Body temperature regulation during acclimation to cold and hypoxia in rats.

Extreme environmental conditions present challenges for thermoregulation in homoeothermic organisms such as mammals. Such challenges are exacerbated when two stressors are experienced simultaneously and each stimulus evokes opposing physiological responses. This is the case of cold, which induces an increase in thermogenesis, and hypoxia, which suppresses metabolism conserving oxygen and preventing hypoxaemia. As an initial approach to understanding the thermoregulatory responses to cold and hypoxia in a small mammal, we explored the effects of acclimation to these two stressors on the body temperature (Tb) and the daily and ultradian Tb variations of Sprague-Dawley rats. As Tb is influenced by sleep-wake cycles, these Tb variations reflect underlying adjustments in set-point and thermosensitivity. The Tb of rats decreased precipitously during initial hypoxic exposure which was more pronounced in cold (Tb=33.4 ± 0.13) than in room temperature (Tb=35.74 ± 0.17) conditions. This decline was followed by an increase in Tb stabilising at a new level ~0.5°C and ~1.4°C below normoxic values at room and cold temperatures, respectively. Daily Tb variations were blunted during hypoxia with a greater effect in the cold. Ultradian Tb variations exhibited daily rhythmicity that disappeared under hypoxia, independent of ambient temperature. The adjustments in Tb during hypoxia and/or cold are in agreement with the hypothesis that an initial decrease in the Tb set-point is followed by its partial re-establishment with chronic hypoxia. This rebound of the Tb set-point might reflect cellular adjustments that would allow animals to better deal with low oxygen conditions, diminishing the drive for a lower Tb set-point. Cold and hypoxia are characteristic of high altitude environments. Understanding how mammals cope with changes in oxygen and temperature will shed light into their ability to colonize new environments along altitudinal clines and increase our understanding of how Tb is regulated under stimuli that impose contrasting physiological constraints.

Postprandial thermogenesis in Bothrops moojeni (Serpentes: Viperidae)

Snakes that can ingest prey that are proportionally large have high metabolic rates during digestion. This great increase in metabolic rate (specific dynamic action - SDA) may create a significant augment in the animal's body temperature. The present study investigated postprandial thermogenesis in Bothrops moojeni. Briefly, two groups of snakes were fed meals equivalent to 17 ± 3 percent and 32 ± 5 percent of their body weight and were observed for 72 hours, in which thermal images of each snake were taken with an infrared camera in a thermostable environment with a constant air temperature of 30ºC. The results showed a significant increase in snake surface temperature, with a thermal peak between 33 and 36 hours after feeding. The meal size had a great impact on the intensity and duration of the thermogenic response. Such increase in temperature appears to be connected with the huge increase in metabolic rates during digestion of relatively large prey by snakes that feed infrequently. The ecologic implication of the thermogenic response is still not well understood; however, it is possible that its presence could affect behaviors associated with the snake digestion, such as postprandial thermophily.

The ventilatory response to environmental hypercarbia in the South American rattlesnake, Crotalus durissus.

To study the effects of environmental hypercarbia on ventilation in snakes, particularly the anomalous hyperpnea that is seen when CO(2) is removed from inspired gas mixtures (post-hypercapnic hyperpnea), gas mixtures of varying concentrations of CO(2) were administered to South American rattlesnakes, Crotalus durissus, breathing through an intact respiratory system or via a tracheal cannula by-passing the upper airways. Exposure to environmental hypercarbia at increasing levels, up to 7% CO(2), produced a progressive decrease in breathing frequency and increase in tidal volume. The net result was that total ventilation increased modestly, up to 5% CO(2) and then declined slightly on 7% CO(2). On return to breathing air there was an immediate but transient increase in breathing frequency and a further increase in tidal volume that produced a marked overshoot in ventilation. The magnitude of this post-hypercapnic hyperpnea was proportional to the level of previously inspired CO(2). Administration of CO(2) to the lungs alone produced effects that were identical to administration to both lungs and upper airways and this effect was removed by vagotomy. Administration of CO(2) to the upper airways alone was without effect. Systemic injection of boluses of CO(2)-rich blood produced an immediate increase in both breathing frequency and tidal volume. These data indicate that the post-hypercapnic hyperpnea resulted from the removal of inhibitory inputs from pulmonary receptors and suggest that while the ventilatory response to environmental hypercarbia in this species is a result of conflicting inputs from different receptor groups, this does not include input from upper airway receptors.

The effect of graded hypoxia on the metabolic rate and buccal activity of a lungless salamander (Desmognathus fuscus).

