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Recovery of catecholamine stores following secretion by the adrenal medulla
lxuiv
Fnontiera in Catecholamine Research
Another characteristic of tyrosine hydroxylase that is markedly dependent on the nature of the cofactor uséd is its amino acid specificity . Although the ability of the enzyme to catalyze the hydroxylation of phenylalanine in the presence of DMPH 4 is poor, .phenylalaniae is an excellent substrate for the enzyme in the presence of tetrahydrobiopterin . These findings suggest that both in adrenal medulla and in the nervous system, phenylalanine is an important precursor of catecholamines .
RECOVERY OF CATECHOLAMINE STORES FOLLOWING SECRETION BY THE ADRENAL MEDULLA N. Kirshner sad T. Slotkin Department of Biochemistry and Department of Physiology and Pharmacol., Duke Univ. Med. Ctr., Durham, N. C. 27710, U.S .A . It is generally accepted that secretion from the adrenal medulla occurs by exocytosis. Evidence from several laboratories has shown that the contents of the adrenal storage vesicle which include catecholamines, adenine nucleotides and proteins, among which is the soluble fraction of dopamine-~i- hydroxylase, appear in perfusates of the stimulated gland in the same relative amounts as found in the soluble fraction upon lysis of the storage vesicles in distilled water. During secretion the membranes of the vesicles remain within the cell as indicated by complete retention of that fraction of dopamine-ß-hydroxylase which is tightly bound to the storage vesicle membrane . During secretion there is no release of typical cytoplasmic enzymes such as lactic dehydrogenase, tyrosine hydroxylase or phenylethanolamine-N-methyltraasferase . More recently we have been concerned with the sequence of events during . recovery of the catecholamine stores . Fundamental to this issue is the state of the catecholamine storage vesicles immediately following secretion . (1) Do they secrete their entire content or only a portion in response to a stimulus? (2) Can vesicles which have secreted their amines retain the ability to takeup end store amines? (3) Are the ghosts of old vesicles reused or does the recovery process involve de r~ao synthesis of new vesicles? (4) If so, are protein components or fragments of the old membranes re-used or are newly synthesized macromolecular components required?
Frontiers in Catecholamine Research
lxxxv
(5) What other factors may be involved in the recovery process? Answers to some of these questions have been obtained . Studies of secretion from rabbit and rat adrenal g~ends show that secretion occurs by an "allrar-none ° release of the total soluble content of the storage vesicles . Using the ratio of dopamine-~i-hydroaylase : catecholamiaes (DBH/CA) of storage vesicles it has been shown is both rabbit and rat adrenal glands, that the storage vesicles isolated by sucrose density centrifugation after various degrees of depletion induced by insulin hypoglycemia have the same DBH/CA ratios and the same buoyant density as vesicles isolated from intact glands. This would be expected only if secretion occurred by an "all-ornone" quantal release of the soluble content. Additional studies of rabbit and rat adrenal glands following stimulation show excellent correlations between the loss of ATP and catecholamines, and between the loss of the ability to take-up exogenous catecholamines and the loss of endogenous amines . Thus immediately folloigg secretion the vesicles no longer retain an important physiologic characteristic. Hetweea 3 and 24', hours following massive secretion from rabbit adrenal gland there is little change in CA or DHH content of the purified vesicles . However, by 48 hours there is complete recovery of the vesicle DBH but only a 3096 recovery of the catecholamiae content, indicatittg that at this time, vesicle formation is complete but 2 to 4 days longer are required for recovery of the catecholamiae stores . In rat adrenals, vesicle formation occurs much more rapidly. Regression curves of DBH vs catecholamines indicate that substantial vesicle formation occurs within 3 hours after initiation of massive secretion. Studies of the recovery of catecholaine stores, ATP content and ability to take up exogenous amines show that recovery of the ATP content and the ability to take up exogenous amines precedes recovery of the catecholamiae content. Thus the rate limiting factor is recovery is not formation of the storage vesicle per se, or the ability to take up and store the amines but appears to be the biosynthesis of the catecholamines . In this respect it is interesting to note that within 48 hours after secretion there is a marked increase in tyrosine hydroxylase activity but no increase in PNMT or dope decarboxylase . The question whether vesicle formation requires de nouo synthesis of membrane protein components or whether components of old membranes are reused has not been resolved but is being actively pursued is this laboratory as well as by others using adrenal glands, salivary glands, pancreas and other secretiry tissues. Hopefully more definitive information will be available in the near future.
