Introduction

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Numerous studies have shown that various forms of stress can cause a leakage of cytoplasmic enzymes into the blood in humans and laboratory animals. Plasma enzymes, including CPK, LDH, GOT and GPT, increase in humans after severe exercise (Kayashima et al., 1995), in rats after restraint alone (Pearl et al., 1966; Sen et al., 1992), restraint with cold exposure (Meltzer, 1971) and prolonged exercise (Altland and Highman, 1961), in sheep after repeated restraint and isolation (Apple et al., 1993) and in dogs after hypothermia (Blair et al., 1961). On the other hand, it is well accepted that stress stimulates the activity of the autonomic nervous system, and its activation is possibly involved in the stress-induced increases in plasma enzymes activity. However, there are few papers demonstrating the role of the sympathetic and parasympathetic nerves in the stress-induced increases in plasma enzymes.

Water immersion stress has been widely used as a technique for making an animal model of gastric lesions, but it is not clear whether the stress influences plasma enzyme activity. The technique involves restraining an animal in a fitted small cage and immersing the animal in 23°C water to the xyphoid process (Takagi et al., 1964). The previous works revealed that either parasympathetic or sympathetic activity is stimulated by this stress. That is, the stress not only stimulates vagal activity (Yano and Harada, 1980) but also raises plasma catecholamine levels (Nakamura et al., 1992) and their urinary excretion (Yano and Harada, 1980).

The present study showed increased activities of plasma enzymes, such as CPK, LDH, GOT and GPT, after water immersion stress in rats. In addition, the plasma levels of urea nitrogen, creatinine and glucose also were elevated by the stress. To clarify the role of the autonomic nervous system in the stress responses, we studied the effects of adrenergic and cholinergic blockers on the stress-induced increases in these biochemical parameters.

	Methods

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Animals. Male Sprague-Dawley rats were purchased from CLEA Japan (Tokyo, Japan) at the age of 6 weeks and kept in our laboratories for 1 week before the experiment under a 12:12 hr light/dark cycle with lights on at 8:00 A.M. The rats were deprived of food for 18 hr, but permitted water ad libitum before use. All animal procedures were carried out as approved by the Animal Care and Use Committee at Fujisawa Pharmaceutical Co. Ltd. (Osaka/Tokyo, Japan).

Stress procedures. Fasted rats weighing 180 to 210 g were subjected to water-immersion-stress as described previously (Takagi et al., 1964). At around 10:00 A.M., the rats were restrained in firmly fitted restraint cages (stainless mesh, 4 × 4 × 14 cm) and vertically immersed in water maintained at 23°C to the level of the xyphoid process. Six hours later, the rats were released and returned to their home cages. Nonstressed rats were kept in their home cages without food and water at 23°C during the period. During the poststress period, the rats were deprived of food but permitted water ad libitum.

Drug study. 6-OHDA, phentolamine hydrochloride, dl-propranolol hydrochloride, timolol maleate, atropine sulfate were purchased from Sigma Chemical Co. (St. Louis, MO). 6-OHDA was dissolved in physiological saline containing 1% (w/v) ascorbic acid and injected i.p. into the rats at a dose of 80 mg/kg in a volume of 2 ml/kg, 7 days before the stress session. Other drugs were dissolved in physiological saline and injected i.p. into the rats in a volume of 2 ml/kg, just before the stress session. Immediately after the 6-hr stress period, blood was collected and used for determining the levels of the plasma parameters by the same procedures as described above.

Catecholamines determination. For determining plasma NE and EPI levels, blood was collected according to the method described by Steffens (1969). Three days before the stress experiment, rats were provided with a silicon heart catheter (outside diameter, 1 mm; inside diameter, 0.5 mm) inserted through the jugular vein and situated at the entrance to the right atrium under pentobarbital anesthesia (50 mg/kg i.p.). One milliliter of blood was collected through the catheter 0, 15, 60 and 360 min after the beginning of stress. After each sampling, the same amount of heparinized blood (20 U/ml) of normal rat was given to compensate the blood loss. NE and EPI in the plasma were extracted by the method of Hallman et al. (1978) and assayed electrochemically by HPLC as described by Watson (1981). The analytical conditions were as follows: HPLC pump, model EP-10 (Eicom, Kyoto, Japan); electrochemical detector, model ECD-100 (Eicom); HPLC column, CA-5ODS, 4.6 × 150 mm (Eicom); mobile phase, 0.1 M sodium phosphate buffer solution (pH 6.0) with 10% (v/v) methanol and 277 µM 1-octanesulfonate (Nakalai Tesque, Kyoto, Japan) and 10 µM disodium ethylenediaminetetraacetic acid (Nakalai Tesque); flow rate, 1 ml/min.

