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Effect of monophenolic amines on glycogen metabolism in the nerve-cord of the american cockroach, Periplaneta americana
Insect Biochem., i973, 3, 53-59. [Scientechnica (Publishers) Ltd.]
53
EFFECT OF M O N O P H E N O L I C AMINES ON GLYCOGEN METABOLISM IN T H E NERVE-CORD OF THE AMERICAN COCKROACH, P E R I P L A N E T A A M E R I C A N A H. A. ROBERTSON ANDJ. E. STEELE Department of Zoology, University of Western Ontario, London 72, Canada (Received 13 July, x972)
ABSTRACT As little as 25 nmoles of tyramine or its ~-hydroxylated derivative octopamine have been shown to have a strong glycogenolytic effect on the cockroach nerve-cord in vivo. This effect results from the activation of phosphorylase. Tyramine, octopamine, and synephrine were shown to activate nerve-cord phosphorylase in vitro at concentrations as low as 5 × I O - ' M. Analogues of the phenolic amines having two hydroxyl groups on the ring (the catecholamines dopamine, norepinephrine, and epinephrine) or those having no hydroxyl groups on the ring (~-phenyIethy]arrdne and ~-hydroxy-~-phenylethylamine) were without effect on phosphorylase. Evidence is presented to show that octopamine is not identical with the corpus cardiacum factor causing glycogenolysis in the nerve-cord.
RECENTLY we reported that octopamine (DL-p-hydroxyphenylethanolamine) could induce glycogenolysis in the nerve-cord of the cockroach (P. americana) in vivo (Robertson and Steele, 1972 ). This glycogenolysis was shown to be the result of increased phosphorylase activity and evidence was presented to suggest that octopamine acted by increasing the rate of synthesis of cyclic adenosine 3',5'-monophosphate (cyclic AMP). Octopamine acts, therefore, in a manner analogous to that of epinephrine and glucagon in mammalian liver (Rail, Sutherland, and Berthet, 1957) and hyperglycaemic hormone in the insect fat body and nerve-cord (Steele, 1963, 1964; Hart and Steele, 1972 ) . It should be pointed out that the factor from the corpus cardiacum which induces glycogenolysis in the nerve-cord may differ from that acting on the fat body, although there is no evidence to support this idea. The mode of action in both instances, however, appears to be the same, involving increased phosphorylase activity resulting from an increase in cyclic AMP levels (Steele, 1963, i964; Hart and Steele, 1972 ). Activation of phosphorylase in cockroach nerve-cord by corpus cardiacum extract, cyclic AMP, and octopamine (Hart and Steele, 1973 ; Robertson and Steele, 1972 ) is the only demonstration of increased phosphorylase activity in nerve tissue resulting from augmented levels of cyclic AMP. Previously, Kakiuchi and Rall (i968a) were able to demonstrate increased levels of cyclic AMP in guinea-pig brain slices after treatment with norepinephrine, histamine, and serotonin. However, these conditions did not result in increased phosphorylase activity (Kakiuchi and Rall, I968b ). The observation that octopamine increased phosphorylase activity in the nerve-cord (Robertson and Steele, 1972) is interesting in view of the reported inactivity of epinephrine (Hart and Steele, i969) , which is known to be effective in activating phosphorylase in fat body (Steele, i972 ). This study is concerned with the effects of some other adrenergic agents
54
ROBERTSON AND STEELE
Insect Biochem.
o n p h o s p h o r y l a s e activity a n d with the relationship b e t w e e n o c t o p a m i n e - i n d u c e d phosphorylase activation a n d that i n d u c e d b y extracts of the corpus c a r d i a c u m . MATERIALS AND METHODS INSECTS
The cockroachesused in this study were obtained from a colonymaintained in our laboratory. They were raised at 25° C. and 7o per cent r.h. on a i2-hour light : 12-hour dark photoperiod. The insects were fed Dog Chow and water ad libitum. Only 2-month-old adult male cockroaches were used in the experiments. CHEMICALS
L-Arterenol bitartrate (norepinephrine), dihydroxyphenylethylamine (dopamine), L-epinephrine bitartrate, DL-p-hydroxyphenylethanolamine hydrochloride (octopamine), I(4-hydroxyphenyl)-2-methylaminoethanol free base (synephrine),p-hydroxyphenylethylamine hydrochloride (tyramine), and chymotrypsin were obtained from Sigma Chemical Co. DL-~-Hydroxy-~-phenylethylamine hydrochloride and ~-phenylethylamine hydrochloride were obtained from K and K Laboratories. EXPERIMENTALPROCEDURES The physiological saline used throughout this study was that of Pringle (i938) with minor modification. Glucose was omitted and 5 m M Tris-HC1 buffer, pH 6'8, included. All dissections and incubations were carried out in this solution. The incubations were performed in Io-ml. beakers containing 2"o ml. of Pringle's solution in a shaking water-bath at 30 ° C.
