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Cardiovascular and sympathetic nervous system responses to an acute stressor in borderline hypertensive rats (BHR)
Physiology& Behavior.Vol. 46, pp. 309-313. ©Pergamon Press plc, 1989. Printed in the U.S.A.
0031-9384/89 $3.00 + .00
Cardiovascular and Sympathetic Nervous System Responses to an Acute Stressor in Borderline Hypertensive Rats (BHR) R O B E R T F. K I R B y , 1 M I C H A E L F. C A L L A H A N , R I C H A R D M c C A R T Y * A N D A L A N K I M J O H N S O N
Departments of Psychology and Pharmacology and the Cardiovascular Center, University of Iowa, Iowa City, IA 52242 and *Department of Psychology, University of Virginia, Charlottesville, VA 22903 R e c e i v e d 1 February 1989
KIRBY, R. F., M. F. CALLAHAN, R. McCARTY AND A. K. JOHNSON. Cardiovascular and sympathetic nervous system responses to an acute stressor in borderline hypertensiverats (BHR). PHYSIOL BEHAV 46(2) 309-313, 1989.--The present study examined cardiovascular and plasma catecholamine responses to acute footshock stress in adult male Wistar-Kyoto (WKY) normotensive, borderline hypertensive (BHR), and spontaneously hypertensive (SHR) rats. Basal mean arterial pressure and heart rate were equivalent for SHRs and BHRs, and levels for both groups were elevated compared to WKYs. Following transfer to the footshock chamber, blood pressure increased to a greater degree in SHRs than in WKYs or BHRs. However, the tachycardia was exaggerated in both BHRs and SHRs compared to WKYs. In response to intermittent footshock stress, all groups had comparable heart rate increases while maintaining blood pressure near baseline levels. SHRs demonstrated a sympathetic hyperresponsiveness to footshock stress, with greater increases in plasma norepinephrine and epinephrine levels than WKYs immediately following footshock. At 5 minutes postfootshock, plasma catecholamines remained elevated in SHRs over both WKYs and BHRs. Plasma catecholamine increases following footshock were comparable at all time points between WKYs and BHRs. The present results demonstrate that sympathetic responsiveness of BHRs to acute footshock stress is more similar to normotensive WKYs than to hypertensive SHRs. Hypertension Plasma catcholamines Borderline hypertensive rat
Stress
Spontaneously hypertensive rat
Cardiovascular regulation
to develop high blood pressure. The BHR model is also of value because it provides an opportunity to investigate the mechanisms that underly the pathogenesis of hypertension in which stress is an etiological factor. Several investigators have concluded that the hypertension evinced by SHRs results from heightened activity or reactivity of the sympathetic nervous system (6, 7, 10). Thus, one hypothesis to account for the development of hypertension in BHRs following chronic stress is that sympathetic responses to environmental stressors may be exaggerated in these animals. The increase in sympathetic outflow would then lead to structural alterations in the vasculature that would contribute to maintenance of hypertension when the initiating factors were no longer present. This sequence of events is consistent with the views of Folkow and other investigators that sympathetic activity plays an important role in the etiology of human essential hypertension (2,4). Plasma catecholamine levels have served as a valuable index for examining sympathetic nervous system function in laboratory rats (10,18). A small percentage of norepinephrine (NE) released from sympathetic nerve terminals reaches the bloodstream unmetabolized. In contrast, epinephrine (EPI) is the primary amine
THE borderline hypertensive rat (BHR) is the first generation progeny of a cross between the spontaneously hypertensive rat (SHR) and its Wistar-Kyoto (WKY) normotensive strain. The BHR model developed by Lawler and his colleagues demonstrates that aversive environmental factors can lead to long-term elevations of blood pressure into the hypertensive range (13-15). The stressor employed by these investigators was a conflict-conflict paradigm, in which no response led to five low intensity cutaneous shocks while a correct response led to only one shock. Following a 3 week training session which was begun at 4 months of age, the shock-shock conflict paradigm was continued for 15 weeks. Systolic blood pressure rose from 150 mmHg to about 190 mmHg during this time and remained elevated for at least 10 weeks following cessation of the conflict paradigm. In addition, BHRs exposed to this prolonged stressor developed cardiac hypertrophy and pathologies consistent with essential hypertension in humans (15). Further studies demonstrated that the increase in blood pressure was specific to BHRs, and could not be elicited in WKY rats exposed to the same stressor (14). These results indicate that environmental stress can lead to the development of hypertension, especially in animals with a genetic susceptibility or predisposition
1Requests for reprints should be addressed to Robert F. Kirby, Department of Psychology, University of Iowa, Iowa City, IA 52242.
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released from the adrenal medulla and this hormone enters the circulation directly. The short half-life of these amines in plasma (approximately 70 seconds in laboratory rats) makes them a useful index for assessing rapid changes in overall sympathetic outflow (10,18). The measurement of plasma catecholamines has been especially valuable for examining sympathetic nervous system responses to acute stress in animals genetically prone to the development of hypertension (10, 11, 18). The pathogenesis of sustained hypertension in BHRs following chronic stress may be due to increased sympathetic responses to stressors which could be inherited from the SHR strain. In order to assess the potential role(s) of sympathetic hyperreactivity in the pathogenesis of stress-induced hypertension in the BHR, the current study examined plasma catecholamine and cardiovascular responses to a brief period of acute footshock stress in adult male WKYs, BHRs, and SHRs. METHOD
Animals Male WKYs, BHRs, and SHRs were born and reared to adulthood in our vivarium. These animals were derived from the SHR and WKY colonies maintained by the University of Iowa Cardiovascular Research Center. The BHRs were the first generation offspring of a cross between SHR females and WKY males (13-15). Within the first 24 hours after birth, litters were culled to eight pups and all pups were raised by their natural mothers. Animals were housed individually from the time of weaning at thirty days postnatal age until tested as adults two months later. Animals were maintained with standard Purina laboratory chow and tap water available ad lib on a 14 hour light/10 hour dark cycle (lights on at 0600).
Surgical Procedures At three months of age, each animal was weighed and anesthetized with Nembutal (pentobarbital sodium, 50 mg/kg). An indwelling PE-50 catheter was surgically placed in the right common carotid artery and guided subcutaneously to exit at the nape of the neck. The catheter was filled with isotonic saline containing 200 IU/ml of heparin and occluded with a sealed 23-gauge needle.
