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Most mammals have a poor tolerance to hypoxia, and prolonged O2 restriction can lead to organ injury, particularly during fetal and early postnatal life. Nevertheless, the llama ( Lama Glama) has evolved efficient mechanisms to adapt to acute and chronic perinatal hypoxia. One striking adaptation is the marked peripheral vasoconstriction measured in the llama fetus in response to acute hypoxia, which allows efficient redistribution of cardiac output toward the fetal heart and adrenal glands. This strong peripheral vasoconstrictor tone is triggered by a carotid body reflex and critically depends on α-adrenergic signaling. A second adaptation is the ability of the llama fetus to protect its brain against hypoxic damage. During hypoxia, in the llama fetus there is no significant increase in brain blood flow. Instead, there is a fall in brain O2 consumption and temperature, together with a decrease of Na+-K+-ATPase activity and Na+ channels expression, protecting against seizures and neuronal death. Finally, the newborn llama does not develop pulmonary hypertension in response to chronic hypoxia. In addition to maintaining basal pulmonary arterial pressure at normal levels the pulmonary arterial pressor response to acute hypoxia is lower in highland than in lowland llamas. The protection against hypoxic pulmonary arterial hypertension and pulmonary contractile hyperreactivity is partly due to increased hemoxygenase-carbon monoxide signaling and decreased Ca2+ sensitization in the newborn llama pulmonary vasculature. These three striking physiological adaptations of the llama allow this species to live and thrive under the chronic influence of the hypobaric hypoxia of life at high altitude.

Nitric oxide (NO) is the main vasodilator agent that drives the rapid decrease of pulmonary vascular resistance for the respiratory onset during the fetal to neonatal transition. Nevertheless, the enhanced NO generation by the neonatal pulmonary arterial endothelium does not prevent development of hypoxic pulmonary hypertension in species without an evolutionary story at high altitude. Therefore, this study aims to describe the limits of the NO function at high-altitude during neonatal life in the sheep as an animal model without tolerance to perinatal hypoxia. We studied the effect of blockade of NO synthesis with l -NAME in the cardiopulmonary response of lowland (580 m) and highland (3600 m) newborn lambs basally and under an episode of acute hypoxia. We also determined the pulmonary expression of proteins that mediate the actions of the NO vasodilator pathway in the pulmonary vasoactive tone and remodeling. We observed an enhanced nitrergic function in highland lambs under basal conditions, evidenced as a markedly greater increase in basal mean pulmonary arterial pressure (mPAP) and resistance (PVR) under blockade of NO synthesis. Further, acute hypoxic challenge in lowland lambs infused with l -NAME markedly increased their mPAP and PVR to values greater than baseline, whilst in highland animals under NO synthesis blockade, these variables did not show additional increase in response to low PO2. Highland animals showed increased pulmonary RhoA expression, decreased PSer188-RhoA fraction, increased PSer311-p65-NFқβ fraction and up-regulated smooth muscle α-actin, relative to lowland controls. Taken together our data suggest that NO-mediated vasodilation is important to keep a low pulmonary vascular resistance under basal conditions and acute hypoxia at low-altitude. At high-altitude, the enhanced nitrergic signaling partially prevents excessive pulmonary hypertension but does not protect against acute hypoxia. The decreased vasodilator efficacy of nitrergic tone in high altitude lambs could be in part due to increased RhoA signaling that opposes to NO action in the hypoxic pulmonary circulation.