Statistical analysis. All results were expressed as mean ± S.E.M. Statistical significance of differences between paired groups was calculated by the Student's t test (two-tailed). Statistically significant effect of drug treatment was analyzed by ANOVA, followed by Dunnett's post hoc analysis when ANOVA was significant. A P value less than .05 was considered as significant.

	Results

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Changes in plasma parameters during and after stress. Figure 1 shows the time-course changes in plasma parameters levels during and after water immersion stress (6 hr). All of the levels stayed constant in the no-stress group, excepting the level of plasma glucose which increased time dependently.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Time-course changes in the levels of plasma parameters during stress and poststress periods. Values are presented as mean ± S.E.M. of eight rats. *P <.05, **P <.01, ***P < .001 versus no-stressed rats at the same period (by Student's t test).

Effects of drugs on the plasma parameters in stressed rats. Figure 2 shows the effect of chemical sympathectomy induced by the pretreatment of 6-OHDA (80 mg/kg i.p.) on the plasma levels of the seven parameters which were significantly increased by the water immersion stress as described above. The stress-induced increases in the plasma CPK, LDH, GOT and GPT activities and urea nitrogen levels, but not the increases in plasma creatinine and glucose levels, were significantly suppressed by 6-OHDA treatment.

View larger version (33K): [in this window] [in a new window]   Fig. 2.   Effects of 6-OHDA on the levels of plasma parameters in stressed rats. *P < .05 versus control rats by Student's t test. Eight rats were used for each group.

View larger version (34K): [in this window] [in a new window]   Fig. 3.   Effects of propranolol on the levels of plasma parameters in stressed rats. ***P < .001 versus the controls (dose 0) by Dunnett's post hoc analysis after ANOVA. Eight rats were used for each group.

View larger version (33K): [in this window] [in a new window]   Fig. 4.   Effects of timolol on the levels of plasma parameters in stressed rats. *P < .05, **P < .01, ***P < .001 versus the controls (dose 0) by Dunnett's post hoc analysis after ANOVA. Eight rats were used for each group.

View larger version (35K): [in this window] [in a new window]   Fig. 5.   Effects of phentolamine on the levels of plasma parameters in stressed rats. *P < .05 versus the controls (dose 0) by Dunnett's post hoc analysis after ANOVA. Eight rats were used for each group.

View larger version (36K): [in this window] [in a new window]   Fig. 6.   Effects of atropine on the levels of plasma parameters in stressed rats. **P < .01 versus the controls (dose 0) by Dunnett's post hoc analysis after ANOVA. Eight rats were used for each group.

Effects of drugs on the plasma parameters in nonstressed rats. As listed in table 1, none of the tested drugs, even at the highest dose, significantly changed plasma parameter levels in the nonstressed rats, except atropine (10 mg/kg), which slightly but significantly decreased plasma urea nitrogen level.

Changes in plasma catecholamine levels in nonstressed and stressed rats with or without 6-OHDA treatment. As shown in figure 7, plasma NE and EPI levels were significantly higher in the stressed rats than in the nonstressed rats throughout the stress period. Plasma NE level in the stressed rats peaked at 15 min after the beginning of stress, when the level was 24-times higher than that in the nonstressed rats. Plasma EPI level in the stressed rats peaked at 60 min, when the level was 16 times higher than that in the no-stressed rats. During the stress period, plasma NE level was lower in the 6-OHDA-treated group than in the control group with statistical significance at 15 and 60 min, whereas the plasma EPI level was hardly changed by the 6-OHDA treatment.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Time-course changes in the levels of plasma norepinephrine and epinephrine in nonstressed and stressed rats with or without 6-OHDA treatment. Values are presented as mean ± S.E.M. of five rats. ***P < .001 versus nonstressed rats; #P < .05, ##P < .01 versus stressed rats without 6-OHDA treatment at the same period (by Student's t test).