Experimental Protocol Two days after surgery, baseline testing was carried out on each animal in its home cage 60-90 minutes after connecting the arterial catheter to a pressure transducer (Century Technology Company). A 10-15 second baseline measure of mean arterial pressure (MAP, mmHg) and heart rate (HR, beats/min) was recorded on a Beckman Dynograph with the animal resting and undisturbed in its home cage. A 0.6 ml blood sample was then collected from the arterial line into a chilled glass tube for measurement of catecholamines. Blood withdrawn from the catheter was immediately replaced by an equal volume of isotonic saline containing 20 IU/ml of heparin. Shortly after basal measures were established, each animal was transferred individually to a clear Plexiglas chamber (25 x 25 x 60 cm) with a stainless steel grid floor while MAP and HR were recorded. The response of each rat to transfer was recorded immediately and 15 seconds after placement in the Plexiglas chamber before the footshock began. A 5 minute period of intermittent footshock (2.5 mA DC, 0.5 sec duration, every 5 sec) was then administered to the animal using a Lehigh Valley constant current shock supply. Mean arterial pressure and HR
were recorded for a 10 second period immediately and 5 minutes following the termination of footshock. Blood samples (0.6 ml) were collected from the arterial catheter at each of these times for analysis of catecholamine levels and the volume was replaced with isotonic saline. At the cessation of testing, animals were euthanized by an overdose of anesthetic infused directly into the arterial line. Footshock testing was Performed between 1100 and 1500 hours; WKY, BHR, and SHR were tested in a randomized order. The chamber was cleaned thoroughly using a mild soap solution after each use.
Biochemical Analysis Blood samples were kept in an ice water bath until centrifuged at 3000 x g for 5 minutes at 4°C. Plasma from each sample was deproteinated by the addition of an equal volume of 0.1 N perchloric acid containing 2% EGTA and 0.2% MgC12 and stored at - 2 0 ° C . NE and EPI were analyzed by a radioenzymatic thin-layer chromatographic procedure (1,23). Samples were incubated for 90 minutes at 37°C with a partially purified preparation of rat liver catechol-O-methyl transferase and S-(3H-methyl) adenosylmethionine. The labeled metanephrine and normetanephrine were extracted into toluene-isoamyl alcohol, back extracted into 0.1 N acetic acid and dried overnight in vacuo. The residues were then dissolved in methanol and spotted onto Whatman LK5DF thin-layer chromatography plates to separate the Omethylated amines. The plates were developed in chloroform: ethanol:ethylamine (60:11.25:7.5 ml) and the bands corresponding to metanephrine and normetanephrine were scraped and placed into glass scintillation vials. The amines were then eluted from the silica gel with 0.1 M ammonium hydroxide, oxidized by treatment with sodium periodate and assayed by liquid scintillation spectrometry. Internal NE and EPI standards as well as plasma blanks were included in each assay. Assay sensitivity was less than 10 pg for each catecholamine and the intra- and interassay coefficients of variation were less than 5%.
Data Analysis Data from each animal were used only if measures of MAP, HR, and plasma catecholamines were available for every time point. Cardiovascular and plasma catecholamine responses to transfer and footshock were analyzed using the General Linear Models procedure for repeated measure analysis of variance with contrast tests run for groups (WKY, BHR, SHR) and time (baseline, transfer, 0 and 5 minutes postfootshock) (SAS Institute, Cary, NC, Version 5). Cardiovascular responses following footshock were compared to the 15 second posttransfer measure which was immediately before the onset of the footshock episode. All results are presented as means ___SEM for 9-13 animals per group. RESULTS
Baseline cardiovascular and plasma catecholamine measures are presented in Table 1. Basal MAP and HR were comparable for BHRs and SHRs, although these measures were elevated for both groups when compared to WKYs. In contrast, baseline plasma catecholamine levels were greatest in BHRs, which had significantly greater NE levels than either WKYs or SHRs. Body weights were also found to differ between the three groups, with BHRs being heavier than WKYs and SHRs. In response to transfer to the footshock chamber, both MAP and HR increased significantly [Fig. 1; F(1,30) = 41.2 and F(1,30) = 179.5, respectively; p's<0.0001]. However, a significant interaction was found for MAP between groups. SHRs had large MAP increases following transfer which were significantly greater than
SYMPATHETIC RESPONSES TO STRESS IN BHR
TABLE 1 BASELINE BLOOD PRESSURES, HEART RATES, PLASMA CATECHOLAMINE LEVELS, AND BODY WEIGHTS IN ADULT MALE WISTAR-KYOTO (WKY), BORDERLINE HYPERTENSIVE (BHR), AND SPONTANEOUSLY HYPERTENSIVE RATS (SHR)
Measure
WKY
MAP (mmHg) HR (beats/min) Plasma NE (pg/ml) Plasma EPI (pg/ml) Body wt. (g)
112 298 299 117 357
BHR
- 4 --- 20 --+ 32 - 20 --- 7
147 352 418 161 407
SHR
± 2* --- 6* +-- 32t +-- 21 _ 7t
146 339 302 113 338
--+ 8* --- 11 : 36 --- 27 --- 6
Results are presented as means --- SEM for 9-13 animals per group. *Significantly different from WKY controls (p<0.05). tSignificantly different from WKYs and SHRs (p<0.05).
those of WKYs and BHRs. In contrast, HR increases following transfer were similar for SHRs and BHRs, and these responses were significantly greater than those of WKYs. The cardiovascular responses to intermittent footshock stress are depicted in Fig. 2. There was a significant effect of footshock on MAP, with a slight decrease immediately following footshock, F ( 1 , 3 0 ) = 15.85, p = 0 . 0 0 0 4 , that reverted to a slight increase at five minutes postfootshock, F(1,30) = 5.35, p = 0 . 0 2 7 7 . There was no significant interaction between groups and time postfootshock on the MAP response. In contrast, footshock produced large increases in HR, F ( 2 , 2 9 ) = 109.4, p < 0 . 0 0 0 1 , but the response varied between groups, F(4,58) = 4.569, p = 0.0028. The increase in HR immediately following footshock was similar for the three groups, but by five minutes postfootshock HRs of BHRs had
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Time Post-Footshock FIG. 2. Cardiovascular responses immediately and 5 minutes postfootshock for Wistar-Kyoto (WKY), borderline hypertensive (BHR), and spontaneously hypertensive (SHR) rats. The mean changes from the prefootshock baseline values --- 1 SEM are presented for MAP (top panel) and HR (bottom panel). Footshock significantly influenced MAP, F(2,29) = 22.08, p<0.001, but there were no interactions between the groups and time postfootshock. The HR response was equivalent across groups immediately following footshock but decreased to a greater extent in BHRs than WKYs or SHRs.
