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Metabolic responses of cardiac, skeletal muscle, and fat cells to catecholamines
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Frontiers in Catecholonine Research
7.
Nelson, P., Ruffner, W. and Nirenberg, M., Proc. Natl. Acad. Sci. US, 64, 1004-1010 (1969) . 8. Harris, A. J. and Dennis, M. J., Science, 167, 1253 (1970) . 9. Falck, B . and Owen, C., Acta Univ . Lund Section II. 7, 1-23 (1965) . 10 . Glenner, G. G., Burtner, J.J. and Brown, G. W., J. Histochem. Cytochem., S, 16731682 (1957) .
METABOLIC RESPONSES OF CARDIAC, SKELETAL MUSCLE, AND FAT CELLS TO CATECHOLAMINES Steven E. Mayer Department of Medicine, University of California, San Diego, La Jolla, California, 92037 U.S.A. One of the best understood functions of cyclic AMP is its role in mediating drug and hormone actions on body energy stores causing increased mobilization of triglycerides as well as of glycogen . Drugs or hormones alter the concentration of `active' cyclic AMP by stimulating or inhibiting receptors in close contact with adenylate cyclase, by altering the rate of cyclic AMP degredation by phosphodiesterase(s), and possibly by causing changes in cyclic AMP binding or sequestration. Cyclic AMP appears to increase the degree of phosphorylation of several proteins via activation of protein kinase. In adipose tissue for example, there are three enzymes that are regulated by covalent modification following phosphorylation : phosphorylase kinase (which in turn phosphorylates glycogen phosphorylase), glycogen synthase and hormone sensitive triglyceride lipase. The question arises: does the elevation of the concentration of `active' cyclic AMP by catecholamines automatically trigger all the cyclic AMP regulated biochemical systems or are there additional levels of control? Allosteric mechanisms controlling the activity of the two forms of muscle phosphorylase (a and b) are well known. In cardiac muscle modification of the ionic composition of the medium alters not only the formation of AMP in response to catecholamines but also the conversion of phosphorylase b to a. In the heart both phosphorylation of phosphorylase kinase and an increased intracellular concentration of Cat+ appear to be necessary for phosphorylase b to a conversion. In skeletal muscle increased Cat + alone appears to be sufficient without conversion of phosphorylase kinase to its phosphorylated, activated, form . In contrast, intracellular Ca 24' appears to play little or no role in the activation of hormone sensitive lipase and phosphorylase and inactivation of glycogen synthase by epinephrine in'fat cells. However, studies on the interaction between catecholamines and insulin in adipocytes suggest another level of control. A low concentration of insulin
Frontiers in Catecholanine Research
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(0 .5 nM) that antagonizes the effect of epinephrine (0 .5 kr.M) on adipocyte phosphorylase, glycogen synthase and lipase only slightly decreases the cAMP level in the cells. In adipocytes prepared from starved rats the antilipolytic action of insulin persists while there is no effect on phosphorylase or glycogen synthase . It may be of interest that consequent to fasting the latter two enzymes are dissociated from the glycogen particle to which they are normally bound . Thus the translation of the catecholamine-receptor interaction into a physiological response may involve changes in the concentration of cyclic AMP but also several other factors such as the permeability of the cell membranes to cations, the catalytic activity of critical enzymes and the association of enzymes and intracellular mediators to larger complexes. To what extent these different are directly influenced by the drug-receptor interaction and to what extent they reflect the physiological state of the tissue remains to be elucidated .
ACTIVITY OF BRAIN PHENOLSULFOTRANSFERASE FOR THE CATECHOLAMINES AND THEIR METABOLITES J. L. Meek and A. Foldes Laboratory of Preclinical Pharmacology, NIMH, Saint Elizabeths Hospital, Washington, D.C . 20032, U.S .A . Metabolites of norepinephrine in brain occur as sulfate conjugates . This conjugation is catalyzed by phenolsulfotransferase (PST) using phosphoadenosine phosphosulfate (PAPS) as a sulfate donor. Although the sulfate conjugation of phenols has been studied extensively in the periphery, little is known about PST in the brain. We have examined the activity in vitro of rat brain PST for phenols derived .from the catecholamines using a colorimetric method (J . Biol . Chem . 229 : 1081, 1957). With a 30,000 g supernatant preparation from whole brain, the compounds most readily sulfurylated were dopamine, methoxyhydroxyphenylglycol (MOPEG), dihydroxyphenylglycol (DOPEG), and the two corresponding neutral dopamine metabolites. In order to purify PST, and to study its distribution in brain we developed a more sensitive isotropic method . The phenols were incubated with enzyme and 35S-PAPS ;then barium hydroxide was used to precipitate the unreacted PAPS . The labelled sulfate esters remained in solution and were decanted into a scintillation vial for measurement. There were large differences in PST levels in the various regions of the brain. Assuming a specific activity of 1 for high speed supernatant from the. rat cerebellum, the specific activities of the other regions were hypothalamus, 13 ; hippocampus, midbrain, and striatum, 8-10 ; medulla-pons, cortex, and spinal
Frontiers in Catecholonine Research
7.
