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Dopamine β-hydroxylase and the regulation of catecholamine biosynthesis
cxvii
Frontiers in Catecholamine Research
cord, 46 ; pituitary, choroid plexus 0.8 ; and pineal, 0 .25. PST activity in the caudal half of the spinal cord did not decrease after spinal section. Therefore, PST does not appear to be localized primarily in aminergic or other descending axons. For kinetic .studies, PST was partially purified by chromatography on DEAE sephadex . The pH optima of PST for phenol MOPEG, DOPEG, dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) ranged from 5.5 to 6.8 . At pH 6.4 the Km's for these substrates ranged from 10-40 AM . The Km for PAPS was 2.5 AM . The Km value for phenol varied sharply with pH and was 16 times greater at pH 5.0 than 6.4 . At pH 6 .4, MOPEG and DOPEG had the highest relative velocity followed by HVA, phenol and DOPAC. Dopamine and norepinephrine were also substrates for PST, but with pH optima of 9. We examined the formation in vivo of sulfate esters by injecting 35S-Na 2S04 intraventricularly . Eccleston (personal communication) has shown that injected sulfate can be "activated" and esterifies phenols injected simultaneously. We used this technique to prepare labelled sulfate esters of the biogenic amines and their metabolites as reference compounds. When 35S-sulfate was injected intraventricularly, conjugation of endogenous phenols occurred . We could detect four conjugates after separation by electrophoresis and two dimensional TLC. The compounds formed were apparently the sulfate esters of MOPEG, DOPEG, HVA, and DOPAC. We conclude that brain phenolsulfotransferase is widely but unevenly distributed in the brain. The enzyme can sulfurylate a variety of compounds related to the catecholamines . In addition to a role in the disposition of norepinephrine metabolites, brain PST may also conjugate DOPAC, HVA and possibly phenolic drugs which enter the brain. DOPAMINE ß-HYDROXYLASE AND THE REGULATION OF CATECHOLAMINE BIOSYNTHESIS Perry B. Moliroff f Department of Pharmacology, University of Colorado School of Medicine, Denver, Colorado 80220, U.S .A . It is probably true that the maximum rate of catecholamine synthesis is limited by the amount of tyrosine hydroxylase (TH) in the cell . On the other hand, the hypothesis that TH is the only regulated step in the biosynthetic This work was supported by a grant from the National Institute of Neurological Disease and Stroke (NS 10206) . t The author is an Established Investigator of the American Heart Association.
Frontiers in Catecholarnine Research
cxvii1
pathway of catecholamines may be an oversimplification for several reasons . In the first place, the substrates and enzymes of this pathway do not have free access to one another since dopamine 6-hydroxylase (DBH) is sequestered in chromaffin granules and in adrenergic vesicles . The lack of free access of the various substrates and enzymes to one another violates one of the important precepts of the rate limiting step concept which is mainly derived from work with biosynthetic pathways in bacteria . Secondly, the catecholamine biosynthetic pathway is branched with some of the dopamine formed from dopa being taken up into vesicles and converted into norepinephrine, and some being metabolized by the intraneuronal enzyme, monoamine oxidase. Approximately one-third of the catecholamine metabolites found in human urine are derived from dopamine (Ceasar, Ruthven, and Sandler, 1969). It thus seems fair to conclude that since competition exists between deamination and ßhydroxylation (or uptake into granules) anything which increases the capacity of the neuron to ß-hydroxylate dopamine, should increase the rate of catecholamine biosynthesis . Additional support for the conclusion that DBH as well as TH is subject to regulation comes from the fact that a number of different procedures that affect TH levels similarly affect DBH levels . These procedures include the administration of drugs like reserpine and 6-rydroxydopamine, various types of stress including immobilization and cold stress, and attack behaviour elicited by hypothalamic stimulation . In the experiments to be discussed, an enzymatic assay for DBH was used to study changes in enzymatic activity that occurred with pharmacologically induced alterations in sympathetic nervous system activity . This assay is based on the conversion of a 8-hydroxylated-product (either phenylethanolamine or octopamine) into its N-methyl derivative, in the presence of radiolabelled. S-adenosyl-methionine (Molinoff, Brimihoin, Weinsholboun and Axelrod, 1971). The activities of DBH (Molinoff, Brimijoin, Weinshilboun and Axelrod, 1970) and of TH (Mueller, Thoenen and Axelrod, 1969) were increased in adrenergic tissues of the rat by agents such as reserpine that are believed to cause a reflex increase in sympathetic tone. After reserpine treatment, DBH activity increased by approximately 50% within 24 hours in sympathetic ganglia. In the heart and, as has also been reported in the adrenal gland (Viveros, Arqueros, Connett and Kirshner, 1969), the increase in activity was preceded by a fall that occurred within a few hours of the injection. The activities of several enzymes not involved in catecholamine biosynthesis were not affected by reserpine. When efferent nerve impulse activity was blocked by either surgical decentralization or by ganglionic blocking agents, the effects of reserpine were