ANALYTICAL METHODS
Phosphorylase activitywas measured in the direction of glycogen synthesis by determining the amount of phosphate released from glucose-I-phosphatein the presence of a small amount of primer glycogen. Tissue samples (2 nerve-cords) were homogenized for I minute in i'o ml. o'oi M E D T A in o'I M N a F using a glass homogenizer with a Teflon pestle. The homogenates were centrifuged for IO minutes at 27,ooo g and o'25 ml. of the supernatant added to o'75 ml. of phosphorylase assay medium (Steele, I963). A second aliquot of supernatant was added to phosphorylase assay medium containing2"5 m M s ' - A M P . I n b o t h i n s t a n c e s o n e - h a l f o f t h e sample was immediately withdrawn to provide a zero-time phosphate determination. The remaining mixture was incubated for I5 minutes at 3o ° C. in a shaking water-bath. In both zero- and 15minute samples the reaction was stopped by the addition of o'5 ml. of 5 per cent trichloroacetic acid. Inorganic phosphate was determined using the method of Fiske and Subbarow (1925). A comparison of phosphorylase activity obtained in the presence and absence of 5'-AMP permitted calculation of the amount of enzyme which was present in the active form. Glycogen was isolated from individual nerve-cords by digesting each cord in I'O ml. of boiling 3o per cent K O H for 2o minutes. The glycogen was precipitated according to the method of Van Handel (1965) and measured with anthrone reagent as described by Carroll, Longley, and Roe (1956).
RESULTS Previous studies (Hart and Steele, i969; Robertson and Steele, 1972) have shown that epinephrine is without an effect on nerve-cord phosphory]ase while octopamine activates at very low concentrations. Because these compounds are structurally similar it was of interest to extend those observations to a number of other related amines. The results are presented in Table I. None of the catecholamines (dopamine, norepinephrine, and epinephrine) tested had any effect on phosphorylase activity, thus confirming the observation of Hart and Steele (1969). In contrast, each of the monophenolic amines (tyramine, octopamine, and synephrine) caused a pronounced activation of nerve-cord phosphorylase (Table 11). There is no evidence to suggest any difference in the response
1973, 3
DRUG EFFECTS ON GLYCOGEN METABOLISM
55
of the enzyme to any particular monophenolic amine, since all produced marked increases in activity at a concentration of I × IO -5 M. The activation of nerve-cord phosphorylase by the monophenolic amines suggested that glycogen metabolism would be altered by the same substances. This is shown in Table I l L Because the catecholamines did not activate phosphorylase, there was no justification for an examination of their effects on glycogen levels within the nerve-cord. One other class of compounds, structurally related to those already investigated, which have not been examined for possible effects on glycogen or phosphorylase, is that including the compounds [3-hydroxy-[3-phenylethylamine and [3-phenylethylamine. These compounds are similar to dopamine and norepinephrine and tyramine and Table / . - - T H E EFFECT OF CATECHOLAMINESON NERVE-CORD PHOSPHORYLASEACTIVITY in vitro (Phosphorylase activity expressed as percentage enzyme in active form) EXPERIMENTAL CONTROL Catecholamines 55"7±3"2 (3) 54"7±2"2 (4) 67"4±3"1 (3)
Dopamine, 5 × IO-S M Norepinephrine, I × IO-4 M Epinephrine, I × lO -4 M
Result 58"8 +2"7 (3) 69"8 ±3"9 (4) 70"0±4"8 (3)
CHANGE (per cent) +5"6 +7"9 +3"8
P
N.S. N.S. N.S.
