Life Sciences, Vol . 25, pp . 747-754 Printed in the U.S .A .
Pergamon Press
STRAIN DIFFERENCES IN RAT ADRENAL BIOSYNTHETIC ENZYMES AND STRESS-INDUCED INCREASES IN PLASMA CATECHOLAMINES Richard McCartyl, Gad M. Gilad2 , Virginia K. Weise, and Irwin J. lcopin 3 Laboratory of Clinical Science National Institute of Mental Health Bethesda, Maryland 20205 (Received in final form July 20, 1979)
We have examined in two inbred rat strains basal and stress-induced increases in plasma levels of epinephrine (EPI) and norepinephrine (NE) and compared these with activities of the adrenal enzymes involved in the synthesis of catecholamines . There were no differences in basal levels of NE and EPI in plasma of adult male rats of the Wistar-Kyoto (WKY) and Brown-Norway (B-N) strains . However, following 5 min. of intermittent footshock, plasma levels of both catecholamines were twice as high in WKY rata as in B-N rata . In the adrenals of unstressed rats, activities of tyrosine hydroxylase and dopamine-beta-hydroxylase were significantly higher in B-N rats . in addition, the adrenal weights and the contents of NE but not EPI were greater in B-N rats . Thus, in these taro rat strains, the capacity of the adrenal gland to synthesize and store catecholaminea appeared to be inversely related to plasma levels of NE and EPI after stress . The differences between the strains appeared to be due to differences in the rates of removal of catecholamines from the peripheral circulation as well as to differences in the rate of release of catecholaminea from the sympatho-adrenal medullary system . Thus biosynthetic enzyme activities need not be related directly to the capacity to release and elevate plasma levels of catecholaminea following stressful stimulation . Major genetic differences have been reported in the activities of the enzymes involved in catecholamine biosynthesis in central and peripheral tissues of inbred strains of rats and mice (1-5) . In addition, the activities of the enzymes tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanolamine-N-methyltransferase (PNMT) are increased by stressful stimulation and changes in enzyme levels have been utilized as indices of the capacity of tissues to synthesize and release catecholaminea (6-9) . Recent studies fro® this laboratory have demonstrated that plasma levels
Research Associate, Pharmacology-Toxicology Training Program, National Institute of General Medical Sciences . Present address : Department of Psychology, University of Virginia, Charlottesville, Virginia 22901 . 2.
Present address : Department of Isotope Research, Weizman Institute of Science, Rehovot, Israel .
3.
To whom requests for reprints should be addressed . 0024-3205/79/090747-0802 .00/0
748
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of epinephrine (EPI) and norepinephrine (NE) provide an accurate index of sympatho-adrenal medullary discharge in response to alerting stimuli (10-12) . In addition, significant differences have been found among rat strains in footshock stress-induced increments in plasma levels of NE and EPI (13) . In the present study, we have examined in two inbred rat strains the relationship between catecholamine biosynthetic enzyme activities in the adrenal gland and stress-induced increases in plasma levels of NE and EPI . Rats of the Brown-Norway (B-N) and Wistar-Kyoto (WKY) strains were selected for study because of the marked differences between these two strains in the increments in plasma catecholamines following stressful stimulation (13) . Methods Male B-N rats were obtained from the Veterinary Resources Branch, National Institutes of Health and male WKY rata were purchased from Taconic Farms, Germantown, New York . Rats of each strain were housed in groups of 4 per cage for at least 1 week prior to use and food and water were available ad lib. The vivarium was maintained on a 12-hour photoperiod (lights on 0600-1800 hrs) at a temperature of 25°C . WRY and B-N rata (300-350 g) were anesthetized with pentobarbital (35-40 mg/kq) and a PE-50 catheter was inserted into the ventral caudal artery of each rat according to the method of