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Organ culture of sympathetic ganglia
F~untiers in Catecholmnine Reaea~h
lxxxvü
ferase, the enzyme which converts serotonin to N~cetylserotonin . This proposal is based on studies is the experimental system used here which have found that aorepinephrine and dibutyryl cyclic AMP increase the activity of Nacetyltransferase 10- to 50-fold and inçrease the net formation of N~cetylserotonin from undetectable levels to 500-1000 picomoles. Large increases in melatonin production also occur. All these effects appear to be regulated by a beta-adrenergic receptor and to be dependent upon protein synthesis. ORGAN CULTURE OF SYMPATHETIC GANGLIA Irwin J. Kopin, Kenneth R. Berv and Jerry G. Webb Laboratory oP Clinical Science, National Institute oP Mental Health, Bethesda, Maryland 20014, U.S .A . The bodies of the neurones of periphal sympathetic nerves are located in paravertebral ganglia and are innervated by cholinergic neurones from the spinal cord. The development and functional state of these neurones is influenced by their cholinergic innervation, but the role of various factors in determining developmental and metabolic responses of the neurones of the Anglia to changes in activity to differentiate in vivo . Sympathetic ganglia can be excised from embryos, neonates, or adult animals and maintained in organ culture in artificial media or transplanted to the anterior chamber of the eye. The morphological, biochemical, and neurophysiolo gical changes which occur in the neurones of_ganglia in organ culture and the effects of various drugs, ions, tissue `factors" and procedures have been examined in a number of laboratories . Soon after excision, neurones in ganglia maintained is organ culture develop one or more processes. The formation of these processes is enhanced by the Nerve Growth Factor described by Levi-Montalcini, but the degree of enhancement appears to vary with the age of the animal . In addition to axonal sprouting, Châuley, et al recently showed that a small proportion of cells have the ability to migrate out of the ganglia. The axonal processes which sprout out from the neurones appear histologically similar to axons from intact ganglia; they contain microtubules and large granular vesicles . During the development of the axonal sprouts there is a striking increase in the ability of the ganglia to take up and retain radioactivtely labelled norepinephrine. Radioautographic examination of ganglia incubated with labelled norepinephrine show that the labelled amine is concentrated in the axonal sprouts. The uptake process in the axonal sprouts is as susceptible to inhibition bY cocaine as is the uptake process for
lzzzvüi
Frontiers in Catecholamine Research
catecholamiaes at the sympathetic serve endings. Furthermore, labelled norepinephrine taken up by the axonal sprouts, but not that taken up into the cell bodies of uncultured ganglia, can be released by depolarization with an electrical field or with high concentrations of potassium. Such release of catecholamines is calcium dependent, is blocked by bretylium, and is enhanced by phenoxybenzamine. The latter effect provides direct evidence for a presynaptic effect of phenoxybenzamine in enhancing catecholamine relise since in this preparation there are no post-synaptic sites for amine uptake. The induction of catecholamine synthesizing enzymes in association with neuronal activation has been termed `transsyaaptic,~ but studies in organ culture have provided abundant evidence that depolarization, presumably associated with catecholamine release, is sufficient to elevate levels of these enzymes. Thus, levels of tyrosine hydroxylase (TH) and dopamine-/3-hydroxylase (DBH) have been found to be elevated in ganglia, as well as adrenal medullae, grown in media containing high concentrations of potassium. This elevation of enzyme level is blocked by inhibition of protein synthesis . Depolarization with high levels of K+ is associated with activation of adenyl cyclase and both DBH and TH levels have been found to be higher in ganglia which have been cultured with dibutyryl ryclic AMP. Theophylline enhances the elevation of DHH and TH by elevated levels of K}. Thus, cyclic ASP appears to play a role in control of the levels of the catecholamine synthesizing enzymes, but the nature of this role remains to be defined. The processes which grow out of the ganglia can ramify among the smooth muscle of irises or other sympathetically inaervated tissues grown in contact with the ganglia. The rate and extent of ramification of the fibres, assessed by the uptake .of labelled norepiaephrine by the irises, appears to be enhanced by NGF and is markedly diminished by drugs, such as colchicine or vinblastine, which disrupt neumtubules. There does not appear to be absolute species specificity for ramification of nerve fibres since in both the anterior chamber of the eye and in organ culture, neuronal fibres from mouse ganglia reinnervate rat or guinea pig iris . The factors controlling the direction of fibre growth and formation of functional junctions with effector cells are not known. In viuo, the levels of TH and of DHB are controlled by their rates of synthesis, degradation, and transport from the cell body, thmugir the axon to the nerve endings. When the axon or nerve endings are damaged, or when axoaal transport is blocked by oolchicine or vinblastine, the levels of enzyme in the ganglia decline and the ability to take up aorepinephrine is enhanced. These changes may take several days to become apparent and seem to involve changes in the priorities of protein synthesis in the cell body. Requirements for protein
Frontiers in Catecholmnine Research
lxxxix
to restore structure are met by a diminution in production of enzymes required to support functions. It is likely that logger tens studies of ganglia in culture will provide insight into the mechanisms controlling the priorities which determine which proteins are required and synthesized.
