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Exposure to high-altitude chronic hypoxia during pregnancy may cause pulmonary hypertension in neonates, as a result of vasoconstriction and vascular remodeling. We hypothesized that susceptibility to pulmonary hypertension, due to an augmented expression and activity of the RhoA/Rho-kinase (ROCK) pathway in these neonates, can be reduced by daily administration of fasudil, a ROCK inhibitor. We studied 10 highland newborn lambs with conception, gestation, and birth at 3,600 m in Putre, Chile. Five highland controls (HLC) were compared with 5 highland lambs treated with fasudil (HL-FAS; 3 mg·kg−1·day−1 iv for 10 days). Ten lowland controls were studied in Lluta (50 m; LLC). During the 10 days of fasudil daily administration, the drug decreased pulmonary arterial pressure (PAP) and resistance (PVR), basally and during a superimposed episode of acute hypoxia. HL-FAS small pulmonary arteries showed diminished muscular area and a reduced contractile response to the thromboxane analog U46619 compared with HLC. Hypoxia, but not fasudil, changed the protein expression pattern of the RhoA/ROCKII pathway. Moreover, HL-FAS lungs expressed less pMYPT1T850 and pMYPT1T696 than HLC, with a potential increase of the myosin light chain phosphatase activity. Finally, hypoxia induced RhoA, ROCKII, and PKG mRNA expression in PASMCs of HLC, but fasudil reduced them (HL-FAS) similarly to LLC. We conclude that fasudil decreases the function of the RhoA/ROCK pathway, reducing the PAP and PVR in chronically hypoxic highland neonatal lambs. The inhibition of ROCKs by fasudil may offer a possible therapeutic tool for the pulmonary hypertension of the neonates.

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.

Compared with lowland species, fetal life for mammalian species whose mothers live in high altitude is demanding. For instance, fetal llamas have to cope with the low fetal arterial PO2 of all species, but also the likely superimposition of hypoxia as a result of the decreased oxygen environment in which the mother lives in the Andean altiplano. When subjected to acute hypoxia the llama fetus responds with an intense peripheral vasoconstriction mediated by alpha-adrenergic mechanisms plus high plasma concentrations of catecholamines and neuropeptide Y (NPY). Endothelial factors such as NO and endothelin-1 also play a role in the regulation of local blood flows. Unlike fetuses of lowland species such as the sheep, the llama fetus shows a profound cerebral hypometabolic response to hypoxia, decreasing cerebral oxygen consumption, Na-K-ATPase activity and temperature, and resulting in an absence of seizures and apoptosis in neural cells. These strategies may have evolved to prevent hypoxic injury to the brain or other organs in the face of the persistent hypobaric hypoxia of life in the Andean altiplano.

Lack of dietary iodine is associated with thyroid insufficiency and its dire consequences including cretinism, yet territories severely deficient in iodine are home to many species of wild animals. The premise of our work is that an adaptation must be in place in order to allow these animals to thrive. We collected phyllotine rodents of the genus Auliscomys from the Altiplanic region of North Chile, an area historically associated with goitre and other manifestations of iodine deficiency disorders. The iodide concentration in the stream water in this locality, at

To mimic the two-signal requirements for T cell activation mediated by ligands, we exposed the superantigens SEA or SEE (signal 1) to T cells incubated with HLA-DR/LFA-3 or HLA-DR/B7-1-CHO transfected cells (signal 2). LFA-3 costimulation was able to induce T cell proliferation as well as IFN-gamma and IL-4 production at similar levels as in cells induced by B7-1. Analysis of the CD28RE of the IL-2 promoter showed specific transcription factor recruitment at the CD28RE element upon induction by B7-1/SEE. Further functional studies with an IL-2 enhancer-promoter carrying either wild type or mutated versions of the CD28RE site revealed that this element is necessary for full activation upon B7-1 costimulation. While both CD28/B7-1 and CD2/LFA-3 costimulation resulted in the up-regulation of IL-4 and IFN-gamma promoters, IL-2 promoter activity and production of IL-2 were only seen after B7-1 costimulation. However, contrary to what has been previously proposed, we show that costimulation with either B7-1 or LFA-3 further enhanced the ERK-2 activity and strongly activated the p38 MAPK pathway, but only B7-1 costimulation induced high levels of JNK-1 activity. These data suggest that the differential effect of CD28 vs. CD2 can be related to the difference in the ability of the two pathways to induce JNK-1 activity.