Chiueh and Kopin (14) . The tubing traversed the subcutaneous tissue of the tail sheath and back to exit at the back of the neck . A 30 cm piece of spring wire was placed over the tubing and secured to the rat with an adhesive tape collar . The tubing was filled with saline containing heparin (500 U/ml) and the end was occluded with a 23G needle and a 1 ml plastic syringe . After surgery, each rat was placed in a clear plastic cage (25 x 25 x 16 cm) that contained bedding material and access to food and water . Each catheter was flushed with 0.5 ml of heparinized saline twice each day. Two days after surgery, blood samples (0 .5 ml) were obtained from the rats while they were resting and undisturbed in their home cages. Each rat was then transferred to a shock chamber (15) and exposed to 5 minutes of inescapable footshock (2 .5 mA, 0.4 sec . duration, every 5 seconds) . A second blood sample was drawn at the end of the interval of footshocks . Blood samples were centrifuged at 4,000 xg for 10 min. at 4°C . The plasma was removed and stored at -20°C until assayed for catecholamines by a radioenzymatic-thin layer chromatographic method (16,17) . The activities of the adrenal enzymes TH, DBH, and PNMT were measured in naive B-N and WRY male rats (250-350 g) . Animals were killed by decapitation and the adrenals were rapidly removed and the pair from each rat were weighed and homogenized in 1 .0 ml of 0 .3 M sucrose . Aliquots were removed for the assay of TH (18), DBH (19), PNMT (20), and catecholamines (16,17) . The rate of removal of exogenously administered 3H-1-NE (24 .7 Ci/mmole, New England Nuclear) was determined in 3 B-N and 3 WKY male rats (375-425 g) . Rats were anesthetized with pentobarbital and the right jugular vein and the 3 H-1-NE was diluted left carotid artery were cannulated with PE-50 tubing . 3 with saline to a concentration of 18 .8 ng/ml (1 .67 ~Ci H-1-NE per ml) and injected rapidly intravenously in a total volume of 0 .5 ml . Arterial blood samples were collected at 15, 30, 45, 60, and 75 seconds after injection. Plasma was obtained fry the blood samples and the 3H=1-NE was absorbed onto altm~ina at pH 8 .6 . The alumina was washed 3 times with buffer (pH 8 .6), the
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3 H-1-NE eluted with 0 .1 N HC1 and the tritium assaye8 by liquid scintillation spectrometry . In another experiment, the levels of 3H-1-NE were determined in plasma of blood obtained at intervals of 0 .67 seconds during the first 15 seconds following the tracer injection, as described by Sapirstein (21) . Results When rats were resting and undisturbed in their home cages, there were no strain differences in plasma levels of NS or EPI . However, after exposure to 5 minutes of intermittent footshock, the levels in plasma of NE and EPI were two-fold higher for WRY rats as compared to B-N rats (Fig . 1) .
BASAL
BTAESB®
Fig . 1 Basal plasma levels (pg/ml) of epinephrine (EPI) and norepinephrine (NE) in undisturbed WILY and H-N rats and levels after 5 minutes of footshock stress . Values are means + SEM for groupa of 6 rats per strain .
In a separate group of unstressed rata, a comparison between strains was made of the adrenal gland content of catecholamines and the levels of catecholamine biosynthetic enzymes . When compared to WRY rats, B-N rats had heavier adrenals which contained significantly more NS but not EPI (Fig . 2) . In addition, the activities of the adrenomedullary enzymes TH and DBH were significantly greater in B-N rats . No strain differences were obaerveä in the activity of PNMT in the adrenal gland (Fig . 3) . To determine if there were significant differences between strains in the rate of removal of catecholamines from the circulation, levels of 3 H-1-NE in plasma were determined after its rapid intravenous injection . At 15 seconds after injection, plasma levels of 3H-1-NE were higher in B-N than in WILY rats, but the rate of decline of the 3H-1-NE in plasma was more rapid in the B-N rats and by 1 min~te there was no significant difference between the attains in the levels of H-1-NE found in the plasma (Fig . 4) .
