Hypoxia is one of the most common and severe challenges to the maintenance of homeostasis. Oxygen sensing is a property of all tissues, and the response to hypoxia is multidimensional involving complicated intracellular networks concerned with the transduction of hypoxia-induced responses. Of all the stresses to which the fetus and newborn infant are subjected, perhaps the most important and clinically relevant is that of hypoxia. Hypoxia during gestation impacts both the mother and fetal development through interactions with an individual’s genetic traits acquired over multiple generations by natural selection and changes in gene expression patterns by altering the epigenetic code. Changes in the epigenome determine “genomic plasticity,” i.e., the ability of genes to be differentially expressed according to environmental cues. The genomic plasticity defined by epigenomic mechanisms including DNA methylation, histone modifications, and noncoding RNAs during development is the mechanistic substrate for phenotypic programming that determines physiological response and risk for healthy or deleterious outcomes. This review explores the impact of gestational hypoxia on maternal health and fetal development, and epigenetic mechanisms of developmental plasticity with emphasis on the uteroplacental circulation, heart development, cerebral circulation, pulmonary development, and the hypothalamic-pituitary-adrenal axis and adipose tissue. The complex molecular and epigenetic interactions that may impact an individual’s physiology and developmental programming of health and disease later in life are discussed.

To examine the nature of inositol 1,4,5-trisphosphate (IP₃)-sensitive and ryanodine (Ryn)-sensitive Ca²⁺stores in isolated canine pulmonary arterial smooth cells (PASMC), agonist-induced changes in global intracellular Ca²⁺concentration ([Ca²⁺]_i) were measured using fura 2-AM fluorescence. Properties of elementary local Ca²⁺release events were characterized using fluo 3-AM or fluo 4-AM, in combination with confocal laser scanning microscopy. In PASMC, depletion of sarcoplasmic reticulum Ca²⁺stores with Ryn (300 μM) and caffeine (Caf; 10 mM) eliminated subsequent Caf-induced intracellular Ca²⁺transients but had little or no effect on the initial IP₃-mediated intracellular Ca²⁺transient induced by ANG II (1 μM). Cyclopiazonic acid (CPA; 10 μM) abolished IP₃-induced intracellular Ca²⁺transients but failed to attenuate the initial Caf-induced intracellular Ca²⁺transient. These results suggest that in canine PASMC, IP₃-, and Ryn-sensitive Ca²⁺stores are organized into spatially distinct compartments while similar experiments in canine renal arterial smooth muscle cells (RASMC) reveal that these Ca²⁺stores are spatially conjoined. In PASMC, spontaneous local intracellular Ca²⁺transients sensitive to modulation by Caf and Ryn were detected, exhibiting spatial-temporal characteristics similar to those previously described for “Ca²⁺sparks” in cardiac and other types of smooth muscle cells. After depletion of Ryn-sensitive Ca²⁺stores, ANG II (8 nM) induced slow, sustained [Ca²⁺]_iincreases originating at sites near the cell surface, which were abolished by depleting IP₃stores. Discrete quantal-like events expected due to the coordinated opening of IP₃receptor clusters (“Ca²⁺puffs”) were not observed. These data provide new information regarding the functional properties and organization of intracellular Ca²⁺stores and elementary Ca²⁺release events in isolated PASMC.

Volume-sensitive osmolyte and anion channels (VSOACs) are activated upon cell swelling in most vertebrate cells. Native VSOACs are believed to be a major pathway for regulatory volume decrease (RVD) through efflux of chloride and organic osmolytes. ClC-3 has been proposed to encode native VSOACs in Xenopus laevis oocytes and in some mammalian cells, including cardiac and vascular smooth muscle cells. The relationship between the ClC-3 chloride channel, the native volume-sensitive osmolyte and anion channel (VSOAC) currents, and cell volume regulation in HeLa cells and X. laevis oocytes was investigated using ClC-3 antisense. In situ hybridization in HeLa cells, semiquantitative and real-time PCR, and immunoblot studies in HeLa cells and X. laevis oocytes demonstrated the presence of ClC-3 mRNA and protein, respectively. Exposing both cell types to hypotonic solutions induced cell swelling and activated native VSOACs. Transient transfection of HeLa cells with ClC-3 antisense oligonucleotide or X. laevis oocytes injected with antisense cRNA abolished the native ClC-3 mRNA transcript and protein and significantly reduced the density of native VSOACs activated by hypotonically induced cell swelling. In addition, antisense against native ClC-3 significantly impaired the ability of HeLa cells and X. laevis oocytes to regulate their volume. These results suggest that ClC-3 is an important molecular component underlying VSOACs and the RVD process in HeLa cells and X. laevis oocytes.