T h e figures given are the means ± SEM. T h e numbers in parentheses represent the number of samples. Each experiment consisted of two nerve-cords incubated in z'o ml. of Pringle's solution buffered to p H 6"8 with 5 m M Tris-HC1, with or without the various catecholamines at the concentrations shown. After I hour at 3o ° C. in a shaking water-bath the nerve-cords were removed and homogenized in i ' o ml. of ioo m M N a F containing io m M E D T A . Th e homo° genate was centrifuged at 2%ooo g for IO minutes and the supernatant used for the determination of phosphorylase activity. Table H . - - T H E EFFECT OF MONOPHENOLIC AMINES ON NERVE-CORD PHOSPHORYLASE ACTIVITY in vitro (expressed as percentage enzyme in active form) EXPERIMENTAL
CHANGE
CONTROL Amine 67"9±3"2 (9) 55"4±5"6 (4) 67.3+2"4 (3) 57"8-4-3"7 (7) 63"3±5"9 (3) 7I-6±1"8 (3) 58"2±1"7 (3) 56'6±4"~ (9) 58"2±5"9 (3) 45"3 ± 2"7 (4) 51"5 ±5"0 (4)
Tyramine, Tyramine, Tyramine, Octopamine, Octopamine, Octopamine, Octopamine, Octopamine, Octopamine, Synephrine, Synephrine,
5 × io-3 M I × IO -4 M I × io -5 M i × lO -3 M i x io -4 M I × lO -5 M 5 × IO-6 M I × io -e M 5 × lO-7 M i × io-3 M i × io -5 M
(per cent)
Result
85"I 4-z'3 7z'4-V4"8 8v6±2"* 87"3 4-2"0 94"I ± o ' 6 90"8 ±0"8 77"1 ± z ' 5 7°'°±4"2 73"4±2"1 81"9±3"5 85"5±1"8
(9) (4) (3) (7) (3) (3) (3) (9) (3) (4) (4)
+25 +3 I +21 +51 +49 +27 +32 +I9 +26 +8~ +66
P
<0.05
T h e values given are the means-4-SEM. T h e figures in parentheses represent the number of samples. Experimental details as in Table I.
56
ROBERTSON AND STEELE
Insect Biochem.
o c t o p a m i n e except for the absence of h y d r o x y l g r o u p s on the p h e n o l ring. I t is apparent f r o m Table I V that these c o m p o u n d s do not have a glycogenolytic effect, consequently it is unlikely that t h e y activate phosphorylase. Table IlL--THE EFFECT O17MONOPHENOLICAMINESON NERVE-CORDGLYCOGENin vivo* TREATMENT
Control Tyramine Octopamine
GLYCOGEN
CHANGE
(~g. per mg.)
(per cent)
6"44-0"4 (24) 5"0 4-0"5 (24) 4"3 ± o ' 3 (24)
--22
P
0"05 0"05
--36
* The figures presented are the means ± SEM with the number of samples in parentheses. Each cockroach was injected with Io ~tl. of Pringle's solution containing 25 nmoles of tyramine or octopamine. Controls received Pringle's solution alone. Nerve-cords were removed 2 hours after injection and glycogen levels determined as described in the text. Table I V . - - T H E EFFECT OF TWO PHENYLETHYLAMINES ON NERVE-CORD GLYCOGEN in vivo*
TREATMENT
Control 13-Phenylethylamine 13-Hydroxy-13-phenylethylamine
GLYCOGEN
CHANGE
(~g. per mg.)
(per cent)
9"o-4-1"9 (5) IO'I ~zo'8 (5) IO"9 4-0"8 (4)
P
N.S.
+7"4 +x5"5
N.S.
* The figures presented are the means 4-SEM. The numbers in parentheses represent the number of samples. Experimental details as in Table III. Table V . - - T H E EFFECT OF CHYMOTRYPSlN DIGESTION ON THE GLYCOGENOLYTIC PROPERTIES OF THE INSECT HYPERGLYCAEMIC HORMONE AND OCTOPAMINE
TREATMENT
Chymotrypsin Hormone Hormone Hormone + chymotrypsin Hormone 4- denatured chymotrypsin Octopamine Octopamine + chymotrypsin
INCUBATIONTIME (minutes) 6o o
60 60 6o
60 6o
GLYCOGEN
CHANGE
0tg. per mg.)
(per cent)
7'174-o"72 (IO) 2"29-4-o"12 (5) 2"6o4-o"17 (5) 8 " 5 7 i i ' 3 o (5)
--68 --64 +20
<0"05 <0.05 N.S.
2"oo4-o"I4 (5) 3"874-0"49 (5) 3"724-0"44 (5)
--72 --48 --46
<0"05 <0"05 <0"05
P
All samples were incubated in phosphate buffer (o'oi M, p H 7"85) at 3°0 C. in a shaking waterbath. Corpus cardiacum extract (IO pairs of glands per ml.) and octopamine (0-948 mg. per ml.) was included where indicated. The concentration of chymotrypsin was 0"5 mg. per ml. Following incubation the protein was heat denatured and removed by centrifugation. Ten-~tl. samples of the supernatant were assayed for glycogenolytic activity by injection into intact cockroaches. Nerve-cords were removed i hour later and the glycogen content determined.
