BIOCHEMICAL MEDICINE 28. 340-346 (1982)

Stress-Induced Changes in in Vivo and in Vitro Dopamine-P- Hydroxylase Activity in Spontaneously Hypertensive Rats

KEISUKE FUJITA, RYOJI TERADAIRA, TAKASHI INOUE, HISAHIDE TAKAHASHI, HIDEHIRO BEPPU, KAORU KAWAI.

KAZUHIRO MARUTA, SHIGERU YAGYU. AND TOSHIHARU NAGATSU

Institute for Comprehensive Medical Science, School of Medicine, Fujitu-Gakuen University, Toyoake, Aichi 470-11. and Laborutory of Cell Physiology.

Department of Life Chemistry, Graduate School at Nagatsuta.

Tokyo institute of Technology, Yokohama 227, Japan

Received November 2, 1981

The hypothesis that the sympathetic nervous system, with its neuro- transmitter norepinephrine, plays an important part in the regulation of blood pressure in patients with hypertension has been extensively studied in animal models of hypertension such as spontaneously hypertensive rats (SHR) (1) or stroke-prone spontaneously hypertensive rats (SHRSP) (2), using, as control, Wistar-Kyoto rats (WKY) from which the SHR and SHRSP were bred. At young ages (3-4 weeks old) before the onset of hypertension, the in vitro activity of dopamine-@-hydroxylase (DBH) (3), which catalyzes the final step of norepinephrine biosynthesis and is the marker enzyme of the sympathetic nerves and adreno-medullary cells, was found to be increased in the mesenteric vessels and serum of SHR (4,5). The higher in vitro DBH activity in the mesenteric vessels and serum was even more pronounced in young SHRSP which had higher blood pressure than SHR at 16 weeks of age (6). However, at 16 weeks of age when hypertension in SHR and SHRSP was fixed, in vitro DBH activity in the mesenteric vessels and serum did not differ significantly among WKY, SHR, and SHRSP (7). In contrast, in vitro DBH activity in the adrenal gland at 16 weeks of age was the highest in SHRSP, followed by SHR and WKY in a decreasing order (7). In accordance with our results, selective activation of in vitro tyrosine hydroxylase activity, which is also an index of sympathetic nerve activity, in the sympathetic nerve ganglia of young SHR was also reported (8). These results on in vitro norepinephrine-related enzymes suggest that the sym-

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STRESS-INDUCED DBH ACTIVITY CHANGES IN RATS 341

pathetic nerve activity may be related to the initiation of hypertension, but not to its maintenance.

Although in vitro DBH activity, which is parallel to the amount of enzyme in the sympathetic neurons, is assumed to be correlated with neuronal activity in vivo, actual in vivo DBH activity may directly in- dicate the sympathetic nerve activity. A new method was recently re- ported to measure in vivo DBH activity in the peripheral sympatho- adrenal medullary system of a whole animal (9).

In the present study in vivo DBH activity in the peripheral sympatho- adrenal tissues of the whole animal and in vitro DBH activity in serum, adrenals, peripheral sympathetically innervated tissues, and brain have been measured simultaneously in order to compare changes and reactivity of the sympathetic nerve activity between SHR and WKY after estab- lishment of hypertension with and without repeated immobilization stress to SHR and WKY.

METHODS

SHR and WKY were raised in our laboratory under the same con- ditions. Immobilization as a model of stress was done in a way similar to that described by Yamori et al. (10). Male WKY and SHR at 12 weeks of age were immobilized by securing their four limbs with strings to a board in a supine position. Blood pressure was determined at 1500 hr in unanesthetized animals using indirect plethysmography (1). Then im- mobilization was carried out from about 1800 hr for 30 min and was repeated again from 1900 hr for another 30 min. Immobilization twice a day was repeated for 7 days, and the animals were killed after ether anesthesia by decapitation 24 hr after the last immobilization.