APPLICATION OF MASS .FRAQNENTOGRAPHY TO THE MEAS~JREMENTS OF ~CATECHOLAMINES llV CLINICAL AND BASZ~C RESEARCH Stephen H. Koslow Laboratory of Preclinical Pharmacology, National Institute of Mental Health, Saint Elizabeths Hospital, Washington, D. C. 20032, U.SA. Mass fragmentography . coupled with gas chromatography has the advantage of rapidly measuring extremely low concentrations (10 -1' -10~' moles) of endogenous catecholamines (norepinephrine (NE) ; Dopamine (DA)) with an extremely high degree of specificity (Koslow, Cattabeni; and Costa, Science, 176' : .177-180, 1972). Formation of the volatile acylated peatafluoropropionic (PFP) derivatives of NE and DA is necessary for their separation by GC, while measurement of the total ion density of a . specific fragment (mass-to-charge, m/e) by mass spectrometer at the compound's GC retention time allows for quantitation . The internal standards, alpha-methyl-norepinephrine (c~M-Ne) and alpha-methyl-dopamine (a-M-DA) are added in the same concentration to both tissue extracts and standard catecholamines, the latter being used to obtain a standard calibration curve. The specificity of the analysis is based on both gas chromatographic retention time and the relative abundance (%) of characteristic fragmènts (m/e) . With all instrumental conditions held constant, the fragmentation pattern of the acylated catecholamines is completely reproducible in terms of the relative abundance (%) of each fragment (m/e). The most abundant fragment (basic peak) is given the value of 1009b and all other fragments are computed as a percentage of the basic peak . BY means of alternating the accelerating voltage, two or three fragments (multiple ion detection, MID) can be consecutively recorded oa the LKB 9000 Gas Chromatograph-Mass Spectrometer . The fragment ratio is first determined for the pure acylated standard and then the tissue extract is subjected to the same MID analysis . Identification of the endogenous compound is established only when the fragment ratio for the tissue extract measured at the compound's retention time is the same as the ratio for the pure standard . MID is not done on internal standards: however, the tissue extracts are run without internal standards to be sure that no `biological background" is present at the masses (m/e) and retention times of the internal standards.
lxxxvü
ferase, the enzyme which converts serotonin to N~cetylserotonin . This proposal is based on studies is the experimental system used here which have found that aorepinephrine and dibutyryl cyclic AMP increase the activity of Nacetyltransferase 10- to 50-fold and inçrease the net formation of N~cetylserotonin from undetectable levels to 500-1000 picomoles. Large increases in melatonin production also occur. All these effects appear to be regulated by a beta-adrenergic receptor and to be dependent upon protein synthesis. ORGAN CULTURE OF SYMPATHETIC GANGLIA Irwin J. Kopin, Kenneth R. Berv and Jerry G. Webb Laboratory oP Clinical Science, National Institute oP Mental Health, Bethesda, Maryland 20014, U.S .A . The bodies of the neurones of periphal sympathetic nerves are located in paravertebral ganglia and are innervated by cholinergic neurones from the spinal cord. The development and functional state of these neurones is influenced by their cholinergic innervation, but the role of various factors in determining developmental and metabolic responses of the neurones of the Anglia to changes in activity to differentiate in vivo . Sympathetic ganglia can be excised from embryos, neonates, or adult animals and maintained in organ culture in artificial media or transplanted to the anterior chamber of the eye. The morphological, biochemical, and neurophysiolo gical changes which occur in the neurones of_ganglia in organ culture and the effects of various drugs, ions, tissue `factors" and procedures have been examined in a number of laboratories . Soon after excision, neurones in ganglia maintained is organ culture develop one or more processes. The formation of these processes is enhanced by the Nerve Growth Factor described by Levi-Montalcini, but the degree of enhancement appears to vary with the age of the animal . In addition to axonal sprouting, Châuley, et al recently showed that a small proportion of cells have the ability to migrate out of the ganglia. The axonal processes which sprout out from the neurones appear histologically similar to axons from intact ganglia; they contain microtubules and large granular vesicles . During the development of the axonal sprouts there is a striking increase in the ability of the ganglia to take up and retain radioactivtely labelled norepinephrine. Radioautographic examination of ganglia incubated with labelled norepinephrine show that the labelled amine is concentrated in the axonal sprouts. The uptake process in the axonal sprouts is as susceptible to inhibition bY cocaine as is the uptake process for
lzzzvüi
Frontiers in Catecholamine Research