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Frontiers in Catecholmnine Research
abolished. Further evidence in support of the idea that nerve activity is involved in this phenomenon came from the fact that DBH activity is increased within 24 hours when sympathetic ganglia are cultured in the presence of increased potassium (Silberstein, Brimijoin, Molinoff, and Lemberger, 1972). The observed increase in DBH activity appears, from several types of study, to represent the synthesis of new enzyme and not the activation of previously existing enzyme . Thus, the Km for phenylethylamine (6 x 10 ,M) and the electrophoretic mobility of DBH on polyacrilamide gels were the same for the enzyme from reserpine treated rats as for that_ from control animals (Ross, Weinshilboun, Molinoff, Vesell and Axelrod, 1972). Furthermore, the increase in DBH activity was blocked either in vivo or in the organ culture system by the protein synthesis inhibitor, cycloheximide. Finally, the incorporation of 'H-leucine into adrenal DBH (measured by immunoadsorption) was increased 2-4 fold in reserpine pre-treated animals (Hartman, Molinoff and Udenfriend, 1970). It is thus concluded that long-tern changes in the level of activity in the sympathetic nervous system results in changes in the amount of DBH in the neuron . These changes, like those observed with TH, appear to be mediated transynaptically and to require protein synthesis. In the adrenergic neuron there are relatively large amounts of DBH compared to the amount of tyrosine hydroxylase. If the changes in DBH activity which have been observed are significantly affecting catecholamine biosynthesis, then some means of limiting the expression of this enzyme activity in vivo must exist. Two possible mechanisms for this limitation are apparent . In the first place, since dopamine ,(3-hydroxylase is sequestered in the storage granules, then dopamine must be transported into these granules before it can be ,8-hydroxylated. Little is known at the present time concerning possible restrictions to the normal transport of dopamine into these granules . However, it has been shown that inhibition of the uptake of dopamine into granules by reserpine results in a marked decrease, in the conversion of dopamine to norepinephrine (Rutledge and Weiner, 1967) . A second potential mechanism for regulating dopamine-,Shydroxylase activity in vivo involves the endogenous inhibitors of this enzyme. These compounds are found in many different tissues and have been partially purified from heart (Chubb, Preston and Austen, 1969) and from the adrenal gland (Duch and Kirshner, 1971) . It has not been thus far possible to define the chemical structure of the endogenous inhibitor of DBH from any tissue . It is very likely that the endogenous inhibitors of DBH are tissue specific . The inhibitory activity in the heart is different in several important respects from that which is found in the spleen or in the adrenal gland. Furthermore,
Frontiers in Catecholarnine Research
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there is apparently more than one inhibitor in each of several adrenergically innervated organs . Most of the inhibitory activity in the rat heart is lost after boiling a homogenate for 5 minutes . A few percent of the inhibitory activity is, however, stable for up to 20 minutes in a boiling water bath . One of the most striking aspects of the endogenous DBH inhibitors is the enormous amount of inhibitory activity which is found . For example, a splenic homogenate at a dilution of one part in four thousand is capable of inhibiting DBH, purified from the bovine adrenal medulla, by over 50%. The effect of the endogenous inhibitors of the spleen, heart, and other tissues can be completely reversed by the addition of an appropriate concentration of Cu ++ . Recent experiments have suggested that a significant percentage of the inhibitor from several adrenergically innervated organs is contained within the adrenergic nerve endings in these organs . Experiments are now being carried out to try to determine the role that these inhibitors are playing in the regulation of DBH activity and of catecholamine biosynthesis . REFERENCES 1. 2. 3. 4. 5.