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50 40 $ 15 i z
10
10
5
0
0 Fig . 2
Paired adrenal weights (pg) and contents norepinephrine (NE) of WKY and B-N rats . of 10 rats per strain .
(pg/`pair) of epinephrine (EPI) and Values are means _+ SEM for groups
Fig . 3 Activities (nmoles product formed per paired adrenals per hr) of tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanolamine-Nmethyltransferase (PNMT) in the paired adrenals of WRY and B-N rats . Values are means + SEM for groups of 10 rats per strain .
Measurements of the levels of 3H-1-NE in plasma during the first 15 seconds after a tracer injection showed that the WRY rata had a greater cardiac output and that the 3H-1-NE was diluted to a greater extent in these rats than in B-N rats (data not shown) . Adrenal glands from B-N and WKY rats were preserved in loi neutral formalin, serially sectioned, and stained with cresyl violet (Fiq . 5) .
Vol . 25, No . 9, 1979
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Rat Strains and CA Responses to Stress
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INTERCEPT SLOPE ~ B-N 20000 t 83D cpm/ml -0.0,61 1 .0008 o WKY 7870 t 260 cpm/ml -0 .0318 t "0007 sec - ~
_v
W Z OL
a
W Z â W OC Z 2 M
Q
N g
a
Fig. 4 Rate of removal of an intravenous injection of 3H-1-norepinephrine from the circulation in 1tP1CY and B-N rats while under pentobarbital anesthesia .
Fig. 5 Photomicrographs of sections (50 microns thick) through the adrenal glands of tü1~CY (A) and B-N (B) rata, stained with cresyl violet . The adrenal gland, including cortex and medulla of 1PRY rats (A) is smaller than that of B-N rats (B) . X 45 .
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Volumes (mm3) calculated from serial sections were : whole gland (B-N : 37 .8, WKY : 30 .2) and adrenal medulla (B-N : 8 .95, WKY: 6 .95) . Thus, the differences between strains in adrenal weights are mainly the result of differences in size of the adrenal cortex . Discussion In the present study, basal plasma levels of NE and EPI were similar in B-N and WKY rats . After 5 minutes of footshock stress, there were significant increases in plasma levels of both catecholamines in B-N and WKY rats . In WKY rats, however, the stress-induced increments in plasma NE and EPI were two-fold greater than in B-N rats . The enzymes TH, DHH, and PNMT are involved in the biosynthesis of catecholamines in the adrenal medulla . In comparing the activities of these enzymes in the adrenal glands, it was found that the strain in which plasma levels of the catecholamines were greater after stress (WKY) had significantly lower activities of TH and DHH than did the less responsive (B-N) strain . Recently, we have replicated and extended these findings of strain differences TH activity (expressed as in catecholamine biosynthetic enzyme activities . units of enzyme activity per mq of tissue) was found to be greater in the adrenal medulla and superior cervical ganglion (SCG) of H-N rats . In contrast, the activities of choline acetyltransferase (CRT) in the adrenals and SCG and catechol-O-methyltransferase (COMT) in liver were greater in the WKY strain (22) . In the B-N and WKY strains, therefore, there appears to be an inverse relationship between activities of catecholamine biosynthetic enzymes in the adrenal medulla and SCG and the stress-induced increments in plasma levels These differences between strains may result from differences of NE and EPI . in the rates of release of NE and EPI into the circulation and/or from differences in the rates of removal of catecholamines from the circulation. The rate of removal of intravenously injected 3H-1-NE from the circulation of anesthetized B-N and WKY rats was examined to evaluate possible differences The level of 3H-1-NE in the in turnover of the catecholamines in plasma . plasma 15 seconds after injection of the labelled amine reflects initial dilution by the cardiac output and distribution to the tissues of the amine . The rate of decline in the plasma levels thereafter reflects the turnover rate of the released endogenous catecholamine . The cardiac output and apparent volume of distribution of the catecholamines appear to be greater in anesthetized WKY rats than in B-N rats since