Key points Perinatal hypoxia causes pulmonary hypertension in neonates, including humans. However, in species adapted to hypoxia, such as the llama, there is protection against pulmonary hypertension. Nitric oxide (NO) is a vasodilatator with an established role in the cardiopulmonary system of many species, but its function in the hypoxic pulmonary vasoconstrictor response in the newborn llama is unknown. Therefore, we studied the role of NO in the cardiopulmonary responses to acute hypoxia in high- and lowland newborn llamas. We show that high- compared to lowland newborn llamas have a reduced pulmonary vasoconstrictor response to acute hypoxia. Protection against excessive pulmonary vasoconstriction in the highland llama is mediated via enhancement of NO pathways, including increased MYPT1 and reduced ROCK expression as well as Ca2+ desensitization. Blunting of pulmonary hypertensive responses to hypoxia through enhanced NO pathways may be an adaptive mechanism to withstand life at high altitude in the newborn llama. Abstract Llamas are born in the Alto Andino with protection against pulmonary hypertension. The physiology underlying protection against pulmonary vasoconstrictor responses to acute hypoxia in highland species is unknown. We determined the role of nitric oxide (NO) in the cardiopulmonary responses to acute hypoxia in high- and lowland newborn llamas. The cardiopulmonary function of newborn llamas born at low (580 m) or high altitude (3600 m) was studied under acute hypoxia, with and without NO blockade. In pulmonary arteries, we measured the reactivity to potassium and sodium nitroprusside (SNP), and in lung we determined the content of cGMP and the expression of the NO-related proteins: BKCa, PDE5, PSer92-PDE5, PKG-1, ROCK1 and 2, MYPT1, PSer695-MYPT1, PThr696-MYPT1, MLC20 and PSer19-MLC20. Pulmonary vascular remodelling was evaluated by morphometry and based on α-actin expression. High- compared to lowland newborn llamas showed lower in vivo pulmonary arterial pressor responses to acute hypoxia. This protection involved enhanced NO function, as NO blockade reverted the effect and the pulmonary arterial dilatator response to SNP was significantly enhanced in highland neonates. The pulmonary expression of ROCK2 and the phosphorylation of MLC20 were lower in high-altitude llamas. Conversely, MYPT1 was up-regulated whilst PSer695-MYPT1 and PThr695-MYPT1 did not change. Enhanced NO-dependent mechanisms were insufficient to prevent pulmonary arterial remodelling. Combined, the data strongly support that in the highland newborn llama reduced ROCK, increased MYPT1 expression and Ca2+ desensitization in pulmonary tissue allow an enhanced NO biology to limit hypoxic pulmonary constrictor responses. Blunting of hypoxic pulmonary hypertensive responses may be an adaptive mechanism to life at high altitude.

KEY POINTS: Perinatal hypoxia causes pulmonary hypertension in neonates, including humans. However, in species adapted to hypoxia, such as the llama, there is protection against pulmonary hypertension. Nitric oxide (NO) is a vasodilatator with an established role in the cardiopulmonary system of many species, but its function in the hypoxic pulmonary vasoconstrictor response in the newborn llama is unknown. Therefore, we studied the role of NO in the cardiopulmonary responses to acute hypoxia in high‐ and lowland newborn llamas. We show that high‐ compared to lowland newborn llamas have a reduced pulmonary vasoconstrictor response to acute hypoxia. Protection against excessive pulmonary vasoconstriction in the highland llama is mediated via enhancement of NO pathways, including increased MYPT1 and reduced ROCK expression as well as Ca(2+) desensitization. Blunting of pulmonary hypertensive responses to hypoxia through enhanced NO pathways may be an adaptive mechanism to withstand life at high altitude in the newborn llama. ABSTRACT: Llamas are born in the Alto Andino with protection against pulmonary hypertension. The physiology underlying protection against pulmonary vasoconstrictor responses to acute hypoxia in highland species is unknown. We determined the role of nitric oxide (NO) in the cardiopulmonary responses to acute hypoxia in high‐ and lowland newborn llamas. The cardiopulmonary function of newborn llamas born at low (580 m) or high altitude (3600 m) was studied under acute hypoxia, with and without NO blockade. In pulmonary arteries, we measured the reactivity to potassium and sodium nitroprusside (SNP), and in lung we determined the content of cGMP and the expression of the NO‐related proteins: BKCa, PDE5, PSer92‐PDE5, PKG‐1, ROCK1 and 2, MYPT1, PSer695‐MYPT1, PThr696‐MYPT1, MLC20 and PSer19‐MLC20. Pulmonary vascular remodelling was evaluated by morphometry and based on α‐actin expression. High‐ compared to lowland newborn llamas showed lower in vivo pulmonary arterial pressor responses to acute hypoxia. This protection involved enhanced NO function, as NO blockade reverted the effect and the pulmonary arterial dilatator response to SNP was significantly enhanced in highland neonates. The pulmonary expression of ROCK2 and the phosphorylation of MLC20 were lower in high‐altitude llamas. Conversely, MYPT1 was up‐regulated whilst PSer695‐MYPT1 and PThr695‐MYPT1 did not change. Enhanced NO‐dependent mechanisms were insufficient to prevent pulmonary arterial remodelling. Combined, the data strongly support that in the highland newborn llama reduced ROCK, increased MYPT1 expression and Ca(2+) desensitization in pulmonary tissue allow an enhanced NO biology to limit hypoxic pulmonary constrictor responses. Blunting of hypoxic pulmonary hypertensive responses may be an adaptive mechanism to life at high altitude.

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.

