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Metabolism of octopamine in vitro by monoamine oxidase in some rat tissues
Life Sciences Vol . 23, pp . 223-230 Printed in the II .S .A .
Pergamon Prese
METABOLISM OF OCTOPAMINE IN VITRO BY MONOAMINE OXIDASE IN SOME RAT TISSUES Geoffrey A . Lyles Department of Biochemistry, University of Tennessee Knoxville, Tennessee 37916 U .S .A . (Received is final form June 5, 1978) Summary The deamination in vitro of DL-octopamine by MAO in rat brain, heart, kidney, liver and vâs ~ e~rens has been studied by a radiochemical method . Kinetic constants for octopamine metabolism, as well as its sensitivity to inhibition by the irreversible MAO inhibitor clorgyline are described for each tissue . On the basis of the inhibition data, it was concluded that octopamine is metabolized preferentially by type A MAO in heart, kidney and vas deferens . However, in brain and liver, type B MAO is also responsible for a significant proportion of total octopamine metabolism . These studies are discussed in relation to current ideas about the regulation of octopamine concentrations in animal tissues, and the possible importance of this amine in mammalian physiology . There is, at present, growin~ interest in the physiological properties of the biogenic amine, octopamine p-hydroxyphenylethanolamine) . The recent development of sensitive assay methods has allowed the measurement of the generally very low concentrations of this compound in a number of mammalian tissues . Its biosynthesis is believed to involve the hydroxylation of p-tyramine in adrenergic storage granules by the enzyme dopamine ß-hydroxylase . Furthermore, other properties of octopamine, such as its ability to be taken up, stored and released from sympathetically innervated tissues, as well as the observations that endogenous levels of this amine are reduced following chemical or surgical denervation of these tissues, all point to a possible, although as yet undefined role of this compound in the process of adrenergic transmission (see 1,2 for reviews) . The detection of deaminated metabolites of octopamine in mammalian urine (3) and the demonstration that some tissue levels of octopamine increase considerably after administration of monoamine oxidase (MAO) inhibitors to experimental animals, have suggested that the enzyme MAO plays a major role in the catabolism of octopamine, and that despite its relatively low endogenous concentrations, the turnover of this amine is extremely rapid (1) . However, to date, little attempt has been made to study directly the properties of octopamine as a substrate for MAO . This outer mitochondrial membrane enzyme is believed to exist in many animal tissues in at least two functional forms (called type A and B MAO), which were originally defined on the basis of their differing relative sensitivities toward the selective irreversible MAO inhibitor, clorgyline (4) . A previous report, in which the utilization of oxygen in the enzymis reaction was used to measure octopamine metabolism by MAO in rat liver, has suggested that octopamine may be a substrate solely for type A MAO (the enzyme species more sensitive to clorgyline) (5) . However, it is clear from recent observations that the substrate specificities of both osoa~96s3/7s/on7~o223So2 .oo/0 Copyright Q 1978 Pergamon Press
224
Metabolism of Octopamine
Vol . 23, No . 3, 1978
types A and B MAO may vary considerably, even in a comparison between tissues from the same animal species (see 6, for review) . For this reason, in the present investigation some properties of octopamine metabolism by MAO, and its sensitivity toward inhibition by clorgyline, have been studied in a variety of rat tissues . In contrast to previous results, the deamination of ôctopamine has been assessed by the use of a radiochemical assay for MAO employing DL-[3H]-octopamine as substrate . Materials and Methods Clorgyline hydrochloride [N-methyl-N-propargyl-3-(2,4-dichlorophenoxy) propylamine, M+B 9302] was a gift from May and Baker Ltd ., Dagenham, Essex, England . DL-[2- 3 H]-octopamine hydrochloride (sp . act . 