Biosci., Vol. 5, Number 3, September 1983, pp. 261 266. Printed in India. The effect of propranolol on rat brain catecholamine biosynthesis «Introduction MADHULIKA SRIVASTAVA and NARINDER Κ. KAPOOR Division of Biophysics, Central Drug Research Institute, Lucknow 226 001 MS received 19 March 1983; revised 14 July 1983 Abstract. The effect of propranolol on the levels of catecholamine in different parts of rat brain has been studied. The catecholamine contents of different regions were lowered by the drug. Dopamine β-hydroxylase activity was also reduced, both in vivo and in vitro. Propranolol is taken up by the brain tissue and the uptake is timedependent. These results suggests that reduction in brain catecholamine levels and dopamine β-hydroxylase activity may be one of the possible ways through which the drug manifests its clinical effects. Keywords. Catecholamine biosynthesis; propranolol; β-blocker; dopamine β- hydroxylase. Propranolol, one of the most popular β-adrenergic blocking agents, has been used effectively in certain clinical cases of hypertension (Buhler et al., 1972; Bravo et al., 1975), cardiac arrhythmias and angina pectoris (Livesley et al., 1973). It easily crosses the blood brain barrier and is concentrated in the central nervous system (CNS) (Masuoka and Hansson, 1967). It is a lipid soluble compound and high concentration of this compound accumulates in the brain of rabbits (Black et al., 1965), rats (Masuoka and Hansson, 1967) and monkeys (Hayes and Cooper, 1971). Catecholamines have been implicated in the pathophysiology of hypertension (de Champlain et al., 1967; Chalmers and Wurtman, 1971; Doba and Reis, 1974). In hypertensive patients, an increased plasma dopamine β-hydroxylase activity has been reported by several investigators (Schanberg et al., 1974; Nagatsu et al, 1976; Lamprecht, 1979). Biochemical effects of propranolol on catecholamine levels and biosynthetic enzyme activities are not well understood. Hence, it was of interest to study the effect of this drug on dopamine β-hydroxylase activity and catecholamine levels in the rat brain. Materials and methods Adult male rats of Charles Foster strain (100-150 g) were obtained from Central Drug Research Institute animal colony. They were maintained on a standard animal house diet. d1-propranolol-hcl was purchased from Sigma Chemical Company, St. Louis, Missouri, USA. For in vivo studies rats were divided into 4 groups of 50 rats each and treated as follows: Abbreviations used: CNS, Central nervous system; E, epinephrine; NE, norepinephrine; DA, dopamine. *C.D.R.I. Communication No. 3053 261
262 Srivastava and Kapoor Group I: Rats administered normal saline served as controls. Group II: Rats received d1-propranolol-hcl (20 mg/kg body wt.) intraperitoneally in normal saline for 3 days at 24 h intervals. Group III: Rats were treated as in group II for 5 days. Group IV: Rats were treated as in group II for 7 days. The rats were killed by cervical dislocation, 1 h after the last dose. The brains were quickly removed, kept on ice and the brain-stem, hypothalamus, and corpus striatum were dissected. Catecholamine estimation The levels of epinephrine (Ε), norepinephrine (NE) and dopamine (DA) of different parts of the brain were estimated as reported earlier according to the method of Malherbe (1971) with certain modifications. The oxidation of catecholamines was carried out by iodine solution as described by Sadavongvivad (1970). Dopamine β-hydroxylase assay The enzyme activity was assayed as described earlier (Srivastava and Kapoor, 1979, 1980) by a modification on the procedure of Nagatsu and Udenfriend (1972) using tyramine as substrate. The reaction product octopamine was cleaved into p-hydroxybenzaldehyde and its absorbance was read measured at 330 nm using a Beckman spectrophotometer. In vitro effect of propranolol on the enzyme activity in brain extracts (prepared in 0.32 Μ sucrose; 0.5 Μ phosphate buffer, ph 6.2 added in the reaction mixture) was studied using different concentrations of the drug. Protein was estimated according to Lowry et al. (1951). Propranolol estimation Tissue propranolol levels were estimated by a slightly modified procedure of Shand et al. (1970). Brain homogenate (16% wt/v) prepared in 0.1 Ν HCl was made alkaline by the addition 1 ml of 1 Ν NaOH (1 to 4 ml homogenate and extracted into 12 ml heptane containing 1.5% isoamylalcohol. After centrifugaion 