The hypothesis that the lungless salamander Desmognathus fuscus responds actively to hypoxia was tested. Patterns of buccal movements [apneic period duration, the duration (min h(-)(1)) of buccal pumping and buccal pumping frequency], heart rate and metabolic rate (rates of oxygen uptake and carbon dioxide output) were determined during a control period (21 % oxygen), a hypoxic period (2, 5, 6.5, 8 or 10 % oxygen) and a recovery period (21 % oxygen). Hypoxic salamanders maintained their rate of oxygen uptake at control levels until a critical oxygen level between 10 and 8 % oxygen was reached. The rate of carbon dioxide output remained constant across all oxygen levels, except for a significant increase during exposure to 5 % oxygen. The buccal activity of lungless salamanders was responsive to environmental hypoxia, with a significant stimulation during exposure to 6.5 % and 5 % oxygen. Buccal pumping frequency was inhibited at 2 % oxygen. Heart rate was stimulated at all hypoxic levels except 2 % O(2). During recovery, metabolic rate and heart rate returned to control levels within 20 min after all hypoxic exposures. The durations of apneic periods increased significantly compared with the hypoxic value during recovery from exposure to 10 %, 6.5 % and 5 % oxygen. Overall, the animals responded actively to hypoxia by increasing the duration of buccal activity as oxygen levels decreased. The ability of these changes to facilitate oxygen uptake is not known. However, the response of the dusky salamander to low levels of oxygen is analogous to the hypoxic ventilatory response observed in lunged vertebrates.

Metabolic depression and enhanced O(2) affinity of mitochondria in hypoxic hypometabolism.

This study examined whether the steady-state hypometabolism seen in overwintering frogs (Rana temporaria) is reflected at the mitochondrial level either by a reduction in their resting (state 4) and active (state 3) respiration rates and/or by increases in O(2) affinity. We isolated mitochondria from the skeletal muscle of cold-submerged frogs at different stages during their hibernation in normoxic and hypoxic water. A modest metabolic depression at the whole animal level (normoxic submergence) was not associated with a reduction in mitochondrial state 4 and state 3 respiration rates. However, mitochondria isolated from frogs that were submerged for 1 mo manifested an increase in their O(2) affinity compared with controls and with animals submerged for 4 mo. Hypometabolism was more pronounced at the whole animal level during hypoxic submergence and was accompanied by 1) a reduction in mitochondrial state 4 and state 3 rates and 2) an increase in the O(2) affinity of mitochondria. These findings demonstrate that metabolic depression can be reflected at all levels of biological organization in hypoxia-tolerant animals.

Role of adenosine in the hypoxia-induced hypothermia of toads.

The concept that hypoxia elicits a drop in body temperature (T(b)) in a wide variety of animals is not new, but the mechanisms remain unclear. We tested the hypothesis that adenosine mediates hypoxia-induced hypothermia in toads. Measurements of selected T(b) were performed using a thermal gradient. Animals were injected (into the lymph sac or intracerebroventricularly) with aminophylline (an adenosine receptor antagonist) followed by an 11-h period of hypoxia (7% O(2)) or normoxia exposure. Control animals received saline injections. Hypoxia elicited a drop in T(b) from 24.8 +/- 0.3 to 19. 5 +/- 1.1 degrees C (P < 0.05). Systemically applied aminophylline (25 mg/kg) did not change T(b) during normoxia, indicating that adenosine does not alter normal thermoregulatory function. However, aminophylline (25 mg/kg) significantly blunted hypoxia-induced hypothermia (P < 0.05). To assess the role of central thermoregulatory mechanisms, a smaller dose of aminophylline (0.25 mg/kg), which did not alter hypoxia-induced hypothermia systemically, was injected into the fourth cerebral ventricle. Intracerebroventricular injection of aminophylline (0.25 mg/kg) caused no significant change in T(b) under normoxia, but it abolished hypoxia-induced hypothermia. The present data indicate that adenosine is a central and possibly peripheral mediator of hypoxia-induced hypothermia.

Constant set points for pH and P(CO2) in cold-submerged skin-breathing frogs.