8 " 4 Ci/mmol) was purchased from New England Nuclear, Boston, Massachusetts, and diluted with an aqueous solution of non-radioactive DL-octopamine hydrochloride (Sigma, St . Louis, Missouri), to give a 100mM stock solution of final specific activity luCi/ For routine studies, aliquots of this stock u~nol, which was stored at -20°C . solution were diluted with appropriate vol~anes of potassium phosphate buffer (0 .2M, pH 7 .8) to obtain substrate-buffer solutions which yielded the desired final octopamine concentrations in the MAO assay described below . Tissues were obtained from three male Charles River rats weighing around 200g . The animals were killed by cervical dislocation and the excised tissues (brain, heart, kidneys, vasa deferentia and liver) from each rat were blotted and then washed in a small volume of saline (0 .9% NaCI w/v in distilled water) to remove excess blood . Adhering fat and connective tissue were removed from the kidneys and vasa deferentia, and the larger blood vessels dissected .away from the heart . Samples of each tissue were weighed, combined and pooled homogenates prepared in potassium phosphate buffer (0 .001 M, pH 7 .8) using a conical ground-glass hand homogenizer, and employing tissue weight (g) to homogenizing buffer volume (ml) ratios of 1 :10 for heart, liver, vas deferens and 1 :5 for brain and kidney . The resulting homogenates were centrifuged at 600g for 10 min and the supernatants decanted . These supernates were divided into suitable portions, and stored frozen at -20°C for a few days, during which time kinetic experiments and inhibition studies using clorgyline were performed on thawed samples . There was no evidence throughout this work that storage of the homogenates in this way significantly affected the activity of MAO towards octopamine since enzyme activities measured in unfrozen samples used immediately after preparation were essentially the same as those in corresponding samples that had been stored frozen . Protein contents of homogenates were determined by the micro-biuret method of Goa (7), using bovine serum albumin as standard . MAO activity was assayed by a radiochemical method, based on that described by Callingham and Laverty (8) . Suitable aliquots (see below) of tissue homogenate and distilled water were added to ice-cooled assay tubes, to give a total combined volume of 50u1 . An additional 50u1 of the appropriate octopamine-buffer solution was then added to give the required octopamine concentration in the total assay volume of 100u1 . All assay tubes were oxygenated, closed with rubber stoppers, and incubated at 37°C in a water bath . After suitable incubation periods (see below), all tubes were rapidly icecooled and the reaction terminated by the addition of 10u1 3N HC1 to each one . Blank assays differed from controls in these procedures only in the respect that the HC1 was added to denature the homogenate protein before the addition of substrate and prior to the subsequent incubation period .
Vol . 23, No . 3, 1978
225
Metabolism of Octopamine
Deaminated metabolites of octopamine were extracted into 0 .5 ml of ethyl acetate : benzene (1 :1 v/v), and after centrifugation of assay tubes to separate the aqueous and organic phase, 0 .4 ml of the upper organic layer was counted for radioactivity in 12 ml 0 .4% butyl-PBD in toluene (w/v) in a Beckman LS-230 liquid scintillation spectrometer . All counts were corrected for quench and converted to dpm for calculation of results . Aliquots (20N1) of the radioactive substrate-buffer solutions were also counted as standards in 10 ml butyl-PBD/toluene containing 2 ml ethoxyethanol, in order to express enzyme specific activities in units of nmoles substrate transformed/hr/mg protei n . In initial experiments using these methods, it was determined that the accumulation of radioactive metabolites from octopamine (at an assay concentration of 1mM) was linear with the use of up to certain quantities of homogenate protein and incubation times in the assay . The upper limits for these parameters were determined for each tissue homogenate (Table 1) and were employed as assay conditions in subsequent kinetic and inhibition studies . In no assays did the net recovered radioactive metabolites represent more than 4% of the total radioactivity added as substrate . TABLE 1 Assay Conditions used for Studying Octopamine Metabolism by MAO TISSUE
HOMOGENATE PROTEIN CONCENTRATION (mg/ml)
ALIQUOT USED (ul)
Brain Heart Liver Kidney vas deferens
16 .5 8 .6 17 .7 11 .4 3 .3
15 20 20 20 20
INCUBATION TIME (min) 20 20 20 45 60