decreased to a greater extent than in either the WKYs, F(1,30) = 14.97, p < 0 . 0 0 0 5 , or the SHRs, F ( 1 , 3 0 ) = 7 . 6 6 , p = 0 . 0 0 9 6 . The plasma NE and EPI responses to footshock were similar in pattern to one another and these responses are depicted in Fig. 3. Plasma catecholamines were elevated following footshock and there was a significant interaction for the NE response with the animal's group and the interaction between groups and time approached significance for the EPI response, F ( 4 , 4 6 ) = 2 . 3 7 , p = 0.066. The increase in plasma NE levels above baseline did not differ significantly between the three groups immediately following footshock, although the increase was twice as large in the SHRs compared to the BHRs and WKYs. At five minutes postfootshock, the plasma NE increase above baseline was significantly greater in the SHR's than either the BHRs or the WKYs. Plasma EPI increases above baseline were also greatest in SHRs immediately following footshock, and were significantly greater than in the WKYs. By five minutes postfootshock, the increase in plasma EPI above baseline was still greatest in SHRs, and the levels were significantly greater in SHRs than in BHRs or WKYs. DISCUSSION
Transfer Response FIG. 1. Cardiovascular responses to transfer to a novel chamber in Wistar-Kyoto (WKY), borderline hypertensive (BHR), and spontaneously hypertensive (SHR) rats. The mean increases above baseline - 1 SEM for MAP (top panel) and HR (bottom panel) are presented. The increase in MAP to transfer was significantly greater in SHRs (**) than either BHRs, F(I,21)= 12.14, p<0.001, or WKYs, F(1,19)= 11.66, p<0.002. However, HR increased to a greater degree (**) in both SHRs, F(1,18) = 20.87, p<0.001, and BHRs, F(1,22)= 17.91, p<0.001, than WKYs.
The SHR is the most extensively studied animal model of human essential hypertension. This strain shares many commonalities with essential hypertension: including a progressive development, responsiveness to similar antihypertensive pharmacological agents, and decreased sensitivity of high pressure baroreceptors (10,11). Two distinct abnormalities in sympathetic nervous system function have been demonstrated in adult SHRs when compared to WKY normotensive rats. These are: a) increased sympathetic terminal outflow of NE in response to stress and; b) heightened responsiveness of the vasculature to released catecholamines
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FIG. 3. Mean norepinephrine and epinephrine levels - 1 SEM in WistarKyoto (WKY), borderline hypertensive (BHR), and spontaneously hypertensive (SHR) rats immediately and 5 minutes postfootshock. Plasma norepinephrine increased significantly following footshock, F(2,29)= 21.89, p<0.001, and the response varied between groups, F(4,50) = 2.60, p<0.05. Norepinephrine levels were significantly greater in SHRs than in BHRs, F(1,22) = 10.33, p<0.01, and WKYs, F(1,20) = 4.32, p<0.05, at 5 minutes postfootshock. Plasma epinephrine levels also increased significantly in response to footshock, F(2,29) = 46.04, p<0.001. The response was greater in SHRs than in WKYs immediately, F(1,20) = 6.39, p<0.05, and 5 minutes, F(1,20)=4.43, p<0.05, following the termination of footshock. The SHR response was significantly greater than the BHR response at 5 minutes postfootshock, F(1,22)=4.28, p<0.05. No differences were found between the WKY and BHR responses to footshock. Results of individual comparisons are denoted by brackets with, *p<0.05 and **p<0.01. (10,11). Together, these produce accentuated elevations in blood pressure and heart rate in response to immobilization, acute " m e n t a l " stress, anticipation of footshock, and placement of animals into a novel environment (10-12, 17, 20). Judy and co-workers have proposed that elevated sympathetic activity is causal to the maintenance of hypertension in the SHR (6,7). This view comes from two lines of evidence. First, activity of pre- and postganglionic sympathetic fiber bundles was greater in SHR than in age-matched normotensive Wistar rats. Treatment with hexamethonium, which blocked ganglionic neurotransmission, decreased blood pressure in both strains and led to equivalent pressures in SHR and Wistar control rats. Second, in the sixth backcross generation of SHRs and normotensive Wistar/Lewis rats, sympathetic nerve activity and resting arterial pressure were highly correlated. These data are very supportive of a causal role for sympathetic activity in the elevated blood pressures of SHRs and their backcrossed progeny. In the present study, increased basal NE levels were found for BHRs. This result may be an indication that elevated tonic sympathetic activity plays a greater role in maintaining basal arterial pressure in BHRs. In contrast, vascular resistance may play a greater role in the elevated pressure of SHRs. Subjects in this experiment were examined at 12 weeks of age. At this age, MAPs of BHRs have peaked but those of SHRs will continue to
increase until approximately 18-22 weeks of age. Although BHRs and SHRs showed comparable basal MAPs in the current study, differences in arterial resistance may be present for these two groups. Elevated arterial resistance of SHRs is not due solely to structural adaptation of the vasculature to increased pressure, but also appears to be due to genetic influences (2,8). Vascular hypertrophy in the SHR still occurs if the development of hypertension is inhibited by early catecholaminergic fiber destruction with 6-OHDA (25). In addition, SHRs develop a greater increase in vascular resistance than genetically normotensive animals which have been hypertensive for an equivalent period (16). Therefore, increased resistance of the vasculature in SHRs is not purely a response to elevated pressure. Taken together, these findings indicate that the similar resting pressures of SHRs and BHRs may be maintained through elevated vascular resistance in the SHR and increased sympathetic drive in the BHR. Transfer to the footshock chamber increased blood pressure in SHRs but not in WKYs or BHRs. This response would be consistent with the known differences in sympathetic responses to acute stress and increased vascular sensitivity of SHRs compared to WKYs (10,11). In contrast, the tachycardic response of SHRs and BHRs to transfer was equivalent, and was significantly greater than that of WKYs. These data suggest that sympathetic drive to the heart increased more in BHRs and SHRs than in normotensive WKYs. Therefore, the mild stress of transfer may unmask elements of disordered cardiovascular responsiveness of BHRs to mild environmental challenges. Plasma catecholamine responses of BHRs to acute footshock stress were more similar to WKYs than to SHRs in the present study. These results indicate that BHRs do not share the sympathetic hyperresponsiveness to acute footshock stress that is characteristic of SHRs. Exaggerated sympathetic responses of SHRs can also be seen in response to immobilization (12), placement in a novel environment (19,21), anticipation of footshock (20), air puff stimulation (9,24), and auditory stimulation (3). Thus, the lack of an exaggerated plasma catecholamine response in BHRs found in the present study is probably not due to the type of stressor employed. This suggests that BHRs and WKYs may exhibit similar sympathetic responses to the conflict-conflict paradigm used by Lawler and co-workers to produce stressinduced hypertension in BHRs (13-15). However, an important distinction between the present study and those of Lawler and co-workers is the duration of the stressor. Habituation of sympathetic responses is normally seen when a stressor is repeated over time (22). It is possible that sympathetic responses in BHRs might not habituate to a repeated stress. Thus, BHRs might ultimately show a response that would be functionally equivalent to an exaggerated sympathetic response if the stressor were presented in repeated sessions. Evidence consistent with this hypothesis has been reported by Hubbard and co-workers (5). After 5 days of conflict-conflict stress, greater plasma NE levels were found in BHRs than in WKYs following stressor presentation. In summary, plasma catecholamine responses of BHRs to acute footshock stress were comparable to WKYs. In contrast, SHRs had consistently greater plasma NE and EPI responses to acute stress than either BHRs or WKYs. ACKNOWLEDGEMENTS This research was supported by U.S. Public Health Service Grants HL33447, HL29906, HL33796, HL14388; NIMH Research Scientist Development Awards MH00064 and MH00529; and by a grant (84-6-19) from the Iowa Affiliate of the American Heart Association. R. F. Kirby was supported by NHLBI postdoctoral fellowship HL07096.
