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Distribution of [14C] epinephrine and [131I] albumin in regions of the brain and other tissues of hibernating citellus lateralis after intraperitoneal injection
I38
Comp. gen. Pharmac., i97i , 2. [Scientechnica (Publishers) Ltd.]
DISTRIBUTION
OF [14C] EPINEPHRINE AND [131I] A L B U M I N IN
REGIONS OF THE BRAIN AND O T H E R TISSUES OF HIBERNATING CITELLUS LATERALIS AFTER INTRAPERITONEAL INJECTION* JANET TWENTE Space Sciences Research Center AND J O H N W. T W E N T E Space Sciences Research Center and Division of Biological Sciences, University of Missouri, Columbia 652oI, U.S.A.
(Received I I Nov., I97O) ABSTRACT i. Hibernating Citellus lateralis were intraperitoneally injected with [131I] human serum albumin prior to the intraperitoneal injection of arousal dosages of [14C] nLepinephrine- 7. Animals were sacrificed during early arousal. 2. Equal distribution of the two isotopes in the hypothalamus, medulla oblongata (with pons), cerebellum, and cerebrum showed that the blood-brain barrier was impermeable to epinephrine in hibernating ground squirrels. 3- Volumes of blood determined for the regions of the brain were: hypothalamus and medulla oblongata (with pons), 2.I per cent; cerebrum, 2"2 per cent; and cerebellum, 2.77 per cent. 4. Significant uptake of epinephrine occurred in the liver, ventricle, and lung. 5. Intraperitoneal injection was proved to be a satisfactory method for the administration of pharmacological agents to hibernating ground squirrels. EVOKED, premature arousal of hibernating, golden-mantled ground squirrels (Citellus lateralis) has been elicited by the intraperitoneal injection of compounds of very diverse chemical structures and molecular weights. These included epinephrine (Twente and Twente, t968a ) and all of the trophic hormones, except prolactin (Twente and Twente, I97o ). Intraperitoneally administered control injections of target-organ secretions, insulin, glucagon, posterior pituitary hormones, and albumin did not cause premature arousal (Twente and Twente, I97O). The technique of intraperitoneal injection allows for pharmacological studies upon unstressed, normally hibernating animals which * Supported by funds from U.S.P.H.S. grant AM-I i32o and from the Space Sciences Research Center.
would seem to be impossible were animals with chronically implanted cannulae to be used. The injection m a y be given with a m i n i m u m of disturbance to the animal; premature arousal is never evoked by the stimulus of the injection itself, if it is administered during the first half of the animal's predicted (normalized) period of hibernation. Increases in cardiac rate resulting from the stimulus of injection are transitory (Twente and Twente, I968b ). The experiment presented here was designed partly to test whether the blood-brain barrier of hibernating ground squirrels was permeable to epinephrine. In addition, the degree of uptake of epinephrine by different tissues as well as the volume of blood of tissues of hibernating animals was determined. Lastly, the validity of the technique of intraperitoneal injection to hibernating animals with respect to whether molecules of varying
I97I, 2, I 3 8 - I 4 4
DISTRIBUTION OF [14C] EPINEPHRINE
molecular weights reached possible sites of action was ascertained. Emphasis was placed upon regional distribution of the labelled compounds in the brain. The possibility that epinephrine m a y reach the central nervous system from the peripheral circulation of hibernating ground squirrels has been proposed (Twente, Cline, and Twente, i97o ). This statement was based on the findings of Twente and Twente (1968a) as follows : - Intraperitoneal injection of epinephrine to hibernating Citellus lateralis resulted in evoked, premature arousal when the dosage, on a log scale, was plotted against the normalized period of hibernation. The latter is calculated as the average duration of an animal's winter pattern periods of hibernation, in hours, expressed as ioo per cent. Fifty gg. L-epinephrine evoked complete arousal, to a body temperature of 35-37 ° C., when administered at the beginning of the normalized period of hibernation (o per cent.). Ten gg. epinephrine caused arousal at approximately 2o per cent; and 2. 5 gg. epinephrine did not effect premature arousal until 33 per cent of the normalized period of hibernation had elapsed. Intermediate dosages fell on a line between these points. Those dosages which did not result in complete arousal caused transient calorigenesis of partial arousals o.5-x i ° C. above the body temperature of hibernation. We considered (Twente and Twente, I968a) that the partial arousals caused by non-arousal dosages of epinephrine were the result of direct or indirect stimulation of the brown fat and were peripheral in nature. We implied that the complete arousals caused by epinephrine were the result of a direct stimulation of the central nervous system. This implication would seem to require that the blood-brain barrier of hibernating ground squirrels be permeable to epinephrine. Although it is commonly conceded that the blood-brain barrier is impermeable to epinephrine, Weil-Malherbe, Whitby, and Axelrod (i 96 I) showed that the blood-brain barrier of cats was permeable to statistically significant quantities of epinephrine and norepinephrine in the region of the
139
hypothalamus. Whether or not the bloodbrain barrier of hibernating animals is more permeable to catecholamines than normothermic animals has not been known. T h e following experiment was based upon the distribution of [14C] epinephrine after intraperitoneal injection to hibernating ground squirrels. The experiment was designed to test whether concentrations of [14C] epinephrine in the organs examined exceeded the values for whole blood. Emphasis was placed upon the regional distribution of the 14C label in the brain in order to ascertain whether or not the bloodbrain barrier of hibernating ground squirrels was permeable to epinephrine. Other tissues were measured for comparative purposes and thereby determined the validity of the method. In order to make meaningful correlations of the measurements, it was necessary to determine the volume of blood in the organs studied. Each animal, therefore, was injected with [131I] h u m a n serum albumin prior to the injection of [14C] epinephrine. This dual labelling technique allowed for the determination of the volume of blood of the different regions of the brain as well as the degree of uptake of the 14C label. Fortuitously, the greatly reduced rate of metabolism of hibernating animals has facilitated determinations which would prove difficult in normothermic animals. Electrocardiographic data showed that cardioacceleration lasted more than 6 hours after a non-arousal dosage of epinephrine (Twente and Twente, i968a ). This physiological demonstration indicates that the half-life of injected epinephrine in hibernating animals is greatly extended. [x31I] H u m a n serum albumin was shown to have retained its integrity in the plasma 2. 5 hours after injection. MATERIALS AND METHODS Adult, hibernating, golden-mantled ground squirrels (Citellus lateralis) of both sexes were maintained singly in stainless steel cages, in constant-temperature environmental rooms at 5° C. Cedar shavings served as bedding; sunflower seeds and cut oranges were available as sources of food and water. Continuous monitoring