The low temperatures encountered by overwintering frogs result in a large downregulation of metabolism and behaviour. However, little is known about acid-base regulation in the extreme cold, especially when frogs become exclusive skin-breathers during their winter submergence. Blood and muscle tissue acid-base parameters (pH, P(CO2), bicarbonate and lactic acid concentrations) were determined in submerged frogs exposed to a range of low temperatures (0.2-7 degrees C). At overwintering temperatures between T = 0.2 and 4 degrees C plasma pH and P(CO2) were maintained constant, whereas intracellular pH regulation resulted in larger pH-temperature slopes occurring in the presumably more active heart muscle (deltapH/deltaT = -0.0313) than in the gastrocnemius muscle (deltapH/deltaT = -0.00799). Although blood pH was not significantly affected by submergence between 0.2 and 4 degrees C (pH = 8.220-8.253), it declined in the 7 degrees C frogs (pH = 8.086), a decrease not linked to the recruitment of anaerobiosis. Plasma P(CO2) and pH in the cold appear to be regulated at constant levels, implying that cutaneous CO2 conductance in submerged frogs is adjusted within the range of overwintering temperatures. This is likely geared toward facilitating the uptake of oxygen under conditions of greater metabolic demand, however there remains the possibility that acid-base balance itself is maintained at a constant set point at the frog's natural overwintering temperatures.

Balancing hypoxia and hypothermia in cold-submerged frogs.

Many animals respond to hypoxic stress by selecting cooler environments, the so-called 'behavioural hypothermia' response. Amphibians overwintering in ice-covered ponds and lakes offer an ecologically relevant test of this response since they must choose between the confounding metabolic effects of profound hypothermia or hypoxia; thermal and chemical conditions can vary from 0 degrees C and normoxic at the ice-water interface to 4 degrees C and markedly hypoxic at depths of 2-4 m. To mimic such environmental conditions, we constructed an experimental chamber that enabled continuous electronic surveillance of an animal's movement along a thermal gradient. When Rana temporaria pre-acclimated to 3.5 degrees C were placed in a normoxic thermal gradient ranging from 0.8 to 8 degrees C, they invariably favoured the warmer end of the chamber. Upon exposure to hypoxia, however, their preferred temperature shifted from a median of 6.8 degrees C (P02 = 158 mmHg; 1 mmHg = 0.133 kPa) to 1.9 degrees C (P02 = 25 mmHg). Metabolic rate measurements from animals exposed simultaneously to acute changes in water temperature and PO2 suggest that movement to colder conditions in hypoxia effects the greatest metabolic savings and prolongs the onset of a plasma lactacidosis.

Hypometabolic homeostasis in overwintering aquatic amphibians.

Many amphibians encounter conditions each winter when their body temperature is so low that normal activities are suspended and the animals enter into a state of torpor. In ice-covered ponds or lakes, oxygen levels may also become limiting, thereby forcing animals to endure prolonged periods of severe hypoxia or anoxia. Certain frogs (e.g. Rana temporaria) can dramatically suppress their metabolism in anoxia but are not as tolerant as other facultative vertebrate anaerobes (e.g. turtle, goldfish) of prolonged periods of complete O2 lack. Many overwintering amphibians do, however, tolerate prolonged bouts of severe hypoxia, relying exclusively on cutaneous gas exchange. Rana temporaria overwintering for 2 months in hypoxic water (PO2 approximately 25 mmHg) at 3 degrees C progressively reduce their blood PCO2 to levels characteristic of water-breathing fish. The result is that blood pH rises and presumably facilitates transcutaneous O2 transfer by increasing Hb O2-affinity. Even after months of severe hypoxia, there is no substantial build-up of lactate as the animals continue to rely on cutaneous gas exchange to satisfy the requirements of a suppressed aerobic metabolism. Our recent experiments have shown that the skeletal muscle of frogs oxyconforms in vitro to the amount of O2 available. The cellular basis for the oxyconformation of skeletal muscle is unknown, but the hypothesis driving our continuing experiments theories that metabolic suppression at a cellular level is synonymous with suppressed ion leak across cellular membranes.

The effects of ambient pH on nitrogen excretion in early life stages of the American toad (Bufo americanus).

Acidification of breeding ponds has been identified as a potential threat to the survival and health of North American amphibian populations. The effects of acid exposure on ion and acid-balance are well known, but there is little information on how environmental water pH influences nitrogen balance in amphibians. The aim of this study was to determine the effects of moderately acidic water (pH 6.0) on nitrogen excretion in early life stages of the toad, Bufo americanus. Acid exposure (pH 6.0, 54 h) resulted in a 20-80% increase in ammonia-N excretion rates in embryos and early, middle and late tadpoles stages, whereas there was no significant effect on urea-N excretion. Tissue ammonia concentrations were significantly higher (+33%) in the embryos and 35-65% lower in the three groups of tadpoles exposed to water of pH 6.0 compared to control animals (pH 8.5). In embryos, ammonia excretion accounted for greater than 90% of total nitrogen excretion (ammonia-N + urea-N), but by the late tadpole stage this value had decreased to approximately 65%. These findings indicate that exposure of embryonic and larval B. americanus to moderately acidic water disrupts nitrogen balance by increasing nitrogen loss as ammonia, with no compensatory decrease in urea excretion.