Journul of Xeirrochemrstr), Raven Press. Ltd., New York 0 1992 international Society for Neurochemistry
Role of the Central Adrenergic System in the Regulation of Prostaglandin Biosynthesis in Rat Brain J. Weidenfeld, K. Kahbha, A. Reches, and *E. Shohami Department of Neurology, Hadassah University Hospital and Laboratory of Endocrinology, Bikur Holim Hospital, and *Department of Pharmacology, Hebrew University-HadassahMedical School, Jerusalem, Israel
cholaminergic neurotoxin 6-hydroxydopamine or vehicle. Other groups of rats were injected intrapentoneally with the tyrosine hydroxylase inhibitor, a-methyl-ptyrosine, or with the inhibitor of dopamine-P-hydroxylase, FLA-63. All these drugs produced a significant depletion of norepinephrine (NE) content in the cortex and hypothalamus. The rats that had lower levels of NE exhibited reduced capacity to synthesize PGEzbut not thromboxane Bzand 6-keto-PGEl, in the cortex
and hypothalamus. However, induced production of PG, stimulated by the bacterial endotoxin lipopolysaccharide (LPS), remained unchanged, namely, a similar (2- to 2.5fold) increase of PG synthesis was noted in control and in NE-depleted rats. We suggest that the regulation of PG synthesis under basal condition requires intact adrenergic input, whereas LPS-induced production of PG is independent of the adrenergic innervation. Key Words: ProstaglandinsCatecholamines-6-Hydroxydopamine-Brain. Weidenfeld J. et al. Role of the central adrenergic system in the regulation of prostaglandin biosynthesis in rat brain. J. Neurochem. 58, 694-699 ( 1992).
It is well established that brain tissue has the capacity to synthesize and release prostaglandins (PGs) (Wolfe, 1982; Chiu and Richardson, 1985). Although the precise functional roles of brain PG in normal and pathological conditions are not known, various stimuli such as trauma (Ellis et al., 1984; Shohami et al., 1987), ischemia (Gaudet et al., 1980; Dempsey et al., 1986), epileptic seizures (Bazan et al., 1986), and hypoglycemia (Shohami et al., 1985) are associated with an increase in brain PG production. Little is known about the endogenous factors that regulate brain PG synthesis, but it is likely that both hormones and neurotransmitters are involved in this mechanism. We have recently provided evidence for the involvement of glucocorticoids in modulating PG synthesis (Weidenfeld et al., 1988, 1989). Several studies have used P-adrenergic blockers to examine the role of catecholamines (CA) in regulating PG release in peripheral tissue following administration of propranolol. Fujimura et al. (1 987) have demonstrated that frusemide-stimulated urinary excretion of PGE2 was reduced by propranolol. Propranolol was also shown to
inhibit the activation of phospholipase A2 by various stimuli in platelets (Vanderhoek and Feinstein, 1979) and the deacylation of phospholipids in mouse lymphoma cells (Levine and Moskowitz, 1979). The antagonistic effect of propranolol was implied in the inhibition of the accumulation of free fatty acids, mainly arachidonic acid (AA), in ischemic dog heart (Miura et al., 1988). PG12 synthesis by rat aorta was investigated by Jeremy et al. (1985); they concluded that it is mediated by an a-adrenoceptor. A role for CA in regulating PG synthesis in neural tissue was also demonstrated (Wolfe et al., 1976). Noradrenaline (NE), dopamine, and adrenaline when added to rat brain synaptosomes significantly stimulated the generation of PGE2 (Hillier et al., 1976). These findings led us to investigate the role of endogenous CA in regulating brain PG levels under basal conditions and following the activation of PG synthesis. We examined the effect of depleting endogenous CA content by (1) intracerebral injection of 6-hydroxydopamine (6-OHDA), a specific CA neurotoxin; (2) inhibition of dopamine-P-hydroxylase (DBH) activity
Received September 26, 1990; revised manuscript received June 8. 1991; accepted July 8. 1991. Address correspondence and reprint requests to Dr. J. Weidenfeld at Department of Neurology, Hadassah University Hospital, Ein Kerern, Jerusalem, Israel. Abbreviations used: AA, arachidonic acid; ANOVA, analysis of
variance: CA, catecholamine; DBH, dopamine P-hydroxylase; FLA63, [bis(4-methyl-i-homopiperazinylthiocarbonyl)]disulfide; LPS, lipopolysaccharide; a - M P T , a-methyl-p-tyrosine; NE, norepinephrine; 6-OHDA, 6-hydroxydopamine; PG, prostaglandin; TH, tyrosine hydroxylase; TXBz, thromboxane Bz ; VNAB, ventral noradrenergic bundle.