14o
TWENTE AND TWENTE
of the core temperature from implanted thermocouples (Twente and Twente, I965), or nest temperatures, by Leeds and Northup Speedomax G recording potentiometers, measured the durations, on time-printed chart paper, of the periods of hibernation, arousal, activity, and induction. The techniques of the measurement and normalization of the periods of hibernation and of injection to hibernating animals without causing evoked arousal have been reported (Twente and Twente, i968b ). Definitions of the terms used pertaining to hibernation have been presented (Twente and Twente, x97o ). Experiments were not initiated until the animals were shown to be demonstrating the constant, and predictable, winter pattern of hibernating behaviour (Twente and Twente, i967). Electrocardiograms were monitored from needle electrodes before, during, and after each injection by means of a Grass Model 7 recording oscillograph. Those animals without implanted thermocouples were fitted with thermocouple belts prior to the injections for measurements of body temperature. These belts, with the sensing element opposed to the ventral surface of the tightly curled, hibernating animal, yielded temperatures essentially identical to those achieved from implanted thermocouples which have the sensing element tied to a lower rib. [131I] H u m a n serum albumin (0. 5 ml., in saline, Mallinckrodt Nuclear, i 5 - i 25 lac.) was intraperitoneally injected i - 5 hours prior to the intraperitoneal injection of the labelled epinephrine in all instances except one (animal number I, Table I). This injection consisted of a mixed [ ~3~I] albumin and [14C] epinephrine solution. No calorigenic or cardiac evidence of arousal occurred in response to the injections of albumin. [14C] DL-Epinephrine-7 (New England Nuclear) was administered intraperitoneally in o.5ml. o.I5 M ' Tes ' buffer (N-tris(hydroxymethyl) methyl-2-aminoethane sulphonic acid, Calbiochem). The p H of the injected solutions of epinephrine was 7'4 at 5 ° C. The dosages of [14C] DL-epinephrine were 4 ° gg. ( Io lac.) to 8o lag. (2o lac.) per animal. These were equivalent to 2o-4o lag. L-epinephrine and were administered at elapsed intervals of the normalized period of hibernation which were calculated to result in complete arousal (Twente and Twente, 1968a ). Electrocardiograms showed that the epinephrine reached the circulation within minutes after injection. The period of latency for the first calorigenesis of arousal after injection ranged from 25 to 3o minutes. H a d the injection itself been the stimulus for arousal, calorigenesis would have been initiated within 12-i 8 minutes and increased cardiac rates would have appeared and accelerated immediately after injection (Twente and Twente, I968b). The 5 animals used in this study were sacrificed by decapitation early in the arousal process after
Comp. gen. Pharmac.
body temperatures had risen from the 5 ° C. temperature of hibernation to 9-1o ° C. (55-97 minutes after injection). Blood was withdrawn immediately prior to decapitation by cardiac puncture with a number 2I hypodermic needle on a 2.5-ml. heparinized syringe. The guidelines of dissection of the brain described by Glowinski and Iversen (1966) were followed. The regions of the brain were dissected on ice. The regions of the brain assayed included the hypothalamus, the medulla oblongata (with pons), the cerebrum (in duplicate samples), and the cerebellum. Blood vessels were stripped from each sample of brain tissue as completely as possible before thorough rinsing with normal saline. Tissues were then blotted and weighed immediately. Duplicate samples of ventricle, lung, liver, interscapular brown fat, and gastrocnemius were then dissected, rinsed, blotted, and weighed. Weights of slices of these tissues ranged from approximately 2oo to 7oo rag. Organs from the peritoneal cavity were contaminated with varying amounts of radioactivity as a result of the site of administration and did not yield meaningful results. Tissues were homogenized, in an ice-bath, in 2 ml. of 6 per cent or IO per cent trichloro-acetic acid with a Polytron homogenizer (PT IO generator). Radioactive iodine was determined by counting each entire homogenate by means of a 3 x 3 inch N a I well-detector connected to a Model 8725 Nuclear of Chicago scaler. The samples were then centrifuged. After 4-6 weeks had elapsed, the small percentage of radioactive iodine in the supernatant that had not been removed by precipitation had decayed to a level which did not affect the determinations of radioactive carbon. The 14C label was determined from aliquots of the supernatants by liquid scintillation counting using Picker Nuclear Liquimat or Beckman Model LS-25o scintillation counters. Aquafluor or Aquasol (New England Nuclear) served as scintillation media. O p t i m u m window settings for the 14C samples were determined and used. Samples were counted twice or more for periods of lO-3O minutes except for those samples with exceedingly high counts. Results from different counts for each sample, as well as duplicate samples, were averaged. The radioactivity of the tissues was expressed as counts per minute per g. of tissue divided by the counts per minute per g. of blood. This fraction was multiplied by ioo so that the values for the tissues could be expressed as a percentage of that of the blood. Samples of plasma were subjected to polyacrylamide gel electrophoresis according to the method of Davis (1964). After electrophoresis, gels were divided into 5-ram. sections and the aaaI activity was determined in these slices. [13aI] H u m a n serum albumin was identified in the
D I S T R I B U T I O N OF
I97I, 2
samples of plasma by comparing the electrophoretic mobility to the iodinated human serum albumin preparations used for injection. Samples of plasma taken from I animal 2'5 hours after injection showed that significant quantities of this protein had not been degraded. RESULTS Dissections were relatively consistent as shown b y the weights of the h y p o t h a l a m u s ,
[14C]
I4I
EPINEPHRINE
the m e d u l l a o b l o n g a t a (with pons), a n d the c e r e b e l l u m (Table I). Weights for all of these regions fell within i s t a n d a r d deviation of those reported for larger samples of these tissues of Citellus lateralis (Twente, Cline, a n d T w e n t e , I97O ). T h e c o n c e n t r a t i o n of lSlI in the tissues, w h e n expressed as a percentage of the conc e n t r a t i o n of the blood, was used as a n index
Table / . - - D I S T R I B U T I O N OF LABELS PROM [t3tI] HUMAN SERUM ALBUMIN AND [ l ' C ]
EPINEPHRINE IN DIFFERENT REGIONS OF THE BRAIN OF GROUND SQUIRRELS AROUSING FROM HIBERNATION ISOTOPE
TISSUE
ANIMAL NUMBER
Blood*
TISSUE WEIGHT (mg.)
