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

Autonomic innervation of the pulmonary vasculature triggers vasomotor contractility predominately through activation of alpha-adrenergic receptors (α-ARs) in the fetal circulation. Long-term hypoxia (LTH) modulates pulmonary vasoconstriction potentially through upregulation of α1-AR in the vasculature. Our study aimed to elucidate the role of α-AR in phenylephrine (PE)-induced pulmonary vascular contractility, comparing the effects of LTH in the fetal and adult periods on α-AR subtypes and PE-mediated Ca2+ responses and contractions. To address this, we performed wire myography, Ca2+ imaging, and mRNA analysis of pulmonary arteries from ewes and fetuses exposed to LTH or normoxia. Postnatal maturation depressed PE-mediated contractile responses. α2-AR activation contracted fetal vessels; however, this was suppressed by LTH. α1A- and α1B-AR subtypes contributed to arterial contractions in all groups. The α1D-AR was also important to contractility in fetal normoxic vessels and LTH mitigated its function. Postnatal maturity increased the number of myocytes with PE-triggered Ca2+ responses while LTH decreased the percentage of fetal myocytes reacting to PE. The difference between myocyte Ca2+ responsiveness and vessel contractility suggests that fetal arteries are sensitized to changes in Ca2+. The results illustrate that α-adrenergic signaling and vascular function change during development and that LTH modifies adrenergic signaling. These changes may represent components in the etiology of pulmonary vascular disease and foretell the therapeutic potential of adrenergic receptor antagonists in the treatment of pulmonary hypertension.

Long-term hypoxia uncouples Ca2+ and eNOS in bradykinin-mediated pulmonary arterial relaxation.

Bradykinin-induced activation of the pulmonary endothelium triggers a rise in intracellular Ca2+ that activates nitric oxide (NO)-dependent vasorelaxation. Chronic hypoxia is commonly associated with increased pulmonary vascular tone, which can cause pulmonary hypertension in responsive individuals. In the present study, we tested the hypothesis that long-term high-altitude hypoxia (LTH) diminishes bradykinin-induced Ca2+ signals and inhibits endothelial nitric oxide synthase (eNOS), prostacyclin (PGI2), and large-conductance K+ (BKCa) channels in sheep, which are moderately responsive to LTH, resulting in decreased pulmonary arterial vasorelaxation. Pulmonary arteries were isolated from ewes kept near sea level (720 m) or at high altitude (3,801 m) for >100 days. Vessel force was measured with wire myography and endothelial intracellular Ca2+ with confocal microscopy. eNOS was inhibited with 100 µM NG-nitro-l-arginine methyl ester (l-NAME), PGI2 production was inhibited with 10 µM indomethacin that inhibits cyclooxygenase, and BKCa channels were blocked with 1 mM tetraethylammonium. Bradykinin-induced endothelial Ca2+ signals increased following LTH, but bradykinin relaxation decreased. Furthermore, some vessels contracted in response to bradykinin after LTH. l-NAME sensitivity decreased, suggesting that eNOS dysfunction played a role in uncoupling Ca2+ signals and bradykinin relaxation. The Ca2+ ionophore A-23187 (10 µM) elicited an enhanced Ca2+ response following LTH while relaxation was unchanged although l-NAME sensitivity increased. Additionally, BKCa function decreased during bradykinin relaxation following LTH. Western analysis showed that BKCa α-subunit expression was increased by LTH while that for the ß1 subunit was unchanged. Overall, these results suggest that those even moderately responsive to LTH can have impaired endothelial function.

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

Long-term hypoxia (LTH) has a profound effect on pulmonary arterial vasoconstriction in the fetus and adult. Dysregulation in Ca2+ signaling is important during the development of LTH-induced pulmonary hypertension. In the present study, we tested the hypothesis that L-type Ca2+ channels (CaL), 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 Ca2+ channel opener FPL 64176 (FPL) in the presence or absence of an inhibitor, Nifedipine (NIF) on arterial contractions, intracellular Ca2+ oscillations, and ryanodine receptor-driven Ca2+ 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 Ca2+ oscillations in myocytes of adult but not fetal arteries, independently of altitude. The FPL effects on Ca2+ oscillations were reversed by NIF in myocytes of hypoxic but not normoxic adults. FPL failed to enhance Ca2+ spark frequency and had little impact on spatiotemporal firing characteristics. These data suggest that CaL-dependent contractions are largely uncoupled from intracellular Ca2+ oscillations and the development of Ca2+ sparks. This raises questions regarding the coupling of pulmonary arterial contractility to membrane depolarization, attendant CaL facilitation, and the related associations with the activation of Ca2+ oscillations and Ca2+ sparks.