I973, 3
DRUG EFFECTS ON GLYCOGEN METABOLISM
57
The glycogenolytic and phosphorylase-activating effect of tyramine and its derivatives raises the important consideration of whether the glycogenolytic factor in the corpus cardiacum is similar to or identical with a monophenolic amine. It was most expedient first to determine whether the active material contained in the corpus cardiacum was a peptide. If it were a peptide, chymotrypsin would be expected to inactivate it but be without an effect should it be a monophenolic amine. In order to ensure that monophenolic amines, if present, would be carried through the experimental procedures, octopamine was incubated with chymotrypsin prior to assay, in the same manner as the corpora cardiaca extracts. In this way the possibility that monophenolic amines would be precipitated with the enzyme or otherwise altered or destroyed was eliminated. On completion of the enzymatic digestion each of the samples and controls was assayed for activity by determining its glycogenolytic effect in the nerve-cord of the intact cockroach. The results are shown in Table 11, conclusively demonstrating that octopamine is not identical with the active factor contained in the corpus cardiacum. DISCUSSION This report confirms the observation by Hart and Steele (1969) that epinephrine has no effect on nerve-cord phosphorylase activity in vitro and at the same time extends these observations to show that its precursors, dopamine and norepinephrine, are similarly without effect. Although a number of authors (Ostlund, 1954; Von Euler, 1961; Frontali and Haggendal, 1969) claim to have demonstrated dopamine and norepinephrine in insect nervous tissues, our observations would seem to preclude a role for them as regulators of glycogen metabolism. The activation of nerve-cord phosphorylase by the monophenolic amines is in contrast to the effect of the catecholamines (Table 1). An examination of the structure of these compounds would suggest that a single hydroxyl group on the ring in the para position is required for activity. The addition of a second hydroxyl group in the meta position results in complete loss of activity. There is now good evidence to suggest that activation of nerve cord phosphorylase by extracts of the corpus cardiacum and monophenolic amines occurs as a result of an accumulation of cyclic adenosine 3',5'-monophosp hate (Hart and Steele, 1973 ; Robertson and Steele, 1972) . These results are important because they appear to be the first report of hormonal or biogenic amine-mediated activation of phosphorylase in a nervous tissue. It is noteworthy that catecholamines, while increasing levels of cyclic AMP in mammalian nervous tissue, do not alter phosphorylase activity (Kakiuchi and Rail, I968b ). The significance of nerve-cord phosphorylase activation, either by the corpus cardiacure factor or monophenolic amines, is uncertain. It may be important to remember that insect nervous tissue contains an order of magnitude more glycogen than does mammalian nerve tissue (Treherne, 1965). Wigglesworth (196o) showed that the prominent food reserve in cockroach nerve-cord is glycogen found in the perineurium cells. At present it is uncertain how the regulation of phosphorylase and the degradation of glycogen is related to the trophic function of these cells. The activation of nerve-cord phosphorylase by the monophenolic amines and the resulting glycogenolysis is most interesting in view of the observations by Carlson (i968a , b) on the firefly light organ. The monophenolic amines were the most active in eliciting a response from the light organ of all adrenergic compounds tested. Smalley (1965) has suggested that the transmitter involved in activation of the light organ controls glycogen metabolism in the end-cells, which is in
58
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Insect Biochem.
agreement with the observation of Smith (1963) that the tracheal end-cells contain large deposits of glycogen. Neither tyramine nor its derivatives have been demonstrated in insect nervous tissue. However, whole insect homogenates have been shown to contain tyramine (Sekeris and Karlson, i962 ). Furthermore, Whitehead (1969) has shown that haemocytes can decarboxylate tyrosine to produce tyramine, while cockroach haemolymph can catalyse the [3-hydroxylation of tyramine to yield octopamine (Lake, Mills, and Brunet, 197o ). In our studies we have obtained evidence to show that cockroach nerve-cord can [3-hydroxylate tyramine (Robertson and Steele, 1973). This is not surprising, since Frontali and Haggendal (1969) have shown that cockroach nerve-cord contains dopamine and norepinephrine, and the same enzyme is known to 13-hydroxylate tyramine as well as dopamine (Kaufman and Friedman, 1965). T h e recent demonstration by Barker, Molinoff, and Kravitz (i972) and Molinoff and Axelrod (i972) that lobster nerve-cord contains relatively large amounts of octopamine is particularly interesting in the light of our observations. In the absence of an unequivocal demonstration of tyramine or its derivatives in insect nerve tissue the physiological significance of the observations reported here must remain in doubt. At the present time various nervous tissues are being analysed for their content of tyramine and derivatives so that, hopefully, a rationale may be provided for the observations made in the present study.