273 I57 327 754 725
Hypothalamus
.~'±S.D.
~±S.D.
Percentage blood
33,8oo
i oo I O0 I oo
17 i , o o o
I O0 IO0 I O0 IO0 IO0
22,200
26,ooo 89,500 12,300 52O
I "5
2900
55° 630 I IOO
2. 5 2"4 1-2
36o
2"9
iooo 54° 850 15 °
I72 223 14o 203
~+S.D.
2.I±O. 7 2"0 i -8 3"0 1.7
3800 950 650 3oo
200
I "8
I50
2.1+o. 5 470 700 750 I3OO 260
5
347 334 i88 269 298 287+63
I "4 3"2 2"9
4500 2IOO 57O
I "5 2"I
I3OO 120
2.6 2' 9 3"7 2"5 I'8 2.74-o. 7
* Results reported are the average of duplicate samples.
2"2 I. 9
2.8 __..,,a_
1.7 2. 7 2.9 2.8 NS>o.o 5
2"6 3"7 2.4 2. 9 2.4 2.8-4-o.5
5ioo I5OO 85o 1700 i6o
NS>o'o5
2"2
2'5±0"5
2.2+0.8 83o 650 97o 2250 220
I'7 1.8
2.I±O- 4
670 4oo 77° 5oo
I7O+45 I 2 3 4
IOO Ioo
56,400 24, I oo 44,900 504°
77 79 9I IO5
II2
~+S.D.
c.p.m. per g.
242 4- 25
Cerebrum*
Cerebellum
Percentage blood
IOI
245 26o 199 256 250
P
c.p.m. per g.
9I±I3
Medulla oblongata (with pons)
14C
lal I
NS>o.o 5
3"o 2"7 3"5 3"8 3.2 3"24-o'4
NS>o.o5
I42
TWENTE
AND
of the volume of the blood (Fig. I). T h e determinations of the volumes of blood reported for the different regions of the brain appear to be good approximations because of the similarity of the percentages of the lSlI label in the different regions of the brain (Fig. I and Table I). Student's t-test showed no statistical significance when the average values for the percentages of the radioactive iodine and carbon were compared for each region of the brain (Table I). These results indicate that the blood-brain barrier is functional in
8 X
Comp. gen. Pharmac.
TWENTE
different animals (Tables I and H). The distribution of this isotope in the tissues, however, was relatively constant when expressed in terms of the concentration in the blood. [14C] Epinephrine was injected inadvertently into the caecum of animal number 5 (Tables I and H). Excessively high counts ( I I × 1 O e c.p.m, per g.) were determined from the caecum of this ground squirrel. The percentages of the 14C label in the brain and other tissues from animal number 5, however, were not unlike the values obtained after definite intraperitoneal injection to other
10" 9-
87654-
a . IL
s2 21-
0
I lll,lll I lllilll
HYPOTHALAMU$ MEDULLA OBLONOATA (WITH PONS)
IIillt
CEREBRUM
CEREBELLUM
Fzo. 1.--Blood volume of 4 regions of the brain of hibernating ground squirrels is shown by the distribution of aslI expressed as a percentage of that isotope in the blood. Similar data for radioactive carbon from [x*C] epinephrine in the same animals show the impermeability of the bloodbrain barrier to epinephrine. The animals used are sequentially arranged for each region of the brain as in TablesI and H (animals numbers i-5 )- i , [ is 1i] Human serum albumin; - - , [ a*C] nL-epinephrine. hibernating and arousing ground squirrels with respect to epinephrine. Values for the two isotopes in other tissues (Table H) tend to verify the validity of the method. These data show that the 14C label from the injected epinephrine is taken up in significant amounts (according to the t-test) by the liver, the lung, and the ventricle during hibernation and early arousal. We cannot interpret the divergent results from animal number I (Table H) which showed high I4C counts in the brown fat and low a4C counts in the liver. T h e excessively high counts of the brown fat from this animal m a y have obscured any statistical significance relating to the uptake of epinephrine by the brown fat of the other animals, T h e concentration of the a4C label in the blood varied more than twentyfold between
animals with respect to the values for the volume of blood and the degree of uptake of the 14C label. DISCUSSION T h e results presented indicate that intraperitoneal injection is a useful and valid technique for pharmacological studies of normally hibernating ground squirrels. Significant quantities of h u m a n serum albumin (M.W. 67,ooo ) entered the circulation and were distributed throughout the circulatory system of the animal without appreciable degradation. The results also show that significant quantities of epinephrine did not leave the circulating blood of hibernating ground squirrels in any of the regions of the brain studied, including the hypothalamus. T h e
197 I , ' ,
DISTRIBUTION OF [14C ] EPINEPHRINE
143
Table //.--DISTRIBUTION OF LABELS FROM [ x a l I ] HUMAN SERUM ALBUMIN AND [14C] EPINEPHRINE IN SLICES OF TISSUES FROM GROUND SQUIRRELS AROUSING FROM HIBERNATION
ISOTOPE TISSUE
Blood
c.p.m. p e r g.
