Mental stress after atropine Research Paper
Clinical Autonomic Research 1, 225--231 (1991)
THE effects of cholinergic blockade on haemodynamic reactivity to standardized mental stress has been studied in nine normotensive males during infusion of atropine (bolus dose 10 #g x kg -1 followed by a constant-rate infusion of 0.02 gg x k g - l x rain -1) or placebo given in a randomized order on two different days. Partial cholinergic blockade increased resting heart rate by 25-30 beats per minute. The magnitude of the heart rate response to stress (reactivity) however was unaffected by the atropine infusion. Also, in four subjects who received a higher dose of atropine (approximately 1.8-1.9 mg), heart rate responses to stress were the same as during placebo infusion. Cholinergic blockade was associated with a small but prolonged increase in diastolic blood pressure. These findings suggest that parasympathetic withdrawal does not contribute to the tachycardia caused by mental arithmetic, and that the pattern of neurogenic activation may differ from that elicited during a classic defence-alarm reaction and by somatomotor activation.
Effect of cholinergic blockade on heart rate, blood pressure and plasma c a t e c h o l a m i n e responses to m e n t a l stress in normal subjects S v e r k e r J e r n , MD, PhD cA, M a r t i n and C h r i s t i n a J e r n , MD, PhD
Pilhall, MD
Department of Clinical Physiology, Ostra Hospital, University of G6teborg, S-416 85 G6teborg, Sweden.
CACorresponding Author
Mental stress, Cholinergic blockade, Atropine, Haemodynamicfactors, Plasma catecholamines
Key words:
Introduction Elevated heart rate at rest is associated with an increased cardiovascular mortality, 1'2 and resting tachycardia predicts future hypertension in young subjects with borderline blood pressure elevation. 3'4 Since increased heart rate is a characteristic part of the defence-alarm reaction, it has been repeatedly suggested that exposure to excessive psychosocial stress and/or increased reactivity to environmental stimuli may be responsible for the resting tachycardia in some individuals, s In support of such a notion, high heart rate reactivity to stress promotes development of coronary atherosclerosis in primates. 6 Although there is a dual neural regulation of heart rate both through sympathetic and cholinergic pathways, studies of stress-induced changes of cardiac rhythm have almost exclusively focused on sympathoadrenal mechanisms] Therefore, little is known about the relative contribution of vagal withdrawal during psychological arousal in man, and the possible influence of cholinergic mechanisms on stress-induced heart rate reactivity. However, understanding of the neural mechanisms underlying the cardiac activation in response to mental stress is important for studies of the possible role of psychosocial factors in the pathogenesis of cardiovascular disorders. In this study we have used a strictly standardized mental arithmetic stress model to characterize physiological stress responses. 8-I° We have recently shown that this test is highly reproducible and can be repeated without adaptation of the stressinduced heart rate responses, u We have therefore © Rapid Communications of Oxford Ltd.
performed two mental stress tests (separated by at least 1 week) during double-blind infusions of atropine and placebo given in randomized order to nine healthy subjects.
Material and Methods Subjects: Nine young, normotensive male subjects participated in the study. All subjects were healthy non-smokers without cardiovascular disease and on no medication. The subjects were recruited from medical students and hospital employees. Their mean age, weight, and height were 26.0 years (range 23-35 years), 76.8 kg (67-90 kg), and 186.9 cm (178-191 cm), respectively. Informed consent was obtained from each subject before inclusion in the study after the rationale, nature, and potential risks of the research had been carefully explained. The protocol was approved by the Ethics Committee of the University of G6teborg. All subjects were asked to refrain from taking methylxanthine-containing products on the day before the experiment, to avoid interference with the catecholamine analyses. The participants were instructed to avoid heavy exercise or emotional excitement, and to be fasting for 4 h prior to the experiment. Experimental design: Each subject participated in randomized order in two experiments separated by at least 1 week. After a 10-min pre-infusion baseline period, they received infusions of either atropine or placebo for 35 min during the first part of the experiment according to a randomized, doubleClinical Autonomic Research.vol 1 • 1991
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S. Jern, M. Pilhall and C. Jern blind schedule. On each day a mental stress test was performed 15 min after start of the infusion. During each stress test, the subjects performed 10 rain of forced mental arithmetic, according to a strictly standardized procedure (see Ref. 11 for reproducibility data on this method). Two investigators participated in the study (M.P. and S.J.), but each subject always met the same investigator during each stress test. The infusion was continued for 10 min after the conclusion of the stress test (post-stress baseline) and was then stopped. Changes of haemodynamics and plasma catecholamines were followed for 10 min immediately after the end of the infusion and for another 10-min period 50-60 min after the infusion.
Procedure: Upon reporting at the laboratory, the subject was placed in a comfortable chair in a semi-recumbent position. One venous cannula (Venflon ®, Viggo, Helsingborg, Sweden) was inserted percutaneously into an antecubital vein of the dominant arm for infusion of atropine. Another venous cannula was inserted into a vein of the dorsum of the hand of the same arm for blood sampling. A 50cm polyethylene catheter was connected to each indwelling cannula, and the catheters were led through the wall to an adjacent control room. This technique allowed us to start and stop the infusions and collect blood samples without the subjects knowing exactly when this was done. The intravenous lines were kept patent by slow saline infusions. A blood pressure cuff was applied on the non-dominant arm, and electrodes for computerized vectorcardiography were fixed to the chest, arms and legs according to a modified Frank system. After a 1-h rest period, the experiment commenced with the first 10-min baseline period with the subject in the quiet and dimly lit room. Then a bolus injection was given over 2 min which was followed by a constant-rate infusion for 35 min. Fifteen minutes after the start of the infusion, the investigator entered the room which was now fully lit and gave an approximately 40-s long oral instruction. The subject then performed forced mental arithmetic for 10 min with serial subtractions of 7 from 700 trying to keep pace with a metronome at a rate of approximately 90/min. The investigator encouraged the subject to increase the speed by short repeat comments. After a positive and reassuring comment, the investigator reduced the light and left the room. The stress task was followed by two 10-min baseline periods during and after the end of the infusion, respectively. The subjects then remained in the same position for another 50 min, at the end of which a second 10-min post-infusion recording was obtained. In a recent study, using the experimental 226
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procedure described above, we have demonstrated that the mental arithmetic stress test is highly reproducible with regard with heart rate, blood pressure, and subjective stress activation levels and reactivity, u When two tests were performed 1 h apart, both activation levels and reactivity during the two tests were closely correlated (R > 0.75 throughout). The coefficients of variation for the attained activation levels ranged between 3.1% and 6.2% for haemodynamic measures and subjective stress ratings. There were no significant differences in stress activation levels attained during the two tests.