A few mammalian species, such as diving mammals and the mammals that live in the low-oxygen environment of high altitudes, e.g. the South American Camelidae, can thrive during prolonged periods of oxygen shortage. To live at high altitudes, Camelidae have evolved strategies that are apparently genetically determined, since they are maintained in animals born and living at sea level. In llamas, a domestic Camelidae, these adaptations include low haemoglobin P50 (partial pressure of oxygen where half of the oxygen binding sites are saturated with oxygen), small elliptical red cells with high haemoglobin concentration, a slight increase in blood haemoglobin concentration, high muscle myoglobin concentration, more efficient oxygen extraction, and high lactic dehydrogenase activity, compared with low-altitude species (Llanos et al. 2003). In addition, the llama maintains a low pulmonary arterial pressure, with an absence of highly muscularized pulmonary arterioles, despite living at very high altitudes (Harris et al. 1982). Fetal life imposes an additional oxygen challenge to high altitude mammals. Fetal arterial partial pressure of oxygen (Pa,O2) is 30 mmHg in the umbilical vein at sea level, and is much lower in high-altitude pregnancies. Compared with fetuses of lowland species, such as sheep, the fetal llama also has low haemoglobin P50 (Moraga et al. 1996), lower basal cardiac output and organ blood flow (Llanos et al. 2003), and a more efficient total oxygen extraction (Benavides et al. 1989). Remarkably, unlike fetuses of lowland species, the llama fetus responds to acute hypoxaemia with only a minor increase in cerebral blood flow (Llanos et al. 2003). Furthermore, the fetal llama shows a progressive fall in brain oxygen delivery and cerebral oxygen consumption with progression of the fetal hypoxaemia (Llanos et al. 2002). We have shown that the electrocorticogram flattens under these conditions of hypoxaemia, and seizure activity does not occur, which suggests that no hypoxaemic damage occurs (Llanos et al. 2003). A pressing question is how the oxygen delivery and consumption can decrease in the fetal llama brain during hypoxaemia, without concomitant damage. One possible explanation is that the neurones undergo hypometabolism during the hypoxaemic episode. In the neurone, more than 50% of energy expenditure is due to the continuous ion pumping needed to maintain the ion electrochemical gradients, which in turn are needed to support the repetitive opening of Na+ and K+ channels, which account for much of the brain's electrical activity (Erecinska & Silver, 1994). In the brain of most vertebrates, ATP rapidly decreases during severe oxygen deprivation; ionic gradients collapse leading to depolarization, followed by a strong and massive intracellular Ca2+ increase, and the onset of the cell death events. Nevertheless, some vertebrates, such as an anoxia-tolerant species of turtles (Trachemys scripta), have developed adaptive brain hypometabolism as a defence response against anoxia. One of the principal features accounting for brain hypometabolism is the reduction in activity of the Na+–K+-ATPase pari passu, with a decreased permeability of Na+ and K+ ions and/or a lesser expression of Na+ and K+ channels in the central nervous system (Hylland et al. 1997; Pek-Scott & Lutz, 1998; Bickler & Buck, 1998; Nilsson & Lutz, 2004). An analogous strategy, imposed by the sustained hypobaric hypoxia of life at extreme altitude, may have evolved in the fetal llama to avoid cerebral damage. In this study, we looked for additional evidence to support the hypothesis that the fetal llama reacts to hypoxaemia with adaptive brain hypometabolism. To further support this contention, we determined whether fetal llama brain temperature decreases, and whether this was associated with a reduction of ion channel density and Na+–K+-ATPase activity, with no signs of cell death in the brain cortex of llama fetuses submitted to prolonged hypoxaemia.

The hypothesis that nitric oxide plays a key role in the regulation of adrenal blood flow and plasma concentrations of cortisol and catecholamines under basal and hypoxaemic conditions in the llama fetus was tested. At 0.6-0.8 of gestation, 11 llama fetuses were surgically prepared for long-term recording under anaesthesia with vascular and amniotic catheters. Following recovery all fetuses underwent an experimental protocol based on 1 h of normoxaemia, 1 h of hypoxaemia and 1 h of recovery. In nine fetuses, the protocol occurred during fetal i.v. infusion with saline and in five fetuses during fetal i.v. treatment with the nitric oxide synthase inhibitor l-NAME. Adrenal blood flow was determined by the radiolabelled microsphere method during each of the experimental periods during saline infusion and treatment with l-NAME. Treatment with l-NAME during normoxaemia led to a marked fall in adrenal blood flow and a pronounced increase in plasma catecholamine concentrations, but it did not affect plasma ACTH or cortisol levels. In saline-infused fetuses, acute hypoxaemia elicited an increase in adrenal blood flow and in plasma ACTH, cortisol, adrenaline and noradrenaline concentrations. Treatment with l-NAME did not affect the increase in fetal plasma ACTH, but prevented the increments in adrenal blood flow and in plasma cortisol and adrenaline concentrations during hypoxaemia in the llama fetus. In contrast, l-NAME further enhanced the increase in fetal plasma noradrenaline. These data support the hypothesis that nitric oxide has important roles in the regulation of adrenal blood flow and adrenal corticomedullary functions during normoxaemia and hypoxaemia functions in the late gestation llama fetus.

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.