Percentage blood IO0 IO0 IO0 I00 I O0
I
33,800
2
22,200
3 4 5
26,000 89,500
I
5800 3ooo 47oo 87oo 84o
Gastrocnemius
2
3 4 5
I2,3OO
17 I4
18 IO
7
~-4-S.D. Ventricle
i 3 4 5
7850 485 ° 48oo
23 23 18 14 18
13,OOO
2050
~+S.D. I
620o
2
77oo 34,000 64oo 3ooo
18 35 38 25 24
~'--S.D.
71
,OOO
56,400 2 1,000
44,900 5o4 ° 17,ooo 11,7OO 71oo 7200 7oo
I 3 4 5
68 60 48 59 39
23,000 I3,3oo 43,ooo I5,4OO 48oo
~-I-S.D.
460,000 lO3,OOO
39,5oo 93,ooo 43oo
46,000 557,ooo 290,000 420,000
94,000
2100
I
m e c h a n i s m b y w h i c h injected e p i n e p h r i n e causes evoked a r o u s a l in these h i b e r n a t i n g animals remains problematical.
I O0 IO0 IO0 IO0 IO0
IO 2I
34 16 14
286,000 107,OOO
88,ooo
27 988 646 I742 I865 <0.05
167 19o I96
25,000
lO4
5200
IO3 152i46
<0"025
9743 I79
4I
IOI,O00
37 42 17
53,000
220
190,000
423
5500
IO 9
35+II
NS > 0.05
269 183 164 207 85
Io54i767
660,000 9ooo 9500 38,000
Percentage blood
I82±67
55+11
3rown fat
P
I9±9
28±8 2
~TiS.D.
I
19:t:4
3 4 5
Lung
c.p.m. per g.
13+5 2
Liver
14C
lZl I
ANIMAL
2135~ 19o 3
NS > o ' o 5
I n d i r e c t evidence suggests t h a t c h e m i c a l l y i n d u c e d p r e m a t u r e arousal is c e n t r a l l y initiated. P e r i p h e r a l effects o f e p i n e p h r i n e
I44
TWENTE AND TWENTE
alone do not seem to constitute sufficient stimuli for arousal. This conclusion is based partly upon data which had been obtained from animals which had been given nonarousal dosages of epinephrine: (x) Profound and prolonged increases in cardiac rate always occurred (Twente and Twente, x968a) ; (2) elevations of core temperature as high as i I ° C. above the temperature of hibernation (partial arousals) resulted in response to non-arousal dosages of epinephrine. These were thought to be caused by thermogenesis of the brown fat when directly stimulated by epinephrine (Twente and Twente, 1968a); and (3) respiratory rates increased markedly (unpublished). In addition, other peripheral effects produced by epinephrine, mimicked by injections of pressor (Twente and Twente, 1968a , x97o ) and lipolytic and hyperglycaemic agents (Twente and Twente, i97o), did not cause premature arousal. Despite the apparent impermeability of the blood-brain barrier to epinephrine as demonstrated by the lack of accumulation from the x4C label of this monoamine in the brain, the possibility cannot be disregarded that small quantities of injected epinephrine, undetectable by the methods employed, actually do cross the blood-brain barrier and stimulate specific neurons to initiate evoked arousal. There is no question that catecholamines are essential to the acute calorigenesis characteristic of the arousal process itself. We emphasize, however, that we are not implying that peripherally released epinephrine is in any way responsible for the apparently, centrally located initiator or ' t r i g g e r ' for arousal, whether spontaneous or mechanically evoked.
REFERENCES DAvis, B. J. (x964) , 'Disc electrophoresis. II. Method and application to human serum proteins ', Ann. W.T. Acad. Sci., x~x, 4o4-427 . GLOWINSKI,J., and IVERSZN,L. (I966), ' Regional studies of catecholamines in rat brain. I. The disposition of (SH) norephinephrine and (SH) dopa in various regions of the brain ', .7. Neurochem., x3, 655-669. TWENTE, J. W., and TWENTE, J. A. (x965), 'Effects of core temperature upon duration of hibernation of CiteUus lateralis ', J. appl. Physiol., ~o, 41 I-4x6. TWENTE, J. W., and TWENTZ, J. A. (I967), ' Seasonal variation in the hibernating behavior of CiteUus lateralis ', in Mammalian Hibernation III (ed. FISHER,K. C., DAWE, A. W., LYMAN,C. P., SCHONBAUM,E., and SOUTH,F. E.), pp. 47-63Edinburgh: Oliver and Boyd. TWENTE, J. W., and TWENTE, J. A. (I968a), ' Effects of epinephrine upon progressive irritability of hibernating Citellus lateralis ', Comp. Biochem. Physiol., 25, 475-483 • TWENTE,J. W., and TWENTE,J. A. (1968b), ' Progressive irritability of hibernating Citellus lateralis ', Comp. Biochem. Physiol., a5, 467-474 • TWENTE, J. W., and TWENTE, J. A. (197o), ' Arousing effects of trophic hormones in hibernating Citellus lateralis ', Comp. gen. Pharmac., x, 43 x-436 . TWENTE,J. W., CLINE, W. H., jun., and TWENTE, J. A. (197o), ' Distribution of epinephrine and norepinephrine in the brain of Citellus lateralis during the hibernating cycle ', Comp. gen. Pharmac., x9 47-53. WEIL-MALHERBE,H., WHITBV,L. G., and AXELROD, J. (x96i), ' T h e blood-brain barrier for catecholamines in different regions of the brain ', in Regional Weurochemistry (ed. KETY, S., and ELKES, J.), pp. ~84-292. Oxford: Pergamon.
Key Word Index: Blood-brain barrier, blood volume of tissues, brain, Citellus lateralis, epinephrine, ground squirrel, hibernation, human serum albumin, intraperitoneal injection.