Intravenous infusions: Atropine was administered as a bolus dose of 10 #g × kg -1 atropine sulphate (ACO, Stockholm, Sweden) given over 2 min, immediately followed by a constant-rate infusion of 0.02 #g × k g - l × min -1 for 35 min. The total individual dose of atropine ranged between 0.72 and 0.96 rag. Placebo (isotonic saline) was administered in the same manner and volume for each subject. Haemodynamic monitoring: Blood pressure was measured at 1-min intervals using an automatic non-invasive oscillometric blood pressure monitor (Dinamap Model 845, Critcon Instruments, Tampa, FL, USA), which has been validated against intra-arterial blood pressure recordings in our laboratory. 12 Heart rate was monitored continuously throughout the experiment by computerized vectorcardiography (MIDA 1000, Ortivus Medical AB, T~iby, Sweden). Blood sampling and biochemical analyses: Blood samples for analysis of plasma catecholamines were obtained on the following time points: at 10, 25, 35, 45, 55 and 105 min (cf. Fig. 1). All blood samples were obtained from the distal indwelling venous cannula. The first 5 ml of blood were always discarded. Eleven millilitres of blood for analysis of catecholamines were drawn into prechilled tubes containing 220 #1 glutathione-EDTA (60 mg/ml and 90 mg/ml, respectively) and immediately placed on melting ice. The samples were centrifuged within 2 min of collection at +4°C and 2 000 g for 5 min. Plasma aliquots were stored at - 7 0 ° C until assay. All coded samples from each study subject were always analysed in the same assay-run. Plasma catecholamines were separated by reversed-phase high performance liquid chromatography (HPLC) and quantified by the highly sensitive electrochemical detector of Waters (Electrochemical Detector 641, Waters, Millipore Ltd, Germany). Dihydroxybenzylamine was used as internal standard. The analytic procedure followed that described by Weicker et al. 13 In our laboratory, the intra-assay coefficients of variation for basal and
Mental stress after atropine
Heart rate bpm
110 90 70 t
t"t--r4
6
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5O Systolic blood pressure mmHg
140 130 120 110 Diastolic blood pressure mmHg
90
80
~,/r
70
,
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60 ~lnfusion
J
I
I"
I
I
I
I
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10
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35
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95
FIG. 1. Haemodynamic changes in response to mental stress during infusion of atropine and placebo. ( infusion, ( - - - ) , placebo. (Mean and SEM).
I
105 min ), Atropine
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S. Jern, M. Pilhall and C. Jern stress levels were 2.5% and 3.3% for noradrenaline (n = 14) and 8.6% and 8.1% (n = 18) for adrenaline, respectively. The detection limit is estimated to 50-100 fmol/1 plasma, when defined as the concentration equivalent to five times the baseline noise. Statistical ana~sis: Standard statistical methods were used. Haemodynamic data from the stress experiment were reduced by dividing baseline and stress periods into 2.5-min epoches, and by computing mean values for heart rate, and systolic and diastolic blood pressure for each period. Data from the plasma catecholamine analyses were not analysed in this manner. Reactivity was defined for each stress experiment as the difference between the mean level during stress and the mean from the corresponding pre-stress baseline period. Overall effects of atropine and placebo were evaluated by a two-factor (two types of Infusion x six Periods) analysis of variance (ANOVA) for repeated measures on individual changes relative to the pre-infusion baseline of each day. When the main effect for type of Infusion or the Infusion x Period interaction term yielded significant F-ratios, differences in levels between the atropine and placebo days were identified on the absolute values by means of Student's t-test for paired comparisons. Further, a separate two-way A N O V A was performed on the stress experiment, i.e. from time 15 min (pre-stress baseline) through 45 min (post-stress baseline). When A N O V A indicated significant Period effects for the stress experiment, possible differences in reactivity were evaluated by t-test of the change from pre-stress baseline (15-25 rain) to stress (25-35 min) during each infusion. In all cases, two-tailed tests were used, and the tests were considered significant at p < 0.05. For all repeated measures analyses, degrees of freedom were corrected according to the conservative Greenhouse and Geisser procedure for possible violation of the assumption of sphericity. 14
infusions was maintained throughout the first part of the experiment, i.e. until 10 rain after the infusions had been stopped. Fifty to 60 min after the end of the infusion, heart rate was still 5 _ 1 bpm [t(8) = 6.4, p = 0.0002] higher after atropine than after placebo. A N O V A also revealed significant effects of the atropine infusion on mean arterial pressure and diastolic blood pressure. Both mean arterial pressure [F(1,8)= 12.5, p = 0.008] and diastolic blood pressure [F(1,8) = 15.4, p = 0.004] were higher on the day of the atropine infusion. However, in contrast to the heart rate changes, the effects of atropine were more pronounced after than during the infusion. Mean arterial and diastolic blood pressures were 4 + 1 mmHg [t(8)= 3.0, p = 0.02] and 6 _ 2 mmHg [t(8) = 3.5, p = 0.008] higher after atropine during the late post-infusion period (i.e. 50-60 min after the infusion). No significant differences between the two infusions were observed for systolic blood pressure or plasma catecholamine levels (Fig. 1 and Table 1). The mental stress test induced highly significant changes in the four haemodynamic variables and also a significant change in plasma noradrenaline concentration, as indicated by significant Period effects of A N O V A [Fs(2,16) > 32.2, p < 0.0002 throughout]. In response to stress, heart rate increased by 18 _+ 2 bpm [t(8) = 7.7, p < 0.0001] and 14 ± 2 bpm [t(8) --- 6.0, p < 0.0003] during the atropine and placebo infusions, respectively (Table 2). The slight difference in stress reactivity between the infusions was not significant. The stress-induced increases in systolic, mean arterial, and diastolic blood pressures ranged between 12 and 14 mmHg [ts(8) > 3.3, p < 0.01 throughout], without any significant differences between the two conditions. In response to the mental stress test, there was significant change of the plasma noradrenaline Table 1. Levels of plasma catecholamines in response to mental stress during infusion of atropine and placebo (mean 4-SEM)
Results
There were no significant differences at rest during the pre-infusion baseline period between the atropine and placebo days for any of the measured variables. Effects of the infusions on haemodynamic variables are shown in Fig. 1. As expected, administration of atropine elevated the heart rate throughout the experiment [F(1,8)= 41.6, p = 0.0002]. Five minutes after the bolus injection and start of the atropine infusion, mean heart rate increased from 58 ± 2 bpm (mean ___ SEM) to 84 + 3 bpm [t(8) = 6.7, p = 0.0001; Fig. 1]. The difference between the atropine and placebo 228
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Atropine infusion
Placebo infusion
p-Noradrenaline nmol/I 10 min 25 min 35 min (stress) 45 min 55 min 105 min
2.52 2.58 2.78 2.22 2.22 2.94
4- 0.29 ___0.37 4- 0.41 4- 0.31 4- 0.38 4- 0.48
2.43 2.42 2.83 2.47 2.50 2.60
4- 0.29 _+ 0.23 4- 0.25 -t- 0.27 + 0.26 4- 0.35
p-Adrenaline nmol/I 10 min 25 min 35 min (stress) 45 min 55 min 105 min
0.33 0,30 0.35 0.27 0.23 0.22
___0.05 4- 0.05 4- 0.06 4- 0.05 4- 0.04 4- 0.03
0.33 0.34 0.35 0.38 0.31 0.22
4- 0.05 +_ 0.03 4- 0.03 4- 0.06 4- 0.06 4- O.04
Mental stress after atropine Table 2. Stress-induced changes (reactivity) of haemodynamics and plasma catecholamines in response to mental stress during infusion of atropine and placebo (mean ± S E M ) Atropine infusion Haemodynamics A Heart rate k Systolic blood pressure mmHg A Mean arterial pressure mmHg k Diastolic blood pressure mmHg
+17.6 +12.4 + 11.6 + 11.7
Plasma catecholamines A p-Noradrenaline nmol/I A p-Adrenaline nmol/I
+ 0 . 2 0 __ 0.13 + 0 . 0 5 ± 0.04
Significance of change from d p < 0.0001.