REFERENCES BARKER,D. L., MOLINOFF,P. B., and KRAVlXZ,E. A. (1972), ' Octopamine in the lobster nervous system', Nature, Lond., (New Biol.), 236, 61-62. CARLSON, A. D. (I968a), 'Effect of adrenergic drugs on the lantern of the larval Photuris firefly', J. exp. Biol., 48, 381-387. CARLSON, A. D. (I968b), 'Effect of drugs on luminescence in larval fireflies', ft. exp. Biol., 49, I95-I99. CARROLL, N. V., LONGLEY,R. W., and ROE, J. H. (1956), 'The determination of glycogen in liver and muscle by use of anthrone reagent', ~. biol. Chem., 220, 583-593. FISKE, C. H., and SUBBAROW,Y. (1925), 'The cholorimetric determination of phosphorus', ft. biol. Chem., 66, 375-4oo. FaONTALI, N., and HAGOENOAL, I. (1969), 'Noradrenaline and dopamine content in the brain of the cockroach, Periplaneta americana', Brain Res., 14, 540-542. HART, D. E., and STEELE,J. E. (I969), ' Inhibition of insect nerve cord phosphorylase activity by 5-hydroxytryptamine', Experientia, 25, 243. HART, D. E., and STEELE,J. E. (I973),ff. Insect Physiol., in the press. KAKIUCHI, S., and RALL, T. W. (I968a), ' The influence of chemical agents on the accumulation of adenosine 3',5' phosphate in slices of rabbit cerebellum', Mol. Pharmac., 4, 367-378. KAKIUCHI, S., and RALL, T. W. (1968b), ' Studies on adenosine 3',5' phosphate in rabbit cerebral cortex', Mol. Pharmac., 4, 379-388. KAUFMAN, S., and FRIEDMAN, S. (I965), 'Dopamine-13-oxidase', Pharmac. Rev., 17, 71-1oo. LAKE, C. R., MILLS R. R., and BRUNET,P. C. J. (i97o), ' ~-Hydroxylation of tyramine by cockroach haemolymph', Biochim. biophys. Acta, 215, 226-228. MOLINOFF, P. B., and AXELROD,J. (I972), ' Distribution and turnover of octopamine in tissues', J. Neurochem., 19, 157-163. OSTLUND, E. (I954), 'The distribution of catecholamines in lower animals and their effect on the heart', Acta physiol, scand., 3I, Suppl. I I2, 1-67. PRINGLE, J. W. S. (I938), 'Proprioception in insects. 1. A new type of mechanical receptor from the palps of the cockroach', ft. exp. Biol., 15~ lOI-113.
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RaLL, T. W., SUTHERLAND,E. W., and BERTHET, J. (x957), ' T h e relationship of epinephrine and glucagon to liver phosphorylase. IV. The effect of epinephrine and glucagon on the reactivation of phosphorylase in liver homogenate', )t. biol. Chem., 224, 463-475. ROBERTSON, H. A., and STEELE,J. E. (I972), 'Activation of insect nerve cord phosphorylase by octopamine and adenosine 3',5' monophosphate', ft. Neurochem., I9~ 16o3-16o6. ROBERTSON,H. A., and STEELE,J. E. (1973), unpublished work. SEKERIS, C. E., and KAaLSON, P. (I962), ' Z u m Tyrosinstoffwechsel der Insekten. VII. Der Katabolische abbau des Tyrosins und die Biogenese der Sklerotisierungssubstanz n-Acetyldopamin. Biochim. biophys. Acta, 62, Io3-I I3. SMALLEY, K. N. (I965), 'Adrenergic transmission in the light organ of the firefly, Photinus pyralis', Comp. Bioehem. Physiol., I6, 467-477. SMITH, D. S. (I963), ' T h e organization and innervation of the luminexent organ in a firefly Photinus pyralis', ft. cell. Biol., I6, 323-359. STEELE, J. E. (I963), ' T h e site of action of insect hyperglycaemic hormone', Gen. comp. Endocrin., 3, 46-52. STEELE, J. E. (i964), ' T h e activation of phosphorylase in an insect by adenosine 3',5'-phosphate and other agents. Am. Zool., 4, 328. STEELe, J. E. (i972), unpublished work. TRmmRNE, J. E. (1965), The neuroehemistry of Arthropods. Cambridge Monographs in Experimental Biology. No. 14 . London: Cambridge University Press. VAN HANDEL, E. (I965), 'Estimation of glycogen in small amounts of tissue', Analyt. Biochem., IX9 256-265. VON EULER, U. S. (I96I), 'Occurrence of catecholamines in acrania and invertebrates', Nature, Lond., 19o~ 17o-171. WHITEHEAD, D. L. (I969), ' N e w evidence for the control mechanism of sclerotization in insects', Nature, Lond., 224, 721-723. WIGGLESWORTH, V. B. (I96O), ' T h e nutrition of the central nervous system in the cockroach Periplaneta americana L. The role of the perineurium and glial cells in the mobilization of reserves',ff, exp. Biol., 37, 5oo--513.