For inhibition studies involving clorgyline, the 50u1 combined volume of homogenate and distilled water also contained appropriate concentrations of the inhibitor . These mixtures were preincubated at 37 ° C for 20 min before the addition of 50u1 octopamine-buffer solution for the assay of MAO activity as described above . All clorgyline concentrations indicated within the text represent preincubation concentrations of this drug . Results Kinet ic cons tants for MAO activity toward oc top amine Deamtnation of octopamine by MAO in the various tissue homogenates was studied using different octopamine concentrations in the assay . The relationship between initial reaction velocities and substrate concentrations was plotted by the double reciprocal Lineweaver-Burk method . These plots (Ftgure 1) were completely linear with octopamine concentrations from 100uM to 2 .5mM, and were fitted to this data by the method of least squares . Furthermore, the plots also appeared to remain linear with octopamine concentrations as low as 25 and 50uM (data not shown), although in these cases, the initial reaction velocities became so lqw as to introduce a greater error in their measurement, and hence a much gneater degree of scatter around the corresponding lines when plotted as reciprocals . On the basis of the observed linearity, the Lineweaver-Burk plots indicated only a single kineticallydistinguishable MAO component acting upon octopamine in each tissue . Kinetic
226
Metabolism of Octopamine
Vol . 23, No . 3, 1978
-s
FI~ . 1 Lineweaver-Burk lots for MAO activit toward octo amine in various rat t ssues . o s : p , eart ; p , ver ; p , vas e erens ; Initial reaction velocities (V) were determined ~r~ " " , kidney . at various substrate concentrations (S) . Each point is the mean of quadruplicate determinations upon a single homogenate prepared from pooled tissues of 3 rats . constants ( . Km and Ymax) for octopamine metabolism, derived from this data, are shown in Table 2 . Stnce the use of octopamine at highep concentrations (5 and 10 mM) resulted to the appearance of some degree of substrate inhibition of MAO activtty (data not shown), the data for Ymax should be considered to represent apparent values only . TABLE 2 Kinetic Constants for MAO Activity Towards Octopamine Tissue
Km (üM)
Vmax (nmoles/(n9 protein/hr)
Brain Heart Liver Ktdney Vas deferens
701 819 843 892 T054
25 47 4i 19 36
Vol . 23, No . 3, 1978
Metabolism of Octopamiae
227
Inhibition by clorgyline of MAO activity toward oçtopamine Tissue homog nate samp1es were preincubated with various concentrations of clorgyline (10 - ~1 to 10 - ~ M) as described in Materials and Methods, before Enzyme addition of octopamine (2 mM) for assay of remaining MAO activity . activities in inhibited samples were expressed as percentages of control activities containing no clorgyline, and were plotted to give the inhibition curves shown in Figures 2 and 3 for the various tissues studied .
~ so, ~oo-
FIG . 2 Inhibition b cloy line of MAO activit toward octo amine in rat heart ~e an .vas e erens . ac po nt s t e mean t s .e . o qua rup cafe eterm na ons upon a single homogenate prepared from Final octopamine concentration was 2 mM. pooled tissues of 3 rats . In heart, kidney and vas deferens homogenates, around 90-100% of enzyme act~,vity wa~ inhibited after preincubation with clorgyline concentrations of 10 - to 10 - M (Figure 2) . From Johnston's original results defining type A or B MAO on the basis of their different sensitivities toward clorgyline (4) the present findings would seem to indicate that octopamine is metabolized predominantly or virtually exclusively by type A MAO in these particular tissues . On the other hand, higher concentrations of clorgyline were required to inhibit completely the MAO activity of rat brain and liver, and the presence of a plateau ~egion in these inhibition curves at clorgyline concentrations of around 10 -7 to 10- 6 M indicated that octopamine is metabolized by both types A and B MAO in these tissues (Figure 31 . Although, from the corresponding curves, type B MAO was responsible for only a relatively small proportion (15-20%) of total octopamine metabolism in rat brain under these assay conditions, this proportion appears to be much greater (around 40%) in rat liver . It was also fpund clorgyline (10 to trol values occurred tion of MAO activity
tha~ in the presence of relatively low concentrations of 10- .M), a small increase in enzyme activity above conin some tissue homogenates . An apparent slight activaby low concentrations of clorgyline has been observed on
22g
Metabolism of Octopamiae
Vol . 23, No . 3, 1978
FIG . 3 Inhibition b clo line of MAO activit bra n an ver . egen as in g.