pre-stress
concentration [F(2,16) = 8.1, p = 0.01], whereas plasma adrenaline was unaffected by stress (Table 1). There were no significant differences between the two types of infusion on levels or patterns of stress-induced changes of either noradrenaline or adrenaline. After conclusion of the mental stress test, there was a rapid return of heart rate and blood pressure to values close to the pre-stress levels of the respective day (Fig. 1). In a second study, a higher dose of atropine (bolus dose of 20 #g x kg -~ infusion of 0.1 #g x k g - l × min -1 body weight) was administered in the same way as during the first study to four subjects (body weight 75-82 kg). After a total dose of approximately 1.8-1.9 mg of atropine, the heart rate responses to stress were the same as during the placebo infusion (Table 3; + 15.1 bpm versus +14.9 bpm after atropine and placebo, respectively).
Discussion The results of the present study show that although the partial cholinergic blockade increased resting heart rate by some 25 to 30 bpm, the magnitude of the heart rate response to stress was unaffected by the atropine infusion. When a higher dose of atropine was given to four subjects (each Table 3. Changes of heart rate in four subjects given the higher dose of atropine (20 #g x kg -1 bolus + 0.1 #g x kg -1 x min -1 body weight) mean ± S E M
Heart rate bpm 0 - 1 0 min 15-25 min 25-35 min (stress) 35-45 min 45-55 min A Heart rate bpm Pre-stress-stress
Atropine infusion
Placebo infusion
56.6 88.5 103.6 87.1 85.8
53.7 52,8 67.7 52.0 51.4
+ 3.4 + 8.8 _+ 9.1 ± 8.7 +__8 4
+ 15.1 ± 3.1
_+ 2.2 _± 3.6 + 2.9 ± 3.0 ± 3.4
+ 14.9 ± 1.8
_+ 2.3 d _+ 2.1 c _+ 2.0 c ± 1.7 d
Placebo infusion
+14.4 +12.3 + 14.0 + 1 2.7
± 2.4 c ± 3.7 b _-t-3.4 b _+ 2.8 b
+ 0 . 4 0 ± 0.13 ~ + 0 . 0 2 ± 0.03
baseline: a p < 0.05,
t-test A vs. P
ns ns ns ns ns ns
b p < 0.01, C p < 0.001,
subject receiving approximately 1.8-1.9 mg atropine), the result was the same; heart rate reactivity to the mental arithmetic stress test was identical to that observed after placebo. Thus, even with a substantial dose of atropine which should cause cardiac cholinergic blockade, heart rate responses to stress were not affected. These findings suggest that parasympathetic withdrawal contributes minimally, if at all, to the tachycardia elicited by a brief mental arithmetic task. If so, it appears that the neural regulatory mechanisms of heart rate responses to this particular form of psychological stress may differ from those of physical exercise, since decreased vagal inhibition is an important part of the tachycardia of somatomotor activation. Is Experimental observations support the notion that this may be a more general characteristic of some psychological arousal states in man, in particular those associated with active coping behaviour. In a study of young, healthy subjects heart rate responses to handgrip and a reaction time task stimulus was applied twice, once before and once after infusion of either placebo, atropine, or propranolol. Whereas the increase in heart rate during handgrip was significantly blocked by both drugs, the heart rate response to the reaction time task was blocked by propranolol but was unaffected by atropine. These data suggest that, in contrast to responses to isometric exercise, cardiac responses to mild active psychological coping during the reaction time tests are not dependent on vagal withdrawal. This has led Obrist lv,18 to hypothesize that the relative influences of sympathetic and parasympathetic mechanisms on heart rate responses during psychological stress will depend on the relative el±citation of somatomotor activation and active coping behaviour. According to this hypothesis, beta-mediated sympathetic cardiac influences would dominate during active psychological coping efforts such as the mental arithmetic task, whereas tasks involving somatomotor activaClinical Autonomic Research, vol 1 • 1991
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tion elicit heart rate elevations by withdrawal of cardiac vagal activity. 17 2o Previous workers have assumed that the cardiac response in man to experimental procedures such as the mental arithmetic task resembles that of the defence-alarm reaction, and that the tachycardia is due to a combination of increased sympathetic stimulation and decreased parasympathetic inhibition. 5'2. This neurogenic pattern has been observed also in rats exposed to various alerting stimuli, such as light, noise and vibration. = It appears, however, that even in this model the vagal component of the responses are variable, especially in normotensive animals. 22 Zanchetti and co-workers 23 have also s h o w n that the behavioural, neurogenic, and haemodynamic responses are complex and diversified in conscious and awake cats in 'natural' settings. Furthermore, there may be important species differences in the reactions to environmental stimuli. It is not unlikely, that in man in whom the complexity of the emotional and behavioural reactions is far .more pronounced, there is an even larger variety of neurogenic responses to stress. In previous human studies, administration of atropine during resting conditions has been shown to increase heart rate and cardiac output, but decrease total peripheral resistance and stroke volume. 24-26 Since chronotropic stimulation per se across a wide range of heart rates does not increase cardiac output or blood pressure, 27 it is assumed that the increased cardiac output may be related to blockade of negative inotropic vagal influences.28 Although the exact mechanism behind the peripheral vasodilation is unknown, it has been commonly assumed that a baroreceptor-mediated reflexogenic decrease in vascular tone has been triggered by the increased cardiac output. However, similar to what has been observed in some previous investigations cholinergic blockade produced slight but significant increases in diastolic blood pressure and mean arterial pressure. 24'2s Interestingly, the increase in diastolic blood pressure was clearly more long-lasting than the effect on heart rate; in fact, the maximal difference in diastolic blood pressure between the atropine and placebo experiments was observed 50-60 min after the end of the infusions. The cause of the pressor effect of atropine is not known, but our findings suggest that different mechanisms may be involved in the effects on heart rate and blood pressure. We have recently shown that high circulating adrenaline levels may amplify the pressor responses to mental stress, z9 Although this effect was confined to the systolic blood pressure response, other investigators have shown an increased heart rate response to other stressors after infusion of adrenaline. 3° Therefore, plasma catecholamine levels must be taken into account in evaluations of haekaodyna230
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mic stress responses. However, in the present study, no differences were observed between the two infusions on either the levels or patterns of stress-induced changes of plasma catecholamines. Thus, there was no evidence of a confounding effect of adrenaline on the heart rate or blood pressure responses to stress after atropine. In conclusion, the results of the present investigation argue against the view that withdrawal of parasympathetic inhibition is an important part of the cardiac response to mental arithmetic in man.