Muscarinic Receptor Activation Affects Pulmonary Artery Contractility in Sheep: The Impact of Maturation and Chronic Hypoxia on Endothelium-Dependent and Endothelium-Independent Function.

Giang, Michael, Demosthenes G. Papamatheakis, Dan Nguyen, Ricardo Paez, Carla Blum Johnston, Joon Kim, Alexander Brunnell, Quintin Blood, Ravi Goyal, Lawrence D. Longo, and Sean M. Wilson. Muscarinic receptor activation affects pulmonary artery contractility in sheep: the impact of maturation and chronic hypoxia on endothelium-dependent and endothelium-independent function. High Alt Med Biol. 17:122-132, 2015.-Muscarinic receptor activation in the pulmonary vasculature can cause endothelium-dependent vasodilation and smooth muscle-dependent vasoconstriction. Chronic hypoxia (CH) can modify both of these responses. This study aimed to assess the combined influence of CH and maturation on endothelium-dependent and endothelium-independent muscarinic-induced vasoreactivity. This was accomplished by performing wire myography on endothelium-intact or endothelium-disrupted pulmonary arterial rings isolated from normoxic or CH fetal and adult sheep. In endothelium-intact arteries, vasodilation was evaluated using cumulative bradykinin doses in phenylephrine and carbachol precontracted pulmonary arterial segments; and vasoconstriction was examined using cumulative doses of carbachol following bradykinin predilation. Effects of nonselective (atropine) and selective M1 (pirenzepine), M2 (AFDX116), and M3 (4-DAMP and Dau5884) muscarinic receptor antagonists were assessed in disrupted arteries. In normoxic arteries, bradykinin relaxation was twofold greater in the adult compared to fetus, while carbachol contraction was fourfold greater. In adult arteries, CH increased bradykinin relaxation and carbachol contraction. In vessels with intact endothelium, maturation and CH augmented maximal response and efficacy for carbachol constriction and bradykinin relaxation. Approximately 50%-80% of adult normoxic and CH endothelium-disrupted arteries contracted to acetylcholine, while ∼50% of fetal normoxic and ∼10% of CH arteries responded. Atropine reduced carbachol-induced contraction in all vessels. Adult normoxic vessels were most responsive to M3 antagonism, fetal to M2 antagonism, while M1 inhibition had no effect. Overall, muscarinic-induced pulmonary arterial contraction is partially endothelium dependent and appears to develop after birth. Fetuses are more reliant on M3 receptors while M2 receptors predominate in adults, whereas CH augments muscarinic-dependent pulmonary vasoconstriction in both.

Microarray gene expression analysis in ovine ductus arteriosus during fetal development and birth transition.

BACKGROUND: Patent ductus arteriosus (PDA) in the newborn is the most common congenital heart anomaly and is significantly more common in preterm infants. Contemporary pharmacological treatment is effective in only 70-80% of the cases. Moreover, indomethacin or ibuprofen, which are used to close a PDA may be accompanied by serious side effects in premature infants. To explore the novel molecular pathways, which may be involved in the maturation and closure of the ductus arteriosus (DA), we used fetal and neonatal sheep to test the hypothesis that maturational development of DA is associated with significant alterations in specific mRNA expression. METHODS: We conducted oligonucleotide microarray experiments on the isolated mRNA from DA and ascending aorta from three study groups (premature fetus-97 ± 0 d, near-term fetus-136 ± 0.8 d, and newborn lamb-12 ± 0 h). We compared the alterations in mRNA expression in DA and aorta to identify genes specifically involved in DA maturation. RESULTS: Results demonstrate significant changes in wingless-integrin1, thrombospondin 1, receptor activator of nuclear factor-kappa B, nitric oxide synthase, and retinoic acid receptor activation signaling pathways. CONCLUSION: We conclude that these pathways may play an important role during both development and postnatal DA closure and warrant further investigation.