Comp. gen. Pharmac., i97i , 2. [Scientechnica (Publishers) Ltd.]
DISTRIBUTION
OF [14C] EPINEPHRINE AND [131I] A L B U M I N IN
REGIONS OF THE BRAIN AND O T H E R TISSUES OF HIBERNATING CITELLUS LATERALIS AFTER INTRAPERITONEAL INJECTION* JANET TWENTE Space Sciences Research Center AND J O H N W. T W E N T E Space Sciences Research Center and Division of Biological Sciences, University of Missouri, Columbia 652oI, U.S.A.
(Received I I Nov., I97O) ABSTRACT i. Hibernating Citellus lateralis were intraperitoneally injected with [131I] human serum albumin prior to the intraperitoneal injection of arousal dosages of [14C] nLepinephrine- 7. Animals were sacrificed during early arousal. 2. Equal distribution of the two isotopes in the hypothalamus, medulla oblongata (with pons), cerebellum, and cerebrum showed that the blood-brain barrier was impermeable to epinephrine in hibernating ground squirrels. 3- Volumes of blood determined for the regions of the brain were: hypothalamus and medulla oblongata (with pons), 2.I per cent; cerebrum, 2"2 per cent; and cerebellum, 2.77 per cent. 4. Significant uptake of epinephrine occurred in the liver, ventricle, and lung. 5. Intraperitoneal injection was proved to be a satisfactory method for the administration of pharmacological agents to hibernating ground squirrels. EVOKED, premature arousal of hibernating, golden-mantled ground squirrels (Citellus lateralis) has been elicited by the intraperitoneal injection of compounds of very diverse chemical structures and molecular weights. These included epinephrine (Twente and Twente, t968a ) and all of the trophic hormones, except prolactin (Twente and Twente, I97o ). Intraperitoneally administered control injections of target-organ secretions, insulin, glucagon, posterior pituitary hormones, and albumin did not cause premature arousal (Twente and Twente, I97O). The technique of intraperitoneal injection allows for pharmacological studies upon unstressed, normally hibernating animals which * Supported by funds from U.S.P.H.S. grant AM-I i32o and from the Space Sciences Research Center.
would seem to be impossible were animals with chronically implanted cannulae to be used. The injection m a y be given with a m i n i m u m of disturbance to the animal; premature arousal is never evoked by the stimulus of the injection itself, if it is administered during the first half of the animal's predicted (normalized) period of hibernation. Increases in cardiac rate resulting from the stimulus of injection are transitory (Twente and Twente, I968b ). The experiment presented here was designed partly to test whether the blood-brain barrier of hibernating ground squirrels was permeable to epinephrine. In addition, the degree of uptake of epinephrine by different tissues as well as the volume of blood of tissues of hibernating animals was determined. Lastly, the validity of the technique of intraperitoneal injection to hibernating animals with respect to whether molecules of varying
I97I, 2, I 3 8 - I 4 4
DISTRIBUTION OF [14C] EPINEPHRINE
molecular weights reached possible sites of action was ascertained. Emphasis was placed upon regional distribution of the labelled compounds in the brain. The possibility that epinephrine m a y reach the central nervous system from the peripheral circulation of hibernating ground squirrels has been proposed (Twente, Cline, and Twente, i97o ). This statement was based on the findings of Twente and Twente (1968a) as follows : - Intraperitoneal injection of epinephrine to hibernating Citellus lateralis resulted in evoked, premature arousal when the dosage, on a log scale, was plotted against the normalized period of hibernation. The latter is calculated as the average duration of an animal's winter pattern periods of hibernation, in hours, expressed as ioo per cent. Fifty gg. L-epinephrine evoked complete arousal, to a body temperature of 35-37 ° C., when administered at the beginning of the normalized period of hibernation (o per cent.). Ten gg. epinephrine caused arousal at approximately 2o per cent; and 2. 5 gg. epinephrine did not effect premature arousal until 33 per cent of the normalized period of hibernation had elapsed. Intermediate dosages fell on a line between these points. Those dosages which did not result in complete arousal caused transient calorigenesis of partial arousals o.5-x i ° C. above the body temperature of hibernation. We considered (Twente and Twente, I968a) that the partial arousals caused by non-arousal dosages of epinephrine were the result of direct or indirect stimulation of the brown fat and were peripheral in nature. We implied that the complete arousals caused by epinephrine were the result of a direct stimulation of the central nervous system. This implication would seem to require that the blood-brain barrier of hibernating ground squirrels be permeable to epinephrine. Although it is commonly conceded that the blood-brain barrier is impermeable to epinephrine, Weil-Malherbe, Whitby, and Axelrod (i 96 I) showed that the blood-brain barrier of cats was permeable to statistically significant quantities of epinephrine and norepinephrine in the region of the
139
hypothalamus. Whether or not the bloodbrain barrier of hibernating animals is more permeable to catecholamines than normothermic animals has not been known. T h e following experiment was based upon the distribution of [14C] epinephrine after intraperitoneal injection to hibernating ground squirrels. The experiment was designed to test whether concentrations of [14C] epinephrine in the organs examined exceeded the values for whole blood. Emphasis was placed upon the regional distribution of the 14C label in the brain in order to ascertain whether or not the bloodbrain barrier of hibernating ground squirrels was permeable to epinephrine. Other tissues were measured for comparative purposes and thereby determined the validity of the method. In order to make meaningful correlations of the measurements, it was necessary to determine the volume of blood in the organs studied. Each animal, therefore, was injected with [131I] h u m a n serum albumin prior to the injection of [14C] epinephrine. This dual labelling technique allowed for the determination of the volume of blood of the different regions of the brain as well as the degree of uptake of the 14C label. Fortuitously, the greatly reduced rate of metabolism of hibernating animals has facilitated determinations which would prove difficult in normothermic animals. Electrocardiographic data showed that cardioacceleration lasted more than 6 hours after a non-arousal dosage of epinephrine (Twente and Twente, i968a ). This physiological demonstration indicates that the half-life of injected epinephrine in hibernating animals is greatly extended. [x31I] H u m a n serum albumin was shown to have retained its integrity in the plasma 2. 5 hours after injection. MATERIALS AND METHODS Adult, hibernating, golden-mantled ground squirrels (Citellus lateralis) of both sexes were maintained singly in stainless steel cages, in constant-temperature environmental rooms at 5° C. Cedar shavings served as bedding; sunflower seeds and cut oranges were available as sources of food and water. Continuous monitoring