References 1. Kannel WB, Kannel C, Paffenbarger RS, Cupples PH, Cupples LA. Heart rate and cardiovascular mortality. The Framingham Study. Am Heart J 1987, 113:1489-1494. 2. Cruickshank JM, Smith JC. The beta-receptor, atheroma and cardiovascular damage. Pharmaco/Ther 1989; 42: 385404. 3. Levy RL, White PD, Stroud WD, Hillman CC. Transient tachycardia: prognostic significance alone and in association with transient hypertension. JAMA 1945; 129: 585. 4. Paffenbarger RS, Jr, Thorne MC, Wing AL. Chronic disease in former college students. VIII. Characteristics in youth predisposing to hypertension in later years. Am J Epidemio/1968; 88: 25. 5. Folkow B. Physiological aspects of primary hypertension. Physiol Rev 1982; 62: 347-504. 6. Clarkson TB, Weingand KW, Kaplan JR, Adams MR. Mechanisms of atherogenesis. Circulation 1987; 76 (suppl I): 1-20. 7. Freyschuss U, Hjemdahl P, Juhlin-Dannfeldt A, Linde B. Cardiovascular and sympathoadrenal responses to mental stress: influence of fl- blockade. Am J Physiol 1988; 255: H 1443-14451. 8. Jern S. Psychological and hemodynamic factors in borderline hypertension. Acta Med Scand 1982; suppl 662: 1-54. 9. Jern C, Eriksson E, Tengborn L, Risberg B, Wadenvik H, Jern S. Changes of plasma coagulation and fibrinolysis in response to mental stress. Thromb Haemostas 1989; 62: 767-771. 10. Manhem K, Jern C, Shanks G, Pilhall M, Hansson L, Jern S. Stress-induced haemodynamic changes during the menstrual cycle. Clin Sci (in press). 11. Jern S, Pilhall M, Jern C. Short-term reproducibility of the mental arithmetic stress test. Clin Sci (in press). 12. Milsom I, Swahn S0, Forsman L, Sivertsson R. An evaluation of automated indirect blood pressure measurements during pregnancy. Acta Obstet Gynecol Scand 1986; 65: 721-725. 13. Weicker H, Feraudi M, H&gele H, Pluto R. Electrochemical detection of catecholamines in urine and plasma after separation with HPLC. Cfin Chim Acta 1984; 141 : 17-25. 14. Vasey MW, Thayer JF. The continuing problem of false positives in repeated measures ANOVA in psychophysiology: a multivariate solution. Psychophysiology 1987; 24: 479-486. 15. Freyschuss U. Cardiovascular adjustment to somatomotor activation. Acta Physiol Scand 1 970; suppl 342: 1-63. 16. Pollak MH, Obrist PA. Effects of autonomic blockade on heart rate responses to reaction time and sustained handgrip tasks. Psychophysiology 1988; 25: 689-695. 17. Obrist PA, Webb RA, Sutterer JR, Howard JL. The cardiac-somatic relationship: some reformulations. Psychophysiology 1970; 6: 569-586. 18. Obrist PA. Cardiovascular psychophysiology. New York: Plenum Press, 1981. 19. Obrist PA, Gaebelein CJ, Teller ES, Langer AW, Grignolo A, Light KC, McCubbin JA. The relationship among heart rate, carotid dP/dt, and blood pressure in humans as a function of the type of stress. Psychophysiology 1 978; 15:102-115. 20. Obrist PA, Light KC, James SA, Strogatzos. Cardiovascular responses to stress: I. Measures of myocardial response and relationship to high resting systolic pressure and parental hypertension. PsychophysioIogy 1987; 24: 65-78. 21. Brod J, Fencte V, Hejl Z, Jirka J. Circulatory changes underlying blood pressure elevations during acute emotional stress (mental arithmetic) in normotensive and hypertensive subjects. Clin Sci 1959; 18: 269-279. 22. Hallb~ck M, Folkow B. Cardiovascular responses to acute mental 'stress' in spontaneously hypertensive rats. Acta Physio/ Scand 1974; 90: 684-698. 23. Zanchetti A, Bachelli G, Mancia G, Ellison GD. Emotion and the cardiovascular system in the cat. In: Physiology, Emotion and
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24. 25.
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Psychosomatic Illness. Ciba Foundation Symposium 8. Amsterdam: Associated Scientific Publishers, 1972; 201-219. Berry JN, Thompsom HK, Miller DE, Mclntosh HD. Changes in cardiac output, stroke volume, and central venous pressure induced by atropine in man. Am Heart J 1959; 58:204-21 3. Gorten R, Gunnells C, Weissler AM, Stead EA. Effects of atropine and isoproterenol on cardiac output, central venous pressure, and mean transit time of indicators placed at three different sites in the venous system. Circ Res 1961 ; 14: 979-983. Daly W J, Ross JC, Behnke RH. The effect of changes in the pulmonary vascular bed produced atropine, pulmonary engorgement, and positive-pressure breathing on diffusing and mechanical properties of the lung. J Clin Invest 1 963; 42:1083-1094. Stein E, Damato AN, Kosowsky BD, Lau SH, Lister JW, The relation of heart rate to cardiovascular dynamics. Circulation 1966; 33: 925-932. Stratton JR, Pfeifer MA, Halter JB. The hemodynamic effects of sympathetic stimulation combined with parasympathetic blockade in man. Circulation 1987; 75: 922-929.
29. Jern S, Pilhall M, Jern C. Infusion of epinephrine augments pressor responses to mental stress. Hypertension, in press. 30. Fellows IW, MacDonald IA, Bennett T, O'Donoghue DP. Effect of intravenous infusion of adrenaline on the cardiovascular responses to distal body subatmospheric pressure in man. C/in Sci 1 988; 75: 389-394. ACKNOWLEDGEMENTS. The skilful technical assistance of Mrs Annika Johansson and Ms Hannele Korhonen is gratefully acknowledged. The present study was supported by a grant and a post-doctoral fellowship from the Swedish Medical Research Council (09046 and 7324, respectively) and by grants from the Swedish Heart-Lung Foundation.
Received 15 April 1991; accepted with revision 9 July 1991.