S-nitrosothiols dilate the mesenteric artery more potently than the femoral artery by a cGMP and L-type calcium channel-dependent mechanism.

S-nitrosothiols (SNOs) are metabolites of NO with potent vasodilatory activity. Our previous studies in sheep indicated that intra-arterially infused SNOs dilate the mesenteric vasculature more than the femoral vasculature. We hypothesized that the mesenteric artery is more responsive to SNO-mediated vasodilation, and investigated various steps along the NO/cGMP pathway to determine the mechanism for this difference. In anesthetized adult sheep, we monitored the conductance of mesenteric and femoral arteries during infusion of S-nitroso-l-cysteine (L-cysNO), and found mesenteric vascular conductance increased (137 ± 3%) significantly more than femoral conductance (26 ± 25%). Similar results were found in wire myography studies of isolated sheep mesenteric and femoral arteries. Vasodilation by SNOs was attenuated in both vessel types by the presence of ODQ (sGC inhibitor), and both YC-1 (sGC agonist) and 8-Br-cGMP (cGMP analog) mediated more potent relaxation in mesenteric arteries than femoral arteries. The vasodilatory difference between mesenteric and femoral arteries was eliminated by antagonists of either protein kinase G or L-type Ca(2+) channels. Western immunoblots showed a larger L-type Ca(2+)/sGC abundance ratio in mesenteric arteries than in femoral arteries. Fetal sheep mesenteric arteries were more responsive to SNOs than adult mesenteric arteries, and had a greater L-Ca(2+)/sGC ratio (p = 0.047 and r = -0.906 for correlation between Emax and L-Ca(2+)/sGC). These results suggest that mesenteric arteries, especially those in fetus, are more responsive to SNO-mediated vasodilation than femoral arteries due to a greater role of the L-type calcium channel in the NO/cGMP pathway.

Vitamin D status and metabolism in an ovine pregnancy model: effect of long-term, high-altitude hypoxia.

Vitamin D status increases during healthy mammalian pregnancy, but the molecular determinants remain uncharacterized. The first objective of this study was to determine the effects of pregnancy, and the second objective was to examine the role of chronic hypoxia on vitamin D status and metabolism in an ovine model. We analyzed the plasma levels of cholecalciferol, 25-OH-D, and 1α,25-(OH)2D in nonpregnant ewes, near-term pregnant ewes, and their fetuses exposed to normoxia (low altitude) or hypoxia (high-altitude) for 100 days. Hypoxic sheep had increased circulating levels of 25-OH-D and 1α,25-(OH)2D compared with normoxic sheep. Hypoxia increases in 25-OH-D were associated with increased expression of renal 25-hydroxylases CYP2R1 and CYP2J. Pregnancy did not increase further the plasma levels of 25-OH-D, but it significantly increased those of the active metabolite, 1α,25-(OH)2D, in both normoxic and hypoxic ewes. Increased bioactivation of vitamin D correlated with increased expression of the vitamin D-activating enzyme CYP27b1 and decreased expression of the inactivating enzyme CYP24a1 in maternal kidneys and placentas. Hypoxia increased parathyroid hormone levels and further increased renal CYP27b1. Pregnancy and hypoxia decreased the expression of vitamin D receptor (VDR) in maternal kidney and lung, with opposite effects on placental VDR. We conclude that ovine pregnancy is a model of increased vitamin D status, and long-term hypoxia further improves vitamin D status due to pregnancy- and hypoxia-specific regulation of VDR and metabolic enzymes.

Postprandial lipids accelerate and redirect nitric oxide consumption in plasma.

Nitric oxide (NO) and O2 are both three-to four-fold more soluble in biological lipids than in aqueous solutions. Their higher concentration within plasma lipids accelerates NO autoxidation to an extent that may be of importance to overall NO bioactivity. This study was undertaken to test the hypothesis that increased plasma lipids after a high-fat meal appreciably accelerate NO metabolism and alter the byproducts formed. We found that plasma collected from subjects after consumption of a single high-fat meal had a higher capacity for NO consumption and consumed NO more rapidly compared to fasting plasma. This increased NO consumption showed a direct correlation with plasma triglyceride concentrations (p = 0.006). The accelerated NO consumption in postprandial plasma was reversed by removal of the lipids from the plasma, was mimicked by the addition of hydrophobic micelles to aqueous buffer, and could not be explained by the presence of either free hemoglobin or ceruloplasmin. The products of NO consumption were shifted in postprandial plasma, with 55% more nitrite (n = 12, p = 0.002) but 50% less SNO (n = 12, p = 0.03) production compared to matched fasted plasma. Modeling calculations indicated that NO autoxidation was accelerated by about 48-fold in the presence of plasma lipids. We conclude that postprandial triglyceride-rich lipoproteins exert a significant influence on NO metabolism in plasma.