The fetal llama ( Lama glama ; a species adapted to live in chronic hypoxia in the highlands of the Andes) did not increase cerebral blood flow and reduce the brain oxygen uptake during hypoxemia. Although nitric oxide (NO) is a normal mediator in the regulation of vascular tone and synaptic transmission, NO overproduction by hypoxemia could produce neuronal damage. We hypothesized that nitric oxide synthase (NOS) activity is either maintained or reduced in the central nervous system of the llama fetuses submitted to chronic hypoxemia. Approximately 85% of the Ca 2+ -dependent NOS activity was soluble, at least 12% was associated with the mitochondrial fraction, and less than 5% remains associated with microsomes. To understand the role of NO in chronic hypoxemia, we determined the effect of 24-h hypoxemia on NOS activity in the central nervous system. No changes in activity or the subcellular distribution of NOS activity in brain tissues after hypoxemia were found. We proposed that the lack of changes in NOS activity in the llama under hypoxemia could be a cytoprotective mechanism inherent to the llama, against possible toxic effects of NO.

The fetal llama has a marked increase in the peripheral vascular resistance and no augmentation of brain blood flow during hypoxemia. In spite of the substantial plasma arginine-vasopressin (AVP) increase during hypoxemia, up to 8 times greater than in fetal sheep, there are no changes of carotid and femoral blood flows during hypoxemia with a V1 receptor blockade, as is seen in the fetal sheep. The aim of this study was to assess the role of AVP function in mediating the combined ventricular output and organ blood flow in the hypoxemic llama fetus. Six fetal llamas at 0.65 of gestation were instrumented under general anesthesia, and cardiorespiratory responses and blood flows determined under normoxemic and hypoxemic conditions. The AVP effect was determined using a V1 antagonist during normoxemic and hypoxemic conditions. Organ blood flows were measured with the radioactive microsphere technique. No significant differences in organ blood flow or in their vascular resistances were seen between the control and treated fetuses during hypoxemia. We conclude that V1 blockade did not have any important role in the cardiovascular response to acute hypoxemia in the llama fetus, in contrast with lowland fetuses. AVP may be playing a role in other regions, possibly in kidney or lung, during hypoxemia.

This study tested the hypothesis that the fetal llama, a species adapted to the chronic hypoxia of life at high altitude, demonstrates a potent carotid chemoreflex influence on adrenocortical responses during acute hypoxemia. Plasma ACTH and cortisol concentrations, and mesencephalic and adrenal blood flows were measured during a 1-h period of acute hypoxemia in six intact and four carotid sinus-denervated llama fetuses at 0.6-0.7 of gestation. Fetal PaO2 was reduced from approximately 23 to about 14 mm Hg in both intact and carotid-denervated groups during acute hypoxemia. During hypoxemia, fetal plasma ACTH, adrenal blood flow, and, therefore, delivery of ACTH to the adrenals increased to similar extents in both intact and carotid-denervated fetal llamas. Despite this, the increase in plasma cortisol in hypoxemia in intact fetuses was absent in carotid-denervated fetuses. In addition, the increase in delivery of cortisol to the mesencephalon calculated in intact fetuses during hypoxemia did not occur in the carotid-denervated group. These data suggest that the integrity of the carotid chemoreceptors is indispensable to cortisol release during acute hypoxemia in the llama fetus, even at 0.6-0.7 of gestation.

Perinatal exposure to chronic hypoxia induces sustained pulmonary hypertension and structural and functional changes in both pulmonary and systemic vascular beds. The aim of this study was to analyze consequences of high-altitude chronic hypoxia during gestation and early after birth in pulmonary and femoral vascular responses in newborn sheep. Lowland (LLNB; 580 m) and highland (HLNB; 3,600 m) newborn lambs were cathetherized under general anesthesia and submitted to acute sustained or stepwise hypoxic episodes. Contractile and dilator responses of isolated pulmonary and femoral small arteries were analyzed in a wire myograph. Under basal conditions, HLNB had a higher pulmonary arterial pressure (PAP; 20.2 ± 2.4 vs. 13.6 ± 0.5 mmHg, P < 0.05) and cardiac output (342 ± 23 vs. 279 ± 13 ml·min−1·kg−1, P < 0.05) compared with LLNB. In small pulmonary arteries, HLNB showed greater contractile capacity and higher sensitivity to nitric oxide. In small femoral arteries, HLNB had lower maximal contraction than LLNB with higher maximal response and sensitivity to noradrenaline and phenylephrine. In acute superimposed hypoxia, HLNB reached higher PAP and femoral vascular resistance than LLNB. Graded hypoxia showed that average PAP was always higher in HLNB compared with LLNB at any Po2. Newborn lambs from pregnancies at high altitude have stronger pulmonary vascular responses to acute hypoxia associated with higher arterial contractile status. In addition, systemic vascular response to acute hypoxia is increased in high-altitude newborns, associated with higher arterial adrenergic responses. These responses determined in intrauterine life and early after birth could be adaptive to chronic hypoxia in the Andean altiplano.