SYMPATHETIC RESPONSES TO STRESS IN BHR
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REFERENCES 1. DaPrada, M.; Zurcher, G. Simultaneous radioenzymatic determination of plasma and tissue adrenaline, noradrenaline, and dopamine within the femtomole range. Life Sci. 19:1161-1174; 1976. 2. Folkow, B. Physiological aspects of primary hypertension. Physiol. Rec. 62:347-504; 1982. 3. Galeno, T. M.; VanHoesen, G. W.; Brody, M. J. Central amygdaloid nucleus lesion attentuates exaggerated hemodynamic responses to noise stress in the spontaneously hypertensive rat. Brain Res. 291: 249-259; 1984. 4. Goldstein, D. S. Plasma catecholamines and essential hypertension: An analytical review. Hypertension 5:86-99; 1983. 5. Hubbard, J. W.; Cox, R. H.; Sanders, B. J. Changes in cardiac output and vascular resistance during behavioral stress in the rat. Am. J. Physiol. 251:R82-R90; 1986. 6. Judy, W. V.; Watanabe, A. M.; Henry, D. P.; Besch, H. R.; Murphy, W. R.; Hockel, G. M. Sympathetic nerve activity: Role in regulation of blood pressure in the spontaneously hypertensive rat. Circ. Res. 38(Suppl. 2):21-29; 1976. 7. Judy, W. V.; Watanabe, A. M.; Murphy, W. R.; Aprison, B. S.; Yu, P. Sympathetic nerve activity and blood pressure in normotensive backcross rats genetically related to the spontaneously hypertensive rat. Hypertension 1:598--604; 1979. 8. Karr-Dulien, V.; Bloomquist, E. I.; Beringer, T.; EI-Bermani, A. I. Flow-pressure relationships in newborn and infant spontaneously hypertensive rats. Blood Vessels 18:245-252; 1981. 9. Koepke, J. P.; DiBona, G. F. High sodium intake enhances renal nerve and antinawiuretic responses to stress in spontaneously hypertensive rats. Hypertension 7:357-363; 1985. 10. Kopin, I. J.; McCarty, R.; Torda, T.; Yamaguchi, I. Catecholamines in plasma and responses to stress. In: Usdin, E.; Kvetnansky, R.; Kopin, I., eds. Catecholamines and stress: Recent advances. New York: Elsevier-North Holland; 1980:197-204. 11. Kopin, I. J.; McCarty, R.; Yamaguchi, I. Plasma catecholamines in human and experimental hypertension. Clin. Exp. Hypertens. 2: 379-394; 1980. 12. Kvetnansky, R.; McCarty, R.; Thoa, N. B.; Lake, C. R.; Kopin, I. J. Sympatho-adrenal responses of spontaneously hypertensive rats to immobilization stress. Am. J. Physiol. 236:H457-H462; 1979. 13. Lawler, J. E.; Barker, G. F.; Hubbard, J. W.; Allen, M. T. The
14.
15. 16.
17. 18.
19.
20. 21. 22.
23. 24. 25.
effects of conflict on tonic levels of blood pressure in the genetically borderline hypertensive rat. Psychophysiology 17:363-370; 1980. Lawler, J. E.; Barker, G. F.; Hubbard, J. W.; Cox, R. H.; Randall, G. W. Blood pressure and plasma renin activity responses to chronic stress in the borderline hypertensive rat. Physiol. Behav. 32:101-105; 1984. Lawler, J. E.; Barker, G. F.; Hubbard, J. W.; Schanb, R. G. Effects of stress on blood pressure and cardiac pathology in rats with borderline hypertension. Hypertension 3:496-505; 1981. Lundgren, Y.; Hallback, M.; Weiss, L.; Folkow, B. Rate and extent of adaptive cardiovascular changes in rats during experimental renal hypertension. Acta Physiol. Scand. 91:103-115; 1974. Lundin, S.; Thoren, P. Renal function and sympathetic activity during mental stress in normotensive and spontaneously hypertensive rats. Acta Physiol. Scand. 115:115-124; 1982. McCarty, R. Stress, behavior and the sympatho-adrenal medullary system. In: Pohorecky, L. A.; Brick, J., eds. Stress and alcohol use. New York: Elsevier; 1983:7-22. McCarty, R.; Chiueh, C. C.; Kopin, I. J. Behavioral and cardiovascular responses of spontaneously hypertensive and normotensive rats to inescapable footshock. Behav. Biol. 22:405-410; 1978. McCarty, R.; Chiueh, C. C.; Kopin, I. J. Spontaneously hypertensive rats: Adrenergic hyperresponsivity to anticipation of electric shock. Behav. Biol. 23:180-188; 1978. McCarty, R.; Kopin, I. J. Alterations in plasma catecholamines and behavior during acute stress in spontaneously hypertensive and WistarKyoto normotensive rats. Life Sci. 22:997-1006; 1978. McCarty, R.; Kvetnansky, R.; Lake, C. R.; Thoa, N. B.; Kopin, I. J. Sympatho-adrenal activity of SHR and WKY rats during recovery from forced immobilization. Physiol. Behav. 21:951-955; 1978. Peuler, J.; Johnson, G. A. Simultaneous single isotope radioenzymatic assay of plasma norepinephrine, epinephrine and dopamine. Life Sci. 21:625-636; 1977. Ricksten, S. E.; Yao, T.; DiBona, G. F.; Thoren, P. Renal nerve activity and exaggerated natriuresis in conscious spontaneously hypertensive rats. Acta Physiol. Scand. 112:161-167; 1981. Weiss, L. Aspects of the relation between functional and structural cardiovascular factors in primary hypertension. Acta Physiol. Scand. [Suppl.] 409:1-58; 1974.
