J. Physiol. (1976), 263, pp. 441-463 With 9 text-ftgurem Printed in Great Britain
441
CYCLIC ADENOSINE
3', 5'-MONOPHOSPHATE IN CEREBROSPINAL FLUID DURING THERMOREGULATION AND FEVER
BY M. J. DASCOMBE AND A. S. MILTON From the Department of Pharmacology, University Medical Buildings, Foresterhill, Aberdeen AB9 2ZD
(Received 21 April 1976) SUMMARY
1. Samples of cerebrospinal fluid (c.s.f.) have been taken from the cisterna magna of unanaesthetized cats, whilst rectal temperature was recorded, during exposure of the animals to various ambient temperatures and during fever induced by pyrogen. The concentration of adenosine 3',5'-monophosphate (cyclic AMP) in samples of c.s.f. has been assayed. 2. Cats exposed to low ambient temperatures (-2 to + 20 C) for 3 h maintained body temperature by both behavioural and autonomic heat gain activity. Exposure of cats to high ambient temperatures (44-45° C) for 3-5 h caused a rise in body temperature of about 2.50 C, despite behavioural and autonomic heat loss activity. Neither cold nor heat stress had a significant effect on c.s.f. cyclic AMP. 3. Fever induced by intravenous Shigella dysenteriae (2 and 20 pg/kg) was associated with a dose-related increase in the concentration of cyclic AMP in c.s.f. Paracetamol (75 mg/kg) injected i.P. before the onset of fever, suppressed the increase in both temperature and c.s.f. cyclic AMP in response to pyrogen. Paracetamol (50 and 100 mg/kg), injected after the onset of fever, caused a fall in temperature, which was not associated with a decrease in the concentration of cyclic AMP in c.s.f. 4. Fever induced in cats by intravenous Shigella dysenteriae (20 ,tg/kg) was associated with an increase in the concentration of cyclic AMP in plasma as well as in c.s.f. 5. The sodium salt of cyclic AMP (0 l-10 mg/kg) injected i.v. into unanaesthetized cats caused a dose-related hypothermia, which was associated with autonomic heat loss activity and a dose-related increase in the concentration of cyclic AMP in cisternal c.s.f., which was not mimicked by adenosine. 6. It is concluded that the raised concentrations of cyclic AMP in c.s.f.,
M. J. DASCOMBE AND A. S. MILTON 442 in response to pyrogen i.v., do not mediate fever in the cat and that the concentration of cyclic AM1P in cisternal c.s.f. may be affected by changes in the plasma concentration of the nucleotide. INTRODUCTION
Noradrenaline and 5-hydroxytryptamine are generally accepted as being involved in the hypothalamic control of body temperature (Feldberg, 1968; Bligh, 1973; Myers, 1974). Adenosine 3',5'-monophosphate (cyclic AMP) is an apparent mediator of these amines in various tissues (Robison, Butcher & Sutherland, 1971) and it is conceivable that this nucleotide may be involved in the central control of normal temperature. This suggestion is supported by the observation that the methylxanthine drugs, caffeine and theophylline, can increase body temperature (Soifer, 1957; Reiman, 1967), by an apparent central action, and also increase brain tissue levels of cyclic AMP (Cheung, 1967; Vernikos-Danellis & Harris, 1968). Prostaglandins of the E series, which Feldberg & Milton (1973) concluded were possible central mediators of fever, have also been found to increase concentrations of cyclic AMP in brain tissue (Wellman & Schwabe, 1973; Zor, Kaneko, Schneider, McCann, Lowe, Bloom, Borland & Field, 1969). Hence, this cyclic nucleotide may also be involved in the central meditation of fever. In the present studies, the possible involvement of cyclic AMP in normal thermoregulation and in fever has been examined by assaying the concentrations of this nucleotide in cisternal cerebrospinal fluid (c.s.f.) from cats during exposure to both low and high ambient temperatures and during fever induced by i.v. injection of a bacterial pyrogen. The concentration of cyclic AMP in plasma during pyrogen-induced fever has also been investigated. In addition the possibility of the blood being a contributory source of cyclic AMP in c.s.f. has been studied. Preliminary reports of this work have already been published (Dascombe & Milton, 1975a, b). Animal
METHODS
Female cats weighing between 1V8 and 3-6 kg, obtained from a single supplier, were used. Animals were individually caged during the experiments, without access to food or water and at an ambient temperature of 20-24° C unless otherwise stated. Experiments were conducted at about the same time each day and each animal was used at intervals of about 1 week.