Uterine vascular tone significantly decreases whereas uterine blood flow dramatically increases during pregnancy. However, the complete molecular mechanisms remain elusive. We hypothesized that increased Ca ²⁺ -activated K ⁺ (BK _Ca ) channel activity contributes to the decreased myogenic tone of uterine arteries in pregnancy. Resistance-sized uterine arteries were isolated from nonpregnant and near-term pregnant sheep. Electrophysiological studies revealed a greater whole-cell K ⁺ current density in pregnant compared with nonpregnant uterine arteries. Tetraethylammonium and iberiotoxin inhibited K ⁺ currents to the same extent in uterine arterial myocytes. The BK _Ca channel current density was significantly increased in pregnant uterine arteries. In accordance, tetraethylammonium significantly increased pressure-induced myogenic tone in pregnant uterine arteries and abolished the difference in myogenic responses between pregnant and nonpregnant uterine arteries. Activation of protein kinase C produced a similar effect to tetraethylammonium by inhibiting BK _Ca channel activity and increasing myogenic tone in pregnant uterine arteries. Chronic treatment of nonpregnant uterine arteries with physiologically relevant concentrations of 17β-estradiol and progesterone caused a significant increase in the BK _Ca channel current density. Western blot analyses demonstrated a significant increase of the β1, but not α, subunit of BK _Ca channels in pregnant uterine arteries. In accordance, steroid treatment of nonpregnant uterine arteries resulted in an upregulation of the β1, but not α, subunit expression. The results indicate that the steroid hormone-mediated upregulation of the β1 subunit and BK _Ca channel activity may play a key role in attenuating myogenic tone of the uterine artery in pregnancy.

Rationale:: T-type (Ca _V 3.1/Ca _V 3.2) Ca ²⁺ channels are expressed in rat cerebral arterial smooth muscle. Although present, their functional significance remains uncertain with findings pointing to a variety of roles. Objective:: This study tested whether Ca _V 3.2 channels mediate a negative feedback response by triggering Ca ²⁺ sparks, discrete events that initiate arterial hyperpolarization by activating large-conductance Ca ²⁺ -activated K ⁺ channels. Methods and Results:: Micromolar Ni ²⁺ , an agent that selectively blocks Ca _V 3.2 but not Ca _V 1.2/Ca _V 3.1, was first shown to depolarize/constrict pressurized rat cerebral arteries; no effect was observed in Ca _V 3.2 ^−/− arteries. Structural analysis using 3-dimensional tomography, immunolabeling, and a proximity ligation assay next revealed the existence of microdomains in cerebral arterial smooth muscle which comprised sarcoplasmic reticulum and caveolae. Within these discrete structures, Ca _V 3.2 and ryanodine receptor resided in close apposition to one another. Computational modeling revealed that Ca ²⁺ influx through Ca _V 3.2 could repetitively activate ryanodine receptor, inducing discrete Ca ²⁺ -induced Ca ²⁺ release events in a voltage-dependent manner. In keeping with theoretical observations, rapid Ca ²⁺ imaging and perforated patch clamp electrophysiology demonstrated that Ni ²⁺ suppressed Ca ²⁺ sparks and consequently spontaneous transient outward K ⁺ currents, large-conductance Ca ²⁺ -activated K ⁺ channel mediated events. Additional functional work on pressurized arteries noted that paxilline, a large-conductance Ca ²⁺ -activated K ⁺ channel inhibitor, elicited arterial constriction equivalent, and not additive, to Ni ²⁺ . Key experiments on human cerebral arteries indicate that Ca _V 3.2 is present and drives a comparable response to moderate constriction. Conclusions:: These findings indicate for the first time that Ca _V 3.2 channels localize to discrete microdomains and drive ryanodine receptor–mediated Ca ²⁺ sparks, enabling large-conductance Ca ²⁺ -activated K ⁺ channel activation, hyperpolarization, and attenuation of cerebral arterial constriction.

Capacitative Ca²⁺ entry (CCE) has been speculated to contribute to Ca²⁺ influx during hypoxic pulmonary vasoconstriction (HPV). The aim of the present study was to directly test if acute hypoxia causes intracellular Ca²⁺ concentration ([Ca²⁺]_i) rises through CCE in canine pulmonary artery smooth muscle cells (PASMCs). In PASMCs loaded with fura‐2, hypoxia produced a transient rise in [Ca²⁺]_i in Ca²⁺‐free solution, indicating Ca²⁺ release from the intracellular Ca²⁺ stores. Subsequent addition of 2 mm Ca²⁺ in hypoxia elicited a sustained rise in [Ca²⁺]_i, which was partially inhibited by 10 μm nisoldipine. The dihydropyridine‐insensitive rise in [Ca²⁺]_i was due to increased Ca²⁺ influx, because it was abolished in Ca²⁺‐free solution and hypoxia was shown to significantly enhance the rate of Mn²⁺ quench of fura‐2 fluorescence. The dihyropyridine‐insensitive rise in [Ca²⁺]_i and the increased rate of Mn²⁺ quench of fura‐2 fluorescence were inhibited by 50 μm SKF 96365 and 500 μm Ni²⁺, but not by 100 μm La³⁺ or 100 μm Gd³⁺, exhibiting pharmacological properties characteristic of CCE. In addition, predepletion of the intracellular Ca²⁺ stores inhibited the rise in [Ca²⁺]_i induced by hypoxia. These results provide the first direct evidence that acute hypoxia, by causing Ca²⁺ release from the intracellular stores, activates CCE in isolated canine PASMCs, which may contribute to HPV.