- Email: [email protected]
Effect of a dopamine β-hydroxylase inhibitor on tissue catecholamine levels in spontaneously hypertensive rats subjected to immobilization-cold stress
EFFECT OF A DOPAMINE /l-HYDROXYLASE INHIBITOR ON TISSUE CATECHOLAMINE LEVELS IN SPONTANEOUSLY HYPERTENSIVE RATS SUBJECTED TO IMMOBILIZATIONCOLD STRESS Y. ISHII, MASAKO HOMMA Research
Laboratories,
Nippon
Kayaku
(Accepted
and A. YOSHIKAWA
Co., Shimo, Kita-ku,
3 October
Tokyo
115, Japan
1974)
Summary-A single administration
of FD-008, a new dopamine /?-hydroxylase inhibitor, lowered norepinephrine levels in the heart, spleen and various brain regions of spontaneously hypertensive rats, which were subjected to immobilization-cold stress, but FD-008 did not affect norepinephrine in the adrenal medulla. The effect of FD-008 was much stronger in stressed than in non-stressed spontaneously hypertensive rats. Dopamine levels in the cortex and striatum of the brain were not significantly affected by FD-008in stressed spontaneously hypertensive rats.
Fusaric acid, 5butylpicolinic acid, which is produced by the fungus Fusarium oxysporum has been shown to be a potent inhibitor of dopamine P-hydroxylase in vitro and in vivo (SUDA, TAKEIJCHI, NAGATSU, MATSUZAKI, MATSUMOTO and UMEZAWA, 1969; NAGATSU, HIDAKA, KUZUYA, TAKEYA, UMEZAWA, TAKEUCHI and SUDA, 1970). During the in vitro screening study of fusaric acid derivatives, 5-(4’chloro)butylpicolinic acid (FD-008) was found to be a more potent dopamine /?-hydroxylase inhibitor than fusaric acid (UMEZAWA, TAKEUCHI, MIYANO, KOSHIGOE and HAMANO, 1973). NAGATSU,KATO and KUZUYA (1973) using these compounds found that the degree of in vivo inhibition was closely comparable to that of in vitro inhibition. ISHII,HOMMA,YOSHIKAWA and UMEZAWA (1975) observed previously that FD-008 decreased endogenous norepinephrine (NE) in the brain and heart of rats but not in the adrenal medulla. It has been reported that serum dopamine fi-hydroxylase activity of rats was elevated by immobilization stress (WEINSHILBOUM, KVETNANSKY, AXELROD and KOPIN, 1971) and that human plasma dopamine fl-hydroxylase activity was enhanced during cold pressor stress and exercise (WOOTENand CARDON, 1973). These reports prompted us to investigate various tissues of spontaneously hypertensive rats after a state of increased sympathetic nerve activity had been induced by acute stress. MATERIALS
AND
METHODS
Male spontaneously hypertensive rats aged 12-15 weeks were used. They were derived from animals originally obtained from the Council for the Spontaneously Hypertensive Rat (Kyoto) and bred in our laboratory. The calcium salt of FD-008 was suspended in 0.5% carboxymethylcellulose and given orally at a dose of 100 mg/kg. Immediately after administration of the drug, animals were subjected to stress according to the method described by TAKAGI and OKABE(1968). The animals were immobilized in wire-mesh restraint cages and immersed to the height of the xiphoid process in water maintained at 23°C. After 6 hours the rats were sacrificed by decapitation and the brain, heart, spleen and adrenal medulla were removed quickly. The cortex, hypothalamus and striatum of the brain were dissected on an iced glass plate. The tissues were frozen in liquid nitrogen and stored at -25°C before use. Norepinephrine and dopamine (DA) were determined using fluorometric procedures described by SHELLENBERGERand GORDON (1971) with a slight modification. Details of the procedures have been reported previously (ISHII rt al., 1975). 155
Y. ISHII. MASAKO HOMMA and A. YOSHIKAWA
156 Table
I. Effect of FD-008 (100 mg/kg. p.0.) on tissue DA and NE levels in spontaneously Concentration Tissue
Control
Amine
Brain cortex
DA NE DA NE NE NE
striatum hypothalamus Heart Spleen
0.79 0.36 13.0 2.1 0.80 0.60
f f k + + i
0.036 0,006 0.65 0.06 0.034 0,034
(pg/g) FD-00X
0.72 0.36 13.0 I.8 0.66 0.57
f + + + + f
0.016 0.005 1.0 0.09* 0.041* 0,046
hypertensive
rats
(“;,)
(91.1) (I 00.0) ( 100.0) (X5.7) (82.5) (950)
The tissues of two animals were pooled for each assay of a region of the brain. The means and standard errors of loassays are given in the table. Percentages of amine after FD-008 administration compared with the control are given. * Statistically significant (P = 0.05) compared with control values.