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.

The fetal llama responds to hypoxemia, with a marked peripheral vasoconstriction but, unlike the sheep, with little or no increase in cerebral blood flow. We tested the hypothesis that the role of nitric oxide (NO) may be increased during hypoxemia in this species, to counterbalance a strong vasoconstrictor effect. Ten fetal llamas were operated under general anesthesia. Mean arterial pressure (MAP), heart rate, cardiac output, total vascular resistance, blood flows, and vascular resistances in cerebral, carotid and femoral vascular beds were determined. Two groups were studied, one with nitric oxide synthase (NOS) blocker NG-nitro-l-arginine methyl ester (l-NAME), and the other with 0.9% NaCl (control group), during normoxemia, hypoxemia, and recovery. During normoxemia, l-NAME produced an increase in fetal MAP and a rapid bradycardia. Cerebral, carotid, and femoral vascular resistance increased and blood flow decreased to carotid and femoral beds, while cerebral blood flow did not change significantly. However, during hypoxemia cerebral and carotid vascular resistance fell by 44% from its value in normoxemia after l-NAME, although femoral vascular resistance progressively increased and remained high during recovery. We conclude that in the llama fetus: 1) NO has an important role in maintaining a vasodilator tone during both normoxemia and hypoxemia in cerebral and femoral vascular beds and 2) during hypoxemia, NOS blockade unmasked the action of other vasodilator agents that contribute, with nitric oxide, to preserving blood flow and oxygen delivery to the tissues.

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.

In the sheep fetus during late gestation, episodes of reduced oxygenation evoke an integrated cardiovascular response that facilitates fetal survival during the period of adversity (Cohn et al. 1974; Giussani et al. 1994a). However, the pattern and magnitude of the fetal defence response to reduced oxygen delivery vary according to the severity of the challenge. In response to acute moderate hypoxaemia, reducing the fetal arterial oxygen pressure (Pa,O2) from ≈24 to ≈12 mmHg, the fetal cardiovascular responses include bradycardia, an increase in arterial blood pressure and an increase in femoral vascular resistance (Boddy et al. 1974; Giussani et al. 1994a). The increase in femoral vascular resistance contributes to peripheral vasoconstriction. This aids the redistribution of the fetal combined ventricular output (CVO) in favour of the adrenal, myocardial and cerebral circulations, which undergo vasodilatation during hypoxaemia (Cohn et al. 1974; Reller et al. 1995; Green et al. 1996; McCrabb & Harding, 1996; van Bel et al. 1997). In response to acute asphyxia, secondary to reduced uterine blood flow, and leading to reductions in the fetal Pa,O2 below 12 mmHg with associated acidaemia, generalised vasoconstriction occurs. This causes reductions in blood flow to even the cerebral, myocardial and adrenal circulations (Yaffe et al. 1987; Jensen et al. 1999; Bennet et al. 2002). Control of the fetal cardiovascular responses to acute hypoxaemia and acute asphyxia involves neural and endocrine components. The bradycardia and the initial femoral vasoconstriction during acute hypoxaemia are triggered by the same carotid chemoreflex, as both can be abolished by selective carotid, but not aortic, chemodenervation (Bartelds et al. 1993; Giussani et al. 1993). During acute asphyxia, selective chemodenervation attenuates the initial fall in heart rate but does not totally prevent bradycardia (Jensen & Hanson, 1995; Jensen et al. 1999). During acute hypoxaemia and acute asphyxia, fetal bradycardia is largely mediated by vagal efferents and peripheral vasoconstriction by α-adrenergic efferents (Cohn et al. 1974; Reuss et al. 1982; Parer, 1984; Giussani et al. 1993; Jensen & Hanson, 1995; Giussani et al. 1997; Jensen et al. 1999; Bennet et al. 2002). Once initiated, fetal peripheral vasoconstriction is augmented during hypoxaemia and asphyxia by slower release into the fetal circulation of classical vasoactive hormones such as adrenaline and arginine vasopressin (Alexander et al. 1974; Jones & Robinson, 1975; Giussani et al. 1994b; Jensen & Hanson, 1995; Jensen et al. 1999; Bennet et al. 2002; Gardner et al. 2002). However, the contribution made by more recently discovered vasoconstrictor agents, such as the neuropeptides, to fetal cardiovascular function either during basal conditions or in response to acute hypoxaemia or asphyxia remains unclear. Neuropeptide Y (NPY) is a 36 amino acid peptide from the family of pancreatic polypeptides. The peptide was discovered and isolated from porcine brain preparations and its molecular structure was reported to be highly conserved across species (Tatemoto et al. 1982). Neuropeptide Y is co-localised with noradrenaline in sympathetic nerve terminals and it is also present in the adrenal medulla in mammals (Allen et al. 1983; Lundberg et al. 1983; Ekblad et al. 1984). At the synaptic terminal, NPY is stored in large dense vesicles and it may be co-released with noradrenaline from sympathetic varicosities depending on the frequency and pattern of nerve stimulation (Bloom et al. 1988). In the adult, NPY is known to have a wide variety of actions; however, one of its earliest actions described in the literature is of potent and long-lasting vasoconstriction, either via a direct effect on NPY-Y1 receptors in peripheral circulations or by potentiation of the vasopressor effects of constrictor agents, such as catecholamines (Edvinsson et al. 1984; Franco-Cereceda et al. 1985; Corder et al. 1986; Waeber, 1990; Balasubramaniam, 1997). One previous study measured changes in fetal plasma NPY levels during basal and acute hypoxaemic conditions (Fletcher et al. 2000). In that study, it was reported that the levels of NPY increased significantly in fetal plasma during acute hypoxaemia. However, the role of this increase in plasma NPY during episodes of reduced oxygenation in the fetus remains unknown. Therefore this study tested the hypothesis that increases in circulating plasma concentrations of NPY play a role in the ovine fetal cardiovascular responses to acute oxygen deprivation during late gestation by: (1) comparing the effects on fetal heart rate, blood pressure and the redistribution of the combined ventricular output of an exogenous infusion of NPY with those of acute moderate or severe hypoxaemia; and (2) determining the effect of fetal intravenous treatment with a selective NPY-Y1 receptor antagonist on the fetal cardiovascular responses to a acute moderate hypoxaemia.