Abstract: The role of endogenous catecholamines in the regulation of brain prostaglandin (PG) synthesis was studied in the rat. Male rats were injected in the brain lateral ventricle or in the ventral noradrenergic bundle with either the cate-
6 94
EFFECT OF CATECHOLAMINES ON BRAIN PROSTAGLANDINS by intraperitoneal administration of [bis(Cmethyl-1homopiperazinylthiocarbonyl)]disulfide(FLA-63); and (3) inhibition of tyrosine hydroxylase (TH) by amethyl-p-tyrosine (a-MPT). PG synthesis and release were measured ex vivo in various brain regions under basal physiological conditions and following the in vivo administration of the bacterial endotoxin lipopolysaccharide (LPS), which is a potent activator of PG production (Lewisand Austen, 1984; Cook et al., 1987). Some of the results reported here have appeared in an abstract (Shohami and Weidenfeld, 1991).
MATERIALS AND METHODS Animals The study was camed out on adult male rats of the Hebrew University strain, weighing approximately 200 g. They were housed in the animal room five or six per cage under artificial illumination between 0600 and 1800 h, at 22-23"C, and were given chow and water ad libitum.
Experimental protocol Rats were anesthetized with sodium pentobarbital (35 mg/ kg) and injected in the lateral ventricle with 300 pg/kg body wt of 6-OHDA (Sigma, St. Louis, MO, U.S.A.) in 20 p1 of saline containing 0.2% ascorbic acid to avoid oxidation or with vehicle. Stereotaxic coordinates with bregma as the reference point were: lateral 1.4, anterior 0.0, and height 4.2 mm. In another group of rats, 6-OHDA was injected directly into the ventral noradrenergic bundle (VNAB) in a dose of 5 pg in 1 ~1of saline. The coordinates for this injection were: lateral k1.6 mm, posterior 4.6 mm, and height 9 mm (bilateral). Six days later, rats were killed to evaluate either their CA content or PG synthetic capacity. Five days after 6-OHDA or vehicle administration, other groups of rats were injected intraperitoneally with 500 pg/kg body wt of LPS in 1 ml of saline or with saline alone. Twenty-four hours later, the rats were decapitated and the production of PG in both groups was determined. To investigate the effect of inhibition of CA production at different steps of their biosynthesis, two other drugs were used (1) a-MPT (Sigma), a specific inhibitor of the enzyme TH, was injected intraperitoneally at a dose of 250 mg/kg body wt in saline; 20 h later the rats were killed. Brain tissue was taken from these rats to measure CA content or PG rate of synthesis: (2) FLA-63 (Regis, Morton Grove, IL, U.S.A.), an inhibitor of DBH, was given intraperitoneally at a dose of 50 mg/kg body wt in saline and 4 h later the rats were killed, for determination of NE content or PG rate of synthesis. In another group of rats, the drug was given 20 h after LPS administration.
Determination of CA content Tissue was sonicated in perchloric acid for 10 s, and aliquots of the homogenate were taken for protein determination (Lowry et al., 1951) and centrifuged at 4°C for 5 min at 13,000 rpm. The supernatant was taken for CA analysis by HPLC with an electrochemical detector as previously described by Felice et al. (1978).
Determination of PG synthesis Following decapitation, brains were rapidly removed, placed on an ice-cold petri dish, and the following brain
695
structures were dissected: hypothalamus, dorsal hippocampus, and a slice (- 10 mg) of the frontal cortex. The tissues were each placed in a tube containing I ml of ice-cold oxygenated (95%02/5% COz) Krebs-Ringer buffer, pH 7.4, as described by Shohami and Gross (1985). The supernatant was immediately decanted and replaced by 1 ml of fresh buffer and, after further oxygenation, the tubes were closed tightly and incubated at 37°C in a shaking water bath for 1 h. The supernatants were then collected and kept at -70°C until assayed for PG. The tissue was homogenized in 1 ml of water, and protein determined on an aliquot of the homogenate according to Lowry et al. (1 95 1). For the determination of the amount of PGE2, thromboxane B2 (TXB2), or 6-ketoPGE,,, aliquots of the supernatant were taken for radioimmunoassay. The assay was camed out in 0.01 M phosphate buffer, pH 7.4, using specific antibodies with cross-reactivity of < 1% with other PGs. Anti-PGE2was purchased from BioYeda, Rehovot, Israel. Anti-TXB2 and 6-keto-PGE,, were obtained from Dr. L. Levine, Brandeis University, Waltham, MA, U.S.A. Tritiated PGE2, TXB2,and 6-keto-PGE,, (100 Ci/mmoI) were purchased from NEN, Boston, MA, U.S.A. Bound and free fractions were separated by dextran-coated charcoal (Norit, Sigma), and radioactivity was counted in a liquid scintillation counter.
