2-AMINOETHYLDIPHENYLBORINATE MODIFIES THE PULMONARY CIRCULATION IN PULMONARY HYPERTENSIVE NEWBORN LAMBS WITH PARTIAL GESTATION AT HIGH ALTITUDE.

Calcium signaling through store operated channels (SOC) is involved in hypoxic pulmonary hypertension. We determined whether a treatment with 2-aminoethyldiphenylborinate (2-APB), a compound with SOC blocker activity, reduces pulmonary hypertension and vascular remodeling. Twelve newborn lambs exposed to perinatal chronic hypoxia were studied, 6 of them received a 2-APB treatment and the other 6 received vehicle treatment, for 10 days in both cases. Throughout this period, we recorded cardiopulmonary variables and on day 11 we evaluated the response to an acute hypoxic challenge. Additionally, we assessed the vasoconstrictor and vasodilator function in isolated pulmonary arteries as well as their remodeling in lung slices. 2-APB reduced pulmonary arterial pressure at the third and tenth days, cardiac output between the fourth and eighth days, and pulmonary vascular resistance at the tenth day of treatment. The pulmonary vasoconstrictor response to acute hypoxia was reduced by the end of treatment. 2-APB also decreased maximal vasoconstrictor response to the thromboxane mimetic U46619 and endothelin-1 and increased maximal relaxation to 8-Br-cGMP. The maximal relaxation and potency to phosphodiesterase-5 and Rho-kinase inhibition with sildenafil and fasudil respectively, were also increased. Finally, 2-APB reduced the medial and adventitial layers' thickness, the expression of α-actin and the percentage of Ki67+ nuclei of small pulmonary arteries. Taken together, our results indicate that 2-APB reduces pulmonary hypertension, vasoconstrictor responses and pathological remodeling in pulmonary hypertensive lambs. We conclude that SOC targeting may be a useful strategy for the treatment of neonatal pulmonary hypertension, however, further testing of specific blockers is needed.

Impact of Maternal Melatonin Suppression on Amount and Functionality of Brown Adipose Tissue (BAT) in the Newborn Sheep.

In human and sheep newborns, brown adipose tissue (BAT) accrued during fetal development is used for newborn thermogenesis. Here, we explored the role of maternal melatonin during gestation on the amount and functionality of BAT in the neonate. We studied BAT from six lambs gestated by ewes exposed to constant light from 63% gestation until delivery to suppress melatonin (LL), six lambs gestated by ewes exposed to LL but receiving daily oral melatonin (12 mg at 1700 h, LL + Mel) and another six control lambs gestated by ewes maintained in 12 h light:12 h dark (LD). Lambs were instrumented at 2 days of age. At 4-6 days of age, they were exposed to 24°C (thermal neutrality conditions) for 1 h, 4°C for 1 h, and 24°C for 1 h. Afterward, lambs were euthanized and BAT was dissected for mRNA measurement, histology, and ex vivo experiments. LL newborns had lower central BAT and skin temperature under thermal neutrality and at 4°C, and higher plasma norepinephrine concentration than LD newborns. In response to 4°C, they had a pronounced decrease in skin temperature and did not increase plasma glycerol. BAT weight in LL newborns was about half of that of LD newborns. Ex vivo, BAT from LL newborns showed increased basal lipolysis and did not respond to NE. In addition, expression of adipogenic/thermogenic genes (UCP1, ADBR3, PPARγ, PPARα, PGC1α, C/EBPß, and perilipin) and of the clock genes Bmal1, Clock, and Per2 was increased. Remarkably, the effects observed in LL newborns were absent in LL + Mel newborns. Thus, our results support that maternal melatonin during gestation is important in determining amount and normal functionality of BAT in the neonate.

Circadian cortisol secretion and circadian adrenal responses to ACTH are maintained in dexamethasone suppressed capuchin monkeys (Cebus apella).