	Discussion

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Various forms of stress cause a leakage of cytoplasmic enzymes into the blood in humans and laboratory animals by causing cell injury or increasing cell membrane permeability (Meltzer, 1971; Sen et al., 1992; Apple et al., 1993; Kayashima, 1995). In agreement with these previous studies, the present study showed significant increases in plasma CPK, LDH, GOT and GPT activities after water immersion stress in rats. In addition, the results with plasma catecholamine levels strongly suggest the acceleration of peripheral sympathetic activity throughout the stress period, and the pretreatment of 6-OHDA (80 mg/kg i.p.) suppressed not only the stress-induced increase in NE level but also that in these plasma enzymes. Another important finding in the present work is that the pretreatment with beta-adrenergic antagonists, as well as 6-OHDA, but not the pretreatment with alpha-antagonist or anticholinergic agent, significantly prevented the stress-induced increases in these plasma enzymes levels without affecting the levels in nonstressed rats. Because propranolol is also characterized by having local anesthetic and membrane-stabilizing effects (Weiner, 1985), such properties might possibly contribute to the antistress action. However, this possibility may be excluded by the result that timolol, which did not have anesthetic or membrane-stabilizing effects (Weiner, 1985), also had an antistress action with similar potency and efficacy to that of propranolol in the present study. Consequently, peripheral sympathetic activation could be involved in the stress-induced increases in the plasma enzymes mediated by beta-adrenoceptors. This speculation is in part supported by the previous findings that an injection of norepinephrine or isoproterenol, a beta-adrenergic agonist, increases plasma CPK, LDH, GOT and GPT activities in rats and dogs (Highman et al., 1959; Wexler, 1970; Van Belle et al., 1992).

Water immersion stress is known to induce gastric lesions in rats (Takagi et al., 1964). It has been reported that the stress-induced gastric lesions are potently prevented by atropine (Harada et al., 1981), but aggravated by propranolol and 6-OHDA (Ray et al., 1987; Shichijo et al., 1993). It is therefore plausible that adrenergic and cholinergic systems play differential roles in the pathogenesis of the stress-induced gastric lesions and increases in plasma CPK, LDH, GOT and GPT activities. In addition, it is likely that the stress damaged not only the stomach but also other tissues, and that most of these plasma enzymes leaked from the latter. Among the four enzymes, plasma CPK was increased most drastically by the stress. The result implies that the stress might cause damage in the CPK-rich tissues, such as the heart and skeletal muscle, which also contain relatively high levels of LDH and GOT. Various forms of stress have been reported to induce focal myocardial necrosis and myofibrillar degeneration (isolation, Raab et al., 1968; restraint, Johansson et al., 1974), which can be prevented by the pretreatment of propranolol (restraint with cold exposure; Lopes et al., 1992). In addition, certain forms of stress cause histopathological changes in the thigh muscle (hypothermia, Knocker, 1955; prolonged exercise, Altland and Highman, 1961).

Our results thus suggest the possibility that excessive peripheral sympathetic activity may directly or indirectly cause cell injury or increase cell membrane permeability in the heart and/or skeletal muscles primarily mediated by beta-adrenoceptors and result in the leakage of their intracellular enzymes such as CPK and LDH into the blood in rats subjected to water immersion stress. This is in accordance with clinical observations; that is, stressful disorders such as subarachnoid hemorrhage and acute head injury are associated with increased plasma NE levels, often accompanied by an increase in plasma CPK activity and cardiac injury which can be inhibited by beta-adrenergic blockade (Neil-Dwyer et al., 1978, 1986; Cruickshank et al., 1987). Myocardial injury has been reported to occur after exogenous administration of NE or isoproterenol (Reichenbach and Benditt, 1970) and, after stimulation of endogenous catecholamines release, induced by tyramine (Downing and Chen, 1985) in experimental animals. Although the detailed mechanism of catecholamine cardiotoxicity is not clear yet, it is in part attributable to intracellular Ca⁺⁺ overload mediated by beta-adrenoceptor (Reichenbach and Benditt, 1970; Mann et al., 1992). In cultured cardiomyocytes, NE exposure leads to abnormality of their cytoskeletal structure and excessive Ca⁺⁺ influx, both of which are attenuated by propranolol but not by phentolamine (Hori et al., 1994), which suggests that beta-adrenergic overstimulation may induce cardiac cell injury. In addition, propranolol has been reported to decrease contractions in cardiac and skeletal muscles and prevent stress-induced excessive oxygen consumption and mobilization of carbohydrate metabolism in these tissues (Jansky et al., 1976; Ahlersova and Ahlers, 1976), such properties possibly increase the tolerance of these tissues against stress. Consequently, the direct and/or indirect effects of beta-adrenergic blockers, i.e., preventing the Ca⁺⁺ overload and/or decreasing the excitability of cardiac and skeletal muscles, possibly account for their antistress action; however, further studies will be needed to elucidate the mechanisms by which beta-adrenergic blockade and chemical sympathectomy prevent the water immersion stress-induced increases in intracellular enzymes in plasma.