toward octo amine in rat
previous occasions (g,~10) and although this effect is not clearly understood, it may be due to an interaction of the lipophilic inhibitor clorgyline with lipid structure around the membrane-bound enzyme (10,11) . Discussion In a direct attempt to investigate whether or not octopamine may undergo deamination in a variety of rat tissues, radioactively-labelled DL-octopamine has been used as the substrate for MAO in a previously described radiochemical assay for the enzyme (8) . It was, indeed, possible by this method to detect octopamine metabolism in each tissue, and it would appear that the radioactive metabolites which were measured, resulted from the action of MAO on this substrate, since the preincubation of tissue homogenates with high concentrations (10 -4 M) of the irreversible MAO inhibitor clorgyline reduced the apparent enzyme activity to blank values . Conditions were determined for each tissue such that the enzymic formation of product was linear with respect to both incubation time and protein concentration within the assay . These conditions were then used in the subsequent estimation of kinetic constants for octopamine deamination in each tissue . Lineweaver-Burk plots for metabolism of octopamine by MAO revealed a single, linear component for each tissue with a Km value (at pH 7 .8) ranging from approximately 0 .70 to 1 .05 x 10 - ~ M . Apparent Vmax values indicated relative enzyme activities in the order heart>liver>vas deferens>brain>kidney . However, MAO activity toward other substrates in tissues such as rat heart (12) and vas deferens (13) has been found to change during adult development of the rat, and thus the relative tissue enzyme activities
Vol . 23, No . 3, 1978
Metabolism of Octopamine
22 9
indicated in the present .study with rats weighing around 200g, may well be different at other ages . Inhibition studies with clorgyline revealed that octopamine is predominantly, 1f not exclusively metabolized by type A MAO in rat heart, vas deferens and kidney, although all of these tissues are known to contain a type B component which acts upon other substrates (9,14 ; Shaffer and Lyles, in preparation) . However, the present results with rat brain and more particularly liver, indicate that octopamine can be metabolized by both MAO forms in these tissues . Our findings with rat liver are in contrast to those of Houslay and Tipton (5), who used an oxygen electrode assay method to study octopamine metabolism by MAO in an outer mitochondrial membrane preparation, and concluded that this amine is a substrate solely for type A MAO in this tissue . There appear to be no obvious reasons for this discrepancy at the moment . Although there is now good evidence that octopamine is associated predominantly with adrenergic neurones in sympathetically innervated tissues (1,2), it is unlikely that the MAO activity toward octopamine measured in the present study solely represents metabolism brought about by intraneuronal enzyme . It has previously been proposed that type A MAO in some tissues (such as vas deferens and pineal gland) may be localized preferentially within adrenergic neurones (14,15) . However, MAO activity in rat heart and liver, organs which contain both types A and B MAO components, is believed to exist almost completely in extraneuronal locations (16,17) . Thus, it seems likely that a variety of cell types possess the capacity to deaminate octopamine . In summary, the present results describe some kinetic properties and some observations on the sensitivity toward inhibition of octopamine deamination in vitro by various rat tissues, several of which have been reported to contâin measurable in vivo concentrations of this amine (1) . Our investigations have been made witht~ racemic mixture of octopamine . To the author's knowledge, this currently appears to be the only form of radiolabelled octopamine which is commercially available from the usual sources, and this material is being widely used in various other studies into the possible biochemical and physiological functions of octopamine . For this reason, information about the potential deamination of this compound is extremely useful . However, MAO has been reported to show a greater specificity toward the naturally-occurring levorotatory isomers of catecholamines such as noradrenaline and adrenaline (18) . Whether or .not such specificity exists also for levo-octopamine is not known at present, but clearly such a possibility should be kept in mind when considering the disposition and metabolic fate of octopamine in experiments using racemic mixtures of this compound . Acknowledgements This work was supported by NIH Grant No . NS 12747-01 . The personal support from a Wellcome Trust Travel Grant, and a Hilton A . Smith Postdoctoral Fellowship, University of Tennessee is gratefully acknowledged . References 1. 2. 3. 4. 5. 6.
P . B . MOLINOFF and S . H . BUCK, in Trace Amines and the Brain 131-160 (Eds . E . Usdin and M . Sandier ; Pub . Marce e er, ew Yor , J . AXELROD and J . M . SAAVEDRA, Nature 265 501-504 (1977) . Y . KAKIMOTO and M . D . ARMSTRONG, J . bid Chem . 237 422-427 (1962) . J . P . JOHNSTON, Biochem . Pharmac . 17 1285-1297 (T4~8) . M . D . HOUSLAY and K . F . TIPTON, Biochem . J . 139 645-652 (1974) . C . J . FOWLER, B . A . CALLINGHAM ., T . J . MANTL~nd K . F . TIPTON, Biochem . Pharmac . 27 97-101 (1978) .
230 7. 8. 9. 10. 11 . 12 . 13 . 14. 15 . 16 . 17 .