at 15 seconds after injection The rate of plasma levels of the labelled amine were lower in the WKY rats . removal of 3H-1-NE was more rapid in B-N than in WKY rats, so that the turnover rates of the catecholamines in plasma of B-N rats appear to be greater The basal plasma levels of catecholamines, however, were than in WKY rats . similar in the two strains of rats . To sustain similar levels with a more rapid turnover, the rate of release of catecholamines must be more rapid in B-N than in WKY rats . This is consistent with the higher levels of the catecholamine biosynthetic enzymes in this strain of rat . After exposure to stress, however, the less rapid rate of removal of released catecholaminea from the circulation of WKY rats contributes to the higher levels of plasma NE and EPI attained in this strain . A greater rate of release of catecholamines during stress by the WKY strain may also contribute to the observed differences. It is unlikely that the rate of O-methylation of circulating NE and EPI would contribute to the strain differences in plasma catecholamines during stress as the strain with the higher COMT activity in the liver also has the higher plasma levels of
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catecholamines . In several studies, the functional activity of the sympathetic-adrenal medullary system has been assessed by measurement of enzyme activities or However, a recent report (25) tissue content of catecholaminea (23,24) . suggests that presynaptic cholinergic activity may influence directly the release of catecholamines during stressful stimulation. This might contribute to the strain differences in the plasma catecholamine response to stress observed in the present study. In summary, we have observed in two inbred rat strains an inverse relationship between plasma catecholamine responses to stress and tissue levels of catecholamines, their biosynthetic enzymes , and size of the adrenal medulla . This strain difference appears to result from differences in rates of removal of catecholamines from the circulation as well as the rates of release of catecholamines from sympathetic nerve endings and the adrenal medulla . The results suggest that activities of TH, DBH, and PNMT may better reflect basal release and utilization of catecholamines than the functional capacity of the sympathetic-adrenal medullary system to respond to stress . Acknowledgement We thank Mr . Lester Carmon for his expert technical assistance . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 .
R. D. CTnunuur.rn, R, HARCHAS, S . KESSLER, and J . D . BARCHAS, Life Sci . _11 565-575 (1972) . R. D. CIARANEIS,O, H . J . HOFFMAN, J . G . M. SHIRE, and J . AXELROD, J . Biol . Chew . _249 4528-4536 (1974) . R. A. ROSS, A. B. JUDD, V . M . PICKEL, T. H. JOH, and D . J. REIS, Nature _264 654-656 (1976) . H. GROBECKER, M . F . ROIZETi, V. WEISE, J. M. SAAVEDRA, and 2 . J . KOPIN, Nature _258 267-268 (1975) . J . T. COYLE, P . WENDER, and A. LIPSKY, Psychopharmacologia _31 25-34 (1973) . R. KVETNANSKY, I. ALBRECHT, T . TORDA, N. SALEH, E. JAHNOVA, and L . MIKULAJ in Catecholamines and Stress (E . Usdin, R. Kvetnansky, and I. J . Kopin, eds.), pp . 237-249, Pergamon Press, Oxford (1976) . R. RVETNANSKY, V. K . WEISE, and I . J. KOPIN, Endocrinology _87 744-749 (1970) . H . THOENEN, Nature _228 861-862 (1970) . D. L. ELY and J . P . HENRY, Horen. Hehav . 10 156-169 (1978) . R. MCCARTY and I . J. KOPIN, Behav. Biol . in press . R. MCCARTY and I . J. KOPIN, Horen. Hehav. _11 248-257 (1978) . R. KVETNANSKY, C. L. SUN, C . R. LAKE, N. THOA, T. TORDA, and I . J . KOPIN, Endocrinology 103 1868-1874 (1978) . R. McCARTY and 2 . J. KOPIN, Physiol. Behau. _21 567-572 (1978) . C . C . CHIUEH and I . J . KOPIN, Am . J. Phyaiol. 234 H690-H695 (1978) . R. McCARTY, C . C. CHIUEH, and I . J . KOPIN, Behav . Biol . _23 180-188 (1978) . V . R. WEISE and I . J . KOPIN, Life Sci. _19 1673-1686 (1976) . J . D. PEULER and G . A . JOHNSON, Life Sci . _21 625-636 (1977) . T . NAGATSU, M. LEVITT, and S. UDENFRIEND, J . Biol . Chew . _239 2910-2917 (1964) . P . H . MOLINOFF, R. WEINSHILHOUM, and J. A3~LRnD, J . Pharmacol . Exp. Ther . 178 425-431 (1971) . . J AxFr.unD, J . Hiol . Chew . 237 1657-1660 (1962) .