Temperature nzeasurement Temperature was measured with a thermistor probe (Yellow Springs Instrument series 400) inserted about 80-100 mm into the rectum and held in position with adhesive tape wrapped round the tail, and monitored on a Jacquet multichannel
CYCLIC AMP AND FEVER
443
recorder. Temperature was recorded continuously. Temperature responses were assessed by integrating the change in temperature (0 C) against time (h) by using computative means to give a thermal response index (TRI), such that a single TRI unit (TRI 10 C. h) is equivalent to a 10 C change in temperature lasting for 1 h (Milton & Wendlandt, 1971). The value of x in TRIX is the period of time (h) for which the temperature response has been assessed. Variation of ambient temperature Cats were placed in an insulated chamber in which the air temperature was maintained at -2 to + 2° C, 24-260 C or 44-46° C. Ambient temperature was varied by heating or cooling ethylene glycol to a pre-set temperature, the glycol was then passed into a heat exchanger by a Churchill Chiller Thermo Circulator. The air in the chamber was circulated, at a speed of 2 km/h, by an electric fan over the heat exchanger and through the section of the chamber housing the animal. The air temperature in the animal section was monitored continuously, using a Yellow Springs Instrument series BEO-401 thermistor. The rectal temperatures of cats were measured as described previously. Pyrogen and drug administration The 'O' somatic antigen of Shigella dysenteriae (Humphrey & Bangham, 1959) was injected in a dose of either 2 or 20 tZg/kg into a saphenous vein of the cat. The minimum period between two injections of pyrogen into any one cat was 7 days to avoid the development of tolerance. Paracetamol (4-acetamidophenol, 4-Ac) was injected i.P. into cats. The paracetamol was dissolved in 1 ml warm propylene glycol and made up to 5 ml with 0.9% NaCl. Adenosine and cyclic AMP were injected i.v. in a solution of 0-9 % NaCl. All drug solutions were passed through a MillexT filter (pore size 220 nm) before administration. All glassware, filters and 0-9 % NaCl ('Steriflex', Allen and Hanburys Ltd.) were sterile and pyrogen-free. Disposable, sterile, pyrogen-free syringes and needles were used to administer the pyrogen and drug solutions.
Collection of c.s.f. samples Samples of c.s.f. were obtained from the cisterna magna of the unanaesthetized cat using the method described by Feldberg, Gupta, Milton & Wendlandt (1973). In an aseptic operation under pentobarbitone sodium anaesthesia (40 mg/kg i.P.) a Collison cannula was fixed to the back of the skull so that the tip of the shaft rested just above the atlanto-occipital membrane. Each animal was allowed to recover for at least two weeks before being used in experiments. After the cat had recovered from the operation samples of c.s.f. were collected without anaesthesia by piercing the occipital membrane with a hollow stainless-steel needle (26-gauge) passed through the cap of the cannula. Attached to the protruding outer end of the needle was a length of fine polyethylene tubing. On keeping the free end of the tubing below the level of the head of the cat, drops of c.s.f. flowed out at a rate 0-03-0-1 ml/min. The needle was left in position throughout the experiment and samples of 0-3-0-6 ml c.s.f. were collected at intervals as indicated in Results. Between collections, loss of c.s.f. from the tubing was prevented by plugging its free end with a pin and attaching the tubing with adhesive tape to the head of the cat. The needle assembly was steam sterilized for 30 min before use. Collection of blood sample In an aseptic operation under pentobarbitone sodium anaesthesia a catheter (4 FG, Portex Ltd.) was implanted into a femoral artery in each cat. The catheter
444
M. J. DASCOMBE
AND A. S. MILTON
was inserted 80-100 mm towards the heart before being tied in. Benzyl-penicilin was sprinkled on to the wound before the skin was sutured and sprayed with NobecutaneB. 300 mg benzylpenicillin was injected I.M. daily for 4 days. Heparin 1000 u. was injected through the catheter post-operatively. Animals were allowed to recover for at least 4 days before being used in experiments. Heparin 500 u. was injected daily through the implanted catheter. Each animal was used at intervals of about 4 days. During experiments, samples of blood (2 ml.) were obtained at the times indicated in Results. Replacement volumes of 0 9% NaCl containing heparin (50 u./ml.) were injected through the catheter.