Statistical analysis Data are represented as means k SEM. Significance of the differences between the mean was calculated using the Student's t test, analysis of variance (ANOVA), and Dunnett's test, as indicated in the appropriate tables.
RESULTS
To verify the effectiveness of various phannacological treatments in reducing brain CA, the levels of NE were measured in the cortex, hypothalamus, and hippocampus. Table 1 depicts NE levels in these structures after 6-OHDA, a-MPT, or FLA-63 as compared with those in control vehicle-treated rats. As expected, intracerebroventricular injection of 6-OHDA significantly reduced NE content by 80 and 72% in the cortex and hypothalamus, respectively. Injection of 6-OHDA TABLE 1. Efects of 6-OHDA, a-MPT, and FLA-63 on the content of NE in bruin tissue (ng/mg ofprotein) Treatment
Frontal cortex
Vehicle (i.c.v.) 6-OHDA (i.c.v.) Vehicle (VNAB) 6-OHDA (VNAB) Vehicle (i.p.) a-MPT (i.p.) FLA-63 (i.p.)
0.5 0.82 k 0.2" 4.3 f 0.4 1.6 f 0.5" 4.6 k 0.4 0.3 2 0.02" 0.4 2 0.02"
Hypothalamus Hippocampus ~~
4.1
_+
32 f 8 9.1 k 3" 35 f I 4.1 f 2" 31 2 6 2.1 ? 0.2" 2.5 t 0.3"
~
5.2 ? 6
Role of the Central Adrenergic System in the Regulation of Prostaglandin Biosynthesis in Rat Brain J. Weidenfeld, K. Kahbha, A. Reches, and *E. Shohami Department of Neurology, Hadassah University Hospital and Laboratory of Endocrinology, Bikur Holim Hospital, and *Department of Pharmacology, Hebrew University-HadassahMedical School, Jerusalem, Israel
cholaminergic neurotoxin 6-hydroxydopamine or vehicle. Other groups of rats were injected intrapentoneally with the tyrosine hydroxylase inhibitor, a-methyl-ptyrosine, or with the inhibitor of dopamine-P-hydroxylase, FLA-63. All these drugs produced a significant depletion of norepinephrine (NE) content in the cortex and hypothalamus. The rats that had lower levels of NE exhibited reduced capacity to synthesize PGEzbut not thromboxane Bzand 6-keto-PGEl, in the cortex
and hypothalamus. However, induced production of PG, stimulated by the bacterial endotoxin lipopolysaccharide (LPS), remained unchanged, namely, a similar (2- to 2.5fold) increase of PG synthesis was noted in control and in NE-depleted rats. We suggest that the regulation of PG synthesis under basal condition requires intact adrenergic input, whereas LPS-induced production of PG is independent of the adrenergic innervation. Key Words: ProstaglandinsCatecholamines-6-Hydroxydopamine-Brain. Weidenfeld J. et al. Role of the central adrenergic system in the regulation of prostaglandin biosynthesis in rat brain. J. Neurochem. 58, 694-699 ( 1992).