Local and systemic vasodilatory effects of low molecular weight S-nitrosothiols.

S-nitrosothiols (SNOs) such as S-nitroso-L-cysteine (L-cysNO) are endogenous compounds with potent vasodilatory activity. During circulation in the blood, the NO moiety can be exchanged among various thiol-containing compounds by S-transnitrosylation, resulting in SNOs with differing capacities to enter the cell (membrane permeability). To determine whether the vasodilating potency of SNOs is dependent upon membrane permeability, membrane-permeable L-cysNO and impermeable S-nitroso-D-cysteine (D-cysNO) and S-nitroso-glutathione (GSNO) were infused into one femoral artery of anesthetized adult sheep while measuring bilateral femoral and systemic vascular conductances. L-cysNO induced vasodilation in the infused hind limb, whereas D-cysNO and GSNO did not. L-cysNO also increased intracellular NO in isolated arterial smooth muscle cells, whereas GSNO did not. The infused SNOs remained predominantly in a low molecular weight form during first-passage through the hind limb vasculature, but were converted into high molecular weight SNOs upon systemic recirculation. At systemic concentrations of ~0.6 µmol/L, all three SNOs reduced mean arterial blood pressure by ~50%, with pronounced vasodilation in the mesenteric bed. Pharmacokinetics of L-cysNO and GSNO were measured in vitro and in vivo and correlated with their hemodynamic effects, membrane permeability, and S-transnitrosylation. These results suggest local vasodilation by SNOs in the hind limb requires membrane permeation, whereas systemic vasodilation does not. The systemic hemodynamic effects of SNOs occur after equilibration of the NO moiety amongst the plasma thiols via S-transnitrosylation.

Developmental acceleration of bradykinin-dependent relaxation by prenatal chronic hypoxia impedes normal development after birth.

Bradykinin-induced activation of the pulmonary endothelium triggers nitric oxide production and other signals that cause vasorelaxation, including stimulation of large-conductance Ca(2+)-activated K(+) (BKCa) channels in myocytes that hyperpolarize the plasma membrane and decrease intracellular Ca(2+). Intrauterine chronic hypoxia (CH) may reduce vasorelaxation in the fetal-to-newborn transition and contribute to pulmonary hypertension of the newborn. Thus we examined the effects of maturation and CH on the role of BKCa channels during bradykinin-induced vasorelaxation by examining endothelial Ca(2+) signals, wire myography, and Western immunoblots on pulmonary arteries isolated from near-term fetal (∼ 140 days gestation) and newborn, 10- to 20-day-old, sheep that lived in normoxia at 700 m or in CH at high altitude (3,801 m) for >100 days. CH enhanced bradykinin-induced relaxation of fetal vessels but decreased relaxation in newborns. Endothelial Ca(2+) responses decreased with maturation but increased with CH. Bradykinin-dependent relaxation was sensitive to 100 µM nitro-L-arginine methyl ester or 10 µM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, supporting roles for endothelial nitric oxide synthase and soluble guanylate cyclase activation. Indomethacin blocked relaxation in CH vessels, suggesting upregulation of PLA2 pathways. BKCa channel inhibition with 1 mM tetraethylammonium reduced bradykinin-induced vasorelaxation in the normoxic newborn and fetal CH vessels. Maturation reduced whole cell BKCa channel α1-subunit expression but increased ß1-subunit expression. These results suggest that CH amplifies the contribution of BKCa channels to bradykinin-induced vasorelaxation in fetal sheep but stunts further development of this vasodilatory pathway in newborns. This involves complex changes in multiple components of the bradykinin-signaling axes.

Sir Joseph Barcroft: one victorian physiologist's contributions to a half century of discovery.