RESULTS
The effect of FD-008 on NE and DA levels in the heart, spleen and various regions of the brain of non-stressed spontaneously hypertensive rats, is shown in Table 1. Norepinephrine levels in the heart and hypothalamus of animals treated with FD-008 (100 mg/ kg, p.o.) were significantly lowered compared with the corresponding controls, while NE or DA levels in other parts were not significantly different from those of control animals.
0.5
0
1.0
1.5
2.0 PPlQ
EiF :.:.:.:.:.: .. 1 7
0
10
5
+
-El
~.:.:.:.:5.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.~.:.:
0
Adrenal
medulla
20 pk!
15
.
control Stress :+x5
15
10
5
Fo.W&5,res*
zopq&mu
..
NE :.> :.* *.v.&
: .*... :.:.:.:.:...=.*
. .
R 5:.:.x
central 51rns FP oQB.Stress
Fig. I. Effect of FD-008 (100 mg/kg, p.0.) on tissue DA and NE levels in spontaneously hypertcnsive rats. which were subjected to stress by immobilization in water at 23°C for 6 hr. A: tissues from two animals were pooled for the assays and I2 animals were used. B: NE in the heart, spleen and adrenal medulla was assayed in the same animals used in A. Statistically significant at P = 0.01 (*)and P = 0.001 (**) when compared with non-stressed rats and at P = 0.01(+) and P = 0.001 (+ +) when compared with stressed rats.
DBH inhibitor
and NE and DA levels in stressed rats
157
As reported previously (ISHII et al., 1975) FD-008 (100 mg/kg) did not affect NE content in the adrenal medulla. The effect of stress alone and stress plus FD-008 on NE and DA levels is presented in Figure 1. Norepinephrine levels in all the analyzed tissues of stressed rats were significantly lower than those of control rats. FD-008 caused further marked reductions of NE content in the hypothalamus, spleen and heart of the stressed rats but did not affect NE in the adrenal medulla. The effect of FD-008 on the reduction of NE in stressed rats was greater than in non-stressed rats. Dopamine levels in the cortex and striatum were increased slightly by stress and a slight further increase was induced by FD-008, though the differences were not statistically significant. DISCUSSION
As reported previously, FD-008 lowered endogeneous NE levels in the heart and brain of normotensive rats and spontaneously hypertensive rats (ISHII et al., 1975). NAGATSUet al. (1973) reported that rat serum dopamine fl-hydroxylase activity was reduced by about 70’%;after an oral administration of 3.5 mg/kg of FD-008 and that the activity of FD-008 was stronger than that of its parent compound, fusaric acid. These experiments were carried out in the daytime when rats seemed to be in a resting state. In the present investigation, NE levels in the heart, spleen, adrenal medulla and various regions of the brain were lowered after stress and FD-008 caused further reductions of NE in all of the analyzed tissues except the adrenal medulla. The effect of FD-008 was much greater in stressed rats than in non-stressed rats. Although increases in DA levels in the cortex and striatum induced by stress or by stress and FD-008 were not statistically significant, they may