We tested the hypothesis that the capuchin monkey adrenal (Cebus apella) gland has oscillatory properties that are independent of adrenocorticotropic hormone (ACTH) by exploring under ACTH suppression by dexamethasone: (i) maintenance of a circadian rhythm of plasma cortisol and (ii) clock time dependency of plasma cortisol response to exogenous ACTH. The capuchin monkey had a clear ACTH and plasma cortisol rhythm. Dexamethasone treatment resulted in low non-rhythmic ACTH levels and decreased cortisol to 1/10 of control values; nevertheless, the circadian rhythm of plasma cortisol persisted. We found that cortisol response to exogenous ACTH was clock time-dependent. The maximal response to ACTH occurred at the acrophase of the cortisol rhythm (0800 h). These results suggest that the capuchin monkey adrenal cortex may possess intrinsic oscillatory properties that participate in the circadian rhythm of adrenal cortisol secretion and in the circadian cortisol response to ACTH.

Carbon monoxide: a novel pulmonary artery vasodilator in neonatal llamas of the Andean altiplano.

AIMS: To study the nitric oxide (NO) and carbon monoxide roles in the regulation of the pulmonary circulation in lowland and highland newborn sheep and llamas. METHODS AND RESULTS: We used neonatal sheep (Ovis aries) and llamas (Lama glama) whose gestation and delivery took place at low (580 m) or high (3600 m) altitude. In vivo, we measured the cardiopulmonary function basally and with a NO synthase (NOS) blockade and calculated the production of carbon monoxide by the lung. In vitro, we determined NOS and soluble guanylate cyclase (sGC) expression, NOS activity, and haemoxygenase (HO) expression in the lung. Pulmonary arterial pressure was elevated at high altitude in sheep but not in llamas. Sheep at high altitude relative to sea level had significantly greater total lung NOS activity and eNOS protein, but reduced sGC and HO expression and carbon monoxide production. In contrast, llamas showed no difference in NO function between altitudes, but a pronounced increase in pulmonary carbon monoxide production and HO expression at high altitude. CONCLUSIONS: In the llama, enhanced pulmonary carbon monoxide, rather than NO, protects against pulmonary hypertension in the newborn period at high altitude. This shift in pulmonary dilator strategy from NO to carbon monoxide has not been previously described, and it may give insight into new treatments for excessive pulmonary vasoconstriction.

Fetal brain hypometabolism during prolonged hypoxaemia in the llama.

In this study we looked for additional evidence to support the hypothesis that fetal llama reacts to hypoxaemia with adaptive brain hypometabolism. We determined fetal llama brain temperature, Na(+) and K(+) channel density and Na(+)-K(+)-ATPase activity. Additionally, we looked to see whether there were signs of cell death in the brain cortex of llama fetuses submitted to prolonged hypoxaemia. Ten fetal llamas were instrumented under general anaesthesia to measure pH, arterial blood gases, mean arterial pressure, heart rate, and brain and core temperatures. Measurements were made 1 h before and every hour during 24 h of hypoxaemia (n = 5), which was imposed by reducing maternal inspired oxygen fraction to reach a fetal arterial partial pressure of oxygen (P(a,O(2))) of about 12 mmHg. A normoxaemic group was the control (n = 5). After 24 h of hypoxaemia, we determined brain cortex Na(+)-K(+)-ATPase activity, ouabain binding, and the expression of NaV1.1, NaV1.2, NaV1.3, NaV1.6, TREK1, TRAAK and K(ATP) channels. The lack of brain cortex damage was assessed as poly ADP-ribose polymerase (PARP) proteolysis. We found a mean decrease of 0.56 degrees C in brain cortex temperature during prolonged hypoxaemia, which was accompanied by a 51% decrease in brain cortex Na(+)-K(+)-ATPase activity, and by a 44% decrease in protein content of NaV1.1, a voltage-gated Na(+) channel. These changes occurred in absence of changes in PARP protein degradation, suggesting that the cell death of the brain was not enhanced in the fetal llama during hypoxaemia. Taken together, these results provide further evidence to support the hypothesis that the fetal llama responds to prolonged hypoxaemia with adaptive brain hypometabolism, partly mediated by decreases in Na(+)-K(+)-ATPase activity and expression of NaV channels.