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Clinical Autonomic Research 1, 225--231 (1991)
THE effects of cholinergic blockade on haemodynamic reactivity to standardized mental stress has been studied in nine normotensive males during infusion of atropine (bolus dose 10 #g x kg -1 followed by a constant-rate infusion of 0.02 gg x k g - l x rain -1) or placebo given in a randomized order on two different days. Partial cholinergic blockade increased resting heart rate by 25-30 beats per minute. The magnitude of the heart rate response to stress (reactivity) however was unaffected by the atropine infusion. Also, in four subjects who received a higher dose of atropine (approximately 1.8-1.9 mg), heart rate responses to stress were the same as during placebo infusion. Cholinergic blockade was associated with a small but prolonged increase in diastolic blood pressure. These findings suggest that parasympathetic withdrawal does not contribute to the tachycardia caused by mental arithmetic, and that the pattern of neurogenic activation may differ from that elicited during a classic defence-alarm reaction and by somatomotor activation.
Effect of cholinergic blockade on heart rate, blood pressure and plasma c a t e c h o l a m i n e responses to m e n t a l stress in normal subjects S v e r k e r J e r n , MD, PhD cA, M a r t i n and C h r i s t i n a J e r n , MD, PhD
Pilhall, MD
Department of Clinical Physiology, Ostra Hospital, University of G6teborg, S-416 85 G6teborg, Sweden.
CACorresponding Author
Mental stress, Cholinergic blockade, Atropine, Haemodynamicfactors, Plasma catecholamines
Key words:
Introduction Elevated heart rate at rest is associated with an increased cardiovascular mortality, 1'2 and resting tachycardia predicts future hypertension in young subjects with borderline blood pressure elevation. 3'4 Since increased heart rate is a characteristic part of the defence-alarm reaction, it has been repeatedly suggested that exposure to excessive psychosocial stress and/or increased reactivity to environmental stimuli may be responsible for the resting tachycardia in some individuals, s In support of such a notion, high heart rate reactivity to stress promotes development of coronary atherosclerosis in primates. 6 Although there is a dual neural regulation of heart rate both through sympathetic and cholinergic pathways, studies of stress-induced changes of cardiac rhythm have almost exclusively focused on sympathoadrenal mechanisms] Therefore, little is known about the relative contribution of vagal withdrawal during psychological arousal in man, and the possible influence of cholinergic mechanisms on stress-induced heart rate reactivity. However, understanding of the neural mechanisms underlying the cardiac activation in response to mental stress is important for studies of the possible role of psychosocial factors in the pathogenesis of cardiovascular disorders. In this study we have used a strictly standardized mental arithmetic stress model to characterize physiological stress responses. 8-I° We have recently shown that this test is highly reproducible and can be repeated without adaptation of the stressinduced heart rate responses, u We have therefore © Rapid Communications of Oxford Ltd.
performed two mental stress tests (separated by at least 1 week) during double-blind infusions of atropine and placebo given in randomized order to nine healthy subjects.
Material and Methods Subjects: Nine young, normotensive male subjects participated in the study. All subjects were healthy non-smokers without cardiovascular disease and on no medication. The subjects were recruited from medical students and hospital employees. Their mean age, weight, and height were 26.0 years (range 23-35 years), 76.8 kg (67-90 kg), and 186.9 cm (178-191 cm), respectively. Informed consent was obtained from each subject before inclusion in the study after the rationale, nature, and potential risks of the research had been carefully explained. The protocol was approved by the Ethics Committee of the University of G6teborg. All subjects were asked to refrain from taking methylxanthine-containing products on the day before the experiment, to avoid interference with the catecholamine analyses. The participants were instructed to avoid heavy exercise or emotional excitement, and to be fasting for 4 h prior to the experiment. Experimental design: Each subject participated in randomized order in two experiments separated by at least 1 week. After a 10-min pre-infusion baseline period, they received infusions of either atropine or placebo for 35 min during the first part of the experiment according to a randomized, doubleClinical Autonomic Research.vol 1 • 1991
225
S. Jern, M. Pilhall and C. Jern blind schedule. On each day a mental stress test was performed 15 min after start of the infusion. During each stress test, the subjects performed 10 rain of forced mental arithmetic, according to a strictly standardized procedure (see Ref. 11 for reproducibility data on this method). Two investigators participated in the study (M.P. and S.J.), but each subject always met the same investigator during each stress test. The infusion was continued for 10 min after the conclusion of the stress test (post-stress baseline) and was then stopped. Changes of haemodynamics and plasma catecholamines were followed for 10 min immediately after the end of the infusion and for another 10-min period 50-60 min after the infusion.
Procedure: Upon reporting at the laboratory, the subject was placed in a comfortable chair in a semi-recumbent position. One venous cannula (Venflon ®, Viggo, Helsingborg, Sweden) was inserted percutaneously into an antecubital vein of the dominant arm for infusion of atropine. Another venous cannula was inserted into a vein of the dorsum of the hand of the same arm for blood sampling. A 50cm polyethylene catheter was connected to each indwelling cannula, and the catheters were led through the wall to an adjacent control room. This technique allowed us to start and stop the infusions and collect blood samples without the subjects knowing exactly when this was done. The intravenous lines were kept patent by slow saline infusions. A blood pressure cuff was applied on the non-dominant arm, and electrodes for computerized vectorcardiography were fixed to the chest, arms and legs according to a modified Frank system. After a 1-h rest period, the experiment commenced with the first 10-min baseline period with the subject in the quiet and dimly lit room. Then a bolus injection was given over 2 min which was followed by a constant-rate infusion for 35 min. Fifteen minutes after the start of the infusion, the investigator entered the room which was now fully lit and gave an approximately 40-s long oral instruction. The subject then performed forced mental arithmetic for 10 min with serial subtractions of 7 from 700 trying to keep pace with a metronome at a rate of approximately 90/min. The investigator encouraged the subject to increase the speed by short repeat comments. After a positive and reassuring comment, the investigator reduced the light and left the room. The stress task was followed by two 10-min baseline periods during and after the end of the infusion, respectively. The subjects then remained in the same position for another 50 min, at the end of which a second 10-min post-infusion recording was obtained. In a recent study, using the experimental 226
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procedure described above, we have demonstrated that the mental arithmetic stress test is highly reproducible with regard with heart rate, blood pressure, and subjective stress activation levels and reactivity, u When two tests were performed 1 h apart, both activation levels and reactivity during the two tests were closely correlated (R > 0.75 throughout). The coefficients of variation for the attained activation levels ranged between 3.1% and 6.2% for haemodynamic measures and subjective stress ratings. There were no significant differences in stress activation levels attained during the two tests.