Key Word Index: Catecholamines, glycogen phosphorylase, hyperglycaemic hormone, insect, octopamine, tyramine.
53
EFFECT OF M O N O P H E N O L I C AMINES ON GLYCOGEN METABOLISM IN T H E NERVE-CORD OF THE AMERICAN COCKROACH, P E R I P L A N E T A A M E R I C A N A H. A. ROBERTSON ANDJ. E. STEELE Department of Zoology, University of Western Ontario, London 72, Canada (Received 13 July, x972)
ABSTRACT As little as 25 nmoles of tyramine or its ~-hydroxylated derivative octopamine have been shown to have a strong glycogenolytic effect on the cockroach nerve-cord in vivo. This effect results from the activation of phosphorylase. Tyramine, octopamine, and synephrine were shown to activate nerve-cord phosphorylase in vitro at concentrations as low as 5 × I O - ' M. Analogues of the phenolic amines having two hydroxyl groups on the ring (the catecholamines dopamine, norepinephrine, and epinephrine) or those having no hydroxyl groups on the ring (~-phenyIethy]arrdne and ~-hydroxy-~-phenylethylamine) were without effect on phosphorylase. Evidence is presented to show that octopamine is not identical with the corpus cardiacum factor causing glycogenolysis in the nerve-cord.
RECENTLY we reported that octopamine (DL-p-hydroxyphenylethanolamine) could induce glycogenolysis in the nerve-cord of the cockroach (P. americana) in vivo (Robertson and Steele, 1972 ). This glycogenolysis was shown to be the result of increased phosphorylase activity and evidence was presented to suggest that octopamine acted by increasing the rate of synthesis of cyclic adenosine 3',5'-monophosphate (cyclic AMP). Octopamine acts, therefore, in a manner analogous to that of epinephrine and glucagon in mammalian liver (Rail, Sutherland, and Berthet, 1957) and hyperglycaemic hormone in the insect fat body and nerve-cord (Steele, 1963, 1964; Hart and Steele, 1972 ) . It should be pointed out that the factor from the corpus cardiacum which induces glycogenolysis in the nerve-cord may differ from that acting on the fat body, although there is no evidence to support this idea. The mode of action in both instances, however, appears to be the same, involving increased phosphorylase activity resulting from an increase in cyclic AMP levels (Steele, 1963, i964; Hart and Steele, 1972 ). Activation of phosphorylase in cockroach nerve-cord by corpus cardiacum extract, cyclic AMP, and octopamine (Hart and Steele, 1973 ; Robertson and Steele, 1972 ) is the only demonstration of increased phosphorylase activity in nerve tissue resulting from augmented levels of cyclic AMP. Previously, Kakiuchi and Rall (i968a) were able to demonstrate increased levels of cyclic AMP in guinea-pig brain slices after treatment with norepinephrine, histamine, and serotonin. However, these conditions did not result in increased phosphorylase activity (Kakiuchi and Rall, I968b ). The observation that octopamine increased phosphorylase activity in the nerve-cord (Robertson and Steele, 1972) is interesting in view of the reported inactivity of epinephrine (Hart and Steele, i969) , which is known to be effective in activating phosphorylase in fat body (Steele, i972 ). This study is concerned with the effects of some other adrenergic agents
54
ROBERTSON AND STEELE
Insect Biochem.
o n p h o s p h o r y l a s e activity a n d with the relationship b e t w e e n o c t o p a m i n e - i n d u c e d phosphorylase activation a n d that i n d u c e d b y extracts of the corpus c a r d i a c u m . MATERIALS AND METHODS INSECTS
The cockroachesused in this study were obtained from a colonymaintained in our laboratory. They were raised at 25° C. and 7o per cent r.h. on a i2-hour light : 12-hour dark photoperiod. The insects were fed Dog Chow and water ad libitum. Only 2-month-old adult male cockroaches were used in the experiments. CHEMICALS
L-Arterenol bitartrate (norepinephrine), dihydroxyphenylethylamine (dopamine), L-epinephrine bitartrate, DL-p-hydroxyphenylethanolamine hydrochloride (octopamine), I(4-hydroxyphenyl)-2-methylaminoethanol free base (synephrine),p-hydroxyphenylethylamine hydrochloride (tyramine), and chymotrypsin were obtained from Sigma Chemical Co. DL-~-Hydroxy-~-phenylethylamine hydrochloride and ~-phenylethylamine hydrochloride were obtained from K and K Laboratories. EXPERIMENTALPROCEDURES The physiological saline used throughout this study was that of Pringle (i938) with minor modification. Glucose was omitted and 5 m M Tris-HC1 buffer, pH 6'8, included. All dissections and incubations were carried out in this solution. The incubations were performed in Io-ml. beakers containing 2"o ml. of Pringle's solution in a shaking water-bath at 30 ° C.