It is well established that brain tissue has the capacity to synthesize and release prostaglandins (PGs) (Wolfe, 1982; Chiu and Richardson, 1985). Although the precise functional roles of brain PG in normal and pathological conditions are not known, various stimuli such as trauma (Ellis et al., 1984; Shohami et al., 1987), ischemia (Gaudet et al., 1980; Dempsey et al., 1986), epileptic seizures (Bazan et al., 1986), and hypoglycemia (Shohami et al., 1985) are associated with an increase in brain PG production. Little is known about the endogenous factors that regulate brain PG synthesis, but it is likely that both hormones and neurotransmitters are involved in this mechanism. We have recently provided evidence for the involvement of glucocorticoids in modulating PG synthesis (Weidenfeld et al., 1988, 1989). Several studies have used P-adrenergic blockers to examine the role of catecholamines (CA) in regulating PG release in peripheral tissue following administration of propranolol. Fujimura et al. (1 987) have demonstrated that frusemide-stimulated urinary excretion of PGE2 was reduced by propranolol. Propranolol was also shown to
inhibit the activation of phospholipase A2 by various stimuli in platelets (Vanderhoek and Feinstein, 1979) and the deacylation of phospholipids in mouse lymphoma cells (Levine and Moskowitz, 1979). The antagonistic effect of propranolol was implied in the inhibition of the accumulation of free fatty acids, mainly arachidonic acid (AA), in ischemic dog heart (Miura et al., 1988). PG12 synthesis by rat aorta was investigated by Jeremy et al. (1985); they concluded that it is mediated by an a-adrenoceptor. A role for CA in regulating PG synthesis in neural tissue was also demonstrated (Wolfe et al., 1976). Noradrenaline (NE), dopamine, and adrenaline when added to rat brain synaptosomes significantly stimulated the generation of PGE2 (Hillier et al., 1976). These findings led us to investigate the role of endogenous CA in regulating brain PG levels under basal conditions and following the activation of PG synthesis. We examined the effect of depleting endogenous CA content by (1) intracerebral injection of 6-hydroxydopamine (6-OHDA), a specific CA neurotoxin; (2) inhibition of dopamine-P-hydroxylase (DBH) activity
Received September 26, 1990; revised manuscript received June 8. 1991; accepted July 8. 1991. Address correspondence and reprint requests to Dr. J. Weidenfeld at Department of Neurology, Hadassah University Hospital, Ein Kerern, Jerusalem, Israel. Abbreviations used: AA, arachidonic acid; ANOVA, analysis of
variance: CA, catecholamine; DBH, dopamine P-hydroxylase; FLA63, [bis(4-methyl-i-homopiperazinylthiocarbonyl)]disulfide; LPS, lipopolysaccharide; a - M P T , a-methyl-p-tyrosine; NE, norepinephrine; 6-OHDA, 6-hydroxydopamine; PG, prostaglandin; TH, tyrosine hydroxylase; TXBz, thromboxane Bz ; VNAB, ventral noradrenergic bundle.
Abstract: The role of endogenous catecholamines in the regulation of brain prostaglandin (PG) synthesis was studied in the rat. Male rats were injected in the brain lateral ventricle or in the ventral noradrenergic bundle with either the cate-
6 94
EFFECT OF CATECHOLAMINES ON BRAIN PROSTAGLANDINS by intraperitoneal administration of [bis(Cmethyl-1homopiperazinylthiocarbonyl)]disulfide(FLA-63); and (3) inhibition of tyrosine hydroxylase (TH) by amethyl-p-tyrosine (a-MPT). PG synthesis and release were measured ex vivo in various brain regions under basal physiological conditions and following the in vivo administration of the bacterial endotoxin lipopolysaccharide (LPS), which is a potent activator of PG production (Lewisand Austen, 1984; Cook et al., 1987). Some of the results reported here have appeared in an abstract (Shohami and Weidenfeld, 1991).
MATERIALS AND METHODS Animals The study was camed out on adult male rats of the Hebrew University strain, weighing approximately 200 g. They were housed in the animal room five or six per cage under artificial illumination between 0600 and 1800 h, at 22-23"C, and were given chow and water ad libitum.