During the first half of the 20th Century, Joseph Barcroft, KBE, FRS of Cambridge University became a world leader in respiratory physiology. He determined the role of neural stimulation in the oxygen consumption of several organs, established many of the factors that regulate the binding of oxygen to haemoglobin, explored the determinants of a human's acclimatization to high altitude and developed the field of fetal cardiovascular physiology. Chair of the Cambridge Department of Physiology from 1925 to 1937, he served as a consultant and member of many UK governmental committees. During World War I, he led a British research unit exploring the effects of poisonous gases on pulmonary function and related problems. In addition to his almost 300 publications, several of his monographs are considered as classics.

Temporal Dissection of Rate Limiting Transcriptional Events Using Pol II ChIP and RNA Analysis of Adrenergic Stress Gene Activation.

In mammals, increasing evidence supports mechanisms of co-transcriptional gene regulation and the generality of genetic control subsequent to RNA polymerase II (Pol II) recruitment. In this report, we use Pol II Chromatin Immunoprecipitation to investigate relationships between the mechanistic events controlling immediate early gene (IEG) activation following stimulation of the α1a-Adrenergic Receptor expressed in rat-1 fibroblasts. We validate our Pol II ChIP assay by comparison to major transcriptional events assessable by microarray and PCR analysis of precursor and mature mRNA. Temporal analysis of Pol II density suggests that reduced proximal pausing often enhances gene expression and was essential for Nr4a3 expression. Nevertheless, for Nr4a3 and several other genes, proximal pausing delayed the time required for initiation of productive elongation, consistent with a role in ensuring transcriptional fidelity. Arrival of Pol II at the 3' cleavage site usually correlated with increased polyadenylated mRNA; however, for Nfil3 and probably Gprc5a expression was delayed and accompanied by apparent pre-mRNA degradation. Intragenic pausing not associated with polyadenylation was also found to regulate and delay Gprc5a expression. Temporal analysis of Nr4a3, Dusp5 and Nfil3 shows that transcription of native IEG genes can proceed at velocities of 3.5 to 4 kilobases/min immediately after activation. Of note, all of the genes studied here also used increased Pol II recruitment as an important regulator of expression. Nevertheless, the generality of co-transcriptional regulation during IEG activation suggests temporal and integrated analysis will often be necessary to distinguish causative from potential rate limiting mechanisms.

Antenatal maternal hypoxia: criterion for fetal growth restriction in rodents.

Rodents are a useful model for life science research. Accumulating evidence suggests that the offspring of mice and rats suffer from similar disorders as humans when exposed to hypoxia during pregnancy. Importantly, with antenatal hypoxic exposure, human neonates demonstrate low birth weight or growth restriction. Similarly, with antenatal hypoxic exposure rodents also demonstrate the fetal growth restriction (FGR). Surprisingly, there is no consensus on the minimum duration or degree of hypoxic exposure required to cause FGR in rodents. Thus, we have reviewed the available literature in an attempt to answer these questions. Based on studies in rats, birth weight reduction of 31% corresponded to 10th percentile reduction in birth weight curve. With the similar criterion (10th percentile), in mice 3 days or more and in rats 7 days or more of 14% or lower hypoxia administration was required to produce statistically significant FGR.

Metabolic Profiles in Ovine Carotid Arteries with Developmental Maturation and Long-Term Hypoxia.

BACKGROUND: Long-term hypoxia (LTH) is an important stressor related to health and disease during development. At different time points from fetus to adult, we are exposed to hypoxic stress because of placental insufficiency, high-altitude residence, smoking, chronic anemia, pulmonary, and heart disorders, as well as cancers. Intrauterine hypoxia can lead to fetal growth restriction and long-term sequelae such as cognitive impairments, hypertension, cardiovascular disorders, diabetes, and schizophrenia. Similarly, prolonged hypoxic exposure during adult life can lead to acute mountain sickness, chronic fatigue, chronic headache, cognitive impairment, acute cerebral and/or pulmonary edema, and death. AIM: LTH also can lead to alteration in metabolites such as fumarate, 2-oxoglutarate, malate, and lactate, which are linked to epigenetic regulation of gene expression. Importantly, during the intrauterine life, a fetus is under a relative hypoxic environment, as compared to newborn or adult. Thus, the changes in gene expression with development from fetus to newborn to adult may be as a consequence of underlying changes in the metabolic profile because of the hypoxic environment along with developmental maturation. To examine this possibility, we examined the metabolic profile in carotid arteries from near-term fetus, newborn, and adult sheep in both normoxic and long-term hypoxic acclimatized groups. RESULTS: Our results demonstrate that LTH differentially regulated glucose metabolism, mitochondrial metabolism, nicotinamide cofactor metabolism, oxidative stress and antioxidants, membrane lipid hydrolysis, and free fatty acid metabolism, each of which may play a role in genetic-epigenetic regulation.