In vivo DBH activity of the whole peripheral sympathetic nerves plus adrenal medulla of WKY and SHR with and without immobilization stress was measured by the method of Hoeldtke and Kaufman (9) with some modifications. At 30 min before decapitation the animals were injected intraperitoneally with 100 @i/kg of [P-‘Hldopamine (sp act 35 Ci/mmole, NET-131). To the control rats fusaric acid (200 mg/kg, dis- solved in saline and adjusted to pH 7 with NaOH), a specific DBH inhibitor (1 l), was injected intraperitoneally 1.25 hr prior to administra- tion of [P-3H]dopamine. Five rats were used for each group of experi- ments, and after ether anesthesia they were sacrificed by decapitation and the blood was collected from the cervical fracture into heparinized beakers. The whole blood was then lyophilized, and tritiated water was determined by counting an aliquot of the collected water in a liquid scintillation counter (Packard Model 2650). Rate of norepinephrine for- mation per 30 min was calculated as

rate of THO release (% conversion/30 min)

= (wt of animal)(0.7)(THO/ml blood) - injected THO x total administered cpm

IO0 3

342 FUJITA ET AL

rate of norepinephrine formation per 30 min

= 2 x (rate of THO released in untreated animals

- rate of THO released in fusaric acid-treated animals).

In this in vivo DBH assay, the reaction (tritium water release accom- panying the formation of norepinephrine from [13-3H]dopamine) was found to be linear for at least 30 min. Since [P-3H]dopamine does not penetrate into brain from blood, DBH activity can only be measured in the peripheral tissue by this method.

In vitro DBH activity in serum and tissues of SHR and WKY without or with stress was measured by the sensitive dual-wavelength spectro- photometric method of Kato et al. (12,13) under saturating concentrations of the substrate, tyramine, and the cofactor, ascorbic acid, to measure the maximum velocity. Rats were anesthetized with ether and decapi- tated, and blood, adrenal gland, vas deferens, heart, and brain were removed quickly and immediately frozen on dry ice. Tissues were ho- mogenized in 0.25 M sucrose, and the homogenates were used for the in vitro DBH assay.

RESULTS

The in vivo DBH activity of the whole animal except brain is shown in Table 1. The rate of norepinephrine formation of unstressed WKY (11.35% per 30 min) was similar to that for Sprague-Dawley rats reported by Hoeldtke and Kaufman (3.36-3.96% per 10 min). However, the rate of norepinephrine formation of unstressed SHR was slightly but signif- icantly lower than that of unstressed WKY.

As also shown in Table 1, in vivo DBH activity of WKY did not change at all even after 7 days of immobilization stress. In contrast, in vivo DBH activity of SHR was significantly increased as compared with that of unstressed SHR.

In vitro DBH activity in serum and tissues (heart, vas deferens, ad- renals, brain stem, and thalamus-hypothalamus) in SHR and WKY with or without stress is shown in Table 2. Before the immobilization stress at 12 weeks of age, SHR had higher DBH activity than WKY only in adrenal glands. After repeated daily immobilization for 7 days, significant elevation of DBH activity in WKY was found only in adrenal glands. In contrast, more pronounced elevation of adrenal DBH was observed in SHR. SHR showed a significant elevation of DBH activity not only in adrenal glands but also in serum, vas deferens, brain stem, and thalamus-hypothalamus. DBH in heart was also increased, but the change was not significant.

As shown in Fig. 1, blood pressure of WKY was nearly constant throughout the period of the experiment from zero time (12 weeks of

STRESS-INDUCED DBH ACTIVITY CHANGES IN RATS 343

TABLE 1 IN VIVO DBH ACTIVITY OF SPONTANEOUSLY HYPERTENSIVE RATS (SHR) AND WISTAR-

KYOTO RATS (WKY) WITH AND WITHOUT REPEATED DAILY IMMOBILIZATION STRESS

In vivo DBH activityb

Rats

Rate of THO release (% of Rate of norepinephrine [B-3H]dopamine converted to formation

THO per 30 min) (%I

WKY Unstressed

Experimental Blank”

Stressed Experimental Blank”

SHR Unstressed

Experimental Blank

Stressed Experimental Blank

7.06 T 0.24 (5) 11.35 k 0.49 (5) 1.38 r 0.68 (5)

7.07 t 0.41 (5) 11.22 + 0.83 (5) 1.46 ? 0.87 (5)

5.76 2 0.34 (5) 9.05 2 0.59’ (5) 1.18 5 0.70 (5)

6.52 2 0.28 (5) 11.34 2 0.73d (5) 1.03 2 0.48 (5)

’ Animals pretreated with fusaric acid. h Values are means + SEM. ‘ Differs from WKY, P < 0.05. d Differs from unstressed SHR, P < 0.01.

age) to the seventh day (13 weeks of age) in unstressed and stressed animals. In contrast, blood pressure of SHR was decreased significantly after 3 days of daily immobilization stress compared either with the blood pressure at zero time or with that of unstressed SHR. This agrees with the results by Kvetnansky et al. (14), who also found a significantly lower mean blood pressure in repeatedly stressed SHR compared with unstressed SHR.