Metabolinm of Octopaminn
vol . 23, No . 3, 1978
J . GOA, Scand . J . clin . lab . Invest . 5 218-222 (1953) . B . A . CALLINGHAM and R . LAYERTY, J . PFarm . Pharmac . 25 940-947 (1973) . G . A . LYLES and B . A . CALLINGHAM, J . Pharm . Pharmac .~_`1 682-691 (1975) . M . D . HOUSLAY, J . Pharm . Pharmac . 29 664-669 (1977) . G . A . LYLES and J . W . GREENAWALT, ~fochem . Pharmac . _26 2269-2274 (1977) . A . HORITA, Nature 215 411-412 (1967) . B . A . CALLINGHAM a~G . A . LYLES, Br . J . Pharmac . _55 305P (1975) . B . JARROTT, J . Neurochem . 18 7-16 (1971) . C . GORIDIS and N . H . NEFF,~europharmac . 10 557-564 (1971) . A . HORITA and M . C . LOWE, Adv . Biochem . Psychopharmac . 5, 227-242 (1972) . K . F . TIPTON, M . D . HOUSLAY and T . J . MANTLE, In Monoamine Oxidase and Its Inhibition, Ciba Foundation Symposium 39 5-16 18 . , and P . A . SHORE, Life Sci . 5 T$73-1378 (1966) .
Pergamon Prese
METABOLISM OF OCTOPAMINE IN VITRO BY MONOAMINE OXIDASE IN SOME RAT TISSUES Geoffrey A . Lyles Department of Biochemistry, University of Tennessee Knoxville, Tennessee 37916 U .S .A . (Received is final form June 5, 1978) Summary The deamination in vitro of DL-octopamine by MAO in rat brain, heart, kidney, liver and vâs ~ e~rens has been studied by a radiochemical method . Kinetic constants for octopamine metabolism, as well as its sensitivity to inhibition by the irreversible MAO inhibitor clorgyline are described for each tissue . On the basis of the inhibition data, it was concluded that octopamine is metabolized preferentially by type A MAO in heart, kidney and vas deferens . However, in brain and liver, type B MAO is also responsible for a significant proportion of total octopamine metabolism . These studies are discussed in relation to current ideas about the regulation of octopamine concentrations in animal tissues, and the possible importance of this amine in mammalian physiology . There is, at present, growin~ interest in the physiological properties of the biogenic amine, octopamine p-hydroxyphenylethanolamine) . The recent development of sensitive assay methods has allowed the measurement of the generally very low concentrations of this compound in a number of mammalian tissues . Its biosynthesis is believed to involve the hydroxylation of p-tyramine in adrenergic storage granules by the enzyme dopamine ß-hydroxylase . Furthermore, other properties of octopamine, such as its ability to be taken up, stored and released from sympathetically innervated tissues, as well as the observations that endogenous levels of this amine are reduced following chemical or surgical denervation of these tissues, all point to a possible, although as yet undefined role of this compound in the process of adrenergic transmission (see 1,2 for reviews) . The detection of deaminated metabolites of octopamine in mammalian urine (3) and the demonstration that some tissue levels of octopamine increase considerably after administration of monoamine oxidase (MAO) inhibitors to experimental animals, have suggested that the enzyme MAO plays a major role in the catabolism of octopamine, and that despite its relatively low endogenous concentrations, the turnover of this amine is extremely rapid (1) . However, to date, little attempt has been made to study directly the properties of octopamine as a substrate for MAO . This outer mitochondrial membrane enzyme is believed to exist in many animal tissues in at least two functional forms (called type A and B MAO), which were originally defined on the basis of their differing relative sensitivities toward the selective irreversible MAO inhibitor, clorgyline (4) . A previous report, in which the utilization of oxygen in the enzymis reaction was used to measure octopamine metabolism by MAO in rat liver, has suggested that octopamine may be a substrate solely for type A MAO (the enzyme species more sensitive to clorgyline) (5) . However, it is clear from recent observations that the substrate specificities of both osoa~96s3/7s/on7~o223So2 .oo/0 Copyright Q 1978 Pergamon Press
224
Metabolism of Octopamine
Vol . 23, No . 3, 1978