Unlike fetal animals of lowland species, the llama fetus does not increase its cerebral blood flow during an episode of acute hypoxaemia. This study tested the hypothesis that the fetal llama brain maintains cerebral hemispheric O2 consumption by increasing cerebral O2 extraction rather than decreasing cerebral oxygen utilisation during acute hypoxaemia. Six llama fetuses were surgically instrumented under general anaesthesia at 217 days of gestation (term ca 350 days) with vascular and amniotic catheters in order to carry out cardiorespiratory studies. Following a control period of 1 h, the llama fetuses underwent 3 x 20 min episodes of progressive hypoxaemia, induced by maternal inhalational hypoxia. During basal conditions and during each of the 20 min of hypoxaemia, fetal cerebral blood flow was measured with radioactive microspheres, cerebral oxygen extraction was calculated, and fetal cerebral hemispheric O2 consumption was determined by the modified Fick principle. During hypoxaemia, fetal arterial O2 tension and fetal pH decreased progressively from 24 +/- 1 to 20 +/- 1 Torr and from 7.36 +/- 0.01 to 7.33 +/- 0.01, respectively, during the first 20 min episode, to 16 +/- 1 Torr and 7.25 +/- 0.05 during the second 20 min episode and to 14 +/- 1 Torr and 7.21 +/- 0.04 during the final 20 min episode. Fetal arterial partial pressure of CO2 (P(a,CO2), 42 +/- 2 Torr) remained unaltered from baseline throughout the experiment. Fetal cerebral hemispheric blood flow and cerebral hemispheric oxygen extraction were unaltered from baseline during progressive hypoxaemia. In contrast, a progressive fall in fetal cerebral hemispheric oxygen consumption occurred during the hypoxaemic challenge. In conclusion, these data do not support the hypothesis that the fetal llama brain maintains cerebral hemispheric O2 consumption by increasing cerebral hemispheric O2 extraction. Rather, the data show that in the llama fetus, a reduction in cerebral hemispheric metabolism occurs during acute hypoxaemia.

Environmental chemicals may be involved in the etiology of breast cancers. Many studies have addressed the association between cancer in humans and agricultural pesticide exposure. Organophosphorous pesticides have been used extensively to control mosquito plagues. Parathion and malathion are organophosphorous pesticides extensively used to control a wide range of sucking and chewing pests of field crops, fruits, and vegetables. They have many structural similarities with naturally occurring compounds, and their primary target of action in insects is the nervous system; they inhibit the release of the enzyme acetylcholinesterase at the synaptic junction. Eserine, parathion, and malathion are cholinesterase inhibitors responsible for the hydrolysis of body choline esters, including acetylcholine at cholinergic synapses. Atropine, a parasympatholytic alkaloid, is used as an antidote to acetylcholinesterase inhibitors. The aim of this study was to examine whether pesticides were able to induce malignant transformation of the rat mammary gland and to determine whether alterations induced by these substances increase the cholinergic activation influencing such transformation. These results showed that eserine, parathion, and malathion increased cell proliferation of terminal end buds of the 44-day-old mammary gland of rats, followed by formation of 8.6, 14.3, and 24.3% of mammary carcinomas, respectively, after about 28 months. At the same time, acetylcholinesterase activity decreased in the serum of these animals from 9.78 +/- 0.78 U/mL in the control animals to 3.05 +/- 0.06 U/mL; 2.57 +/- 0.15 U/mL; and 3.88 +/- 0.44 U/mL in the eserine-, parathion-, and malathion-treated groups, respectively. However, atropine alone induced a significant (p < 0.05) decrease in the acetylcholinesterase activity from the control value of 9.78 +/- 0.78 to 4.38 +/- 0.10 for atropine alone, to 1.32 +/- 0.06 for atropine in combination with eserine, and 2.39 +/- 0.29 for atropine with malathion, and there was no mammary tumor formation. These results indicate that organophosphorous pesticides induce changes in the epithelium of mammary gland influencing the process of carcinogenesis, and such alterations occur at the level of nervous system by increasing the cholinergic stimulation.