0031-9384/89 $3.00 + .00
Cardiovascular and Sympathetic Nervous System Responses to an Acute Stressor in Borderline Hypertensive Rats (BHR) R O B E R T F. K I R B y , 1 M I C H A E L F. C A L L A H A N , R I C H A R D M c C A R T Y * A N D A L A N K I M J O H N S O N
Departments of Psychology and Pharmacology and the Cardiovascular Center, University of Iowa, Iowa City, IA 52242 and *Department of Psychology, University of Virginia, Charlottesville, VA 22903 R e c e i v e d 1 February 1989
KIRBY, R. F., M. F. CALLAHAN, R. McCARTY AND A. K. JOHNSON. Cardiovascular and sympathetic nervous system responses to an acute stressor in borderline hypertensiverats (BHR). PHYSIOL BEHAV 46(2) 309-313, 1989.--The present study examined cardiovascular and plasma catecholamine responses to acute footshock stress in adult male Wistar-Kyoto (WKY) normotensive, borderline hypertensive (BHR), and spontaneously hypertensive (SHR) rats. Basal mean arterial pressure and heart rate were equivalent for SHRs and BHRs, and levels for both groups were elevated compared to WKYs. Following transfer to the footshock chamber, blood pressure increased to a greater degree in SHRs than in WKYs or BHRs. However, the tachycardia was exaggerated in both BHRs and SHRs compared to WKYs. In response to intermittent footshock stress, all groups had comparable heart rate increases while maintaining blood pressure near baseline levels. SHRs demonstrated a sympathetic hyperresponsiveness to footshock stress, with greater increases in plasma norepinephrine and epinephrine levels than WKYs immediately following footshock. At 5 minutes postfootshock, plasma catecholamines remained elevated in SHRs over both WKYs and BHRs. Plasma catecholamine increases following footshock were comparable at all time points between WKYs and BHRs. The present results demonstrate that sympathetic responsiveness of BHRs to acute footshock stress is more similar to normotensive WKYs than to hypertensive SHRs. Hypertension Plasma catcholamines Borderline hypertensive rat
Stress
Spontaneously hypertensive rat
Cardiovascular regulation
to develop high blood pressure. The BHR model is also of value because it provides an opportunity to investigate the mechanisms that underly the pathogenesis of hypertension in which stress is an etiological factor. Several investigators have concluded that the hypertension evinced by SHRs results from heightened activity or reactivity of the sympathetic nervous system (6, 7, 10). Thus, one hypothesis to account for the development of hypertension in BHRs following chronic stress is that sympathetic responses to environmental stressors may be exaggerated in these animals. The increase in sympathetic outflow would then lead to structural alterations in the vasculature that would contribute to maintenance of hypertension when the initiating factors were no longer present. This sequence of events is consistent with the views of Folkow and other investigators that sympathetic activity plays an important role in the etiology of human essential hypertension (2,4). Plasma catecholamine levels have served as a valuable index for examining sympathetic nervous system function in laboratory rats (10,18). A small percentage of norepinephrine (NE) released from sympathetic nerve terminals reaches the bloodstream unmetabolized. In contrast, epinephrine (EPI) is the primary amine
THE borderline hypertensive rat (BHR) is the first generation progeny of a cross between the spontaneously hypertensive rat (SHR) and its Wistar-Kyoto (WKY) normotensive strain. The BHR model developed by Lawler and his colleagues demonstrates that aversive environmental factors can lead to long-term elevations of blood pressure into the hypertensive range (13-15). The stressor employed by these investigators was a conflict-conflict paradigm, in which no response led to five low intensity cutaneous shocks while a correct response led to only one shock. Following a 3 week training session which was begun at 4 months of age, the shock-shock conflict paradigm was continued for 15 weeks. Systolic blood pressure rose from 150 mmHg to about 190 mmHg during this time and remained elevated for at least 10 weeks following cessation of the conflict paradigm. In addition, BHRs exposed to this prolonged stressor developed cardiac hypertrophy and pathologies consistent with essential hypertension in humans (15). Further studies demonstrated that the increase in blood pressure was specific to BHRs, and could not be elicited in WKY rats exposed to the same stressor (14). These results indicate that environmental stress can lead to the development of hypertension, especially in animals with a genetic susceptibility or predisposition
1Requests for reprints should be addressed to Robert F. Kirby, Department of Psychology, University of Iowa, Iowa City, IA 52242.
309
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310
released from the adrenal medulla and this hormone enters the circulation directly. The short half-life of these amines in plasma (approximately 70 seconds in laboratory rats) makes them a useful index for assessing rapid changes in overall sympathetic outflow (10,18). The measurement of plasma catecholamines has been especially valuable for examining sympathetic nervous system responses to acute stress in animals genetically prone to the development of hypertension (10, 11, 18). The pathogenesis of sustained hypertension in BHRs following chronic stress may be due to increased sympathetic responses to stressors which could be inherited from the SHR strain. In order to assess the potential role(s) of sympathetic hyperreactivity in the pathogenesis of stress-induced hypertension in the BHR, the current study examined plasma catecholamine and cardiovascular responses to a brief period of acute footshock stress in adult male WKYs, BHRs, and SHRs. METHOD
Animals Male WKYs, BHRs, and SHRs were born and reared to adulthood in our vivarium. These animals were derived from the SHR and WKY colonies maintained by the University of Iowa Cardiovascular Research Center. The BHRs were the first generation offspring of a cross between SHR females and WKY males (13-15). Within the first 24 hours after birth, litters were culled to eight pups and all pups were raised by their natural mothers. Animals were housed individually from the time of weaning at thirty days postnatal age until tested as adults two months later. Animals were maintained with standard Purina laboratory chow and tap water available ad lib on a 14 hour light/10 hour dark cycle (lights on at 0600).
Surgical Procedures At three months of age, each animal was weighed and anesthetized with Nembutal (pentobarbital sodium, 50 mg/kg). An indwelling PE-50 catheter was surgically placed in the right common carotid artery and guided subcutaneously to exit at the nape of the neck. The catheter was filled with isotonic saline containing 200 IU/ml of heparin and occluded with a sealed 23-gauge needle.