FIG. 1. Effect of daily immobilization stress on blood pressure of spontaneously hy- pertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) at 12 weeks of age. Blood pressure was measured 3 hr before immobilization every day. Results are the means + SEM of five rats. *Statistically significant compared to the unstressed controls at the same experimental day, P < 0.05.

344 FUJITA ET AL.

DISCUSSION

Decreased in vivo DBH activity suggests that SHR at 12 weeks of age may have decreased sympathetic nerve activity as compared with WKY. In contrast, in vitro DBH activity did not differ significantly between SHR and WKY.

The increase in in vivo DBH activity after stress only in SHR also suggests that SHR have a higher response to stress than WKY, and agrees with the finding that after immobilization stress in vitro DBH activity, which is related to the amount of enzyme, was also significantly increased in the adrenal gland, serum, and tissues of SHR (Table 2). The increase of in vivo DBH activity after stress observed in SHR may be due to the induction of the DBH molecule in the peripheral sympatho- adrenomedullary system. A small but significant increase of in vitro DBH activity in the adrenal glands of WKY after stress was also observed. However, since in vivo DBH activity of WKY did not change at all after stress, this small increase of in vitro DBH in the adrenal medulla of WKY may not affect the total in vivo enzyme activity in peripheral tissues.

The discrepancy between in vivo and in vitro DBH activities may suggest that in vitro DBH activity, which is parallel to the DBH amount, may not necessarily indicate in vivo DBH activity, which may be directly correlated with the actual sympathetic nerve activity.

Since the present results suggest elevated sympathetic nerve activity after stress in SHR as compared with WKY, a more pronounced elevation

TABLE 2 IN VITRO DOPAMINE-P-HYDROXYLASE (DBH) ACTIVITY OF SFONTANEOUSLY HYPERTENSIVE

RATS (SHR) AND WISTAR-KYOTO RATS (WKY) WITH AND WITHOUT REPEATED

DAILY IMMOBILIZATION STRESS ___.--.-

DBH activity (nmole/midmg tissue)

Tissues (n = 7)

Serum Heart Vas deferens Adrenals Brain stem Thalamus-

hypothalamus

SHR WKY

Unstressed Stressed

0.179 f 0.007 0.250 ?I 0.011*,‘+ 1.04 + 0.20 1.40 k 0.26 5.09 * 0.77 8.57 -r- l.OP

44.81 -r- 4.62 84.70 ‘- 11.35*,” 0.81 2 0.11 1.15 * O.lO”,’

0.59 2 0.12 1.09 k 0.14”,’ 0.60 2 0.14 0.63 2 0.08

Unstressed Stressed

0.178 t 0.017 0.175 e 0.017 1.14 + 0.19 0.96 + 0.16 3.58 f 0.55 3.87 ‘- 0.78

29.79 IT 3.06 42.64 1?1 1.49’ 0.94 k 0.13 0.89 2 0.05

a.’ Difference from unstressed SHR: y P c 0.05, b P < 0.01. r.d Difference from stressed WKY: ’ P < 0.05, ‘P < 0.01. ‘.’ Difference from unstressed WKY: ’ P < 0.05, f P < 0.01.

STRESS-INDUCED DBH ACTIVITY CHANGES IN RATS 345

in blood pressure of SHR may be expected after stress. On the contrary, blood pressure in SHR was decreased after repeated immobilization stress.

Kvetfiansky et al. (12) also reported decrease in blood pressure after immobilization stress. Kvetnansky et al. (12) suggested that the decrease in blood pressure of SHR after immobilization stress could be due to violent struggling of SHR. However, the mechanism of blood pressure decrease after repeated immobilization stress in SHR is not clear.

The results may indicate that the response of adrenal glands and sym- pathetic nerves to stress is more elevated in SHR than in WKY, and such responses to stress may be different genetically in various strains of rats.

It is of interest that the DBH level was increased in SHR after repeated immobilization not only in the vas deferens and adrenals but also in the brain. This suggests that SHR also have higher response in central nor- adrenergic neurons than WKY, and that the linkage between central and peripheral noradrenergic neurons may also be altered in SHR.

The significance of elevated activation of the sympatho-adrenal system after repeated immobilization stress in SHR remains a topic for further study. Since blood pressure was decreased by immobilization of SHR, increased sympatho-adrenal activity may be a compensatory enhance- ment via vasoreceptor reflex. This possibility is also supported by the finding of Kvetiiansky et al. (12) that plasma levels of norepinephrine and epinephrine are higher in SHR than WKY after immobilization.