Intravenous infusions: Atropine was administered as a bolus dose of 10 #g × kg -1 atropine sulphate (ACO, Stockholm, Sweden) given over 2 min, immediately followed by a constant-rate infusion of 0.02 #g × k g - l × min -1 for 35 min. The total individual dose of atropine ranged between 0.72 and 0.96 rag. Placebo (isotonic saline) was administered in the same manner and volume for each subject. Haemodynamic monitoring: Blood pressure was measured at 1-min intervals using an automatic non-invasive oscillometric blood pressure monitor (Dinamap Model 845, Critcon Instruments, Tampa, FL, USA), which has been validated against intra-arterial blood pressure recordings in our laboratory. 12 Heart rate was monitored continuously throughout the experiment by computerized vectorcardiography (MIDA 1000, Ortivus Medical AB, T~iby, Sweden). Blood sampling and biochemical analyses: Blood samples for analysis of plasma catecholamines were obtained on the following time points: at 10, 25, 35, 45, 55 and 105 min (cf. Fig. 1). All blood samples were obtained from the distal indwelling venous cannula. The first 5 ml of blood were always discarded. Eleven millilitres of blood for analysis of catecholamines were drawn into prechilled tubes containing 220 #1 glutathione-EDTA (60 mg/ml and 90 mg/ml, respectively) and immediately placed on melting ice. The samples were centrifuged within 2 min of collection at +4°C and 2 000 g for 5 min. Plasma aliquots were stored at - 7 0 ° C until assay. All coded samples from each study subject were always analysed in the same assay-run. Plasma catecholamines were separated by reversed-phase high performance liquid chromatography (HPLC) and quantified by the highly sensitive electrochemical detector of Waters (Electrochemical Detector 641, Waters, Millipore Ltd, Germany). Dihydroxybenzylamine was used as internal standard. The analytic procedure followed that described by Weicker et al. 13 In our laboratory, the intra-assay coefficients of variation for basal and
Mental stress after atropine
Heart rate bpm
110 90 70 t
t"t--r4
6
?--~-?
0
5O Systolic blood pressure mmHg
140 130 120 110 Diastolic blood pressure mmHg
90
80
~,/r
70
,
r>Ti
t"T-T T
60 ~lnfusion
J
I
I"
I
I
I
I
0
10
25
35
45
55
"'"
I
95
FIG. 1. Haemodynamic changes in response to mental stress during infusion of atropine and placebo. ( infusion, ( - - - ) , placebo. (Mean and SEM).
I
105 min ), Atropine
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227
S. Jern, M. Pilhall and C. Jern stress levels were 2.5% and 3.3% for noradrenaline (n = 14) and 8.6% and 8.1% (n = 18) for adrenaline, respectively. The detection limit is estimated to 50-100 fmol/1 plasma, when defined as the concentration equivalent to five times the baseline noise. Statistical ana~sis: Standard statistical methods were used. Haemodynamic data from the stress experiment were reduced by dividing baseline and stress periods into 2.5-min epoches, and by computing mean values for heart rate, and systolic and diastolic blood pressure for each period. Data from the plasma catecholamine analyses were not analysed in this manner. Reactivity was defined for each stress experiment as the difference between the mean level during stress and the mean from the corresponding pre-stress baseline period. Overall effects of atropine and placebo were evaluated by a two-factor (two types of Infusion x six Periods) analysis of variance (ANOVA) for repeated measures on individual changes relative to the pre-infusion baseline of each day. When the main effect for type of Infusion or the Infusion x Period interaction term yielded significant F-ratios, differences in levels between the atropine and placebo days were identified on the absolute values by means of Student's t-test for paired comparisons. Further, a separate two-way A N O V A was performed on the stress experiment, i.e. from time 15 min (pre-stress baseline) through 45 min (post-stress baseline). When A N O V A indicated significant Period effects for the stress experiment, possible differences in reactivity were evaluated by t-test of the change from pre-stress baseline (15-25 rain) to stress (25-35 min) during each infusion. In all cases, two-tailed tests were used, and the tests were considered significant at p < 0.05. For all repeated measures analyses, degrees of freedom were corrected according to the conservative Greenhouse and Geisser procedure for possible violation of the assumption of sphericity. 14
infusions was maintained throughout the first part of the experiment, i.e. until 10 rain after the infusions had been stopped. Fifty to 60 min after the end of the infusion, heart rate was still 5 _ 1 bpm [t(8) = 6.4, p = 0.0002] higher after atropine than after placebo. A N O V A also revealed significant effects of the atropine infusion on mean arterial pressure and diastolic blood pressure. Both mean arterial pressure [F(1,8)= 12.5, p = 0.008] and diastolic blood pressure [F(1,8) = 15.4, p = 0.004] were higher on the day of the atropine infusion. However, in contrast to the heart rate changes, the effects of atropine were more pronounced after than during the infusion. Mean arterial and diastolic blood pressures were 4 + 1 mmHg [t(8)= 3.0, p = 0.02] and 6 _ 2 mmHg [t(8) = 3.5, p = 0.008] higher after atropine during the late post-infusion period (i.e. 50-60 min after the infusion). No significant differences between the two infusions were observed for systolic blood pressure or plasma catecholamine levels (Fig. 1 and Table 1). The mental stress test induced highly significant changes in the four haemodynamic variables and also a significant change in plasma noradrenaline concentration, as indicated by significant Period effects of A N O V A [Fs(2,16) > 32.2, p < 0.0002 throughout]. In response to stress, heart rate increased by 18 _+ 2 bpm [t(8) = 7.7, p < 0.0001] and 14 ± 2 bpm [t(8) --- 6.0, p < 0.0003] during the atropine and placebo infusions, respectively (Table 2). The slight difference in stress reactivity between the infusions was not significant. The stress-induced increases in systolic, mean arterial, and diastolic blood pressures ranged between 12 and 14 mmHg [ts(8) > 3.3, p < 0.01 throughout], without any significant differences between the two conditions. In response to the mental stress test, there was significant change of the plasma noradrenaline Table 1. Levels of plasma catecholamines in response to mental stress during infusion of atropine and placebo (mean 4-SEM)
Results
There were no significant differences at rest during the pre-infusion baseline period between the atropine and placebo days for any of the measured variables. Effects of the infusions on haemodynamic variables are shown in Fig. 1. As expected, administration of atropine elevated the heart rate throughout the experiment [F(1,8)= 41.6, p = 0.0002]. Five minutes after the bolus injection and start of the atropine infusion, mean heart rate increased from 58 ± 2 bpm (mean ___ SEM) to 84 + 3 bpm [t(8) = 6.7, p = 0.0001; Fig. 1]. The difference between the atropine and placebo 228
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Atropine infusion
Placebo infusion
p-Noradrenaline nmol/I 10 min 25 min 35 min (stress) 45 min 55 min 105 min
2.52 2.58 2.78 2.22 2.22 2.94
4- 0.29 ___0.37 4- 0.41 4- 0.31 4- 0.38 4- 0.48
2.43 2.42 2.83 2.47 2.50 2.60
4- 0.29 _+ 0.23 4- 0.25 -t- 0.27 + 0.26 4- 0.35
p-Adrenaline nmol/I 10 min 25 min 35 min (stress) 45 min 55 min 105 min
0.33 0,30 0.35 0.27 0.23 0.22
___0.05 4- 0.05 4- 0.06 4- 0.05 4- 0.04 4- 0.03
0.33 0.34 0.35 0.38 0.31 0.22
4- 0.05 +_ 0.03 4- 0.03 4- 0.06 4- 0.06 4- O.04
Mental stress after atropine Table 2. Stress-induced changes (reactivity) of haemodynamics and plasma catecholamines in response to mental stress during infusion of atropine and placebo (mean ± S E M ) Atropine infusion Haemodynamics A Heart rate k Systolic blood pressure mmHg A Mean arterial pressure mmHg k Diastolic blood pressure mmHg
+17.6 +12.4 + 11.6 + 11.7
Plasma catecholamines A p-Noradrenaline nmol/I A p-Adrenaline nmol/I
+ 0 . 2 0 __ 0.13 + 0 . 0 5 ± 0.04
Significance of change from d p < 0.0001.