types A and B MAO may vary considerably, even in a comparison between tissues from the same animal species (see 6, for review) . For this reason, in the present investigation some properties of octopamine metabolism by MAO, and its sensitivity toward inhibition by clorgyline, have been studied in a variety of rat tissues . In contrast to previous results, the deamination of ôctopamine has been assessed by the use of a radiochemical assay for MAO employing DL-[3H]-octopamine as substrate . Materials and Methods Clorgyline hydrochloride [N-methyl-N-propargyl-3-(2,4-dichlorophenoxy) propylamine, M+B 9302] was a gift from May and Baker Ltd ., Dagenham, Essex, England . DL-[2- 3 H]-octopamine hydrochloride (sp . act . 8 " 4 Ci/mmol) was purchased from New England Nuclear, Boston, Massachusetts, and diluted with an aqueous solution of non-radioactive DL-octopamine hydrochloride (Sigma, St . Louis, Missouri), to give a 100mM stock solution of final specific activity luCi/ For routine studies, aliquots of this stock u~nol, which was stored at -20°C . solution were diluted with appropriate vol~anes of potassium phosphate buffer (0 .2M, pH 7 .8) to obtain substrate-buffer solutions which yielded the desired final octopamine concentrations in the MAO assay described below . Tissues were obtained from three male Charles River rats weighing around 200g . The animals were killed by cervical dislocation and the excised tissues (brain, heart, kidneys, vasa deferentia and liver) from each rat were blotted and then washed in a small volume of saline (0 .9% NaCI w/v in distilled water) to remove excess blood . Adhering fat and connective tissue were removed from the kidneys and vasa deferentia, and the larger blood vessels dissected .away from the heart . Samples of each tissue were weighed, combined and pooled homogenates prepared in potassium phosphate buffer (0 .001 M, pH 7 .8) using a conical ground-glass hand homogenizer, and employing tissue weight (g) to homogenizing buffer volume (ml) ratios of 1 :10 for heart, liver, vas deferens and 1 :5 for brain and kidney . The resulting homogenates were centrifuged at 600g for 10 min and the supernatants decanted . These supernates were divided into suitable portions, and stored frozen at -20°C for a few days, during which time kinetic experiments and inhibition studies using clorgyline were performed on thawed samples . There was no evidence throughout this work that storage of the homogenates in this way significantly affected the activity of MAO towards octopamine since enzyme activities measured in unfrozen samples used immediately after preparation were essentially the same as those in corresponding samples that had been stored frozen . Protein contents of homogenates were determined by the micro-biuret method of Goa (7), using bovine serum albumin as standard . MAO activity was assayed by a radiochemical method, based on that described by Callingham and Laverty (8) . Suitable aliquots (see below) of tissue homogenate and distilled water were added to ice-cooled assay tubes, to give a total combined volume of 50u1 . An additional 50u1 of the appropriate octopamine-buffer solution was then added to give the required octopamine concentration in the total assay volume of 100u1 . All assay tubes were oxygenated, closed with rubber stoppers, and incubated at 37°C in a water bath . After suitable incubation periods (see below), all tubes were rapidly icecooled and the reaction terminated by the addition of 10u1 3N HC1 to each one . Blank assays differed from controls in these procedures only in the respect that the HC1 was added to denature the homogenate protein before the addition of substrate and prior to the subsequent incubation period .
Vol . 23, No . 3, 1978
225
Metabolism of Octopamine
Deaminated metabolites of octopamine were extracted into 0 .5 ml of ethyl acetate : benzene (1 :1 v/v), and after centrifugation of assay tubes to separate the aqueous and organic phase, 0 .4 ml of the upper organic layer was counted for radioactivity in 12 ml 0 .4% butyl-PBD in toluene (w/v) in a Beckman LS-230 liquid scintillation spectrometer . All counts were corrected for quench and converted to dpm for calculation of results . Aliquots (20N1) of the radioactive substrate-buffer solutions were also counted as standards in 10 ml butyl-PBD/toluene containing 2 ml ethoxyethanol, in order to express enzyme specific activities in units of nmoles substrate transformed/hr/mg protei n . In initial experiments using these methods, it was determined that the accumulation of radioactive metabolites from octopamine (at an assay concentration of 1mM) was linear with the use of up to certain quantities of homogenate protein and incubation times in the assay . The upper limits for these parameters were determined for each tissue homogenate (Table 1) and were employed as assay conditions in subsequent kinetic and inhibition studies . In no assays did the net recovered radioactive metabolites represent more than 4% of the total radioactivity added as substrate . TABLE 1 Assay Conditions used for Studying Octopamine Metabolism by MAO TISSUE
HOMOGENATE PROTEIN CONCENTRATION (mg/ml)
ALIQUOT USED (ul)
Brain Heart Liver Kidney vas deferens
16 .5 8 .6 17 .7 11 .4 3 .3
15 20 20 20 20
INCUBATION TIME (min) 20 20 20 45 60