14o
TWENTE AND TWENTE
of the core temperature from implanted thermocouples (Twente and Twente, I965), or nest temperatures, by Leeds and Northup Speedomax G recording potentiometers, measured the durations, on time-printed chart paper, of the periods of hibernation, arousal, activity, and induction. The techniques of the measurement and normalization of the periods of hibernation and of injection to hibernating animals without causing evoked arousal have been reported (Twente and Twente, i968b ). Definitions of the terms used pertaining to hibernation have been presented (Twente and Twente, x97o ). Experiments were not initiated until the animals were shown to be demonstrating the constant, and predictable, winter pattern of hibernating behaviour (Twente and Twente, i967). Electrocardiograms were monitored from needle electrodes before, during, and after each injection by means of a Grass Model 7 recording oscillograph. Those animals without implanted thermocouples were fitted with thermocouple belts prior to the injections for measurements of body temperature. These belts, with the sensing element opposed to the ventral surface of the tightly curled, hibernating animal, yielded temperatures essentially identical to those achieved from implanted thermocouples which have the sensing element tied to a lower rib. [131I] H u m a n serum albumin (0. 5 ml., in saline, Mallinckrodt Nuclear, i 5 - i 25 lac.) was intraperitoneally injected i - 5 hours prior to the intraperitoneal injection of the labelled epinephrine in all instances except one (animal number I, Table I). This injection consisted of a mixed [ ~3~I] albumin and [14C] epinephrine solution. No calorigenic or cardiac evidence of arousal occurred in response to the injections of albumin. [14C] DL-Epinephrine-7 (New England Nuclear) was administered intraperitoneally in o.5ml. o.I5 M ' Tes ' buffer (N-tris(hydroxymethyl) methyl-2-aminoethane sulphonic acid, Calbiochem). The p H of the injected solutions of epinephrine was 7'4 at 5 ° C. The dosages of [14C] DL-epinephrine were 4 ° gg. ( Io lac.) to 8o lag. (2o lac.) per animal. These were equivalent to 2o-4o lag. L-epinephrine and were administered at elapsed intervals of the normalized period of hibernation which were calculated to result in complete arousal (Twente and Twente, 1968a ). Electrocardiograms showed that the epinephrine reached the circulation within minutes after injection. The period of latency for the first calorigenesis of arousal after injection ranged from 25 to 3o minutes. H a d the injection itself been the stimulus for arousal, calorigenesis would have been initiated within 12-i 8 minutes and increased cardiac rates would have appeared and accelerated immediately after injection (Twente and Twente, I968b). The 5 animals used in this study were sacrificed by decapitation early in the arousal process after
Comp. gen. Pharmac.
body temperatures had risen from the 5 ° C. temperature of hibernation to 9-1o ° C. (55-97 minutes after injection). Blood was withdrawn immediately prior to decapitation by cardiac puncture with a number 2I hypodermic needle on a 2.5-ml. heparinized syringe. The guidelines of dissection of the brain described by Glowinski and Iversen (1966) were followed. The regions of the brain were dissected on ice. The regions of the brain assayed included the hypothalamus, the medulla oblongata (with pons), the cerebrum (in duplicate samples), and the cerebellum. Blood vessels were stripped from each sample of brain tissue as completely as possible before thorough rinsing with normal saline. Tissues were then blotted and weighed immediately. Duplicate samples of ventricle, lung, liver, interscapular brown fat, and gastrocnemius were then dissected, rinsed, blotted, and weighed. Weights of slices of these tissues ranged from approximately 2oo to 7oo rag. Organs from the peritoneal cavity were contaminated with varying amounts of radioactivity as a result of the site of administration and did not yield meaningful results. Tissues were homogenized, in an ice-bath, in 2 ml. of 6 per cent or IO per cent trichloro-acetic acid with a Polytron homogenizer (PT IO generator). Radioactive iodine was determined by counting each entire homogenate by means of a 3 x 3 inch N a I well-detector connected to a Model 8725 Nuclear of Chicago scaler. The samples were then centrifuged. After 4-6 weeks had elapsed, the small percentage of radioactive iodine in the supernatant that had not been removed by precipitation had decayed to a level which did not affect the determinations of radioactive carbon. The 14C label was determined from aliquots of the supernatants by liquid scintillation counting using Picker Nuclear Liquimat or Beckman Model LS-25o scintillation counters. Aquafluor or Aquasol (New England Nuclear) served as scintillation media. O p t i m u m window settings for the 14C samples were determined and used. Samples were counted twice or more for periods of lO-3O minutes except for those samples with exceedingly high counts. Results from different counts for each sample, as well as duplicate samples, were averaged. The radioactivity of the tissues was expressed as counts per minute per g. of tissue divided by the counts per minute per g. of blood. This fraction was multiplied by ioo so that the values for the tissues could be expressed as a percentage of that of the blood. Samples of plasma were subjected to polyacrylamide gel electrophoresis according to the method of Davis (1964). After electrophoresis, gels were divided into 5-ram. sections and the aaaI activity was determined in these slices. [13aI] H u m a n serum albumin was identified in the
D I S T R I B U T I O N OF
I97I, 2
samples of plasma by comparing the electrophoretic mobility to the iodinated human serum albumin preparations used for injection. Samples of plasma taken from I animal 2'5 hours after injection showed that significant quantities of this protein had not been degraded. RESULTS Dissections were relatively consistent as shown b y the weights of the h y p o t h a l a m u s ,
[14C]
I4I
EPINEPHRINE
the m e d u l l a o b l o n g a t a (with pons), a n d the c e r e b e l l u m (Table I). Weights for all of these regions fell within i s t a n d a r d deviation of those reported for larger samples of these tissues of Citellus lateralis (Twente, Cline, a n d T w e n t e , I97O ). T h e c o n c e n t r a t i o n of lSlI in the tissues, w h e n expressed as a percentage of the conc e n t r a t i o n of the blood, was used as a n index
Table / . - - D I S T R I B U T I O N OF LABELS PROM [t3tI] HUMAN SERUM ALBUMIN AND [ l ' C ]
EPINEPHRINE IN DIFFERENT REGIONS OF THE BRAIN OF GROUND SQUIRRELS AROUSING FROM HIBERNATION ISOTOPE
TISSUE
ANIMAL NUMBER
Blood*
TISSUE WEIGHT (mg.)