have been caused by the combination of stress and FD-008. It is known that dopamine P-hydroxylase is highly localized in the heart, splenic nerves and adrenal medulla. In the brain, dopamine P-hydroxylase activity is high in the hypothalamus and brainstem and low in the striatum. The fact that FD-008 caused marked reductions of NE not only in an increased sympathetic state but also in the regions where dopamine /Chydroxylase activity is high, confirms that the reductions of NE by FD-008 result from dopamine /?-hydroxylase inhibition. However, the physiological significance of the reduction of NE by dopamine j&hydroxylase inhibition during acute stress remains to be determined. REFERENCES ISHII. Y.. HOMMA, M.. YOSHIKAWA, A. and UMEZAWA, H. (1975). Pharmacological actions of FD-008, a new dopamint /Ghydroxylase inhibitor. 111 Effects on endogenous biogenic amine levels in rats. Ar~neimittel-Forsch. (In press). NAGATSLI.T.. HIDAKA. H.. KL~ZLYA, H.. TAKEYA, K.. UMEZAWA. H., TAKEUCHI. T. and SUDA, H. (1970). Inhibition of dopamine [Ghydroxylase by fusaric acid (5-butylpicolinic acid) in vitro and in uiro. Biochem. Pharmuc. 19: 35-44. NAGATSIJ.T.. KATO, T. and KUZUYA, H. (1973). New inhibitors of microbial origin for dopamine fi-hydroxylase. Frontirrs in Catrcholarninr Research, pp. 83-86. Pergamon Press, New York. SH~LL~W~RGER. M. K. and GORDON, J. H. (1971). A rapid, simplified procedure for simultaneous assay of norepinephrine, dopamine and 5-hydroxytryptamine from discrete brain areas. A~alyr. Biochrm. 39: 356372. SCIDA.H., TAKEWHI. T., NAGATSU, T., MA~SUZAKI, M., MATSUMOTO, I. and UMEZAWA. H. (1969). Inhibition of dopamine /Ghydroxylase by 5-alkylpicolinic acid and their hypotensive effects. Chrm. plzarm. Bull. 17: 23772380. TAKAGI, K. and OKABE. S. (1968). The effects of drugs on the production and recovery of the stress ulcer. Japan. J. Pl7cutnuc. 18: 9-18. UMWAWA. H., TAKEIXHI. T., MIYANO. K., KOSHIGOE. T. and HAMANO, H. (1973). Fusaric acid derivatives: the effect on dopamine fl-hydroxylase. J. Antihiot. (Tokyo). 26: 189. WIXNSHILBOLM, R. M.. KVETNANSKY, R.. AXELROD. J. and KOPIN, I. J. (1971). Elevation of serum dopamine /J’hydroxylase activity with forced immobilization. Nufure Nrw Biol. 230: 287-288. WOOTEN. G. F. and CARDON, P. V. (1973). Plasma dopamine [Ghydroxylase activity: elevation in man during cold pressor test and exercise. Arch. Nrurol. 28: 103-106.
Laboratories,
Nippon
Kayaku
(Accepted
and A. YOSHIKAWA
Co., Shimo, Kita-ku,
3 October
Tokyo
115, Japan
1974)
Summary-A single administration
of FD-008, a new dopamine /?-hydroxylase inhibitor, lowered norepinephrine levels in the heart, spleen and various brain regions of spontaneously hypertensive rats, which were subjected to immobilization-cold stress, but FD-008 did not affect norepinephrine in the adrenal medulla. The effect of FD-008 was much stronger in stressed than in non-stressed spontaneously hypertensive rats. Dopamine levels in the cortex and striatum of the brain were not significantly affected by FD-008in stressed spontaneously hypertensive rats.