Assay for cyclic AMP in c.8.f. Concentrations of cyclic AMP were measured using the competitive protein binding assay described by Tovey, Oldham & Whelan (1974). Assay reagents were obtained in freeze-dried form from the Radiochemical Centre, Amersham. Samples of c.s.f. were assayed routinely after being stored in sealed ampoules at - 200 C in the dark for 18-96 h, in the absence of an inhibitor of phosphodiesterase activity. No significant change in content of cyclic AMP was determined after storage. All samples were deproteinized, before assaying for cyclic AMP content, by the following procedure. Absolute ethanol 500 Iul was added to 250 pl aliquots of c.s.f., mixed and allowed to stand for 5 min at room temperature (20-240 C). After centrifuging at 1250 g for 5 min in an unrefrigerated centrifuge, the supernatant was removed using a pasteur pipette. The precipitate was washed with three aliquots of 250 M1 absolute ethanol, centrifuged and all the supernatant fractions combined. The alcoholic solution was evaporated to dryness, under a stream of nitrogen, in a water bath at 550 C. The residue was dissolved in 125 p1. assay buffer and after centrifugation (as described earlier in this paragraph), 50 pl. samples of the resultant solution were assayed for cyclic AMP.
Assay for cyclic AMP in plasma Plasma was prepared for assay using a method based on that of Tovey et al. (1974). A 2 ml. sample of blood was added immediately to 2% of its volume of 0.25 M EDTA pH 7-5 contained in a cooled centrifuge tube and the contents immediately mixed. The EDTA-treated blood was centrifuged at 1500 g for 5 min in an unrefrigerated centrifuge. The plasma was removed and stored in sealed ampoules in the dark at -20° C; 250 1I. aliquots were deproteinized and assayed for cyclic AMP as described for c.s.f. The results are expressed as pmol cyclic AMP/ml. c.s.f. or plasma, and are uncorrected for losses during recovery (about 5 and 15% respectively) or for apparent losses (about 24 %) due to interference in the assay of plasma samples. Liquid scintilaation counting The radioactivity of samples was measured using a Packard Tri-Carb Liquid Scintillation Spectrometer 2425. The radioactivity in 200 pl. samples of assay supernatants was counted for 10 or 20 min. The efficiency of counting each sample was monitored by the channels ratio method (Baillie, 1960). The variations in counting efficiencies (about 28 %) for the six to eight samples of any one experiment were small (standard deviation of the mean < 1 %), hence, counts per minute rather than disintegrations per minute were used in the calculation of results. The scintillation fluid was a mixture of toluene and Triton X-100 (Turner, 1969). Five g PPO (2,5-diphenyloxazole) and 0-1 g POPOP (1,4-di-2-(5-phenyloxazolyl)benzene) were dissolved in 1 1 toluene and 500 ml Triton X-100 were added.
CYCLIC AMP AND FEVER
445
In later experiments, the use of POPOP as a secondary scintillant was found to be unnecessary for the liquid scintillation spectrometer used, and the POPOP was omitted from the scintillant fluid, without loss of counting efficiency.
Material u8ed 1,4-di-2-(5-phenyloxazolyl)-benzene (B.D.H. Chemicals Ltd.). Heparin ('Pularin', Evans Medical Ltd.). Paracetamol, toluene, Triton X-100 and 2,5-diphenyloxazole (Koch Light). Reagents for cyclic AMP assay (Radiochemical Centre, Amersham, England). Adenosine and adenosine 3',5'-monophosphoric acid monosodium salt (Sigma Chemical Company). Stati8tical anoly8i8 Results are expressed as the mean + s.E. of n experiments in groups of four to six cats. The probability (P) of the significance of the difference between different results was assessed by either a paired or an unpaired t test as appropriate (Armitage, 1971). RESULTS
Cyclic AMP in c.8.f. during heat and cold stress Four cats were exposed to a low ambient temperature of -2 to + 20 C or a high ambient temperature of 44-46° C. After a control period of 1 h at an ambient temperature of 24-26° C, the temperature in the chamber was set for either low or high ambient temperature. Changes in air temperature were complete within 60 and 30 min respectively. 4 h after initiating these changes, the air temperature was changed back to 24..G 260 C, this being attained within 30 min. Exposure of cats to low ambient temperature caused the adoption of a crouched posture, peripheral vasoconstriction, piloerection and almost continual shivering. This activity resulted in an increase in rectal temperature above control values assessed as the TRI (P