Sixty-three pregnant llamas of known breeding date were used in this study. Forty-six of them were submitted to surgery between 186 and 320 days of gestation (52-91% of average gestation period, respectively). Under general anesthesia their fetuses were exteriorized and fetal weight (W), biparietal diameter (BPD) and femoral (F), tarsus-hoof (T-H), tibial (T)) and fronto-occipital (F-O) length were determined. Additionally, the same variables were determined on 16 newborn llamas. The weight was measured in kg and the length in cm. All the collected data was entered into a spreadsheet and different regression analyses as a function of gestational age (GA) were assessed. The best fit equations and their correlation for linear regression were the following: GA=169.448+16.66(*)W, r=0.99; GA=-51.713+44.77(*)BPD, r=0.88; GA=-72.139+39.48(*)F-O, r=0.71; GA=39.304+8.35(*)T-H, r=0.97; GA=91.276+8.23(*)T, r=0.86; GA=102.029+9.94(*)F, r=0.91. For multiple regression, the dependent variable GA can be predicted by the following equation: GA=67.462+11.163(*)W+20.297(*)BPD. Results of the present study indicated measured variables to be highly correlated with GA. This could be useful on daily basis in clinical examination of the neonates, in assessment of fetal growth and well being with cesarean sections, in the determination of GA in late gestation abortions, and in perinatal and reproductive research in the llama.

Carbon monoxide (CO) is produced by the action of the heme oxygenase (HO) complex through the oxidation of heme. CO, like nitric oxide (NO), is a molecular gas that among other actions stimulates guanylyl cyclase and increases cGMP levels in smooth muscle cells, regulating the vascular tone. Acute hypoxia generates pulmonary hypertension and increases the expression of inducible HO isoform (HO-1) in the vascular endothelium. Inhaled NO causes a potent pulmonary vasodilation. We hypothesized that inhaled CO might produce similar actions as NO on pulmonary vascular resistance (PVR). To test our contention, we studied the effects of inhaled CO (40 ppm) in the augmented PVR observed during hypoxemia. Five chronically instrumented German Merino sheep were submitted to a protocol consisting of 20 min of normoxemia (N), 20 min of isocapnic hypoxemia (H20), 20 min of isocapnic hypoxemia plus CO 40 ppm (H40), and 20 min of recovery (R). In the control protocol, we did not administer inhaled CO. Arterial gases and pH, percentage of carboxyhemoglobin (COHb), systemic and pulmonary arterial pressure, systemic and pulmonary vascular resistance, and cardiac output were measured during each period. During H20 period, there was a significant increase in cardiac output and PVR in sheep submitted to both protocols. The sheep treated with inhaled CO (H40 + CO) showed a modest but significant decrease (16%) in the elevated PVR. Our data indicate that inhaled CO decreases pulmonary vascular resistance associated with acute hypoxemia in adult sheep.