Fusaric acid, 5butylpicolinic acid, which is produced by the fungus Fusarium oxysporum has been shown to be a potent inhibitor of dopamine P-hydroxylase in vitro and in vivo (SUDA, TAKEIJCHI, NAGATSU, MATSUZAKI, MATSUMOTO and UMEZAWA, 1969; NAGATSU, HIDAKA, KUZUYA, TAKEYA, UMEZAWA, TAKEUCHI and SUDA, 1970). During the in vitro screening study of fusaric acid derivatives, 5-(4’chloro)butylpicolinic acid (FD-008) was found to be a more potent dopamine /?-hydroxylase inhibitor than fusaric acid (UMEZAWA, TAKEUCHI, MIYANO, KOSHIGOE and HAMANO, 1973). NAGATSU,KATO and KUZUYA (1973) using these compounds found that the degree of in vivo inhibition was closely comparable to that of in vitro inhibition. ISHII,HOMMA,YOSHIKAWA and UMEZAWA (1975) observed previously that FD-008 decreased endogenous norepinephrine (NE) in the brain and heart of rats but not in the adrenal medulla. It has been reported that serum dopamine fi-hydroxylase activity of rats was elevated by immobilization stress (WEINSHILBOUM, KVETNANSKY, AXELROD and KOPIN, 1971) and that human plasma dopamine fl-hydroxylase activity was enhanced during cold pressor stress and exercise (WOOTENand CARDON, 1973). These reports prompted us to investigate various tissues of spontaneously hypertensive rats after a state of increased sympathetic nerve activity had been induced by acute stress. MATERIALS
AND
METHODS
Male spontaneously hypertensive rats aged 12-15 weeks were used. They were derived from animals originally obtained from the Council for the Spontaneously Hypertensive Rat (Kyoto) and bred in our laboratory. The calcium salt of FD-008 was suspended in 0.5% carboxymethylcellulose and given orally at a dose of 100 mg/kg. Immediately after administration of the drug, animals were subjected to stress according to the method described by TAKAGI and OKABE(1968). The animals were immobilized in wire-mesh restraint cages and immersed to the height of the xiphoid process in water maintained at 23°C. After 6 hours the rats were sacrificed by decapitation and the brain, heart, spleen and adrenal medulla were removed quickly. The cortex, hypothalamus and striatum of the brain were dissected on an iced glass plate. The tissues were frozen in liquid nitrogen and stored at -25°C before use. Norepinephrine and dopamine (DA) were determined using fluorometric procedures described by SHELLENBERGERand GORDON (1971) with a slight modification. Details of the procedures have been reported previously (ISHII rt al., 1975). 155
Y. ISHII. MASAKO HOMMA and A. YOSHIKAWA
156 Table
I. Effect of FD-008 (100 mg/kg. p.0.) on tissue DA and NE levels in spontaneously Concentration Tissue
Control
Amine
Brain cortex
DA NE DA NE NE NE
striatum hypothalamus Heart Spleen
0.79 0.36 13.0 2.1 0.80 0.60
f f k + + i
0.036 0,006 0.65 0.06 0.034 0,034
(pg/g) FD-00X
0.72 0.36 13.0 I.8 0.66 0.57
f + + + + f
0.016 0.005 1.0 0.09* 0.041* 0,046
hypertensive
rats
(“;,)
(91.1) (I 00.0) ( 100.0) (X5.7) (82.5) (950)
The tissues of two animals were pooled for each assay of a region of the brain. The means and standard errors of loassays are given in the table. Percentages of amine after FD-008 administration compared with the control are given. * Statistically significant (P = 0.05) compared with control values.
RESULTS
The effect of FD-008 on NE and DA levels in the heart, spleen and various regions of the brain of non-stressed spontaneously hypertensive rats, is shown in Table 1. Norepinephrine levels in the heart and hypothalamus of animals treated with FD-008 (100 mg/ kg, p.o.) were significantly lowered compared with the corresponding controls, while NE or DA levels in other parts were not significantly different from those of control animals.
0.5
0
1.0
1.5
2.0 PPlQ
EiF :.:.:.:.:.: .. 1 7
0
10
5
+
-El
~.:.:.:.:5.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.:.~.:.:
0
Adrenal
medulla
20 pk!
15
.
control Stress :+x5
15
10
5
Fo.W&5,res*
zopq&mu
..
NE :.> :.* *.v.&
: .*... :.:.:.:.:...=.*
. .
R 5:.:.x
central 51rns FP oQB.Stress
Fig. I. Effect of FD-008 (100 mg/kg, p.0.) on tissue DA and NE levels in spontaneously hypertcnsive rats. which were subjected to stress by immobilization in water at 23°C for 6 hr. A: tissues from two animals were pooled for the assays and I2 animals were used. B: NE in the heart, spleen and adrenal medulla was assayed in the same animals used in A. Statistically significant at P = 0.01 (*)and P = 0.001 (**) when compared with non-stressed rats and at P = 0.01(+) and P = 0.001 (+ +) when compared with stressed rats.