441
CYCLIC ADENOSINE
3', 5'-MONOPHOSPHATE IN CEREBROSPINAL FLUID DURING THERMOREGULATION AND FEVER
BY M. J. DASCOMBE AND A. S. MILTON From the Department of Pharmacology, University Medical Buildings, Foresterhill, Aberdeen AB9 2ZD
(Received 21 April 1976) SUMMARY
1. Samples of cerebrospinal fluid (c.s.f.) have been taken from the cisterna magna of unanaesthetized cats, whilst rectal temperature was recorded, during exposure of the animals to various ambient temperatures and during fever induced by pyrogen. The concentration of adenosine 3',5'-monophosphate (cyclic AMP) in samples of c.s.f. has been assayed. 2. Cats exposed to low ambient temperatures (-2 to + 20 C) for 3 h maintained body temperature by both behavioural and autonomic heat gain activity. Exposure of cats to high ambient temperatures (44-45° C) for 3-5 h caused a rise in body temperature of about 2.50 C, despite behavioural and autonomic heat loss activity. Neither cold nor heat stress had a significant effect on c.s.f. cyclic AMP. 3. Fever induced by intravenous Shigella dysenteriae (2 and 20 pg/kg) was associated with a dose-related increase in the concentration of cyclic AMP in c.s.f. Paracetamol (75 mg/kg) injected i.P. before the onset of fever, suppressed the increase in both temperature and c.s.f. cyclic AMP in response to pyrogen. Paracetamol (50 and 100 mg/kg), injected after the onset of fever, caused a fall in temperature, which was not associated with a decrease in the concentration of cyclic AMP in c.s.f. 4. Fever induced in cats by intravenous Shigella dysenteriae (20 ,tg/kg) was associated with an increase in the concentration of cyclic AMP in plasma as well as in c.s.f. 5. The sodium salt of cyclic AMP (0 l-10 mg/kg) injected i.v. into unanaesthetized cats caused a dose-related hypothermia, which was associated with autonomic heat loss activity and a dose-related increase in the concentration of cyclic AMP in cisternal c.s.f., which was not mimicked by adenosine. 6. It is concluded that the raised concentrations of cyclic AMP in c.s.f.,
M. J. DASCOMBE AND A. S. MILTON 442 in response to pyrogen i.v., do not mediate fever in the cat and that the concentration of cyclic AM1P in cisternal c.s.f. may be affected by changes in the plasma concentration of the nucleotide. INTRODUCTION
Noradrenaline and 5-hydroxytryptamine are generally accepted as being involved in the hypothalamic control of body temperature (Feldberg, 1968; Bligh, 1973; Myers, 1974). Adenosine 3',5'-monophosphate (cyclic AMP) is an apparent mediator of these amines in various tissues (Robison, Butcher & Sutherland, 1971) and it is conceivable that this nucleotide may be involved in the central control of normal temperature. This suggestion is supported by the observation that the methylxanthine drugs, caffeine and theophylline, can increase body temperature (Soifer, 1957; Reiman, 1967), by an apparent central action, and also increase brain tissue levels of cyclic AMP (Cheung, 1967; Vernikos-Danellis & Harris, 1968). Prostaglandins of the E series, which Feldberg & Milton (1973) concluded were possible central mediators of fever, have also been found to increase concentrations of cyclic AMP in brain tissue (Wellman & Schwabe, 1973; Zor, Kaneko, Schneider, McCann, Lowe, Bloom, Borland & Field, 1969). Hence, this cyclic nucleotide may also be involved in the central meditation of fever. In the present studies, the possible involvement of cyclic AMP in normal thermoregulation and in fever has been examined by assaying the concentrations of this nucleotide in cisternal cerebrospinal fluid (c.s.f.) from cats during exposure to both low and high ambient temperatures and during fever induced by i.v. injection of a bacterial pyrogen. The concentration of cyclic AMP in plasma during pyrogen-induced fever has also been investigated. In addition the possibility of the blood being a contributory source of cyclic AMP in c.s.f. has been studied. Preliminary reports of this work have already been published (Dascombe & Milton, 1975a, b). Animal
METHODS
Female cats weighing between 1V8 and 3-6 kg, obtained from a single supplier, were used. Animals were individually caged during the experiments, without access to food or water and at an ambient temperature of 20-24° C unless otherwise stated. Experiments were conducted at about the same time each day and each animal was used at intervals of about 1 week.