1. The effects of fetal intravenous treatment with phentolamine or a vasopressinergic V1-receptor antagonist on the fetal cardiovascular responses to acute hypoxaemia in the llama were investigated. 2. Six llama fetuses were surgically prepared between 60 and 70 % of gestation under general halothane anaesthesia with vascular catheters and transit-time ultrasonic flow probes around a carotid artery and a femoral artery. At least 4 days after surgery all fetuses were subjected to a 3 h experiment: 1 h of normoxia, 1 h of hypoxaemia and 1 h of recovery while on slow i.v. infusion with saline. On separate days this experiment was repeated with fetal i.v. treatment with either phentolamine or a V1-receptor antagonist dissolved in saline. 3. During saline infusion all llama fetuses responded to acute hypoxaemia with intense femoral vasoconstriction. Phentolamine during normoxia produced hypotension, tachycardia and vasodilatation in both the carotid and the femoral circulations. During hypoxaemia, fetuses treated with phentolamine did not elicit the pronounced femoral vasoconstriction and all died within 20 min of the onset of hypoxaemia. A V1-receptor antagonist produced a femoral vasodilatation during normoxia but did not affect the fetal cardiovascular responses to acute hypoxaemia. 4. In conclusion, alpha-adrenergic and V1-vasopressinergic mechanisms contribute to a basal vasoconstrictor tone in the femoral circulation in the llama fetus. The enhanced femoral vasoconstriction during acute hypoxaemia in the llama fetus is not mediated by stimulation of V1-vasopressin receptors, but is dependent on alpha-adrenergic receptor stimulation. Such alpha-adrenergic efferent mechanisms are indispensable to fetal survival during hypoxaemia in the llama since their abolition leads to cardiovascular collapse and death.

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.

We tested the hypothesis that the llama fetus has a blunted cardiovascular chemoreflex response to hypoxemia by investigating the effects of acute hypoxemia on perfusion pressure, heart rate, and the distribution of the combined ventricular output in 10 chronically instrumented fetal llamas at 0.6-0.7 gestation. Four llama fetuses had the carotid sinus nerves sectioned. In the intact fetuses, there was a marked bradycardia, an increase in perfusion pressure, and a pronounced peripheral vasoconstriction during hypoxemia. These cardiovascular responses during hypoxemia in intact fetuses were accompanied by a pronounced increase in plasma vasopressin, but not in plasma angiotensin II concentrations. Carotid denervation prevented the bradycardia at the onset of hypoxemia, but it did not affect the intense vasoconstriction during hypoxemia. Plasma vasopressin and angiotensin II levels were not measured in carotid-denervated fetuses. Our results do not support the hypothesis that the carotid chemoreflex during hypoxemia is blunted in the llama fetus. However, they emphasize that other mechanisms, such as increased vasopressin concentrations, operate to produce an intense vasoconstriction in hypoxemia. This intense vasoconstriction in the llama fetus during hypoxemia may reflect the influence of chronic exposure to the hypoxia of high altitude on the magnitude and gain of fetal cardiovascular responses to a superimposed acute episode of hypoxemia.

Samples of maternal and fetal plasma, fetal urine, and amniotic fluid were collected from 8 chronically cannulated pregnant llamas, in the last third of gestation. The samples were obtained for up to 18 days post-surgery. Osmolality, sodium (Na), potassium (K), chloride (Cl), and urea were measured on 40 samples collected on days 1, 2, 3, 4-5, 6-7, 8-9, and 10-19. The osmolalities of maternal and fetal plasma, fetal urine and amniotic fluid, averaged over these 7 time periods, were, respectively, 312 +/- 2, 311 +/- 1, 484 +/- 14, and 317 +/- 1 mosmol kg-1. Values are given as mean +/- s.e. The major differences from fetal fluid values in the ovine fetus (from previously published values) were the higher osmolality and urea concentration of llama fetal urine. Urine flow rate measured in 6 fetuses, 4.5-6.5 kg body weight, was 5.8 +/- 0.4 mliter h-1; urea clearance rate was 55.5 +/- 11.8 mliter h-1. Glomerular filtration rate (GFR), measured with 51Cr-EDTA in 5 fetuses on 1-4 occasions, was 111.4 +/- 23.3 mliter h-1. Fractional reabsorptions (FR) of Na, K and Cl were 97.9 +/- 1, 75.9 +/- 13.5 and 97.7 +/- 0.4% respectively. The GFR (25 mliter kg-1 h-1) and urine flow rate (1 mL kg-1 h-1) were less than half and about one-tenth the respective values in ovine fetuses. As Na reabsorption is the major oxygen-consuming activity of the kidney, the llama fetal kidney requires only half the oxygen needed by the ovine fetal kidney to reabsorb the filtered sodium load. The reason for the formation of hypertonic, rather than hypotonic, urine in the fetal llama may be due to both greater morphological maturity of the kidney and the excretion of as yet unidentified osmotically active organic substances.