ANALYTICAL METHODS
Phosphorylase activitywas measured in the direction of glycogen synthesis by determining the amount of phosphate released from glucose-I-phosphatein the presence of a small amount of primer glycogen. Tissue samples (2 nerve-cords) were homogenized for I minute in i'o ml. o'oi M E D T A in o'I M N a F using a glass homogenizer with a Teflon pestle. The homogenates were centrifuged for IO minutes at 27,ooo g and o'25 ml. of the supernatant added to o'75 ml. of phosphorylase assay medium (Steele, I963). A second aliquot of supernatant was added to phosphorylase assay medium containing2"5 m M s ' - A M P . I n b o t h i n s t a n c e s o n e - h a l f o f t h e sample was immediately withdrawn to provide a zero-time phosphate determination. The remaining mixture was incubated for I5 minutes at 3o ° C. in a shaking water-bath. In both zero- and 15minute samples the reaction was stopped by the addition of o'5 ml. of 5 per cent trichloroacetic acid. Inorganic phosphate was determined using the method of Fiske and Subbarow (1925). A comparison of phosphorylase activity obtained in the presence and absence of 5'-AMP permitted calculation of the amount of enzyme which was present in the active form. Glycogen was isolated from individual nerve-cords by digesting each cord in I'O ml. of boiling 3o per cent K O H for 2o minutes. The glycogen was precipitated according to the method of Van Handel (1965) and measured with anthrone reagent as described by Carroll, Longley, and Roe (1956).
RESULTS Previous studies (Hart and Steele, i969; Robertson and Steele, 1972) have shown that epinephrine is without an effect on nerve-cord phosphory]ase while octopamine activates at very low concentrations. Because these compounds are structurally similar it was of interest to extend those observations to a number of other related amines. The results are presented in Table I. None of the catecholamines (dopamine, norepinephrine, and epinephrine) tested had any effect on phosphorylase activity, thus confirming the observation of Hart and Steele (1969). In contrast, each of the monophenolic amines (tyramine, octopamine, and synephrine) caused a pronounced activation of nerve-cord phosphorylase (Table 11). There is no evidence to suggest any difference in the response
1973, 3
DRUG EFFECTS ON GLYCOGEN METABOLISM
55
of the enzyme to any particular monophenolic amine, since all produced marked increases in activity at a concentration of I × IO -5 M. The activation of nerve-cord phosphorylase by the monophenolic amines suggested that glycogen metabolism would be altered by the same substances. This is shown in Table I l L Because the catecholamines did not activate phosphorylase, there was no justification for an examination of their effects on glycogen levels within the nerve-cord. One other class of compounds, structurally related to those already investigated, which have not been examined for possible effects on glycogen or phosphorylase, is that including the compounds [3-hydroxy-[3-phenylethylamine and [3-phenylethylamine. These compounds are similar to dopamine and norepinephrine and tyramine and Table / . - - T H E EFFECT OF CATECHOLAMINESON NERVE-CORD PHOSPHORYLASEACTIVITY in vitro (Phosphorylase activity expressed as percentage enzyme in active form) EXPERIMENTAL CONTROL Catecholamines 55"7±3"2 (3) 54"7±2"2 (4) 67"4±3"1 (3)
Dopamine, 5 × IO-S M Norepinephrine, I × IO-4 M Epinephrine, I × lO -4 M
Result 58"8 +2"7 (3) 69"8 ±3"9 (4) 70"0±4"8 (3)
CHANGE (per cent) +5"6 +7"9 +3"8
P
N.S. N.S. N.S.