10 ml of the organic phase was extracted into 1.5 ml of 0.1 Ν HCl and the fluorescence of the acid phase was measured in an Aminco-Bowman spectrophotofluorometer (excitation maximum 295 nm; emission maximum 360 nm, uncorrected). Results As shown in table 1 a progressive decrease in the levels of E, NE and DA in the brain stem was seen. While the Ε and NE levels remained almost unchanged even after 5 days of treatment with d1-propranolol. Dopamine β-hydroxylase activity was also inhibited progressively showing 23 % inhibition at 7 days. In hypothalamus, Ε and DA decreased progressively, (table 2) upto 5 days, while NE level declined gradually at all periods of treatment. Dopamine β-hydroxylase activity was maximally inhibited (29%) at 7 days. NE and DA contents of corpus striatum were altered similarly as in the brain-stem while the levels of Ε was not significantly affected (table 3). Dopamine β-hydroxylase activity showed marginal inhibition (19%) at 7 days. The in vitro treatment, addition of propranolol inhibited the enzyme maximally by (32%) at 1 mm (table 4). The decrease in catecholamine
Propranolol and brain catecholamine biosynthesis 263 Table 1. Effect of d1-propranolol treatment on catecholamine level and dopamine β-hydroxylase activity of rat brain-stem. *nmol octopamine formed per mg protein per min. All values are mean ± S.E.M. of 6 observations. Numbers in parenthesis indicate the number of brain-stems pooled. P<0.01. Table 2. Effect of d1-propranolol treatment on catecholamine level and dopamine β-hydroxylase activity of rat hypothalamus. *nmol octopamine formed per mg protein per min. All values are mean ± S.E.M. of 5 observations. Number of hypothalamus pooled is indicated in parenthesis. P>0.01. levels in various regions of the brain may be correlated with the decreased activity of the enzyme upon treatment with propranolol. Propranolol was also taken up by the brain. The drug levels were 0.93, 1.70 and 2.58 µg/g wet tissue at 3, 5 and 7 days of treatment, respectively. Discussion In the present investigations, it is observed that propranolol administration results in progressive decline in the levels of catecholamines in different parts of the brain viz. brain-stem, hypothalamus and corpus striatum. Similarly, dopamine β- hydroxylase activity was also decreased in these parts of the brain. In the brainstem, levels of Ε were decreased maximally at 5 days of propranolol administration. A continued decrease in the level of NE was observed in the three parts of the brain till the 7th day after the drug administration. Although the decrease in the
264 Srivastava and Kapoor Table 3. Effect of d1-propranolol treatment on catecholamine level and dopamine β-hydroxylase activity of rat corpus striatum. *nmol octopamine formed per mg protein per min. All values are mean ± S.E.M. of 6 observations. Number of corpus-striatum pooled is indicated in parenthesis. P< 0.01. Table 4. In vitro effect of d1-propranolol on rat brain dopamine β-hydroxylase activity. nmol octopamine formed per mg protein per min. Values are mean ± S.E.M. of 6 observations. P>0.001. synthesis of NE could be due to the decreased activity of dopamine β-hydroxylase, that other enzymes like monoamine oxidase and catecholomethyl transferase have a role in the regulation of catecholamine levels cannot be ruled out. The decrease in DA level could also be due to the involvement of tyrosine hydroxylase. Bhagat (1979) has explained the relationship between decreased adrenal tyrosome hydroxylase activity and lowering of blood pressure. It was proposed that a decrease in this enzyme activity resulted in lowered NE synthesis leading eventually to a decrease in the amount of amine available for release. A considerable amount of information has accumulated in the recent years to suggest that β-adrenoceptor blocking action of propranolol leads to hypotension and other therapeutic actions (Kelliher and Buckley, 1970; Srivastava et al.,1973; Sharma et al., 1979; Dollery et al., 1973; Planz and Planz, 1981; Virtanen et al., 1982; Bianchetti et al., 1982), however, some investigators have presented evidence to suggest that these actions are independent of the β-adrenergic blocking activity of the compound (Leszkovszky and Tardos, 1965; Murmann et al., 1966; Grana and Sossi, 1967). Several antihypertensive drugs have been reported to
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