Temperature nzeasurement Temperature was measured with a thermistor probe (Yellow Springs Instrument series 400) inserted about 80-100 mm into the rectum and held in position with adhesive tape wrapped round the tail, and monitored on a Jacquet multichannel
CYCLIC AMP AND FEVER
443
recorder. Temperature was recorded continuously. Temperature responses were assessed by integrating the change in temperature (0 C) against time (h) by using computative means to give a thermal response index (TRI), such that a single TRI unit (TRI 10 C. h) is equivalent to a 10 C change in temperature lasting for 1 h (Milton & Wendlandt, 1971). The value of x in TRIX is the period of time (h) for which the temperature response has been assessed. Variation of ambient temperature Cats were placed in an insulated chamber in which the air temperature was maintained at -2 to + 2° C, 24-260 C or 44-46° C. Ambient temperature was varied by heating or cooling ethylene glycol to a pre-set temperature, the glycol was then passed into a heat exchanger by a Churchill Chiller Thermo Circulator. The air in the chamber was circulated, at a speed of 2 km/h, by an electric fan over the heat exchanger and through the section of the chamber housing the animal. The air temperature in the animal section was monitored continuously, using a Yellow Springs Instrument series BEO-401 thermistor. The rectal temperatures of cats were measured as described previously. Pyrogen and drug administration The 'O' somatic antigen of Shigella dysenteriae (Humphrey & Bangham, 1959) was injected in a dose of either 2 or 20 tZg/kg into a saphenous vein of the cat. The minimum period between two injections of pyrogen into any one cat was 7 days to avoid the development of tolerance. Paracetamol (4-acetamidophenol, 4-Ac) was injected i.P. into cats. The paracetamol was dissolved in 1 ml warm propylene glycol and made up to 5 ml with 0.9% NaCl. Adenosine and cyclic AMP were injected i.v. in a solution of 0-9 % NaCl. All drug solutions were passed through a MillexT filter (pore size 220 nm) before administration. All glassware, filters and 0-9 % NaCl ('Steriflex', Allen and Hanburys Ltd.) were sterile and pyrogen-free. Disposable, sterile, pyrogen-free syringes and needles were used to administer the pyrogen and drug solutions.
Collection of c.s.f. samples Samples of c.s.f. were obtained from the cisterna magna of the unanaesthetized cat using the method described by Feldberg, Gupta, Milton & Wendlandt (1973). In an aseptic operation under pentobarbitone sodium anaesthesia (40 mg/kg i.P.) a Collison cannula was fixed to the back of the skull so that the tip of the shaft rested just above the atlanto-occipital membrane. Each animal was allowed to recover for at least two weeks before being used in experiments. After the cat had recovered from the operation samples of c.s.f. were collected without anaesthesia by piercing the occipital membrane with a hollow stainless-steel needle (26-gauge) passed through the cap of the cannula. Attached to the protruding outer end of the needle was a length of fine polyethylene tubing. On keeping the free end of the tubing below the level of the head of the cat, drops of c.s.f. flowed out at a rate 0-03-0-1 ml/min. The needle was left in position throughout the experiment and samples of 0-3-0-6 ml c.s.f. were collected at intervals as indicated in Results. Between collections, loss of c.s.f. from the tubing was prevented by plugging its free end with a pin and attaching the tubing with adhesive tape to the head of the cat. The needle assembly was steam sterilized for 30 min before use. Collection of blood sample In an aseptic operation under pentobarbitone sodium anaesthesia a catheter (4 FG, Portex Ltd.) was implanted into a femoral artery in each cat. The catheter
444
M. J. DASCOMBE
AND A. S. MILTON
was inserted 80-100 mm towards the heart before being tied in. Benzyl-penicilin was sprinkled on to the wound before the skin was sutured and sprayed with NobecutaneB. 300 mg benzylpenicillin was injected I.M. daily for 4 days. Heparin 1000 u. was injected through the catheter post-operatively. Animals were allowed to recover for at least 4 days before being used in experiments. Heparin 500 u. was injected daily through the implanted catheter. Each animal was used at intervals of about 4 days. During experiments, samples of blood (2 ml.) were obtained at the times indicated in Results. Replacement volumes of 0 9% NaCl containing heparin (50 u./ml.) were injected through the catheter.