For inhibition studies involving clorgyline, the 50u1 combined volume of homogenate and distilled water also contained appropriate concentrations of the inhibitor . These mixtures were preincubated at 37 ° C for 20 min before the addition of 50u1 octopamine-buffer solution for the assay of MAO activity as described above . All clorgyline concentrations indicated within the text represent preincubation concentrations of this drug . Results Kinet ic cons tants for MAO activity toward oc top amine Deamtnation of octopamine by MAO in the various tissue homogenates was studied using different octopamine concentrations in the assay . The relationship between initial reaction velocities and substrate concentrations was plotted by the double reciprocal Lineweaver-Burk method . These plots (Ftgure 1) were completely linear with octopamine concentrations from 100uM to 2 .5mM, and were fitted to this data by the method of least squares . Furthermore, the plots also appeared to remain linear with octopamine concentrations as low as 25 and 50uM (data not shown), although in these cases, the initial reaction velocities became so lqw as to introduce a greater error in their measurement, and hence a much gneater degree of scatter around the corresponding lines when plotted as reciprocals . On the basis of the observed linearity, the Lineweaver-Burk plots indicated only a single kineticallydistinguishable MAO component acting upon octopamine in each tissue . Kinetic
226
Metabolism of Octopamine
Vol . 23, No . 3, 1978
-s
FI~ . 1 Lineweaver-Burk lots for MAO activit toward octo amine in various rat t ssues . o s : p , eart ; p , ver ; p , vas e erens ; Initial reaction velocities (V) were determined ~r~ " " , kidney . at various substrate concentrations (S) . Each point is the mean of quadruplicate determinations upon a single homogenate prepared from pooled tissues of 3 rats . constants ( . Km and Ymax) for octopamine metabolism, derived from this data, are shown in Table 2 . Stnce the use of octopamine at highep concentrations (5 and 10 mM) resulted to the appearance of some degree of substrate inhibition of MAO activtty (data not shown), the data for Ymax should be considered to represent apparent values only . TABLE 2 Kinetic Constants for MAO Activity Towards Octopamine Tissue
Km (üM)
Vmax (nmoles/(n9 protein/hr)
Brain Heart Liver Ktdney Vas deferens
701 819 843 892 T054
25 47 4i 19 36
Vol . 23, No . 3, 1978
Metabolism of Octopamiae
227
Inhibition by clorgyline of MAO activity toward oçtopamine Tissue homog nate samp1es were preincubated with various concentrations of clorgyline (10 - ~1 to 10 - ~ M) as described in Materials and Methods, before Enzyme addition of octopamine (2 mM) for assay of remaining MAO activity . activities in inhibited samples were expressed as percentages of control activities containing no clorgyline, and were plotted to give the inhibition curves shown in Figures 2 and 3 for the various tissues studied .
~ so, ~oo-
FIG . 2 Inhibition b cloy line of MAO activit toward octo amine in rat heart ~e an .vas e erens . ac po nt s t e mean t s .e . o qua rup cafe eterm na ons upon a single homogenate prepared from Final octopamine concentration was 2 mM. pooled tissues of 3 rats . In heart, kidney and vas deferens homogenates, around 90-100% of enzyme act~,vity wa~ inhibited after preincubation with clorgyline concentrations of 10 - to 10 - M (Figure 2) . From Johnston's original results defining type A or B MAO on the basis of their different sensitivities toward clorgyline (4) the present findings would seem to indicate that octopamine is metabolized predominantly or virtually exclusively by type A MAO in these particular tissues . On the other hand, higher concentrations of clorgyline were required to inhibit completely the MAO activity of rat brain and liver, and the presence of a plateau ~egion in these inhibition curves at clorgyline concentrations of around 10 -7 to 10- 6 M indicated that octopamine is metabolized by both types A and B MAO in these tissues (Figure 31 . Although, from the corresponding curves, type B MAO was responsible for only a relatively small proportion (15-20%) of total octopamine metabolism in rat brain under these assay conditions, this proportion appears to be much greater (around 40%) in rat liver . It was also fpund clorgyline (10 to trol values occurred tion of MAO activity
tha~ in the presence of relatively low concentrations of 10- .M), a small increase in enzyme activity above conin some tissue homogenates . An apparent slight activaby low concentrations of clorgyline has been observed on
22g
Metabolism of Octopamiae
Vol . 23, No . 3, 1978
FIG . 3 Inhibition b clo line of MAO activit bra n an ver . egen as in g.