Experimental protocol Rats were anesthetized with sodium pentobarbital (35 mg/ kg) and injected in the lateral ventricle with 300 pg/kg body wt of 6-OHDA (Sigma, St. Louis, MO, U.S.A.) in 20 p1 of saline containing 0.2% ascorbic acid to avoid oxidation or with vehicle. Stereotaxic coordinates with bregma as the reference point were: lateral 1.4, anterior 0.0, and height 4.2 mm. In another group of rats, 6-OHDA was injected directly into the ventral noradrenergic bundle (VNAB) in a dose of 5 pg in 1 ~1of saline. The coordinates for this injection were: lateral k1.6 mm, posterior 4.6 mm, and height 9 mm (bilateral). Six days later, rats were killed to evaluate either their CA content or PG synthetic capacity. Five days after 6-OHDA or vehicle administration, other groups of rats were injected intraperitoneally with 500 pg/kg body wt of LPS in 1 ml of saline or with saline alone. Twenty-four hours later, the rats were decapitated and the production of PG in both groups was determined. To investigate the effect of inhibition of CA production at different steps of their biosynthesis, two other drugs were used (1) a-MPT (Sigma), a specific inhibitor of the enzyme TH, was injected intraperitoneally at a dose of 250 mg/kg body wt in saline; 20 h later the rats were killed. Brain tissue was taken from these rats to measure CA content or PG rate of synthesis: (2) FLA-63 (Regis, Morton Grove, IL, U.S.A.), an inhibitor of DBH, was given intraperitoneally at a dose of 50 mg/kg body wt in saline and 4 h later the rats were killed, for determination of NE content or PG rate of synthesis. In another group of rats, the drug was given 20 h after LPS administration.
Determination of CA content Tissue was sonicated in perchloric acid for 10 s, and aliquots of the homogenate were taken for protein determination (Lowry et al., 1951) and centrifuged at 4°C for 5 min at 13,000 rpm. The supernatant was taken for CA analysis by HPLC with an electrochemical detector as previously described by Felice et al. (1978).
Determination of PG synthesis Following decapitation, brains were rapidly removed, placed on an ice-cold petri dish, and the following brain
695
structures were dissected: hypothalamus, dorsal hippocampus, and a slice (- 10 mg) of the frontal cortex. The tissues were each placed in a tube containing I ml of ice-cold oxygenated (95%02/5% COz) Krebs-Ringer buffer, pH 7.4, as described by Shohami and Gross (1985). The supernatant was immediately decanted and replaced by 1 ml of fresh buffer and, after further oxygenation, the tubes were closed tightly and incubated at 37°C in a shaking water bath for 1 h. The supernatants were then collected and kept at -70°C until assayed for PG. The tissue was homogenized in 1 ml of water, and protein determined on an aliquot of the homogenate according to Lowry et al. (1 95 1). For the determination of the amount of PGE2, thromboxane B2 (TXB2), or 6-ketoPGE,,, aliquots of the supernatant were taken for radioimmunoassay. The assay was camed out in 0.01 M phosphate buffer, pH 7.4, using specific antibodies with cross-reactivity of < 1% with other PGs. Anti-PGE2was purchased from BioYeda, Rehovot, Israel. Anti-TXB2 and 6-keto-PGE,, were obtained from Dr. L. Levine, Brandeis University, Waltham, MA, U.S.A. Tritiated PGE2, TXB2,and 6-keto-PGE,, (100 Ci/mmoI) were purchased from NEN, Boston, MA, U.S.A. Bound and free fractions were separated by dextran-coated charcoal (Norit, Sigma), and radioactivity was counted in a liquid scintillation counter.
Statistical analysis Data are represented as means k SEM. Significance of the differences between the mean was calculated using the Student's t test, analysis of variance (ANOVA), and Dunnett's test, as indicated in the appropriate tables.
RESULTS
To verify the effectiveness of various phannacological treatments in reducing brain CA, the levels of NE were measured in the cortex, hypothalamus, and hippocampus. Table 1 depicts NE levels in these structures after 6-OHDA, a-MPT, or FLA-63 as compared with those in control vehicle-treated rats. As expected, intracerebroventricular injection of 6-OHDA significantly reduced NE content by 80 and 72% in the cortex and hypothalamus, respectively. Injection of 6-OHDA TABLE 1. Efects of 6-OHDA, a-MPT, and FLA-63 on the content of NE in bruin tissue (ng/mg ofprotein) Treatment
Frontal cortex
Vehicle (i.c.v.) 6-OHDA (i.c.v.) Vehicle (VNAB) 6-OHDA (VNAB) Vehicle (i.p.) a-MPT (i.p.) FLA-63 (i.p.)
0.5 0.82 k 0.2" 4.3 f 0.4 1.6 f 0.5" 4.6 k 0.4 0.3 2 0.02" 0.4 2 0.02"
Hypothalamus Hippocampus ~~
4.1
_+
32 f 8 9.1 k 3" 35 f I 4.1 f 2" 31 2 6 2.1 ? 0.2" 2.5 t 0.3"
~
5.2 ? 6