Assay for cyclic AMP in c.8.f. Concentrations of cyclic AMP were measured using the competitive protein binding assay described by Tovey, Oldham & Whelan (1974). Assay reagents were obtained in freeze-dried form from the Radiochemical Centre, Amersham. Samples of c.s.f. were assayed routinely after being stored in sealed ampoules at - 200 C in the dark for 18-96 h, in the absence of an inhibitor of phosphodiesterase activity. No significant change in content of cyclic AMP was determined after storage. All samples were deproteinized, before assaying for cyclic AMP content, by the following procedure. Absolute ethanol 500 Iul was added to 250 pl aliquots of c.s.f., mixed and allowed to stand for 5 min at room temperature (20-240 C). After centrifuging at 1250 g for 5 min in an unrefrigerated centrifuge, the supernatant was removed using a pasteur pipette. The precipitate was washed with three aliquots of 250 M1 absolute ethanol, centrifuged and all the supernatant fractions combined. The alcoholic solution was evaporated to dryness, under a stream of nitrogen, in a water bath at 550 C. The residue was dissolved in 125 p1. assay buffer and after centrifugation (as described earlier in this paragraph), 50 pl. samples of the resultant solution were assayed for cyclic AMP.
Assay for cyclic AMP in plasma Plasma was prepared for assay using a method based on that of Tovey et al. (1974). A 2 ml. sample of blood was added immediately to 2% of its volume of 0.25 M EDTA pH 7-5 contained in a cooled centrifuge tube and the contents immediately mixed. The EDTA-treated blood was centrifuged at 1500 g for 5 min in an unrefrigerated centrifuge. The plasma was removed and stored in sealed ampoules in the dark at -20° C; 250 1I. aliquots were deproteinized and assayed for cyclic AMP as described for c.s.f. The results are expressed as pmol cyclic AMP/ml. c.s.f. or plasma, and are uncorrected for losses during recovery (about 5 and 15% respectively) or for apparent losses (about 24 %) due to interference in the assay of plasma samples. Liquid scintilaation counting The radioactivity of samples was measured using a Packard Tri-Carb Liquid Scintillation Spectrometer 2425. The radioactivity in 200 pl. samples of assay supernatants was counted for 10 or 20 min. The efficiency of counting each sample was monitored by the channels ratio method (Baillie, 1960). The variations in counting efficiencies (about 28 %) for the six to eight samples of any one experiment were small (standard deviation of the mean < 1 %), hence, counts per minute rather than disintegrations per minute were used in the calculation of results. The scintillation fluid was a mixture of toluene and Triton X-100 (Turner, 1969). Five g PPO (2,5-diphenyloxazole) and 0-1 g POPOP (1,4-di-2-(5-phenyloxazolyl)benzene) were dissolved in 1 1 toluene and 500 ml Triton X-100 were added.
CYCLIC AMP AND FEVER
445
In later experiments, the use of POPOP as a secondary scintillant was found to be unnecessary for the liquid scintillation spectrometer used, and the POPOP was omitted from the scintillant fluid, without loss of counting efficiency.
Material u8ed 1,4-di-2-(5-phenyloxazolyl)-benzene (B.D.H. Chemicals Ltd.). Heparin ('Pularin', Evans Medical Ltd.). Paracetamol, toluene, Triton X-100 and 2,5-diphenyloxazole (Koch Light). Reagents for cyclic AMP assay (Radiochemical Centre, Amersham, England). Adenosine and adenosine 3',5'-monophosphoric acid monosodium salt (Sigma Chemical Company). Stati8tical anoly8i8 Results are expressed as the mean + s.E. of n experiments in groups of four to six cats. The probability (P) of the significance of the difference between different results was assessed by either a paired or an unpaired t test as appropriate (Armitage, 1971). RESULTS
Cyclic AMP in c.8.f. during heat and cold stress Four cats were exposed to a low ambient temperature of -2 to + 20 C or a high ambient temperature of 44-46° C. After a control period of 1 h at an ambient temperature of 24-26° C, the temperature in the chamber was set for either low or high ambient temperature. Changes in air temperature were complete within 60 and 30 min respectively. 4 h after initiating these changes, the air temperature was changed back to 24..G 260 C, this being attained within 30 min. Exposure of cats to low ambient temperature caused the adoption of a crouched posture, peripheral vasoconstriction, piloerection and almost continual shivering. This activity resulted in an increase in rectal temperature above control values assessed as the TRI (P