toward octo amine in rat
previous occasions (g,~10) and although this effect is not clearly understood, it may be due to an interaction of the lipophilic inhibitor clorgyline with lipid structure around the membrane-bound enzyme (10,11) . Discussion In a direct attempt to investigate whether or not octopamine may undergo deamination in a variety of rat tissues, radioactively-labelled DL-octopamine has been used as the substrate for MAO in a previously described radiochemical assay for the enzyme (8) . It was, indeed, possible by this method to detect octopamine metabolism in each tissue, and it would appear that the radioactive metabolites which were measured, resulted from the action of MAO on this substrate, since the preincubation of tissue homogenates with high concentrations (10 -4 M) of the irreversible MAO inhibitor clorgyline reduced the apparent enzyme activity to blank values . Conditions were determined for each tissue such that the enzymic formation of product was linear with respect to both incubation time and protein concentration within the assay . These conditions were then used in the subsequent estimation of kinetic constants for octopamine deamination in each tissue . Lineweaver-Burk plots for metabolism of octopamine by MAO revealed a single, linear component for each tissue with a Km value (at pH 7 .8) ranging from approximately 0 .70 to 1 .05 x 10 - ~ M . Apparent Vmax values indicated relative enzyme activities in the order heart>liver>vas deferens>brain>kidney . However, MAO activity toward other substrates in tissues such as rat heart (12) and vas deferens (13) has been found to change during adult development of the rat, and thus the relative tissue enzyme activities
Vol . 23, No . 3, 1978
Metabolism of Octopamine
22 9
indicated in the present .study with rats weighing around 200g, may well be different at other ages . Inhibition studies with clorgyline revealed that octopamine is predominantly, 1f not exclusively metabolized by type A MAO in rat heart, vas deferens and kidney, although all of these tissues are known to contain a type B component which acts upon other substrates (9,14 ; Shaffer and Lyles, in preparation) . However, the present results with rat brain and more particularly liver, indicate that octopamine can be metabolized by both MAO forms in these tissues . Our findings with rat liver are in contrast to those of Houslay and Tipton (5), who used an oxygen electrode assay method to study octopamine metabolism by MAO in an outer mitochondrial membrane preparation, and concluded that this amine is a substrate solely for type A MAO in this tissue . There appear to be no obvious reasons for this discrepancy at the moment . Although there is now good evidence that octopamine is associated predominantly with adrenergic neurones in sympathetically innervated tissues (1,2), it is unlikely that the MAO activity toward octopamine measured in the present study solely represents metabolism brought about by intraneuronal enzyme . It has previously been proposed that type A MAO in some tissues (such as vas deferens and pineal gland) may be localized preferentially within adrenergic neurones (14,15) . However, MAO activity in rat heart and liver, organs which contain both types A and B MAO components, is believed to exist almost completely in extraneuronal locations (16,17) . Thus, it seems likely that a variety of cell types possess the capacity to deaminate octopamine . In summary, the present results describe some kinetic properties and some observations on the sensitivity toward inhibition of octopamine deamination in vitro by various rat tissues, several of which have been reported to contâin measurable in vivo concentrations of this amine (1) . Our investigations have been made witht~ racemic mixture of octopamine . To the author's knowledge, this currently appears to be the only form of radiolabelled octopamine which is commercially available from the usual sources, and this material is being widely used in various other studies into the possible biochemical and physiological functions of octopamine . For this reason, information about the potential deamination of this compound is extremely useful . However, MAO has been reported to show a greater specificity toward the naturally-occurring levorotatory isomers of catecholamines such as noradrenaline and adrenaline (18) . Whether or .not such specificity exists also for levo-octopamine is not known at present, but clearly such a possibility should be kept in mind when considering the disposition and metabolic fate of octopamine in experiments using racemic mixtures of this compound . Acknowledgements This work was supported by NIH Grant No . NS 12747-01 . The personal support from a Wellcome Trust Travel Grant, and a Hilton A . Smith Postdoctoral Fellowship, University of Tennessee is gratefully acknowledged . References 1. 2. 3. 4. 5. 6.
P . B . MOLINOFF and S . H . BUCK, in Trace Amines and the Brain 131-160 (Eds . E . Usdin and M . Sandier ; Pub . Marce e er, ew Yor , J . AXELROD and J . M . SAAVEDRA, Nature 265 501-504 (1977) . Y . KAKIMOTO and M . D . ARMSTRONG, J . bid Chem . 237 422-427 (1962) . J . P . JOHNSTON, Biochem . Pharmac . 17 1285-1297 (T4~8) . M . D . HOUSLAY and K . F . TIPTON, Biochem . J . 139 645-652 (1974) . C . J . FOWLER, B . A . CALLINGHAM ., T . J . MANTL~nd K . F . TIPTON, Biochem . Pharmac . 27 97-101 (1978) .
230 7. 8. 9. 10. 11 . 12 . 13 . 14. 15 . 16 . 17 .
Metabolinm of Octopaminn
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