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21 . 22 . 23 . 24 . 25 .
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L . A . SAPIRSTEIN, Am . J . Physiol . 193 161-170 (1958) . G . M . GILAD, R . McCARTY, V . K . WEISE, and I . J . KOPIN, Brain Res . in press . J . SLATER, D . A . HLIZZARD, and L . A . POHORECKY, Pharmacol . Biochem . Behav . _6 511-520 (1977) . J . D . HARCHAS, R . D . CIARANEISA, J . A . DOMINIC, T . DEGUCHI, E . K . ORENBERG, J . RENSON, and S . RESSLER, J . Psychiat . Res . 11 347-360 (1974) . I . H . ULUS, M . C . SCALLY, and R . J . WURTMAN, J . Phasmacol . Exp . Ther . 20 4 676-682 (1978) .
Pergamon Press
STRAIN DIFFERENCES IN RAT ADRENAL BIOSYNTHETIC ENZYMES AND STRESS-INDUCED INCREASES IN PLASMA CATECHOLAMINES Richard McCartyl, Gad M. Gilad2 , Virginia K. Weise, and Irwin J. lcopin 3 Laboratory of Clinical Science National Institute of Mental Health Bethesda, Maryland 20205 (Received in final form July 20, 1979)
We have examined in two inbred rat strains basal and stress-induced increases in plasma levels of epinephrine (EPI) and norepinephrine (NE) and compared these with activities of the adrenal enzymes involved in the synthesis of catecholamines . There were no differences in basal levels of NE and EPI in plasma of adult male rats of the Wistar-Kyoto (WKY) and Brown-Norway (B-N) strains . However, following 5 min. of intermittent footshock, plasma levels of both catecholamines were twice as high in WKY rata as in B-N rata . In the adrenals of unstressed rats, activities of tyrosine hydroxylase and dopamine-beta-hydroxylase were significantly higher in B-N rats . in addition, the adrenal weights and the contents of NE but not EPI were greater in B-N rats . Thus, in these taro rat strains, the capacity of the adrenal gland to synthesize and store catecholaminea appeared to be inversely related to plasma levels of NE and EPI after stress . The differences between the strains appeared to be due to differences in the rates of removal of catecholamines from the peripheral circulation as well as to differences in the rate of release of catecholaminea from the sympatho-adrenal medullary system . Thus biosynthetic enzyme activities need not be related directly to the capacity to release and elevate plasma levels of catecholaminea following stressful stimulation . Major genetic differences have been reported in the activities of the enzymes involved in catecholamine biosynthesis in central and peripheral tissues of inbred strains of rats and mice (1-5) . In addition, the activities of the enzymes tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanolamine-N-methyltransferase (PNMT) are increased by stressful stimulation and changes in enzyme levels have been utilized as indices of the capacity of tissues to synthesize and release catecholaminea (6-9) . Recent studies fro® this laboratory have demonstrated that plasma levels
Research Associate, Pharmacology-Toxicology Training Program, National Institute of General Medical Sciences . Present address : Department of Psychology, University of Virginia, Charlottesville, Virginia 22901 . 2.
Present address : Department of Isotope Research, Weizman Institute of Science, Rehovot, Israel .
3.