DBH inhibitor
and NE and DA levels in stressed rats
157
As reported previously (ISHII et al., 1975) FD-008 (100 mg/kg) did not affect NE content in the adrenal medulla. The effect of stress alone and stress plus FD-008 on NE and DA levels is presented in Figure 1. Norepinephrine levels in all the analyzed tissues of stressed rats were significantly lower than those of control rats. FD-008 caused further marked reductions of NE content in the hypothalamus, spleen and heart of the stressed rats but did not affect NE in the adrenal medulla. The effect of FD-008 on the reduction of NE in stressed rats was greater than in non-stressed rats. Dopamine levels in the cortex and striatum were increased slightly by stress and a slight further increase was induced by FD-008, though the differences were not statistically significant. DISCUSSION
As reported previously, FD-008 lowered endogeneous NE levels in the heart and brain of normotensive rats and spontaneously hypertensive rats (ISHII et al., 1975). NAGATSUet al. (1973) reported that rat serum dopamine fl-hydroxylase activity was reduced by about 70’%;after an oral administration of 3.5 mg/kg of FD-008 and that the activity of FD-008 was stronger than that of its parent compound, fusaric acid. These experiments were carried out in the daytime when rats seemed to be in a resting state. In the present investigation, NE levels in the heart, spleen, adrenal medulla and various regions of the brain were lowered after stress and FD-008 caused further reductions of NE in all of the analyzed tissues except the adrenal medulla. The effect of FD-008 was much greater in stressed rats than in non-stressed rats. Although increases in DA levels in the cortex and striatum induced by stress or by stress and FD-008 were not statistically significant, they may have been caused by the combination of stress and FD-008. It is known that dopamine P-hydroxylase is highly localized in the heart, splenic nerves and adrenal medulla. In the brain, dopamine P-hydroxylase activity is high in the hypothalamus and brainstem and low in the striatum. The fact that FD-008 caused marked reductions of NE not only in an increased sympathetic state but also in the regions where dopamine /Chydroxylase activity is high, confirms that the reductions of NE by FD-008 result from dopamine /?-hydroxylase inhibition. However, the physiological significance of the reduction of NE by dopamine j&hydroxylase inhibition during acute stress remains to be determined. REFERENCES ISHII. Y.. HOMMA, M.. YOSHIKAWA, A. and UMEZAWA, H. (1975). Pharmacological actions of FD-008, a new dopamint /Ghydroxylase inhibitor. 111 Effects on endogenous biogenic amine levels in rats. Ar~neimittel-Forsch. (In press). NAGATSLI.T.. HIDAKA. H.. KL~ZLYA, H.. TAKEYA, K.. UMEZAWA. H., TAKEUCHI. T. and SUDA, H. (1970). Inhibition of dopamine [Ghydroxylase by fusaric acid (5-butylpicolinic acid) in vitro and in uiro. Biochem. Pharmuc. 19: 35-44. NAGATSIJ.T.. KATO, T. and KUZUYA, H. (1973). New inhibitors of microbial origin for dopamine fi-hydroxylase. Frontirrs in Catrcholarninr Research, pp. 83-86. Pergamon Press, New York. SH~LL~W~RGER. M. K. and GORDON, J. H. (1971). A rapid, simplified procedure for simultaneous assay of norepinephrine, dopamine and 5-hydroxytryptamine from discrete brain areas. A~alyr. Biochrm. 39: 356372. SCIDA.H., TAKEWHI. T., NAGATSU, T., MA~SUZAKI, M., MATSUMOTO, I. and UMEZAWA. H. (1969). Inhibition of dopamine /Ghydroxylase by 5-alkylpicolinic acid and their hypotensive effects. Chrm. plzarm. Bull. 17: 23772380. TAKAGI, K. and OKABE. S. (1968). The effects of drugs on the production and recovery of the stress ulcer. Japan. J. Pl7cutnuc. 18: 9-18. UMWAWA. H., TAKEIXHI. T., MIYANO. K., KOSHIGOE. T. and HAMANO, H. (1973). Fusaric acid derivatives: the effect on dopamine fl-hydroxylase. J. Antihiot. (Tokyo). 26: 189. WIXNSHILBOLM, R. M.. KVETNANSKY, R.. AXELROD. J. and KOPIN, I. J. (1971). Elevation of serum dopamine /J’hydroxylase activity with forced immobilization. Nufure Nrw Biol. 230: 287-288. WOOTEN. G. F. and CARDON, P. V. (1973). Plasma dopamine [Ghydroxylase activity: elevation in man during cold pressor test and exercise. Arch. Nrurol. 28: 103-106.