pre-stress
concentration [F(2,16) = 8.1, p = 0.01], whereas plasma adrenaline was unaffected by stress (Table 1). There were no significant differences between the two types of infusion on levels or patterns of stress-induced changes of either noradrenaline or adrenaline. After conclusion of the mental stress test, there was a rapid return of heart rate and blood pressure to values close to the pre-stress levels of the respective day (Fig. 1). In a second study, a higher dose of atropine (bolus dose of 20 #g x kg -~ infusion of 0.1 #g x k g - l × min -1 body weight) was administered in the same way as during the first study to four subjects (body weight 75-82 kg). After a total dose of approximately 1.8-1.9 mg of atropine, the heart rate responses to stress were the same as during the placebo infusion (Table 3; + 15.1 bpm versus +14.9 bpm after atropine and placebo, respectively).
Discussion The results of the present study show that although the partial cholinergic blockade increased resting heart rate by some 25 to 30 bpm, the magnitude of the heart rate response to stress was unaffected by the atropine infusion. When a higher dose of atropine was given to four subjects (each Table 3. Changes of heart rate in four subjects given the higher dose of atropine (20 #g x kg -1 bolus + 0.1 #g x kg -1 x min -1 body weight) mean ± S E M
Heart rate bpm 0 - 1 0 min 15-25 min 25-35 min (stress) 35-45 min 45-55 min A Heart rate bpm Pre-stress-stress
Atropine infusion
Placebo infusion
56.6 88.5 103.6 87.1 85.8
53.7 52,8 67.7 52.0 51.4
+ 3.4 + 8.8 _+ 9.1 ± 8.7 +__8 4
+ 15.1 ± 3.1
_+ 2.2 _± 3.6 + 2.9 ± 3.0 ± 3.4
+ 14.9 ± 1.8
_+ 2.3 d _+ 2.1 c _+ 2.0 c ± 1.7 d
Placebo infusion
+14.4 +12.3 + 14.0 + 1 2.7
± 2.4 c ± 3.7 b _-t-3.4 b _+ 2.8 b
+ 0 . 4 0 ± 0.13 ~ + 0 . 0 2 ± 0.03
baseline: a p < 0.05,
t-test A vs. P
ns ns ns ns ns ns
b p < 0.01, C p < 0.001,
subject receiving approximately 1.8-1.9 mg atropine), the result was the same; heart rate reactivity to the mental arithmetic stress test was identical to that observed after placebo. Thus, even with a substantial dose of atropine which should cause cardiac cholinergic blockade, heart rate responses to stress were not affected. These findings suggest that parasympathetic withdrawal contributes minimally, if at all, to the tachycardia elicited by a brief mental arithmetic task. If so, it appears that the neural regulatory mechanisms of heart rate responses to this particular form of psychological stress may differ from those of physical exercise, since decreased vagal inhibition is an important part of the tachycardia of somatomotor activation. Is Experimental observations support the notion that this may be a more general characteristic of some psychological arousal states in man, in particular those associated with active coping behaviour. In a study of young, healthy subjects heart rate responses to handgrip and a reaction time task stimulus was applied twice, once before and once after infusion of either placebo, atropine, or propranolol. Whereas the increase in heart rate during handgrip was significantly blocked by both drugs, the heart rate response to the reaction time task was blocked by propranolol but was unaffected by atropine. These data suggest that, in contrast to responses to isometric exercise, cardiac responses to mild active psychological coping during the reaction time tests are not dependent on vagal withdrawal. This has led Obrist lv,18 to hypothesize that the relative influences of sympathetic and parasympathetic mechanisms on heart rate responses during psychological stress will depend on the relative el±citation of somatomotor activation and active coping behaviour. According to this hypothesis, beta-mediated sympathetic cardiac influences would dominate during active psychological coping efforts such as the mental arithmetic task, whereas tasks involving somatomotor activaClinical Autonomic Research, vol 1 • 1991
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S. Jern, M. Pilhall and C. Jern
tion elicit heart rate elevations by withdrawal of cardiac vagal activity. 17 2o Previous workers have assumed that the cardiac response in man to experimental procedures such as the mental arithmetic task resembles that of the defence-alarm reaction, and that the tachycardia is due to a combination of increased sympathetic stimulation and decreased parasympathetic inhibition. 5'2. This neurogenic pattern has been observed also in rats exposed to various alerting stimuli, such as light, noise and vibration. = It appears, however, that even in this model the vagal component of the responses are variable, especially in normotensive animals. 22 Zanchetti and co-workers 23 have also s h o w n that the behavioural, neurogenic, and haemodynamic responses are complex and diversified in conscious and awake cats in 'natural' settings. Furthermore, there may be important species differences in the reactions to environmental stimuli. It is not unlikely, that in man in whom the complexity of the emotional and behavioural reactions is far .more pronounced, there is an even larger variety of neurogenic responses to stress. In previous human studies, administration of atropine during resting conditions has been shown to increase heart rate and cardiac output, but decrease total peripheral resistance and stroke volume. 24-26 Since chronotropic stimulation per se across a wide range of heart rates does not increase cardiac output or blood pressure, 27 it is assumed that the increased cardiac output may be related to blockade of negative inotropic vagal influences.28 Although the exact mechanism behind the peripheral vasodilation is unknown, it has been commonly assumed that a baroreceptor-mediated reflexogenic decrease in vascular tone has been triggered by the increased cardiac output. However, similar to what has been observed in some previous investigations cholinergic blockade produced slight but significant increases in diastolic blood pressure and mean arterial pressure. 24'2s Interestingly, the increase in diastolic blood pressure was clearly more long-lasting than the effect on heart rate; in fact, the maximal difference in diastolic blood pressure between the atropine and placebo experiments was observed 50-60 min after the end of the infusions. The cause of the pressor effect of atropine is not known, but our findings suggest that different mechanisms may be involved in the effects on heart rate and blood pressure. We have recently shown that high circulating adrenaline levels may amplify the pressor responses to mental stress, z9 Although this effect was confined to the systolic blood pressure response, other investigators have shown an increased heart rate response to other stressors after infusion of adrenaline. 3° Therefore, plasma catecholamine levels must be taken into account in evaluations of haekaodyna230
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mic stress responses. However, in the present study, no differences were observed between the two infusions on either the levels or patterns of stress-induced changes of plasma catecholamines. Thus, there was no evidence of a confounding effect of adrenaline on the heart rate or blood pressure responses to stress after atropine. In conclusion, the results of the present investigation argue against the view that withdrawal of parasympathetic inhibition is an important part of the cardiac response to mental arithmetic in man.