To whom requests for reprints should be addressed . 0024-3205/79/090747-0802 .00/0
748
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1979
of epinephrine (EPI) and norepinephrine (NE) provide an accurate index of sympatho-adrenal medullary discharge in response to alerting stimuli (10-12) . In addition, significant differences have been found among rat strains in footshock stress-induced increments in plasma levels of NE and EPI (13) . In the present study, we have examined in two inbred rat strains the relationship between catecholamine biosynthetic enzyme activities in the adrenal gland and stress-induced increases in plasma levels of NE and EPI . Rats of the Brown-Norway (B-N) and Wistar-Kyoto (WKY) strains were selected for study because of the marked differences between these two strains in the increments in plasma catecholamines following stressful stimulation (13) . Methods Male B-N rats were obtained from the Veterinary Resources Branch, National Institutes of Health and male WKY rata were purchased from Taconic Farms, Germantown, New York . Rats of each strain were housed in groups of 4 per cage for at least 1 week prior to use and food and water were available ad lib. The vivarium was maintained on a 12-hour photoperiod (lights on 0600-1800 hrs) at a temperature of 25°C . WRY and B-N rata (300-350 g) were anesthetized with pentobarbital (35-40 mg/kq) and a PE-50 catheter was inserted into the ventral caudal artery of each rat according to the method of Chiueh and Kopin (14) . The tubing traversed the subcutaneous tissue of the tail sheath and back to exit at the back of the neck . A 30 cm piece of spring wire was placed over the tubing and secured to the rat with an adhesive tape collar . The tubing was filled with saline containing heparin (500 U/ml) and the end was occluded with a 23G needle and a 1 ml plastic syringe . After surgery, each rat was placed in a clear plastic cage (25 x 25 x 16 cm) that contained bedding material and access to food and water . Each catheter was flushed with 0.5 ml of heparinized saline twice each day. Two days after surgery, blood samples (0 .5 ml) were obtained from the rats while they were resting and undisturbed in their home cages. Each rat was then transferred to a shock chamber (15) and exposed to 5 minutes of inescapable footshock (2 .5 mA, 0.4 sec . duration, every 5 seconds) . A second blood sample was drawn at the end of the interval of footshocks . Blood samples were centrifuged at 4,000 xg for 10 min. at 4°C . The plasma was removed and stored at -20°C until assayed for catecholamines by a radioenzymatic-thin layer chromatographic method (16,17) . The activities of the adrenal enzymes TH, DBH, and PNMT were measured in naive B-N and WRY male rats (250-350 g) . Animals were killed by decapitation and the adrenals were rapidly removed and the pair from each rat were weighed and homogenized in 1 .0 ml of 0 .3 M sucrose . Aliquots were removed for the assay of TH (18), DBH (19), PNMT (20), and catecholamines (16,17) . The rate of removal of exogenously administered 3H-1-NE (24 .7 Ci/mmole, New England Nuclear) was determined in 3 B-N and 3 WKY male rats (375-425 g) . Rats were anesthetized with pentobarbital and the right jugular vein and the 3 H-1-NE was diluted left carotid artery were cannulated with PE-50 tubing . 3 with saline to a concentration of 18 .8 ng/ml (1 .67 ~Ci H-1-NE per ml) and injected rapidly intravenously in a total volume of 0 .5 ml . Arterial blood samples were collected at 15, 30, 45, 60, and 75 seconds after injection. Plasma was obtained fry the blood samples and the 3H=1-NE was absorbed onto altm~ina at pH 8 .6 . The alumina was washed 3 times with buffer (pH 8 .6), the
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3 H-1-NE eluted with 0 .1 N HC1 and the tritium assaye8 by liquid scintillation spectrometry . In another experiment, the levels of 3H-1-NE were determined in plasma of blood obtained at intervals of 0 .67 seconds during the first 15 seconds following the tracer injection, as described by Sapirstein (21) . Results When rats were resting and undisturbed in their home cages, there were no strain differences in plasma levels of NS or EPI . However, after exposure to 5 minutes of intermittent footshock, the levels in plasma of NE and EPI were two-fold higher for WRY rats as compared to B-N rats (Fig . 1) .