Antenatal maternal low protein diet: ACE-2 in the mouse lung and sexually dimorphic programming of hypertension.

Elevated blood pressure is an important global health problem, and in-utero under-nutrition may be an important factor in the pathogenesis of hypertension. In the present study, we tested the hypothesis that antenatal maternal low protein diet (MLPD) leads to sexually dimorphic developmental programming of the components of the pulmonary renin-angiotensin system. This may be important in the antenatal MLPD-associated development of hypertension. In pregnant mice, we administered normal (control) and isocaloric 50% protein restricted diet, commencing one week before mating and continuing until delivery of the pups. From the 18th to 24th week postnatal, we measured blood pressure in the offspring by use of a non-invasive tail-cuff method. In the same mice, we examined the mRNA and protein expression of the key components of the pulmonary renin-angiotensin system. Also, we examined microRNA complementary to angiotensin converting enzymes (ACE) 2 in the offspring lungs. Our results demonstrate that as a consequence of antenatal MLPD: 1) pup birthweight was significantly reduced in both sexes. 2) female offspring developed hypertension, but males did not. 3) In female offspring, ACE-2 protein expression was significantly reduced without any change in the mRNA levels. 4) miRNA 429, which has a binding site on ACE-2 - 3' UTR was significantly upregulated in the female antenatal MLPD offspring. 5) In males, ACE-2 mRNA and protein expression were unaltered. We conclude that in the mouse, antenatal MLPD-induced reduction of ACE-2 in the female offspring lung may be an important mechanisms in sexually dimorphic programming of hypertension.

Macrophage gene expression associated with remodeling of the prepartum rat cervix: microarray and pathway analyses.

As the critical gatekeeper for birth, prepartum remodeling of the cervix is associated with increased resident macrophages (Mφ), proinflammatory processes, and extracellular matrix degradation. This study tested the hypothesis that expression of genes unique to Mφs characterizes the prepartum from unremodeled nonpregnant cervix. Perfused cervix from prepartum day 21 postbreeding (D21) or nonpregnant (NP) rats, with or without Mφs, had RNA extracted and whole genome microarray analysis performed. By subtractive analyses, expression of 194 and 120 genes related to Mφs in the cervix from D21 rats were increased and decreased, respectively. In both D21 and NP groups, 158 and 57 Mφ genes were also more or less up- or down-regulated, respectively. Mφ gene expression patterns were most strongly correlated within groups and in 5 major clustering patterns. In the cervix from D21 rats, functional categories and canonical pathways of increased expression by Mφ gene related to extracellular matrix, cell proliferation, differentiation, as well as cell signaling. Pathways were characteristic of inflammation and wound healing, e.g., CD163, CD206, and CCR2. Signatures of only inflammation pathways, e.g., CSF1R, EMR1, and MMP12 were common to both D21 and NP groups. Thus, a novel and complex balance of Mφ genes and clusters differentiated the degraded extracellular matrix and cellular genomic activities in the cervix before birth from the unremodeled state. Predicted Mφ activities, pathways, and networks raise the possibility that expression patterns of specific genes characterize and promote prepartum remodeling of the cervix for parturition at term and with preterm labor.

Long-term hypoxia increases calcium affinity of BK channels in ovine fetal and adult cerebral artery smooth muscle.