Experimental Protocol Two days after surgery, baseline testing was carried out on each animal in its home cage 60-90 minutes after connecting the arterial catheter to a pressure transducer (Century Technology Company). A 10-15 second baseline measure of mean arterial pressure (MAP, mmHg) and heart rate (HR, beats/min) was recorded on a Beckman Dynograph with the animal resting and undisturbed in its home cage. A 0.6 ml blood sample was then collected from the arterial line into a chilled glass tube for measurement of catecholamines. Blood withdrawn from the catheter was immediately replaced by an equal volume of isotonic saline containing 20 IU/ml of heparin. Shortly after basal measures were established, each animal was transferred individually to a clear Plexiglas chamber (25 x 25 x 60 cm) with a stainless steel grid floor while MAP and HR were recorded. The response of each rat to transfer was recorded immediately and 15 seconds after placement in the Plexiglas chamber before the footshock began. A 5 minute period of intermittent footshock (2.5 mA DC, 0.5 sec duration, every 5 sec) was then administered to the animal using a Lehigh Valley constant current shock supply. Mean arterial pressure and HR
were recorded for a 10 second period immediately and 5 minutes following the termination of footshock. Blood samples (0.6 ml) were collected from the arterial catheter at each of these times for analysis of catecholamine levels and the volume was replaced with isotonic saline. At the cessation of testing, animals were euthanized by an overdose of anesthetic infused directly into the arterial line. Footshock testing was Performed between 1100 and 1500 hours; WKY, BHR, and SHR were tested in a randomized order. The chamber was cleaned thoroughly using a mild soap solution after each use.
Biochemical Analysis Blood samples were kept in an ice water bath until centrifuged at 3000 x g for 5 minutes at 4°C. Plasma from each sample was deproteinated by the addition of an equal volume of 0.1 N perchloric acid containing 2% EGTA and 0.2% MgC12 and stored at - 2 0 ° C . NE and EPI were analyzed by a radioenzymatic thin-layer chromatographic procedure (1,23). Samples were incubated for 90 minutes at 37°C with a partially purified preparation of rat liver catechol-O-methyl transferase and S-(3H-methyl) adenosylmethionine. The labeled metanephrine and normetanephrine were extracted into toluene-isoamyl alcohol, back extracted into 0.1 N acetic acid and dried overnight in vacuo. The residues were then dissolved in methanol and spotted onto Whatman LK5DF thin-layer chromatography plates to separate the Omethylated amines. The plates were developed in chloroform: ethanol:ethylamine (60:11.25:7.5 ml) and the bands corresponding to metanephrine and normetanephrine were scraped and placed into glass scintillation vials. The amines were then eluted from the silica gel with 0.1 M ammonium hydroxide, oxidized by treatment with sodium periodate and assayed by liquid scintillation spectrometry. Internal NE and EPI standards as well as plasma blanks were included in each assay. Assay sensitivity was less than 10 pg for each catecholamine and the intra- and interassay coefficients of variation were less than 5%.
Data Analysis Data from each animal were used only if measures of MAP, HR, and plasma catecholamines were available for every time point. Cardiovascular and plasma catecholamine responses to transfer and footshock were analyzed using the General Linear Models procedure for repeated measure analysis of variance with contrast tests run for groups (WKY, BHR, SHR) and time (baseline, transfer, 0 and 5 minutes postfootshock) (SAS Institute, Cary, NC, Version 5). Cardiovascular responses following footshock were compared to the 15 second posttransfer measure which was immediately before the onset of the footshock episode. All results are presented as means ___SEM for 9-13 animals per group. RESULTS
Baseline cardiovascular and plasma catecholamine measures are presented in Table 1. Basal MAP and HR were comparable for BHRs and SHRs, although these measures were elevated for both groups when compared to WKYs. In contrast, baseline plasma catecholamine levels were greatest in BHRs, which had significantly greater NE levels than either WKYs or SHRs. Body weights were also found to differ between the three groups, with BHRs being heavier than WKYs and SHRs. In response to transfer to the footshock chamber, both MAP and HR increased significantly [Fig. 1; F(1,30) = 41.2 and F(1,30) = 179.5, respectively; p's<0.0001]. However, a significant interaction was found for MAP between groups. SHRs had large MAP increases following transfer which were significantly greater than
SYMPATHETIC RESPONSES TO STRESS IN BHR
TABLE 1 BASELINE BLOOD PRESSURES, HEART RATES, PLASMA CATECHOLAMINE LEVELS, AND BODY WEIGHTS IN ADULT MALE WISTAR-KYOTO (WKY), BORDERLINE HYPERTENSIVE (BHR), AND SPONTANEOUSLY HYPERTENSIVE RATS (SHR)
Measure
WKY
MAP (mmHg) HR (beats/min) Plasma NE (pg/ml) Plasma EPI (pg/ml) Body wt. (g)
112 298 299 117 357
BHR
- 4 --- 20 --+ 32 - 20 --- 7
147 352 418 161 407
SHR
± 2* --- 6* +-- 32t +-- 21 _ 7t
146 339 302 113 338
--+ 8* --- 11 : 36 --- 27 --- 6
Results are presented as means --- SEM for 9-13 animals per group. *Significantly different from WKY controls (p<0.05). tSignificantly different from WKYs and SHRs (p<0.05).
those of WKYs and BHRs. In contrast, HR increases following transfer were similar for SHRs and BHRs, and these responses were significantly greater than those of WKYs. The cardiovascular responses to intermittent footshock stress are depicted in Fig. 2. There was a significant effect of footshock on MAP, with a slight decrease immediately following footshock, F ( 1 , 3 0 ) = 15.85, p = 0 . 0 0 0 4 , that reverted to a slight increase at five minutes postfootshock, F(1,30) = 5.35, p = 0 . 0 2 7 7 . There was no significant interaction between groups and time postfootshock on the MAP response. In contrast, footshock produced large increases in HR, F ( 2 , 2 9 ) = 109.4, p < 0 . 0 0 0 1 , but the response varied between groups, F(4,58) = 4.569, p = 0.0028. The increase in HR immediately following footshock was similar for the three groups, but by five minutes postfootshock HRs of BHRs had
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Time Post-Footshock FIG. 2. Cardiovascular responses immediately and 5 minutes postfootshock for Wistar-Kyoto (WKY), borderline hypertensive (BHR), and spontaneously hypertensive (SHR) rats. The mean changes from the prefootshock baseline values --- 1 SEM are presented for MAP (top panel) and HR (bottom panel). Footshock significantly influenced MAP, F(2,29) = 22.08, p<0.001, but there were no interactions between the groups and time postfootshock. The HR response was equivalent across groups immediately following footshock but decreased to a greater extent in BHRs than WKYs or SHRs.