Nelson, P., Ruffner, W. and Nirenberg, M., Proc. Natl. Acad. Sci. US, 64, 1004-1010 (1969) . 8. Harris, A. J. and Dennis, M. J., Science, 167, 1253 (1970) . 9. Falck, B . and Owen, C., Acta Univ . Lund Section II. 7, 1-23 (1965) . 10 . Glenner, G. G., Burtner, J.J. and Brown, G. W., J. Histochem. Cytochem., S, 16731682 (1957) .
METABOLIC RESPONSES OF CARDIAC, SKELETAL MUSCLE, AND FAT CELLS TO CATECHOLAMINES Steven E. Mayer Department of Medicine, University of California, San Diego, La Jolla, California, 92037 U.S.A. One of the best understood functions of cyclic AMP is its role in mediating drug and hormone actions on body energy stores causing increased mobilization of triglycerides as well as of glycogen . Drugs or hormones alter the concentration of `active' cyclic AMP by stimulating or inhibiting receptors in close contact with adenylate cyclase, by altering the rate of cyclic AMP degredation by phosphodiesterase(s), and possibly by causing changes in cyclic AMP binding or sequestration. Cyclic AMP appears to increase the degree of phosphorylation of several proteins via activation of protein kinase. In adipose tissue for example, there are three enzymes that are regulated by covalent modification following phosphorylation : phosphorylase kinase (which in turn phosphorylates glycogen phosphorylase), glycogen synthase and hormone sensitive triglyceride lipase. The question arises: does the elevation of the concentration of `active' cyclic AMP by catecholamines automatically trigger all the cyclic AMP regulated biochemical systems or are there additional levels of control? Allosteric mechanisms controlling the activity of the two forms of muscle phosphorylase (a and b) are well known. In cardiac muscle modification of the ionic composition of the medium alters not only the formation of AMP in response to catecholamines but also the conversion of phosphorylase b to a. In the heart both phosphorylation of phosphorylase kinase and an increased intracellular concentration of Cat+ appear to be necessary for phosphorylase b to a conversion. In skeletal muscle increased Cat + alone appears to be sufficient without conversion of phosphorylase kinase to its phosphorylated, activated, form . In contrast, intracellular Ca 24' appears to play little or no role in the activation of hormone sensitive lipase and phosphorylase and inactivation of glycogen synthase by epinephrine in'fat cells. However, studies on the interaction between catecholamines and insulin in adipocytes suggest another level of control. A low concentration of insulin
Frontiers in Catecholanine Research
cxvi
(0 .5 nM) that antagonizes the effect of epinephrine (0 .5 kr.M) on adipocyte phosphorylase, glycogen synthase and lipase only slightly decreases the cAMP level in the cells. In adipocytes prepared from starved rats the antilipolytic action of insulin persists while there is no effect on phosphorylase or glycogen synthase . It may be of interest that consequent to fasting the latter two enzymes are dissociated from the glycogen particle to which they are normally bound . Thus the translation of the catecholamine-receptor interaction into a physiological response may involve changes in the concentration of cyclic AMP but also several other factors such as the permeability of the cell membranes to cations, the catalytic activity of critical enzymes and the association of enzymes and intracellular mediators to larger complexes. To what extent these different are directly influenced by the drug-receptor interaction and to what extent they reflect the physiological state of the tissue remains to be elucidated .
ACTIVITY OF BRAIN PHENOLSULFOTRANSFERASE FOR THE CATECHOLAMINES AND THEIR METABOLITES J. L. Meek and A. Foldes Laboratory of Preclinical Pharmacology, NIMH, Saint Elizabeths Hospital, Washington, D.C . 20032, U.S .A . Metabolites of norepinephrine in brain occur as sulfate conjugates . This conjugation is catalyzed by phenolsulfotransferase (PST) using phosphoadenosine phosphosulfate (PAPS) as a sulfate donor. Although the sulfate conjugation of phenols has been studied extensively in the periphery, little is known about PST in the brain. We have examined the activity in vitro of rat brain PST for phenols derived .from the catecholamines using a colorimetric method (J . Biol . Chem . 229 : 1081, 1957). With a 30,000 g supernatant preparation from whole brain, the compounds most readily sulfurylated were dopamine, methoxyhydroxyphenylglycol (MOPEG), dihydroxyphenylglycol (DOPEG), and the two corresponding neutral dopamine metabolites. In order to purify PST, and to study its distribution in brain we developed a more sensitive isotropic method . The phenols were incubated with enzyme and 35S-PAPS ;then barium hydroxide was used to precipitate the unreacted PAPS . The labelled sulfate esters remained in solution and were decanted into a scintillation vial for measurement. There were large differences in PST levels in the various regions of the brain. Assuming a specific activity of 1 for high speed supernatant from the. rat cerebellum, the specific activities of the other regions were hypothalamus, 13 ; hippocampus, midbrain, and striatum, 8-10 ; medulla-pons, cortex, and spinal