catecholamiaes at the sympathetic serve endings. Furthermore, labelled norepinephrine taken up by the axonal sprouts, but not that taken up into the cell bodies of uncultured ganglia, can be released by depolarization with an electrical field or with high concentrations of potassium. Such release of catecholamines is calcium dependent, is blocked by bretylium, and is enhanced by phenoxybenzamine. The latter effect provides direct evidence for a presynaptic effect of phenoxybenzamine in enhancing catecholamine relise since in this preparation there are no post-synaptic sites for amine uptake. The induction of catecholamine synthesizing enzymes in association with neuronal activation has been termed `transsyaaptic,~ but studies in organ culture have provided abundant evidence that depolarization, presumably associated with catecholamine release, is sufficient to elevate levels of these enzymes. Thus, levels of tyrosine hydroxylase (TH) and dopamine-/3-hydroxylase (DBH) have been found to be elevated in ganglia, as well as adrenal medullae, grown in media containing high concentrations of potassium. This elevation of enzyme level is blocked by inhibition of protein synthesis . Depolarization with high levels of K+ is associated with activation of adenyl cyclase and both DBH and TH levels have been found to be higher in ganglia which have been cultured with dibutyryl ryclic AMP. Theophylline enhances the elevation of DHH and TH by elevated levels of K}. Thus, cyclic ASP appears to play a role in control of the levels of the catecholamine synthesizing enzymes, but the nature of this role remains to be defined. The processes which grow out of the ganglia can ramify among the smooth muscle of irises or other sympathetically inaervated tissues grown in contact with the ganglia. The rate and extent of ramification of the fibres, assessed by the uptake .of labelled norepiaephrine by the irises, appears to be enhanced by NGF and is markedly diminished by drugs, such as colchicine or vinblastine, which disrupt neumtubules. There does not appear to be absolute species specificity for ramification of nerve fibres since in both the anterior chamber of the eye and in organ culture, neuronal fibres from mouse ganglia reinnervate rat or guinea pig iris . The factors controlling the direction of fibre growth and formation of functional junctions with effector cells are not known. In viuo, the levels of TH and of DHB are controlled by their rates of synthesis, degradation, and transport from the cell body, thmugir the axon to the nerve endings. When the axon or nerve endings are damaged, or when axoaal transport is blocked by oolchicine or vinblastine, the levels of enzyme in the ganglia decline and the ability to take up aorepinephrine is enhanced. These changes may take several days to become apparent and seem to involve changes in the priorities of protein synthesis in the cell body. Requirements for protein
Frontiers in Catecholmnine Research
lxxxix
to restore structure are met by a diminution in production of enzymes required to support functions. It is likely that logger tens studies of ganglia in culture will provide insight into the mechanisms controlling the priorities which determine which proteins are required and synthesized.
APPLICATION OF MASS .FRAQNENTOGRAPHY TO THE MEAS~JREMENTS OF ~CATECHOLAMINES llV CLINICAL AND BASZ~C RESEARCH Stephen H. Koslow Laboratory of Preclinical Pharmacology, National Institute of Mental Health, Saint Elizabeths Hospital, Washington, D. C. 20032, U.SA. Mass fragmentography . coupled with gas chromatography has the advantage of rapidly measuring extremely low concentrations (10 -1' -10~' moles) of endogenous catecholamines (norepinephrine (NE) ; Dopamine (DA)) with an extremely high degree of specificity (Koslow, Cattabeni; and Costa, Science, 176' : .177-180, 1972). Formation of the volatile acylated peatafluoropropionic (PFP) derivatives of NE and DA is necessary for their separation by GC, while measurement of the total ion density of a . specific fragment (mass-to-charge, m/e) by mass spectrometer at the compound's GC retention time allows for quantitation . The internal standards, alpha-methyl-norepinephrine (c~M-Ne) and alpha-methyl-dopamine (a-M-DA) are added in the same concentration to both tissue extracts and standard catecholamines, the latter being used to obtain a standard calibration curve. The specificity of the analysis is based on both gas chromatographic retention time and the relative abundance (%) of characteristic fragmènts (m/e) . With all instrumental conditions held constant, the fragmentation pattern of the acylated catecholamines is completely reproducible in terms of the relative abundance (%) of each fragment (m/e). The most abundant fragment (basic peak) is given the value of 1009b and all other fragments are computed as a percentage of the basic peak . BY means of alternating the accelerating voltage, two or three fragments (multiple ion detection, MID) can be consecutively recorded oa the LKB 9000 Gas Chromatograph-Mass Spectrometer . The fragment ratio is first determined for the pure acylated standard and then the tissue extract is subjected to the same MID analysis . Identification of the endogenous compound is established only when the fragment ratio for the tissue extract measured at the compound's retention time is the same as the ratio for the pure standard . MID is not done on internal standards: however, the tissue extracts are run without internal standards to be sure that no `biological background" is present at the masses (m/e) and retention times of the internal standards.