decreased to a greater extent than in either the WKYs, F(1,30) = 14.97, p < 0 . 0 0 0 5 , or the SHRs, F ( 1 , 3 0 ) = 7 . 6 6 , p = 0 . 0 0 9 6 . The plasma NE and EPI responses to footshock were similar in pattern to one another and these responses are depicted in Fig. 3. Plasma catecholamines were elevated following footshock and there was a significant interaction for the NE response with the animal's group and the interaction between groups and time approached significance for the EPI response, F ( 4 , 4 6 ) = 2 . 3 7 , p = 0.066. The increase in plasma NE levels above baseline did not differ significantly between the three groups immediately following footshock, although the increase was twice as large in the SHRs compared to the BHRs and WKYs. At five minutes postfootshock, the plasma NE increase above baseline was significantly greater in the SHR's than either the BHRs or the WKYs. Plasma EPI increases above baseline were also greatest in SHRs immediately following footshock, and were significantly greater than in the WKYs. By five minutes postfootshock, the increase in plasma EPI above baseline was still greatest in SHRs, and the levels were significantly greater in SHRs than in BHRs or WKYs. DISCUSSION
Transfer Response FIG. 1. Cardiovascular responses to transfer to a novel chamber in Wistar-Kyoto (WKY), borderline hypertensive (BHR), and spontaneously hypertensive (SHR) rats. The mean increases above baseline - 1 SEM for MAP (top panel) and HR (bottom panel) are presented. The increase in MAP to transfer was significantly greater in SHRs (**) than either BHRs, F(I,21)= 12.14, p<0.001, or WKYs, F(1,19)= 11.66, p<0.002. However, HR increased to a greater degree (**) in both SHRs, F(1,18) = 20.87, p<0.001, and BHRs, F(1,22)= 17.91, p<0.001, than WKYs.
The SHR is the most extensively studied animal model of human essential hypertension. This strain shares many commonalities with essential hypertension: including a progressive development, responsiveness to similar antihypertensive pharmacological agents, and decreased sensitivity of high pressure baroreceptors (10,11). Two distinct abnormalities in sympathetic nervous system function have been demonstrated in adult SHRs when compared to WKY normotensive rats. These are: a) increased sympathetic terminal outflow of NE in response to stress and; b) heightened responsiveness of the vasculature to released catecholamines
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FIG. 3. Mean norepinephrine and epinephrine levels - 1 SEM in WistarKyoto (WKY), borderline hypertensive (BHR), and spontaneously hypertensive (SHR) rats immediately and 5 minutes postfootshock. Plasma norepinephrine increased significantly following footshock, F(2,29)= 21.89, p<0.001, and the response varied between groups, F(4,50) = 2.60, p<0.05. Norepinephrine levels were significantly greater in SHRs than in BHRs, F(1,22) = 10.33, p<0.01, and WKYs, F(1,20) = 4.32, p<0.05, at 5 minutes postfootshock. Plasma epinephrine levels also increased significantly in response to footshock, F(2,29) = 46.04, p<0.001. The response was greater in SHRs than in WKYs immediately, F(1,20) = 6.39, p<0.05, and 5 minutes, F(1,20)=4.43, p<0.05, following the termination of footshock. The SHR response was significantly greater than the BHR response at 5 minutes postfootshock, F(1,22)=4.28, p<0.05. No differences were found between the WKY and BHR responses to footshock. Results of individual comparisons are denoted by brackets with, *p<0.05 and **p<0.01. (10,11). Together, these produce accentuated elevations in blood pressure and heart rate in response to immobilization, acute " m e n t a l " stress, anticipation of footshock, and placement of animals into a novel environment (10-12, 17, 20). Judy and co-workers have proposed that elevated sympathetic activity is causal to the maintenance of hypertension in the SHR (6,7). This view comes from two lines of evidence. First, activity of pre- and postganglionic sympathetic fiber bundles was greater in SHR than in age-matched normotensive Wistar rats. Treatment with hexamethonium, which blocked ganglionic neurotransmission, decreased blood pressure in both strains and led to equivalent pressures in SHR and Wistar control rats. Second, in the sixth backcross generation of SHRs and normotensive Wistar/Lewis rats, sympathetic nerve activity and resting arterial pressure were highly correlated. These data are very supportive of a causal role for sympathetic activity in the elevated blood pressures of SHRs and their backcrossed progeny. In the present study, increased basal NE levels were found for BHRs. This result may be an indication that elevated tonic sympathetic activity plays a greater role in maintaining basal arterial pressure in BHRs. In contrast, vascular resistance may play a greater role in the elevated pressure of SHRs. Subjects in this experiment were examined at 12 weeks of age. At this age, MAPs of BHRs have peaked but those of SHRs will continue to
increase until approximately 18-22 weeks of age. Although BHRs and SHRs showed comparable basal MAPs in the current study, differences in arterial resistance may be present for these two groups. Elevated arterial resistance of SHRs is not due solely to structural adaptation of the vasculature to increased pressure, but also appears to be due to genetic influences (2,8). Vascular hypertrophy in the SHR still occurs if the development of hypertension is inhibited by early catecholaminergic fiber destruction with 6-OHDA (25). In addition, SHRs develop a greater increase in vascular resistance than genetically normotensive animals which have been hypertensive for an equivalent period (16). Therefore, increased resistance of the vasculature in SHRs is not purely a response to elevated pressure. Taken together, these findings indicate that the similar resting pressures of SHRs and BHRs may be maintained through elevated vascular resistance in the SHR and increased sympathetic drive in the BHR. Transfer to the footshock chamber increased blood pressure in SHRs but not in WKYs or BHRs. This response would be consistent with the known differences in sympathetic responses to acute stress and increased vascular sensitivity of SHRs compared to WKYs (10,11). In contrast, the tachycardic response of SHRs and BHRs to transfer was equivalent, and was significantly greater than that of WKYs. These data suggest that sympathetic drive to the heart increased more in BHRs and SHRs than in normotensive WKYs. Therefore, the mild stress of transfer may unmask elements of disordered cardiovascular responsiveness of BHRs to mild environmental challenges. Plasma catecholamine responses of BHRs to acute footshock stress were more similar to WKYs than to SHRs in the present study. These results indicate that BHRs do not share the sympathetic hyperresponsiveness to acute footshock stress that is characteristic of SHRs. Exaggerated sympathetic responses of SHRs can also be seen in response to immobilization (12), placement in a novel environment (19,21), anticipation of footshock (20), air puff stimulation (9,24), and auditory stimulation (3). Thus, the lack of an exaggerated plasma catecholamine response in BHRs found in the present study is probably not due to the type of stressor employed. This suggests that BHRs and WKYs may exhibit similar sympathetic responses to the conflict-conflict paradigm used by Lawler and co-workers to produce stressinduced hypertension in BHRs (13-15). However, an important distinction between the present study and those of Lawler and co-workers is the duration of the stressor. Habituation of sympathetic responses is normally seen when a stressor is repeated over time (22). It is possible that sympathetic responses in BHRs might not habituate to a repeated stress. Thus, BHRs might ultimately show a response that would be functionally equivalent to an exaggerated sympathetic response if the stressor were presented in repeated sessions. Evidence consistent with this hypothesis has been reported by Hubbard and co-workers (5). After 5 days of conflict-conflict stress, greater plasma NE levels were found in BHRs than in WKYs following stressor presentation. In summary, plasma catecholamine responses of BHRs to acute footshock stress were comparable to WKYs. In contrast, SHRs had consistently greater plasma NE and EPI responses to acute stress than either BHRs or WKYs. ACKNOWLEDGEMENTS This research was supported by U.S. Public Health Service Grants HL33447, HL29906, HL33796, HL14388; NIMH Research Scientist Development Awards MH00064 and MH00529; and by a grant (84-6-19) from the Iowa Affiliate of the American Heart Association. R. F. Kirby was supported by NHLBI postdoctoral fellowship HL07096.