273 I57 327 754 725
Hypothalamus
.~'±S.D.
~±S.D.
Percentage blood
33,8oo
i oo I O0 I oo
17 i , o o o
I O0 IO0 I O0 IO0 IO0
22,200
26,ooo 89,500 12,300 52O
I "5
2900
55° 630 I IOO
2. 5 2"4 1-2
36o
2"9
iooo 54° 850 15 °
I72 223 14o 203
~+S.D.
2.I±O. 7 2"0 i -8 3"0 1.7
3800 950 650 3oo
200
I "8
I50
2.1+o. 5 470 700 750 I3OO 260
5
347 334 i88 269 298 287+63
I "4 3"2 2"9
4500 2IOO 57O
I "5 2"I
I3OO 120
2.6 2' 9 3"7 2"5 I'8 2.74-o. 7
* Results reported are the average of duplicate samples.
2"2 I. 9
2.8 __..,,a_
1.7 2. 7 2.9 2.8 NS>o.o 5
2"6 3"7 2.4 2. 9 2.4 2.8-4-o.5
5ioo I5OO 85o 1700 i6o
NS>o'o5
2"2
2'5±0"5
2.2+0.8 83o 650 97o 2250 220
I'7 1.8
2.I±O- 4
670 4oo 77° 5oo
I7O+45 I 2 3 4
IOO Ioo
56,400 24, I oo 44,900 504°
77 79 9I IO5
II2
~+S.D.
c.p.m. per g.
242 4- 25
Cerebrum*
Cerebellum
Percentage blood
IOI
245 26o 199 256 250
P
c.p.m. per g.
9I±I3
Medulla oblongata (with pons)
14C
lal I
NS>o.o 5
3"o 2"7 3"5 3"8 3.2 3"24-o'4
NS>o.o5
I42
TWENTE
AND
of the volume of the blood (Fig. I). T h e determinations of the volumes of blood reported for the different regions of the brain appear to be good approximations because of the similarity of the percentages of the lSlI label in the different regions of the brain (Fig. I and Table I). Student's t-test showed no statistical significance when the average values for the percentages of the radioactive iodine and carbon were compared for each region of the brain (Table I). These results indicate that the blood-brain barrier is functional in
8 X
Comp. gen. Pharmac.
TWENTE
different animals (Tables I and H). The distribution of this isotope in the tissues, however, was relatively constant when expressed in terms of the concentration in the blood. [14C] Epinephrine was injected inadvertently into the caecum of animal number 5 (Tables I and H). Excessively high counts ( I I × 1 O e c.p.m, per g.) were determined from the caecum of this ground squirrel. The percentages of the 14C label in the brain and other tissues from animal number 5, however, were not unlike the values obtained after definite intraperitoneal injection to other
10" 9-
87654-
a . IL
s2 21-
0
I lll,lll I lllilll
HYPOTHALAMU$ MEDULLA OBLONOATA (WITH PONS)
IIillt
CEREBRUM
CEREBELLUM
Fzo. 1.--Blood volume of 4 regions of the brain of hibernating ground squirrels is shown by the distribution of aslI expressed as a percentage of that isotope in the blood. Similar data for radioactive carbon from [x*C] epinephrine in the same animals show the impermeability of the bloodbrain barrier to epinephrine. The animals used are sequentially arranged for each region of the brain as in TablesI and H (animals numbers i-5 )- i , [ is 1i] Human serum albumin; - - , [ a*C] nL-epinephrine. hibernating and arousing ground squirrels with respect to epinephrine. Values for the two isotopes in other tissues (Table H) tend to verify the validity of the method. These data show that the 14C label from the injected epinephrine is taken up in significant amounts (according to the t-test) by the liver, the lung, and the ventricle during hibernation and early arousal. We cannot interpret the divergent results from animal number I (Table H) which showed high I4C counts in the brown fat and low a4C counts in the liver. T h e excessively high counts of the brown fat from this animal m a y have obscured any statistical significance relating to the uptake of epinephrine by the brown fat of the other animals, T h e concentration of the a4C label in the blood varied more than twentyfold between
animals with respect to the values for the volume of blood and the degree of uptake of the 14C label. DISCUSSION T h e results presented indicate that intraperitoneal injection is a useful and valid technique for pharmacological studies of normally hibernating ground squirrels. Significant quantities of h u m a n serum albumin (M.W. 67,ooo ) entered the circulation and were distributed throughout the circulatory system of the animal without appreciable degradation. The results also show that significant quantities of epinephrine did not leave the circulating blood of hibernating ground squirrels in any of the regions of the brain studied, including the hypothalamus. T h e
197 I , ' ,
DISTRIBUTION OF [14C ] EPINEPHRINE
143
Table //.--DISTRIBUTION OF LABELS FROM [ x a l I ] HUMAN SERUM ALBUMIN AND [14C] EPINEPHRINE IN SLICES OF TISSUES FROM GROUND SQUIRRELS AROUSING FROM HIBERNATION
ISOTOPE TISSUE
Blood
c.p.m. p e r g.