Increased responses of SHR to environmental stress and the resultant increased secretion of both epinephrine from adrenal glands and nor- epinephrine from sympathetic nerves could contribute to the vascular changes and development of arteriosclerosis in SHR.

Serum DBH activity in patients with essential hypertension had been reported to be higher than that of normal controls (15), but later reports could not confirm this elevation in serum DBH in essential hypertension (16). Serum DBH activity is questioned as an index of peripheral sym- pathetic nerve activity. The in vivo DBH activity in hypertensive patients may be of significance in elucidation of the role of sympathetic nerve activity either at the onset or in maintenance of hypertension.

The elevated response in the sympatho-adrenal system in SHR, as expressed in the elevation of both in vivo and in vitro DBH activity, is of interest, since stress is assumed to be one factor for the onset and maintenance of human essential hypertension.

SUMMARY

The in vivo dopamine+-hydroxylase (DBH) activity in peripheral tis- sues of the whole animal and the in vitro DBH activity in serum, adrenal

346 FUJITA ET AL..

glands, peripheral sympathetically innervated tissue, and brain were ex- amined in spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) at 12 weeks of age before and after daily immobilization stress. In vitro DBH activity did not differ significantly between SHR and WKY before stress, but in viva DBH activity in SHR was significantly lower than that in WKY. After repeated immobilization stress, in vivo DBH activity was significantly elevated only in SHR but not in WKY. In vitro DBH activity in serum, adrenal gland, vas deferens, and brain was also significantly increased in SHR, whereas that in WKY was increased only in adrenal glands. These results suggest that SHR at 12 weeks of age, when hypertension is established, may have even lower sympathetic nerve activity than WKY, and that after repeated stress the response of the sympatho-adrenal system is higher in SHR than in WKY.

REFERENCES

1. Okamoto, K., and Aoki, K., Jupnn. Circ. J. 27, 282 (1963).

2. Okamoto, K., Yamori, Y., and Nagaoka, A., Circ. Res. 34/35, 143 (1974). 3. Levin, E. Y., Levenberg, B., and Kaufman, S., J. Biol. Chem. 235, 2080 (1960).

4. Nagatsu, T., Kato, T., Numata (Sudo), Y., Ikuta. K., Umezawa, H., Matsuzaki, M.. and Takeuchi. T.. Nature (London) 251, 630 (1974).

5. Nagatsu, T., Ikuta, K., Numata (Sudo). Y., Kato, T., Sano, M., Nagatsu, I.. Umezawa, H., Matsuzaki, M., and Takeuchi. T., Science 191, 290 (1976).

6. Nagatsu, T.. Kato, T., Hashimoto, Y., Numata (Sudo). Y., Yamori, Y., and Okamoto. K., Experientin 34, 305 (1978).

7. Nagatsu, T., Kato, T., Numata (Sudo), Y., Ikuta, K.. Sane, IA., Nagatsu, I.. Umezawa, H., Matsuzaki, M., and Takeuchi. T., Japan. J. Pharmacol. 27, 531 (1977).

8. Nakamura, K.. and Nakamura, K.. Nature (London) 266, 265 (1977).

9. Hoeldtke, R., and Kaufman, S., Biochem. Pharmacol. 27, 249 (1978). 10. Yamori, Y., Matsumoto. M.. Yamabe. H., and Okamoto, K., Japan. Circ. J. 33, 399

(1969). 11. Hidaka, H., Nagatsu, T.. Takeya, K., Takeuchi, T., Suds, H., Kojiri, K., Matsuzaki,

M., and Umezawa, H., J. Antibiofics 22, 228 (1969).

12. Kato, T., Kuzuya, H., and Nagatsu, T., Biochem. Med. 10, 320 (1974). 13. Kato, T.. Wakui, Y., Nagatsu, T.. and Ohnishi. T.. Biochem. Phnrmacol. 27, 829

(1978). 14. Kvetnansky, R., McCarty, R., Thoa, N. B., Raymond Lake, C., and Kopin, I. J..

Amer. J. Physiol. 236, H457 (1979).

15. Stone, R. A., Gunnells, J. C., Robinson, R. R., Schanberg, S. M., and Kirshner, N.. Circ. Res. 34135, Suppl. I, I-47 (1974).

16. Lake, C. R., Ziegler, M. G., Coleman, M., and Kopin, 1. J., Circ. Res. 41, 865 (1977).