Chubb, I. W., Preston, B. N. and Austin, L., Biochem . J ., 111, 245 (1969) . Ceasar, P. M., Ruthuen, C. R. J. and Sandler, M., Br. J. Pharnac ., 36, 70 (1969) . Hartman, B. K., Molinoff, P. B. and Udenfriend, S., The Phann ., 12, 470 (l970). Duch, D. S. and Kirshner, N., BBA., 236, 628 (1971) . Molinoff, P. B., Brimijoin, S., Weirshilboun, R. and Axelrod, J., Proc. Nat. Acad. Sci. U.S.A ., 66, 453, (1970) . 6. Moliroff, P. B., Weinshilboun, R. and Axelrod, J., J. Pharmac. Exp . Ther., (in press), (1971) . 7. Mueller, R. A., Thoenen, H. and Axelrod, J., J. Pharm. Exp . Ther., 169, 74 (l969) . 8. Ross, S. B., Weinshilboun, R., Moliroff, P. B., Vesell, E. S. and Axelrod, J., Mot . Pho-m ., 8, 50 (1972) . 9. Rutledge, C. O. and Weiner, N., J. Pharrn. Exp . Ther., 157, 290 (1967) . 10 . Silberstein, S., Brimijoin, S., Molirhoff, P. B. and Lemberger, L., J. Neurochem ., 19, 919 (1972) . 11 . Viveros, O. H., Arqueros, L., Cornett, R. J. and Kirshner, N., Molec. Phannac ., S, 69 (1969) . TECHNICAL STRATEGIES FOR THE STUDY OF CATECHOLAMINES IN MAN Dennis L. Murphy Laboratory of Clinical Science, NIMH Bethesda, Maryland, U.S.A . This presentation will briefly reviéw some methods for catecholamine investigation in man, focusing in particular on attempts to study catecholaminerelated processes at the human cellular level, with examples from current studies . Unlike animal preparations in which catecholamine metabolism can be
Frontiers in Catecholamine Research
cord, 46 ; pituitary, choroid plexus 0.8 ; and pineal, 0 .25. PST activity in the caudal half of the spinal cord did not decrease after spinal section. Therefore, PST does not appear to be localized primarily in aminergic or other descending axons. For kinetic .studies, PST was partially purified by chromatography on DEAE sephadex . The pH optima of PST for phenol MOPEG, DOPEG, dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) ranged from 5.5 to 6.8 . At pH 6.4 the Km's for these substrates ranged from 10-40 AM . The Km for PAPS was 2.5 AM . The Km value for phenol varied sharply with pH and was 16 times greater at pH 5.0 than 6.4 . At pH 6 .4, MOPEG and DOPEG had the highest relative velocity followed by HVA, phenol and DOPAC. Dopamine and norepinephrine were also substrates for PST, but with pH optima of 9. We examined the formation in vivo of sulfate esters by injecting 35S-Na 2S04 intraventricularly . Eccleston (personal communication) has shown that injected sulfate can be "activated" and esterifies phenols injected simultaneously. We used this technique to prepare labelled sulfate esters of the biogenic amines and their metabolites as reference compounds. When 35S-sulfate was injected intraventricularly, conjugation of endogenous phenols occurred . We could detect four conjugates after separation by electrophoresis and two dimensional TLC. The compounds formed were apparently the sulfate esters of MOPEG, DOPEG, HVA, and DOPAC. We conclude that brain phenolsulfotransferase is widely but unevenly distributed in the brain. The enzyme can sulfurylate a variety of compounds related to the catecholamines . In addition to a role in the disposition of norepinephrine metabolites, brain PST may also conjugate DOPAC, HVA and possibly phenolic drugs which enter the brain. DOPAMINE ß-HYDROXYLASE AND THE REGULATION OF CATECHOLAMINE BIOSYNTHESIS Perry B. Moliroff f Department of Pharmacology, University of Colorado School of Medicine, Denver, Colorado 80220, U.S .A . It is probably true that the maximum rate of catecholamine synthesis is limited by the amount of tyrosine hydroxylase (TH) in the cell . On the other hand, the hypothesis that TH is the only regulated step in the biosynthetic This work was supported by a grant from the National Institute of Neurological Disease and Stroke (NS 10206) . t The author is an Established Investigator of the American Heart Association.