References 1. Kannel WB, Kannel C, Paffenbarger RS, Cupples PH, Cupples LA. Heart rate and cardiovascular mortality. The Framingham Study. Am Heart J 1987, 113:1489-1494. 2. Cruickshank JM, Smith JC. The beta-receptor, atheroma and cardiovascular damage. Pharmaco/Ther 1989; 42: 385404. 3. Levy RL, White PD, Stroud WD, Hillman CC. Transient tachycardia: prognostic significance alone and in association with transient hypertension. JAMA 1945; 129: 585. 4. Paffenbarger RS, Jr, Thorne MC, Wing AL. Chronic disease in former college students. VIII. Characteristics in youth predisposing to hypertension in later years. Am J Epidemio/1968; 88: 25. 5. Folkow B. Physiological aspects of primary hypertension. Physiol Rev 1982; 62: 347-504. 6. Clarkson TB, Weingand KW, Kaplan JR, Adams MR. Mechanisms of atherogenesis. Circulation 1987; 76 (suppl I): 1-20. 7. Freyschuss U, Hjemdahl P, Juhlin-Dannfeldt A, Linde B. Cardiovascular and sympathoadrenal responses to mental stress: influence of fl- blockade. Am J Physiol 1988; 255: H 1443-14451. 8. Jern S. Psychological and hemodynamic factors in borderline hypertension. Acta Med Scand 1982; suppl 662: 1-54. 9. Jern C, Eriksson E, Tengborn L, Risberg B, Wadenvik H, Jern S. Changes of plasma coagulation and fibrinolysis in response to mental stress. Thromb Haemostas 1989; 62: 767-771. 10. Manhem K, Jern C, Shanks G, Pilhall M, Hansson L, Jern S. Stress-induced haemodynamic changes during the menstrual cycle. Clin Sci (in press). 11. Jern S, Pilhall M, Jern C. Short-term reproducibility of the mental arithmetic stress test. Clin Sci (in press). 12. Milsom I, Swahn S0, Forsman L, Sivertsson R. An evaluation of automated indirect blood pressure measurements during pregnancy. Acta Obstet Gynecol Scand 1986; 65: 721-725. 13. Weicker H, Feraudi M, H&gele H, Pluto R. Electrochemical detection of catecholamines in urine and plasma after separation with HPLC. Cfin Chim Acta 1984; 141 : 17-25. 14. Vasey MW, Thayer JF. The continuing problem of false positives in repeated measures ANOVA in psychophysiology: a multivariate solution. Psychophysiology 1987; 24: 479-486. 15. Freyschuss U. Cardiovascular adjustment to somatomotor activation. Acta Physiol Scand 1 970; suppl 342: 1-63. 16. Pollak MH, Obrist PA. Effects of autonomic blockade on heart rate responses to reaction time and sustained handgrip tasks. Psychophysiology 1988; 25: 689-695. 17. Obrist PA, Webb RA, Sutterer JR, Howard JL. The cardiac-somatic relationship: some reformulations. Psychophysiology 1970; 6: 569-586. 18. Obrist PA. Cardiovascular psychophysiology. New York: Plenum Press, 1981. 19. Obrist PA, Gaebelein CJ, Teller ES, Langer AW, Grignolo A, Light KC, McCubbin JA. The relationship among heart rate, carotid dP/dt, and blood pressure in humans as a function of the type of stress. Psychophysiology 1 978; 15:102-115. 20. Obrist PA, Light KC, James SA, Strogatzos. Cardiovascular responses to stress: I. Measures of myocardial response and relationship to high resting systolic pressure and parental hypertension. PsychophysioIogy 1987; 24: 65-78. 21. Brod J, Fencte V, Hejl Z, Jirka J. Circulatory changes underlying blood pressure elevations during acute emotional stress (mental arithmetic) in normotensive and hypertensive subjects. Clin Sci 1959; 18: 269-279. 22. Hallb~ck M, Folkow B. Cardiovascular responses to acute mental 'stress' in spontaneously hypertensive rats. Acta Physio/ Scand 1974; 90: 684-698. 23. Zanchetti A, Bachelli G, Mancia G, Ellison GD. Emotion and the cardiovascular system in the cat. In: Physiology, Emotion and
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Psychosomatic Illness. Ciba Foundation Symposium 8. Amsterdam: Associated Scientific Publishers, 1972; 201-219. Berry JN, Thompsom HK, Miller DE, Mclntosh HD. Changes in cardiac output, stroke volume, and central venous pressure induced by atropine in man. Am Heart J 1959; 58:204-21 3. Gorten R, Gunnells C, Weissler AM, Stead EA. Effects of atropine and isoproterenol on cardiac output, central venous pressure, and mean transit time of indicators placed at three different sites in the venous system. Circ Res 1961 ; 14: 979-983. Daly W J, Ross JC, Behnke RH. The effect of changes in the pulmonary vascular bed produced atropine, pulmonary engorgement, and positive-pressure breathing on diffusing and mechanical properties of the lung. J Clin Invest 1 963; 42:1083-1094. Stein E, Damato AN, Kosowsky BD, Lau SH, Lister JW, The relation of heart rate to cardiovascular dynamics. Circulation 1966; 33: 925-932. Stratton JR, Pfeifer MA, Halter JB. The hemodynamic effects of sympathetic stimulation combined with parasympathetic blockade in man. Circulation 1987; 75: 922-929.
29. Jern S, Pilhall M, Jern C. Infusion of epinephrine augments pressor responses to mental stress. Hypertension, in press. 30. Fellows IW, MacDonald IA, Bennett T, O'Donoghue DP. Effect of intravenous infusion of adrenaline on the cardiovascular responses to distal body subatmospheric pressure in man. C/in Sci 1 988; 75: 389-394. ACKNOWLEDGEMENTS. The skilful technical assistance of Mrs Annika Johansson and Ms Hannele Korhonen is gratefully acknowledged. The present study was supported by a grant and a post-doctoral fellowship from the Swedish Medical Research Council (09046 and 7324, respectively) and by grants from the Swedish Heart-Lung Foundation.
Received 15 April 1991; accepted with revision 9 July 1991.
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