Acclimatization to high-altitude, long-term hypoxia (LTH) reportedly alters cerebral artery contraction-relaxation responses associated with changes in K(+) channel activity. We hypothesized that to maintain oxygenation during LTH, basilar arteries (BA) in the ovine adult and near-term fetus would show increased large-conductance Ca(2+) activated potassium (BK) channel activity. We measured BK channel activity, expression, and cell surface distribution by use of patch-clamp electrophysiology, flow cytometry, and confocal microscopy, respectively, in myocytes from normoxic control and LTH adult and near-term fetus BA. Electrophysiological data showed that BK channels in LTH myocytes exhibited 1) lowered Ca(2+) set points, 2) left-shifted activation voltages, and 3) longer dwell times. BK channels in LTH myocytes also appeared to be more dephosphorylated. These differences collectively make LTH BK channels more sensitive to activation. Studies using flow cytometry showed that the LTH fetus exhibited increased BK ß1 subunit surface expression. In addition, in both fetal groups confocal microscopy revealed increased BK channel clustering and colocalization to myocyte lipid rafts. We conclude that increased BK channel activity in LTH BA occurred in association with increased channel affinity for Ca(2+) and left-shifted voltage activation. Increased cerebrovascular BK channel activity may be a mechanism by which LTH adult and near-term fetal sheep can acclimatize to long-term high altitude hypoxia. Our findings suggest that increasing BK channel activity in cerebral myocytes may be a therapeutic target to ameliorate the adverse effects of high altitude in adults or of intrauterine hypoxia in the fetus.

Cerebral artery alpha-1 AR subtypes: high altitude long-term acclimatization responses.

In response to hypoxia and other stress, the sympathetic (adrenergic) nervous system regulates arterial contractility and blood flow, partly through differential activities of the alpha1 (α1) - adrenergic receptor (AR) subtypes (α1A-, α1B-, and α1D-AR). Thus, we tested the hypothesis that with acclimatization to long-term hypoxia (LTH), contractility of middle cerebral arteries (MCA) is regulated by changes in expression and activation of the specific α1-AR subtypes. We conducted experiments in MCA from adult normoxic sheep maintained near sea level (300 m) and those exposed to LTH (110 days at 3801 m). Following acclimatization to LTH, ovine MCA showed a 20% reduction (n = 5; P<0.05) in the maximum tension achieved by 10-5 M phenylephrine (PHE). LTH-acclimatized cerebral arteries also demonstrated a statistically significant (P<0.05) inhibition of PHE-induced contractility in the presence of specific α1-AR subtype antagonists. Importantly, compared to normoxic vessels, there was significantly greater (P<0.05) α1B-AR subtype mRNA and protein levels in LTH acclimatized MCA. Also, our results demonstrate that extracellular regulated kinase 1 and 2 (ERK1/2)-mediated negative feedback regulation of PHE-induced contractility is modulated by α1B-AR subtype. Overall, in ovine MCA, LTH produces profound effects on α1-AR subtype expression and function.

"Surprised by joy"*: four decades of contributions to developmental physiology.

Presented at the 40th Anniversary Celebration of the Center for Perinatal Biology of Loma Linda University School of Medicine, honoring Dr. Longo for his 40 years of extraordinary leadership and service, February 11, 2013.

Acclimatization to long-term hypoxia: gene expression in ovine carotid arteries.

Exposure to acute high-altitude hypoxia is associated with an increase in cerebral blood flow (CBF) as a consequence of low arterial O2 tension. However, in response to high altitude acclimatization, CBF returns to levels similar to those at sea level, and tissue blood flow is maintained by an increase in angiogenesis. Of consequence, dysregulation of the acclimatization responses and CBF can result in acute mountain sickness, acute cerebral and/or pulmonary edema. To elucidate the signal transduction pathways involved in successful acclimatization to high altitude, in ovine carotid arteries, we tested the hypothesis that high altitude-associated long-term hypoxia results in changes in gene expression of critical signaling pathways. We acclimatized nonpregnant adult sheep to 3,801 m altitude for ∼110 days and conducted oligonucleotide microarray experiments on carotid arteries. Of a total of 116 regulated genes, 58 genes were significantly upregulated and 58 genes were significantly downregulated (each >2-fold, P < 0.05). Major upregulated genes included suprabasin and myelin basic protein, whereas downregulated genes included BAG2. Several of these genes are known to activate the ERK canonical signal transduction pathway and the process of angiogenesis. We conclude that among other changes, the altered signal transduction molecules involved in high-altitude acclimatization are associated ERK activation and angiogenesis.