BASAL
BTAESB®
Fig . 1 Basal plasma levels (pg/ml) of epinephrine (EPI) and norepinephrine (NE) in undisturbed WILY and H-N rats and levels after 5 minutes of footshock stress . Values are means + SEM for groupa of 6 rats per strain .
In a separate group of unstressed rata, a comparison between strains was made of the adrenal gland content of catecholamines and the levels of catecholamine biosynthetic enzymes . When compared to WRY rats, B-N rats had heavier adrenals which contained significantly more NS but not EPI (Fig . 2) . In addition, the activities of the adrenomedullary enzymes TH and DBH were significantly greater in B-N rats . No strain differences were obaerveä in the activity of PNMT in the adrenal gland (Fig . 3) . To determine if there were significant differences between strains in the rate of removal of catecholamines from the circulation, levels of 3 H-1-NE in plasma were determined after its rapid intravenous injection . At 15 seconds after injection, plasma levels of 3H-1-NE were higher in B-N than in WILY rats, but the rate of decline of the 3H-1-NE in plasma was more rapid in the B-N rats and by 1 min~te there was no significant difference between the attains in the levels of H-1-NE found in the plasma (Fig . 4) .
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10
10
5
0
0 Fig . 2
Paired adrenal weights (pg) and contents norepinephrine (NE) of WKY and B-N rats . of 10 rats per strain .
(pg/`pair) of epinephrine (EPI) and Values are means _+ SEM for groups
Fig . 3 Activities (nmoles product formed per paired adrenals per hr) of tyrosine hydroxylase (TH), dopamine-beta-hydroxylase (DBH), and phenylethanolamine-Nmethyltransferase (PNMT) in the paired adrenals of WRY and B-N rats . Values are means + SEM for groups of 10 rats per strain .
Measurements of the levels of 3H-1-NE in plasma during the first 15 seconds after a tracer injection showed that the WRY rata had a greater cardiac output and that the 3H-1-NE was diluted to a greater extent in these rats than in B-N rats (data not shown) . Adrenal glands from B-N and WKY rats were preserved in loi neutral formalin, serially sectioned, and stained with cresyl violet (Fiq . 5) .
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INTERCEPT SLOPE ~ B-N 20000 t 83D cpm/ml -0.0,61 1 .0008 o WKY 7870 t 260 cpm/ml -0 .0318 t "0007 sec - ~
_v
W Z OL
a
W Z â W OC Z 2 M
Q
N g
a
Fig. 4 Rate of removal of an intravenous injection of 3H-1-norepinephrine from the circulation in 1tP1CY and B-N rats while under pentobarbital anesthesia .
Fig. 5 Photomicrographs of sections (50 microns thick) through the adrenal glands of tü1~CY (A) and B-N (B) rata, stained with cresyl violet . The adrenal gland, including cortex and medulla of 1PRY rats (A) is smaller than that of B-N rats (B) . X 45 .