Percentage blood IO0 IO0 IO0 I00 I O0
I
33,800
2
22,200
3 4 5
26,000 89,500
I
5800 3ooo 47oo 87oo 84o
Gastrocnemius
2
3 4 5
I2,3OO
17 I4
18 IO
7
~-4-S.D. Ventricle
i 3 4 5
7850 485 ° 48oo
23 23 18 14 18
13,OOO
2050
~+S.D. I
620o
2
77oo 34,000 64oo 3ooo
18 35 38 25 24
~'--S.D.
71
,OOO
56,400 2 1,000
44,900 5o4 ° 17,ooo 11,7OO 71oo 7200 7oo
I 3 4 5
68 60 48 59 39
23,000 I3,3oo 43,ooo I5,4OO 48oo
~-I-S.D.
460,000 lO3,OOO
39,5oo 93,ooo 43oo
46,000 557,ooo 290,000 420,000
94,000
2100
I
m e c h a n i s m b y w h i c h injected e p i n e p h r i n e causes evoked a r o u s a l in these h i b e r n a t i n g animals remains problematical.
I O0 IO0 IO0 IO0 IO0
IO 2I
34 16 14
286,000 107,OOO
88,ooo
27 988 646 I742 I865 <0.05
167 19o I96
25,000
lO4
5200
IO3 152i46
<0"025
9743 I79
4I
IOI,O00
37 42 17
53,000
220
190,000
423
5500
IO 9
35+II
NS > 0.05
269 183 164 207 85
Io54i767
660,000 9ooo 9500 38,000
Percentage blood
I82±67
55+11
3rown fat
P
I9±9
28±8 2
~TiS.D.
I
19:t:4
3 4 5
Lung
c.p.m. per g.
13+5 2
Liver
14C
lZl I
ANIMAL
2135~ 19o 3
NS > o ' o 5
I n d i r e c t evidence suggests t h a t c h e m i c a l l y i n d u c e d p r e m a t u r e arousal is c e n t r a l l y initiated. P e r i p h e r a l effects o f e p i n e p h r i n e
I44
TWENTE AND TWENTE
alone do not seem to constitute sufficient stimuli for arousal. This conclusion is based partly upon data which had been obtained from animals which had been given nonarousal dosages of epinephrine: (x) Profound and prolonged increases in cardiac rate always occurred (Twente and Twente, x968a) ; (2) elevations of core temperature as high as i I ° C. above the temperature of hibernation (partial arousals) resulted in response to non-arousal dosages of epinephrine. These were thought to be caused by thermogenesis of the brown fat when directly stimulated by epinephrine (Twente and Twente, 1968a); and (3) respiratory rates increased markedly (unpublished). In addition, other peripheral effects produced by epinephrine, mimicked by injections of pressor (Twente and Twente, 1968a , x97o ) and lipolytic and hyperglycaemic agents (Twente and Twente, i97o), did not cause premature arousal. Despite the apparent impermeability of the blood-brain barrier to epinephrine as demonstrated by the lack of accumulation from the x4C label of this monoamine in the brain, the possibility cannot be disregarded that small quantities of injected epinephrine, undetectable by the methods employed, actually do cross the blood-brain barrier and stimulate specific neurons to initiate evoked arousal. There is no question that catecholamines are essential to the acute calorigenesis characteristic of the arousal process itself. We emphasize, however, that we are not implying that peripherally released epinephrine is in any way responsible for the apparently, centrally located initiator or ' t r i g g e r ' for arousal, whether spontaneous or mechanically evoked.
REFERENCES DAvis, B. J. (x964) , 'Disc electrophoresis. II. Method and application to human serum proteins ', Ann. W.T. Acad. Sci., x~x, 4o4-427 . GLOWINSKI,J., and IVERSZN,L. (I966), ' Regional studies of catecholamines in rat brain. I. The disposition of (SH) norephinephrine and (SH) dopa in various regions of the brain ', .7. Neurochem., x3, 655-669. TWENTE, J. W., and TWENTE, J. A. (x965), 'Effects of core temperature upon duration of hibernation of CiteUus lateralis ', J. appl. Physiol., ~o, 41 I-4x6. TWENTE, J. W., and TWENTZ, J. A. (I967), ' Seasonal variation in the hibernating behavior of CiteUus lateralis ', in Mammalian Hibernation III (ed. FISHER,K. C., DAWE, A. W., LYMAN,C. P., SCHONBAUM,E., and SOUTH,F. E.), pp. 47-63Edinburgh: Oliver and Boyd. TWENTE, J. W., and TWENTE, J. A. (I968a), ' Effects of epinephrine upon progressive irritability of hibernating Citellus lateralis ', Comp. Biochem. Physiol., 25, 475-483 • TWENTE,J. W., and TWENTE,J. A. (1968b), ' Progressive irritability of hibernating Citellus lateralis ', Comp. Biochem. Physiol., a5, 467-474 • TWENTE, J. W., and TWENTE, J. A. (197o), ' Arousing effects of trophic hormones in hibernating Citellus lateralis ', Comp. gen. Pharmac., x, 43 x-436 . TWENTE,J. W., CLINE, W. H., jun., and TWENTE, J. A. (197o), ' Distribution of epinephrine and norepinephrine in the brain of Citellus lateralis during the hibernating cycle ', Comp. gen. Pharmac., x9 47-53. WEIL-MALHERBE,H., WHITBV,L. G., and AXELROD, J. (x96i), ' T h e blood-brain barrier for catecholamines in different regions of the brain ', in Regional Weurochemistry (ed. KETY, S., and ELKES, J.), pp. ~84-292. Oxford: Pergamon.
Key Word Index: Blood-brain barrier, blood volume of tissues, brain, Citellus lateralis, epinephrine, ground squirrel, hibernation, human serum albumin, intraperitoneal injection.