SYMPATHETIC RESPONSES TO STRESS IN BHR
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REFERENCES 1. DaPrada, M.; Zurcher, G. Simultaneous radioenzymatic determination of plasma and tissue adrenaline, noradrenaline, and dopamine within the femtomole range. Life Sci. 19:1161-1174; 1976. 2. Folkow, B. Physiological aspects of primary hypertension. Physiol. Rec. 62:347-504; 1982. 3. Galeno, T. M.; VanHoesen, G. W.; Brody, M. J. Central amygdaloid nucleus lesion attentuates exaggerated hemodynamic responses to noise stress in the spontaneously hypertensive rat. Brain Res. 291: 249-259; 1984. 4. Goldstein, D. S. Plasma catecholamines and essential hypertension: An analytical review. Hypertension 5:86-99; 1983. 5. Hubbard, J. W.; Cox, R. H.; Sanders, B. J. Changes in cardiac output and vascular resistance during behavioral stress in the rat. Am. J. Physiol. 251:R82-R90; 1986. 6. Judy, W. V.; Watanabe, A. M.; Henry, D. P.; Besch, H. R.; Murphy, W. R.; Hockel, G. M. Sympathetic nerve activity: Role in regulation of blood pressure in the spontaneously hypertensive rat. Circ. Res. 38(Suppl. 2):21-29; 1976. 7. Judy, W. V.; Watanabe, A. M.; Murphy, W. R.; Aprison, B. S.; Yu, P. Sympathetic nerve activity and blood pressure in normotensive backcross rats genetically related to the spontaneously hypertensive rat. Hypertension 1:598--604; 1979. 8. Karr-Dulien, V.; Bloomquist, E. I.; Beringer, T.; EI-Bermani, A. I. Flow-pressure relationships in newborn and infant spontaneously hypertensive rats. Blood Vessels 18:245-252; 1981. 9. Koepke, J. P.; DiBona, G. F. High sodium intake enhances renal nerve and antinawiuretic responses to stress in spontaneously hypertensive rats. Hypertension 7:357-363; 1985. 10. Kopin, I. J.; McCarty, R.; Torda, T.; Yamaguchi, I. Catecholamines in plasma and responses to stress. In: Usdin, E.; Kvetnansky, R.; Kopin, I., eds. Catecholamines and stress: Recent advances. New York: Elsevier-North Holland; 1980:197-204. 11. Kopin, I. J.; McCarty, R.; Yamaguchi, I. Plasma catecholamines in human and experimental hypertension. Clin. Exp. Hypertens. 2: 379-394; 1980. 12. Kvetnansky, R.; McCarty, R.; Thoa, N. B.; Lake, C. R.; Kopin, I. J. Sympatho-adrenal responses of spontaneously hypertensive rats to immobilization stress. Am. J. Physiol. 236:H457-H462; 1979. 13. Lawler, J. E.; Barker, G. F.; Hubbard, J. W.; Allen, M. T. The
14.
15. 16.
17. 18.
19.
20. 21. 22.
23. 24. 25.
effects of conflict on tonic levels of blood pressure in the genetically borderline hypertensive rat. Psychophysiology 17:363-370; 1980. Lawler, J. E.; Barker, G. F.; Hubbard, J. W.; Cox, R. H.; Randall, G. W. Blood pressure and plasma renin activity responses to chronic stress in the borderline hypertensive rat. Physiol. Behav. 32:101-105; 1984. Lawler, J. E.; Barker, G. F.; Hubbard, J. W.; Schanb, R. G. Effects of stress on blood pressure and cardiac pathology in rats with borderline hypertension. Hypertension 3:496-505; 1981. Lundgren, Y.; Hallback, M.; Weiss, L.; Folkow, B. Rate and extent of adaptive cardiovascular changes in rats during experimental renal hypertension. Acta Physiol. Scand. 91:103-115; 1974. Lundin, S.; Thoren, P. Renal function and sympathetic activity during mental stress in normotensive and spontaneously hypertensive rats. Acta Physiol. Scand. 115:115-124; 1982. McCarty, R. Stress, behavior and the sympatho-adrenal medullary system. In: Pohorecky, L. A.; Brick, J., eds. Stress and alcohol use. New York: Elsevier; 1983:7-22. McCarty, R.; Chiueh, C. C.; Kopin, I. J. Behavioral and cardiovascular responses of spontaneously hypertensive and normotensive rats to inescapable footshock. Behav. Biol. 22:405-410; 1978. McCarty, R.; Chiueh, C. C.; Kopin, I. J. Spontaneously hypertensive rats: Adrenergic hyperresponsivity to anticipation of electric shock. Behav. Biol. 23:180-188; 1978. McCarty, R.; Kopin, I. J. Alterations in plasma catecholamines and behavior during acute stress in spontaneously hypertensive and WistarKyoto normotensive rats. Life Sci. 22:997-1006; 1978. McCarty, R.; Kvetnansky, R.; Lake, C. R.; Thoa, N. B.; Kopin, I. J. Sympatho-adrenal activity of SHR and WKY rats during recovery from forced immobilization. Physiol. Behav. 21:951-955; 1978. Peuler, J.; Johnson, G. A. Simultaneous single isotope radioenzymatic assay of plasma norepinephrine, epinephrine and dopamine. Life Sci. 21:625-636; 1977. Ricksten, S. E.; Yao, T.; DiBona, G. F.; Thoren, P. Renal nerve activity and exaggerated natriuresis in conscious spontaneously hypertensive rats. Acta Physiol. Scand. 112:161-167; 1981. Weiss, L. Aspects of the relation between functional and structural cardiovascular factors in primary hypertension. Acta Physiol. Scand. [Suppl.] 409:1-58; 1974.