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Volumes (mm3) calculated from serial sections were : whole gland (B-N : 37 .8, WKY : 30 .2) and adrenal medulla (B-N : 8 .95, WKY: 6 .95) . Thus, the differences between strains in adrenal weights are mainly the result of differences in size of the adrenal cortex . Discussion In the present study, basal plasma levels of NE and EPI were similar in B-N and WKY rats . After 5 minutes of footshock stress, there were significant increases in plasma levels of both catecholamines in B-N and WKY rats . In WKY rats, however, the stress-induced increments in plasma NE and EPI were two-fold greater than in B-N rats . The enzymes TH, DHH, and PNMT are involved in the biosynthesis of catecholamines in the adrenal medulla . In comparing the activities of these enzymes in the adrenal glands, it was found that the strain in which plasma levels of the catecholamines were greater after stress (WKY) had significantly lower activities of TH and DHH than did the less responsive (B-N) strain . Recently, we have replicated and extended these findings of strain differences TH activity (expressed as in catecholamine biosynthetic enzyme activities . units of enzyme activity per mq of tissue) was found to be greater in the adrenal medulla and superior cervical ganglion (SCG) of H-N rats . In contrast, the activities of choline acetyltransferase (CRT) in the adrenals and SCG and catechol-O-methyltransferase (COMT) in liver were greater in the WKY strain (22) . In the B-N and WKY strains, therefore, there appears to be an inverse relationship between activities of catecholamine biosynthetic enzymes in the adrenal medulla and SCG and the stress-induced increments in plasma levels These differences between strains may result from differences of NE and EPI . in the rates of release of NE and EPI into the circulation and/or from differences in the rates of removal of catecholamines from the circulation. The rate of removal of intravenously injected 3H-1-NE from the circulation of anesthetized B-N and WKY rats was examined to evaluate possible differences The level of 3H-1-NE in the in turnover of the catecholamines in plasma . plasma 15 seconds after injection of the labelled amine reflects initial dilution by the cardiac output and distribution to the tissues of the amine . The rate of decline in the plasma levels thereafter reflects the turnover rate of the released endogenous catecholamine . The cardiac output and apparent volume of distribution of the catecholamines appear to be greater in anesthetized WKY rats than in B-N rats since at 15 seconds after injection The rate of plasma levels of the labelled amine were lower in the WKY rats . removal of 3H-1-NE was more rapid in B-N than in WKY rats, so that the turnover rates of the catecholamines in plasma of B-N rats appear to be greater The basal plasma levels of catecholamines, however, were than in WKY rats . similar in the two strains of rats . To sustain similar levels with a more rapid turnover, the rate of release of catecholamines must be more rapid in B-N than in WKY rats . This is consistent with the higher levels of the catecholamine biosynthetic enzymes in this strain of rat . After exposure to stress, however, the less rapid rate of removal of released catecholaminea from the circulation of WKY rats contributes to the higher levels of plasma NE and EPI attained in this strain . A greater rate of release of catecholamines during stress by the WKY strain may also contribute to the observed differences. It is unlikely that the rate of O-methylation of circulating NE and EPI would contribute to the strain differences in plasma catecholamines during stress as the strain with the higher COMT activity in the liver also has the higher plasma levels of
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catecholamines . In several studies, the functional activity of the sympathetic-adrenal medullary system has been assessed by measurement of enzyme activities or However, a recent report (25) tissue content of catecholaminea (23,24) . suggests that presynaptic cholinergic activity may influence directly the release of catecholamines during stressful stimulation. This might contribute to the strain differences in the plasma catecholamine response to stress observed in the present study. In summary, we have observed in two inbred rat strains an inverse relationship between plasma catecholamine responses to stress and tissue levels of catecholamines, their biosynthetic enzymes , and size of the adrenal medulla . This strain difference appears to result from differences in rates of removal of catecholamines from the circulation as well as the rates of release of catecholamines from sympathetic nerve endings and the adrenal medulla . The results suggest that activities of TH, DBH, and PNMT may better reflect basal release and utilization of catecholamines than the functional capacity of the sympathetic-adrenal medullary system to respond to stress . Acknowledgement We thank Mr . Lester Carmon for his expert technical assistance . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 .
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L . A . SAPIRSTEIN, Am . J . Physiol . 193 161-170 (1958) . G . M . GILAD, R . McCARTY, V . K . WEISE, and I . J . KOPIN, Brain Res . in press . J . SLATER, D . A . HLIZZARD, and L . A . POHORECKY, Pharmacol . Biochem . Behav . _6 511-520 (1977) . J . D . HARCHAS, R . D . CIARANEISA, J . A . DOMINIC, T . DEGUCHI, E . K . ORENBERG, J . RENSON, and S . RESSLER, J . Psychiat . Res . 11 347-360 (1974) . I . H . ULUS, M . C . SCALLY, and R . J . WURTMAN, J . Phasmacol . Exp . Ther . 20 4 676-682 (1978) .