Frontiers in Catecholarnine Research
cxvii1
pathway of catecholamines may be an oversimplification for several reasons . In the first place, the substrates and enzymes of this pathway do not have free access to one another since dopamine 6-hydroxylase (DBH) is sequestered in chromaffin granules and in adrenergic vesicles . The lack of free access of the various substrates and enzymes to one another violates one of the important precepts of the rate limiting step concept which is mainly derived from work with biosynthetic pathways in bacteria . Secondly, the catecholamine biosynthetic pathway is branched with some of the dopamine formed from dopa being taken up into vesicles and converted into norepinephrine, and some being metabolized by the intraneuronal enzyme, monoamine oxidase. Approximately one-third of the catecholamine metabolites found in human urine are derived from dopamine (Ceasar, Ruthven, and Sandler, 1969). It thus seems fair to conclude that since competition exists between deamination and ßhydroxylation (or uptake into granules) anything which increases the capacity of the neuron to ß-hydroxylate dopamine, should increase the rate of catecholamine biosynthesis . Additional support for the conclusion that DBH as well as TH is subject to regulation comes from the fact that a number of different procedures that affect TH levels similarly affect DBH levels . These procedures include the administration of drugs like reserpine and 6-rydroxydopamine, various types of stress including immobilization and cold stress, and attack behaviour elicited by hypothalamic stimulation . In the experiments to be discussed, an enzymatic assay for DBH was used to study changes in enzymatic activity that occurred with pharmacologically induced alterations in sympathetic nervous system activity . This assay is based on the conversion of a 8-hydroxylated-product (either phenylethanolamine or octopamine) into its N-methyl derivative, in the presence of radiolabelled. S-adenosyl-methionine (Molinoff, Brimihoin, Weinsholboun and Axelrod, 1971). The activities of DBH (Molinoff, Brimijoin, Weinshilboun and Axelrod, 1970) and of TH (Mueller, Thoenen and Axelrod, 1969) were increased in adrenergic tissues of the rat by agents such as reserpine that are believed to cause a reflex increase in sympathetic tone. After reserpine treatment, DBH activity increased by approximately 50% within 24 hours in sympathetic ganglia. In the heart and, as has also been reported in the adrenal gland (Viveros, Arqueros, Connett and Kirshner, 1969), the increase in activity was preceded by a fall that occurred within a few hours of the injection. The activities of several enzymes not involved in catecholamine biosynthesis were not affected by reserpine. When efferent nerve impulse activity was blocked by either surgical decentralization or by ganglionic blocking agents, the effects of reserpine were
cxiz
Frontiers in Catecholmnine Research
abolished. Further evidence in support of the idea that nerve activity is involved in this phenomenon came from the fact that DBH activity is increased within 24 hours when sympathetic ganglia are cultured in the presence of increased potassium (Silberstein, Brimijoin, Molinoff, and Lemberger, 1972). The observed increase in DBH activity appears, from several types of study, to represent the synthesis of new enzyme and not the activation of previously existing enzyme . Thus, the Km for phenylethylamine (6 x 10 ,M) and the electrophoretic mobility of DBH on polyacrilamide gels were the same for the enzyme from reserpine treated rats as for that_ from control animals (Ross, Weinshilboun, Molinoff, Vesell and Axelrod, 1972). Furthermore, the increase in DBH activity was blocked either in vivo or in the organ culture system by the protein synthesis inhibitor, cycloheximide. Finally, the incorporation of 'H-leucine into adrenal DBH (measured by immunoadsorption) was increased 2-4 fold in reserpine pre-treated animals (Hartman, Molinoff and Udenfriend, 1970). It is thus concluded that long-tern changes in the level of activity in the sympathetic nervous system results in changes in the amount of DBH in the neuron . These changes, like those observed with TH, appear to be