Fnontiera in Catecholamine Research
Another characteristic of tyrosine hydroxylase that is markedly dependent on the nature of the cofactor uséd is its amino acid specificity . Although the ability of the enzyme to catalyze the hydroxylation of phenylalanine in the presence of DMPH 4 is poor, .phenylalaniae is an excellent substrate for the enzyme in the presence of tetrahydrobiopterin . These findings suggest that both in adrenal medulla and in the nervous system, phenylalanine is an important precursor of catecholamines .
RECOVERY OF CATECHOLAMINE STORES FOLLOWING SECRETION BY THE ADRENAL MEDULLA N. Kirshner sad T. Slotkin Department of Biochemistry and Department of Physiology and Pharmacol., Duke Univ. Med. Ctr., Durham, N. C. 27710, U.S .A . It is generally accepted that secretion from the adrenal medulla occurs by exocytosis. Evidence from several laboratories has shown that the contents of the adrenal storage vesicle which include catecholamines, adenine nucleotides and proteins, among which is the soluble fraction of dopamine-~i- hydroxylase, appear in perfusates of the stimulated gland in the same relative amounts as found in the soluble fraction upon lysis of the storage vesicles in distilled water. During secretion the membranes of the vesicles remain within the cell as indicated by complete retention of that fraction of dopamine-ß-hydroxylase which is tightly bound to the storage vesicle membrane . During secretion there is no release of typical cytoplasmic enzymes such as lactic dehydrogenase, tyrosine hydroxylase or phenylethanolamine-N-methyltraasferase . More recently we have been concerned with the sequence of events during . recovery of the catecholamine stores . Fundamental to this issue is the state of the catecholamine storage vesicles immediately following secretion . (1) Do they secrete their entire content or only a portion in response to a stimulus? (2) Can vesicles which have secreted their amines retain the ability to takeup end store amines? (3) Are the ghosts of old vesicles reused or does the recovery process involve de r~ao synthesis of new vesicles? (4) If so, are protein components or fragments of the old membranes re-used or are newly synthesized macromolecular components required?
Frontiers in Catecholamine Research
lxxxv
(5) What other factors may be involved in the recovery process? Answers to some of these questions have been obtained . Studies of secretion from rabbit and rat adrenal g~ends show that secretion occurs by an "allrar-none ° release of the total soluble content of the storage vesicles . Using the ratio of dopamine-~i-hydroaylase : catecholamiaes (DBH/CA) of storage vesicles it has been shown is both rabbit and rat adrenal glands, that the storage vesicles isolated by sucrose density centrifugation after various degrees of depletion induced by insulin hypoglycemia have the same DBH/CA ratios and the same buoyant density as vesicles isolated from intact glands. This would be expected only if secretion occurred by an "all-ornone" quantal release of the soluble content. Additional studies of rabbit and rat adrenal glands following stimulation show excellent correlations between the loss of ATP and catecholamines, and between the loss of the ability to take-up exogenous catecholamines and the loss of endogenous amines . Thus immediately folloigg secretion the vesicles no longer retain an important physiologic characteristic. Hetweea 3 and 24', hours following massive secretion from rabbit adrenal gland there is little change in CA or DHH content of the purified vesicles . However, by 48 hours there is complete recovery of the vesicle DBH but only a 3096 recovery of the catecholamiae content, indicatittg that at this time, vesicle formation is complete but 2 to 4 days longer are required for recovery of the catecholamiae stores . In rat adrenals, vesicle formation occurs much more rapidly. Regression curves of DBH vs catecholamines indicate that substantial vesicle formation occurs within 3 hours after initiation of massive secretion. Studies of the recovery of catecholaine stores, ATP content and ability to take up exogenous amines show that recovery of the ATP content and the ability to take up exogenous amines precedes recovery of the catecholamiae content. Thus the rate limiting factor is recovery is not formation of the storage vesicle per se, or the ability to take up and store the amines but appears to be the biosynthesis of the catecholamines . In this respect it is interesting to note that within 48 hours after secretion there is a marked increase in tyrosine hydroxylase activity but no increase in PNMT or dope decarboxylase . The question whether vesicle formation requires de nouo synthesis of membrane protein components or whether components of old membranes are reused has not been resolved but is being actively pursued is this laboratory as well as by others using adrenal glands, salivary glands, pancreas and other secretiry tissues. Hopefully more definitive information will be available in the near future.