La mortalidad por cancer de mama ha ido aumentando en Arica Chile, donde ha sobrepasado las tasas nacionales 11 veces entre los anos 1990 y 2010. La ciudad de Arica recibio aspersiones del pesticida organofosforado malation, con el fin de controlar la mosca mediterranea, por primera vez hace 33 anos. Por otra parte hemos demostrado que un tratamiento con malation induce la formacion de carcinomas mamarios en ratas hembras Sprague Dowley. El objetivo de este trabajo es encontrar una relacion entre las aspersiones con malation y el aumento en la tasa de mortalidad por cancer de mama que se ha observado en Arica en los ultimos anos. Se extrajeron de bases de datos, los casos de cancer mamario diagnosticados entre 1995 y 2005, en los Hospitales Dr. Juan Noe Crevani de Arica y Ernesto Torres de Iquique. El numero de pacientes diagnosticados con cancer de mama fue 100 en Arica y 58 en Iquique, ciudad que nunca fue fumigada con malation y con una poblacion similar a la de Arica durante esos anos. El analisis estadistico de las caracteristicas de la muestra, en relacion a los factores de riesgo de cancer mamario, mostro que no hay diferencia significativa entre las mujeres de Arica y de Iquique. Sin embargo, las mujeres con mayor tiempo de exposicion al malation fueron 5,7 veces mas propensas a ser diagnosticadas con cancer de mama (OR = 5,7, p

We determined whether store-operated channels (SOC) are involved in neonatal pulmonary artery function under conditions of acute and chronic hypoxia, using newborn sheep gestated and born either at high altitude (HA, 3,600 m) or low altitude (LA, 520 m). Cardiopulmonary variables were recorded in vivo, with and without SOC blockade by 2-aminoethyldiphenylborinate (2-APB), during basal or acute hypoxic conditions. 2-APB did not have effects on basal mean pulmonary arterial pressure (mPAP), cardiac output, systemic arterial blood pressure, or systemic vascular resistance in both groups of neonates. During acute hypoxia 2-APB reduced mPAP and pulmonary vascular resistance in LA and HA, but this reduction was greater in HA. In addition, isolated pulmonary arteries mounted in a wire myograph were assessed for vascular reactivity. HA arteries showed a greater relaxation and sensitivity to SOC blockers than LA arteries. The pulmonary expression of two SOC-forming subunits, TRPC4 and STIM1, was upregulated in HA. Taken together, our results show that SOC contribute to hypoxic pulmonary vasoconstriction in newborn sheep and that SOC are upregulated by chronic hypoxia. Therefore, SOC may contribute to the development of neonatal pulmonary hypertension. We propose SOC channels could be potential targets to treat neonatal pulmonary hypertension.

Pulmonary arterial hypertension is one of the most serious pathologies that can affect the 140 million people living at altitudes over 2500 m. The primary emphasis of this review is pulmonary artery hypertension in mammals (sheep and llamas) at high altitude, with specific focus on the heme oxygenase and carbon monoxide (HO-CO) system. We highlight the fact that the neonatal llama has neither pulmonary artery hypertension nor pulmonary vascular remodeling in the Andean altiplano. These neonates have an enhanced HO-CO system function, increasing the HO-1 protein expression and CO production by the pulmonary vessels, when compared to llamas raised at low altitude, or neonatal sheep raised at high altitude. The neonatal sheep has high altitude pulmonary artery hypertension in spite of enhancement of the NO system, with high eNOS protein expression and NO production by the lung. The gasotransmitters NO and CO are important in the regulation of the pulmonary vascular function at high altitudes in both high altitude acclimatized species, such as the sheep, and high altitude adapted species, such as the llama.