The llama fetus, a high altitude species has its basal peripheral vascular resistance (PVR), two times higher than fetuses of lowland species. AVP is an important regulator of the PVR in the latter. The aim of this study was to assess the AVP role in the maintenance of the femoral (FVR) and carotid (CVR) vascular resistance in normoxic conditions in the Ilama fetus. To that effect, we instrumented 5 fetal llamas at 0.59-0.72 of gestation, in which we continously determined the FVR and CVT before (45min) and after (15min) the administration of an VI receptor blocker d (CH2) 5-Try (Me) AVP (bolus of 240 ug×Kg plus infusion of 0.4ug × Kg × min). Four days before the experiment, we implanted ultrasonic flow transducers around the fetal femoral and carotid arteries and catheters were placed in femoral arteries and veins and in the amniotic cavities. The vascular resistance was calculated dividing the perfusion pressure by the blood flow. The antiAVP administration reduced the FVR from 2.36 ± 0.31 to 1.89 ± 0.22 mmHg × min/ml (× ± SEM; p

The fetal llama, in contrast to fetuses of lowland species, does not increase or increases discreetly total brain blood flow during acute hypoxemia, decreasing brain O2 delivery. Since the fetal llama maintains cardiovascular function during acute hypoxemia, we postulate that regions of the medulla, involved in cardiocirculatory regulation, could importantly increase the blood flow with maintenance of O2 delivery. To answer this question 10 fetal llama 0.6-0.7 of gestation were chronically catheterized. Total and regional blood flows (radiolabelled microspheres) and O2 deliveries were determined during normoxemia (N) and after 45-60 min of severe hypoxemia (H) (

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

Although the species has evolved at high altitudes, adult llamas have low hematocrits (small red cells plus a high number of erythrocytes). Hemaglobin concentrations, are similar to species that have evolved in lowlands. Preliminary data obtained from llama fetuses show a P50 lower than the human. Since there is no information regarding red cells characteristics during intrauterine life in llamas we studied red blood cell counts per mm, hematocrit, hemoglobin concentration, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCV) and mean corpuscular hemoglobin concentration (MCHC) in arterial samples of 25 pregnant llamas and their fetuses (purchased in Parinacota, 4400m above sea level), in the last half of gestation. These red blood cell characteristics (high numbers of small sizes, high hemoglobin concentrations) may allow optimal blood flow and increased oxygen availability and extraction by the tissues in hypoxia. (Grant Fondecyt # 89-1080 - Chile).

Administration of TRH and B to mothers decreases the incidence of bronchopulmonary dysplasia in premature babies. Fetal administration of TRH and Cortisol at 0.85 of gestation increases lung compliance in premature lambs. Since it is not known whether maternal administration of TRH plus B in sheep (in similar doses as in humans) modifies fetal lung compliance, we measured the changes of fetal lung volume induced “in vitro” by pressures of 40, 10, and 5 cm of H2O into the fetal trachea (V40, V10, V5). Twenty one fetal sheep at 0.83 of gestation were divided in three groups: Group TRH + B (n=5) the mothers received TRH (400 ug q 8 h for 6 doses, i.v.) plus B (12 mg q day for 2 doses, i.m.), mothers in Group B (n=5) received only B (same doses), while Group C were 11 control fetuses. The results are expressed as ml air/g of wet fetal lung (ml/g).

NPY, a 36 aminoacid peptide, is one of the most active vasocon-trictor agents in the systemic and pulmonary circulation. NPY plasma concentration in adults increases during stress. Human newborns have high plasma NPY concentrations, but it is unknown whether it participates as mediator of the cardiovascular response to stress in the neonatal period. We measured cardiac output (radiactive microspheres) and aortic and pulmonary artery blood pressures and calculated systemic and pulmonary vascular resistances in 6 newborn lambs (22.8±2.9 days) before and during NPY administration, placing catheters in the pulmonary artery, descending aorta, left ventricle and inferior vena cava. A bolus of 4 nM/kg of NPY followed by an infusion of 0.2 nM/min × kg for 30 min was given 1 or 2 days after surgery after a basal period of 20 min. Aortic and pulmonary blood pressures were registered continously and cardiac output was measured at 0 min, 15 and 30 min during NPY infusion. The results were (X±SEM; *p>0.05, Friedman test): NPY increased systemic and pulmonary vascular resistance. These changes along with the possible decrease of coronary blood flow could explain the fall in cardiac output. This will results in a decrease in pulmonary and systemic perfusion. These results suggest that NPY could be a mediator in the blood flow redistribution observed during stress. If its action become exagered, failure of some of the compensatory cardiorespiratory mechanisms during stress in the neonatal period can occur causing the death of the newborn.