512 × 512 pixels were made over 234 s (roughly 780 ms/frame). In order to make sure that the smooth muscle intracellular Ca 2+ was recorded, the pinhole was set at an imaging depth of 5.4 μm, which is roughly the depth of an individual smooth muscle cell based on examination of both fixed and live preparations ( Hadley et al, 2012 ; Shen et al, 2018 ). This thicker optical sectioning of samples was performed to mitigate the effects of sample ruffling, allowing for visualization of more myocytes than would otherwise be possible at other depths.…”

Gestational Hypoxia and Programing of Lung Metabolism

“…Fetuses and lambs born at high altitude have other abnormalities that parallel infants with hypoxia-induced pulmonary hypertension (Berger and Konduri, 2006; Abman, 2007; Konduri and Kim, 2009), including elevated pulmonary pressures, exacerbated hypoxic-induced pulmonary vasoconstriction (Herrera et al, 2007, 2010; Blood et al, 2013), impaired vasodilation (Blum-Johnston et al, 2016), arterial remodeling (Bixby et al, 2007), and right ventricular hypertrophy (Herrera et al, 2007). Studies further demonstrate that Ca 2+ signals are disrupted in endothelium and smooth muscle and this involves modifications in the expression and function of multiple receptor signaling systems and ion channels (Herrera et al, 2007, 2010; Goyal et al, 2011; Hadley et al, 2012; Blood et al, 2013; Blum-Johnston et al, 2016; Shen et al, 2018).…”