We determined whether postnatal pulmonary hypertension induced by 70% of pregnancy at high altitude (HA) persists once the offspring return to sea level and investigated pulmonary vascular mechanisms operating under these circumstances. Pregnant ewes were divided into two groups: conception, pregnancy, and delivery at low altitude (580 m, LLL) and conception at low altitude, pregnancy at HA (3,600 m) from 30% of gestation until delivery, and return to lowland (LHL). Pulmonary arterial pressure (PAP) was measured in vivo. Vascular reactivity and morphometry were assessed in small pulmonary arteries (SPA). Protein expression of vascular mediators was determined. LHL lambs had higher basal PAP and a greater increment in PAP after NG-nitro-l-arginine methyl ester (20.9 ± 1.1 vs. 13.7 ± 0.5 mmHg; 39.9 ± 5.0 vs. 18.3 ± 1.3 mmHg, respectively). SPA from LHL had a greater maximal contraction to K+ (1.34 ± 0.05 vs. 1.16 ± 0.05 N/m), higher sensitivity to endothelin-1 and nitroprusside, and persistence of dilatation following blockade of soluble guanylate cyclase. The heart ratio of the right ventricle-to-left ventricle plus septum was higher in the LHL relative to LLL. The muscle area of SPA (29.3 ± 2.9 vs. 21.1 ± 1.7%) and the protein expression of endothelial nitric oxide synthase (1.7 ± 0.1 vs. 1.1 ± 0.2), phosphodiesterase (1.4 ± 0.1 vs. 0.7 ± 0.1), and Ca2+-activated K+ channel (0.76 ± 0.16 vs. 0.30 ± 0.01) were greater in LHL compared with LLL lambs. In contrast, LHL had decreased heme oxygenase-1 expression (0.82 ± 0.26 vs. 2.22 ± 0.44) and carbon monoxide production (all P < 0.05). Postnatal pulmonary hypertension induced by 70% of pregnancy at HA promotes cardiopulmonary remodeling that persists at sea level.

The pregnant llama (Lama glama) has walked for millions of years through the thin oxygen trail of the Andean altiplano. We hypothesize that a pool of genes has been selected in the llama that express efficient mechanisms to withstand this low-oxygen milieu. The llama fetus responds to acute hypoxia with an intense peripheral vasoconstriction that is not affected by bilateral section of the carotid sinus nerves. Moreover, the increase in fetal plasma concentrations of vasoconstrictor hormones, such as catecholamines, neuropeptide Y, and vasopressin, is much greater in the llama than in the sheep fetus. Furthermore, treatment of fetal llamas with an alpha-adrenergic antagonist abolished the peripheral vasoconstriction and resulted in fetal cardiovascular collapse and death during acute hypoxia, suggesting an indispensable upregulation of alpha-adrenergic mechanisms in this high altitude species. Local endothelial factors such as nitric oxide (NO) also play a key role in the regulation of fetal adrenal blood flow and in the adrenal secretion of catecholamines and cortisol. Interestingly, in contrast to the human or sheep fetus, the llama fetus showed a small increase in brain blood flow during acute hypoxia, with no increase in oxygen extraction across the brain, and thereby a decrease in brain oxygen consumption. These results suggest that the llama fetus responds to acute hypoxia with hypometabolism. How this reduction in metabolism is produced and how the cells are preserved during this condition remain to be elucidated.

The adult llama ( Lama glama ) has several compensatory mechanisms that allow it to successfully survive at high altitude. Llama fetuses at 0.6–0.7 of gestation, and near-term llama fetuses studied close to surgery, did not increase cerebral blood flow and decreased cerebral oxygen delivery during acute hypoxemia. It is not known whether these responses were the result of immaturity or surgical stress. The aim of this study was to determine whether the lack of increase in cerebral blood flow and the decrease in cerebral oxygen delivery during hypoxemia in the fetal llama is characteristic of this high-altitude species near term, and under nonstressed conditions. We chronically catheterized 7 llamas and their fetuses near to term, at 0.7–0.9 of gestation. Fetal cardiac output, cerebral and regional blood flows, systemic blood pressure, heart rate, pH, and blood gases, organ vascular resistances and organ oxygen deliveries were determined at least 4 days after surgery, both during the basal state and after 1 hr of acute fetal hypoxemia. During hypoxemia the llama fetus did not increase cerebral blood flow and markedly decreased its cerebral oxygen delivery. There was also a marked decrease in kidney blood flow and oxygen delivery. These results indicate that, in contrast to fetuses of lowland species, the fetal llama does not increase the cerebral blood flow during hypoxemia, suggesting specific cellular mechanisms to preserve brain integrity during oxygen limitation.

Fetal sheep responds to H with blood hypertension, bradycardia, and cardiac output redistribution. Since the response to H in fetuses from species that evolved at high altitudes is not fully known, we studied the cardiovascular response to H in chronically catheterized fetal llamas. we determined in 7 fetal llamas (4 post surgery days) arterial gases, mean arterial pressure, heart rate, cardiac output and its distribution (radiolabelled microspheres) basally (50%Sat Hb descending aorta) and after 20 min of H (14% Sat Hb). The results were: X±SEM,*p