We also found that restraint alone (immobilizing rats in a restraint cage for 6 hr without water immersion) significantly (P < .01) increased plasma CPK activity in the rats, but the level was remarkably lower than that in restraint rats with water immersion (no-stress, 154 ± 8; restraint alone, 619 ± 135; water immersion stress, 18,636 ± 3,450 U/l; mean ± S.E.M. of 8 rats). Since it has been reported that immersing rats at 25°C for 6 hr decreases their body temperature to 28°C (Yano and Harada, 1980), hypothermia seems to be one of the factor involved in the water immersion stress-induced increase in plasma enzymes. Indeed, increased membrane permeability with increased Ca⁺⁺ influx and leakage of intracellular enzymes has been demonstrated in cultured cardiomyocytes under hypothermic conditions (Hryshko et al., 1989; Orita et al., 1993), which suggests that hypothermia itself may impair the membrane barrier. In addition, hypothermia is known to stimulate catecholamine secretion. This evidence combined with the present results also would explain the previous report showing that plasma CPK activity is positively correlated with the extent of hypothermia in rats following restraint with cold exposure (Meltzer, 1971).

Impairment of renal functions might occur after the water immersion stress, which was suggested by the present data that showed significant increases in plasma urea nitrogen and creatinine. Other forms of stress, such as hypothermia and prolonged exercise, have been reported to elevate plasma urea nitrogen (Altland and Highman, 1961; Sen et al., 1992) and induce histological abnormalities in the kidney (Knocker, 1955). Phentolamine significantly prevented the water immersion stress-induced increase in plasma urea nitrogen, although the drug at even the highest dose (10 mg/kg i.p.) prevented the stress response by only 35% (plasma urea nitrogen levels as follows; stress group, 46 ± 2; phentolamine-treated stress group, 35 ± 3; no-stress group, 15 ± 1 mg/dl) and had no effect on the rise in plasma creatinine levels. Consequently, alpha-adrenoceptors may play a part in the stress-induced renal dysfunction, and other mechanisms may be involved. It has been reported that an alpha-1 adrenergic blocker, prazosin, antagonizes the contractile effect of norepinephrine on the intrarenal artery (Owen, 1993). Because the plasma norepinephrine level rises remarkably after water immersion stress, we therefore speculate that phentolamine could protect kidney against the stress through its vasodilating effect.

Water immersion stress has been reported to elevate plasma glucose levels in rats (Yano et al., 1976); this was confirmed in the present study. Extending this finding, the present data suggest that alpha-adrenoceptors and cholinergic nerves may contribute to the stress-induced hyperglycemia. It is well established that alpha-adrenoceptor overfunctions contribute to the hyperglycemia induced by various forms of stress (Surwit, 1992), while the role of the cholinergic nerves is not fully understood. However, our speculation is partially supported by the previous findings that restraint stress stimulates the synthesis and release of acetylcholine in brain (Finkelstein et al., 1985; Imperato et al., 1991) and an intracerebral injection of cholinergic agents induces hyperglycemia (Iguchi et al., 1990; Kunoh et al., 1992).

In conclusion, the present study clearly showed that water immersion stress causes the massive increase in intracellular enzymes, such as CPK, LDH, GOT and GPT, in the plasma of rats. Excessive peripheral sympathetic activity possibly plays a role in the stress-induced increases in these plasma enzymes levels primarily mediated by beta-adrenoceptors, while alpha-adrenoceptors and the cholinergic nerves may be involved in the stress-induced renal dysfunction and/or hyperglycemia.