mediated transynaptically and to require protein synthesis. In the adrenergic neuron there are relatively large amounts of DBH compared to the amount of tyrosine hydroxylase. If the changes in DBH activity which have been observed are significantly affecting catecholamine biosynthesis, then some means of limiting the expression of this enzyme activity in vivo must exist. Two possible mechanisms for this limitation are apparent . In the first place, since dopamine ,(3-hydroxylase is sequestered in the storage granules, then dopamine must be transported into these granules before it can be ,8-hydroxylated. Little is known at the present time concerning possible restrictions to the normal transport of dopamine into these granules . However, it has been shown that inhibition of the uptake of dopamine into granules by reserpine results in a marked decrease, in the conversion of dopamine to norepinephrine (Rutledge and Weiner, 1967) . A second potential mechanism for regulating dopamine-,Shydroxylase activity in vivo involves the endogenous inhibitors of this enzyme. These compounds are found in many different tissues and have been partially purified from heart (Chubb, Preston and Austen, 1969) and from the adrenal gland (Duch and Kirshner, 1971) . It has not been thus far possible to define the chemical structure of the endogenous inhibitor of DBH from any tissue . It is very likely that the endogenous inhibitors of DBH are tissue specific . The inhibitory activity in the heart is different in several important respects from that which is found in the spleen or in the adrenal gland. Furthermore,
Frontiers in Catecholarnine Research
cxx
there is apparently more than one inhibitor in each of several adrenergically innervated organs . Most of the inhibitory activity in the rat heart is lost after boiling a homogenate for 5 minutes . A few percent of the inhibitory activity is, however, stable for up to 20 minutes in a boiling water bath . One of the most striking aspects of the endogenous DBH inhibitors is the enormous amount of inhibitory activity which is found . For example, a splenic homogenate at a dilution of one part in four thousand is capable of inhibiting DBH, purified from the bovine adrenal medulla, by over 50%. The effect of the endogenous inhibitors of the spleen, heart, and other tissues can be completely reversed by the addition of an appropriate concentration of Cu ++ . Recent experiments have suggested that a significant percentage of the inhibitor from several adrenergically innervated organs is contained within the adrenergic nerve endings in these organs . Experiments are now being carried out to try to determine the role that these inhibitors are playing in the regulation of DBH activity and of catecholamine biosynthesis . REFERENCES 1. 2. 3. 4. 5.
Chubb, I. W., Preston, B. N. and Austin, L., Biochem . J ., 111, 245 (1969) . Ceasar, P. M., Ruthuen, C. R. J. and Sandler, M., Br. J. Pharnac ., 36, 70 (1969) . Hartman, B. K., Molinoff, P. B. and Udenfriend, S., The Phann ., 12, 470 (l970). Duch, D. S. and Kirshner, N., BBA., 236, 628 (1971) . Molinoff, P. B., Brimijoin, S., Weirshilboun, R. and Axelrod, J., Proc. Nat. Acad. Sci. U.S.A ., 66, 453, (1970) . 6. Moliroff, P. B., Weinshilboun, R. and Axelrod, J., J. Pharmac. Exp . Ther., (in press), (1971) . 7. Mueller, R. A., Thoenen, H. and Axelrod, J., J. Pharm. Exp . Ther., 169, 74 (l969) . 8. Ross, S. B., Weinshilboun, R., Moliroff, P. B., Vesell, E. S. and Axelrod, J., Mot . Pho-m ., 8, 50 (1972) . 9. Rutledge, C. O. and Weiner, N., J. Pharrn. Exp . Ther., 157, 290 (1967) . 10 . Silberstein, S., Brimijoin, S., Molirhoff, P. B. and Lemberger, L., J. Neurochem ., 19, 919 (1972) . 11 . Viveros, O. H., Arqueros, L., Cornett, R. J. and Kirshner, N., Molec. Phannac ., S, 69 (1969) . TECHNICAL STRATEGIES FOR THE STUDY OF CATECHOLAMINES IN MAN Dennis L. Murphy Laboratory of Clinical Science, NIMH Bethesda, Maryland, U.S.A . This presentation will briefly reviéw some methods for catecholamine investigation in man, focusing in particular on attempts to study catecholaminerelated processes at the human cellular level, with examples from current studies . Unlike animal preparations in which catecholamine metabolism can be