Nitric oxide synthase activity in brain tissues from llama fetuses submitted to hypoxemia

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Abstract

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 Ca2+-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.

Introduction

The umbilical vein Po2 of fetuses of several species at sea level is 70% lower than maternal aortic Po2, which is equivalent to Po2 values obtained from the aorta in human climbers at very high altitudes (West et al., 1983). In their natural environment, the Andes mountains, llamas’ Po2 is lower than low land mammals. This peculiar hypoxemic condition has exerted upon the llama species (Lama glama) strong selective pressures allowing the fetal llama to develop mechanisms to cope with hypoxemia, as illustrated by the cardiovascular and metabolic responses to hypoxemia (Llanos et al., 1995, Giussani et al., 1996, Riquelme et al., 1997, Llanos et al., 2000). Compared to the fetal sheep, the llama fetus submitted to hypoxemia does not exhibit carotid and cerebral artery vasodilatation although it does present an intense peripheral vasoconstriction (Giussani et al., 1996). Therefore, cerebral blood flow does not increase and there is a marked reduction in femoral and carcass blood flow during hypoxemia (Llanos et al., 1995, Giussani et al., 1996). Contemporary with this response, cerebral oxygen delivery and consumption decrease from the very beginning of the hypoxemic insult (Llanos et al., 2000). To our knowledge, this is the only species whose fetuses do not protect their central nervous system by a substantial increase in the cerebral blood flow during hypoxemia (Llanos et al., 1995). Moreover, this is the only species whose fetuses do not maintain the cerebral oxygen consumption during an episode of moderate or severe hypoxemia (Riquelme et al., 1997, Llanos et al., 2000). Therefore, there is some basis to speculate that the llama fetus has defensive mechanisms in order to avoid hypoxemic neuronal damage. Among the mechanisms that could produce neuronal injury in hypoxemia is the overproduction of nitric oxide (NO) (Iadecola, 1997). Although NO is a normal mediator in the regulation of vascular tone and synaptic transmission, chronic hypoxemia induces an increase in either nitric oxide synthase (NOS) activity or mRNA expression in the cerebral tissue of sheep fetuses, a lowland species suggesting NO overproduction (Aguan et al., 1998). Furthermore, NOS blockade preserves neuronal integrity in fetuses and neonates of lowland species submitted to chronic hypoxemia (Malyshev et al., 1999). Gunn et al. (1992) examined the histologic and electrophysiologic changes associated with asphyxia due to uterine artery occlusion of varying duration in fetal sheep. They found that asphyxia for prolonged periods of up to 2 h might be accompanied by neuronal death. Their data support the suggestion that good cerebral perfusion is critical for surviving asphyxia without damage. Also neonatal mice lacking nNOS have been shown to be less vulnerable to acute hypoxic–ischemic injury (Ferriero et al., 1996). Both data suggest a role for NO in eliciting cerebral damage during hypoxemia.

Since there is no increase in cerebral blood flow and there is a marked reduction in cerebral oxygen consumption during hypoxemia in the fetal llama, we hypothesized that NOS activity is either maintained or reduced in the central nervous system of llama fetuses submitted to chronic hypoxemia.

Section snippets

Use of animals

Time-dated pregnant llamas (10), were obtained from the University of Chile farm at the Rinconada de Maipú (580 m above sea level). Upon arrival to the laboratory in Santiago, also at 585 m above sea level, the llamas were housed in an open yard with access to food and water ad libitum and they were familiarized with the study metabolic cage and the laboratory conditions for 1–2 weeks prior to surgery.

Surgical preparation

Maternal surgery was carried out using well-established techniques previously described in

Results

The ten fetal llamas had a mean weight of 2979±1516 g, means±S.E.M., which corresponds to 54–76% of gestation (gestational age approx. 350 days, where term is 7000–8000 g) (Fowler, 1989).

Discussion

The results from this study were consistent with the hypothesis that NOS activity is either maintained or reduced in the central nervous system of llama fetuses submitted to chronic hypoxemia, because NOS activity did not change with chronic hypoxemia (24 h) in the two tissues studied. We found differences in NOS activity in the cerebral cortex and in the cerebellum, being significantly higher in the cerebral cortex of the fetal llama.

This is the first report on NOS activity, measured as

Acknowledgements

This work has been supported by Fondecyt 1970236, Chile.

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