T h e figures given are the means ± SEM. T h e numbers in parentheses represent the number of samples. Each experiment consisted of two nerve-cords incubated in z'o ml. of Pringle's solution buffered to p H 6"8 with 5 m M Tris-HC1, with or without the various catecholamines at the concentrations shown. After I hour at 3o ° C. in a shaking water-bath the nerve-cords were removed and homogenized in i ' o ml. of ioo m M N a F containing io m M E D T A . Th e homo° genate was centrifuged at 2%ooo g for IO minutes and the supernatant used for the determination of phosphorylase activity. Table H . - - T H E EFFECT OF MONOPHENOLIC AMINES ON NERVE-CORD PHOSPHORYLASE ACTIVITY in vitro (expressed as percentage enzyme in active form) EXPERIMENTAL
CHANGE
CONTROL Amine 67"9±3"2 (9) 55"4±5"6 (4) 67.3+2"4 (3) 57"8-4-3"7 (7) 63"3±5"9 (3) 7I-6±1"8 (3) 58"2±1"7 (3) 56'6±4"~ (9) 58"2±5"9 (3) 45"3 ± 2"7 (4) 51"5 ±5"0 (4)
Tyramine, Tyramine, Tyramine, Octopamine, Octopamine, Octopamine, Octopamine, Octopamine, Octopamine, Synephrine, Synephrine,
5 × io-3 M I × IO -4 M I × io -5 M i × lO -3 M i x io -4 M I × lO -5 M 5 × IO-6 M I × io -e M 5 × lO-7 M i × io-3 M i × io -5 M
(per cent)
Result
85"I 4-z'3 7z'4-V4"8 8v6±2"* 87"3 4-2"0 94"I ± o ' 6 90"8 ±0"8 77"1 ± z ' 5 7°'°±4"2 73"4±2"1 81"9±3"5 85"5±1"8
(9) (4) (3) (7) (3) (3) (3) (9) (3) (4) (4)
+25 +3 I +21 +51 +49 +27 +32 +I9 +26 +8~ +66
P
<0.05
T h e values given are the means-4-SEM. T h e figures in parentheses represent the number of samples. Experimental details as in Table I.
56
ROBERTSON AND STEELE
Insect Biochem.
o c t o p a m i n e except for the absence of h y d r o x y l g r o u p s on the p h e n o l ring. I t is apparent f r o m Table I V that these c o m p o u n d s do not have a glycogenolytic effect, consequently it is unlikely that t h e y activate phosphorylase. Table IlL--THE EFFECT O17MONOPHENOLICAMINESON NERVE-CORDGLYCOGENin vivo* TREATMENT
Control Tyramine Octopamine
GLYCOGEN
CHANGE
(~g. per mg.)
(per cent)
6"44-0"4 (24) 5"0 4-0"5 (24) 4"3 ± o ' 3 (24)
--22
P
0"05 0"05
--36
* The figures presented are the means ± SEM with the number of samples in parentheses. Each cockroach was injected with Io ~tl. of Pringle's solution containing 25 nmoles of tyramine or octopamine. Controls received Pringle's solution alone. Nerve-cords were removed 2 hours after injection and glycogen levels determined as described in the text. Table I V . - - T H E EFFECT OF TWO PHENYLETHYLAMINES ON NERVE-CORD GLYCOGEN in vivo*
TREATMENT
Control 13-Phenylethylamine 13-Hydroxy-13-phenylethylamine
GLYCOGEN
CHANGE
(~g. per mg.)
(per cent)
9"o-4-1"9 (5) IO'I ~zo'8 (5) IO"9 4-0"8 (4)
P
N.S.
+7"4 +x5"5
N.S.
* The figures presented are the means 4-SEM. The numbers in parentheses represent the number of samples. Experimental details as in Table III. Table V . - - T H E EFFECT OF CHYMOTRYPSlN DIGESTION ON THE GLYCOGENOLYTIC PROPERTIES OF THE INSECT HYPERGLYCAEMIC HORMONE AND OCTOPAMINE
TREATMENT
Chymotrypsin Hormone Hormone Hormone + chymotrypsin Hormone 4- denatured chymotrypsin Octopamine Octopamine + chymotrypsin
INCUBATIONTIME (minutes) 6o o
60 60 6o
60 6o
GLYCOGEN
CHANGE
0tg. per mg.)
(per cent)
7'174-o"72 (IO) 2"29-4-o"12 (5) 2"6o4-o"17 (5) 8 " 5 7 i i ' 3 o (5)
--68 --64 +20
<0"05 <0.05 N.S.
2"oo4-o"I4 (5) 3"874-0"49 (5) 3"724-0"44 (5)
--72 --48 --46
<0"05 <0"05 <0"05
P
All samples were incubated in phosphate buffer (o'oi M, p H 7"85) at 3°0 C. in a shaking waterbath. Corpus cardiacum extract (IO pairs of glands per ml.) and octopamine (0-948 mg. per ml.) was included where indicated. The concentration of chymotrypsin was 0"5 mg. per ml. Following incubation the protein was heat denatured and removed by centrifugation. Ten-~tl. samples of the supernatant were assayed for glycogenolytic activity by injection into intact cockroaches. Nerve-cords were removed i hour later and the glycogen content determined.
I973, 3
DRUG EFFECTS ON GLYCOGEN METABOLISM
57
The glycogenolytic and phosphorylase-activating effect of tyramine and its derivatives raises the important consideration of whether the glycogenolytic factor in the corpus cardiacum is similar to or identical with a monophenolic amine. It was most expedient first to determine whether the active material contained in the corpus cardiacum was a peptide. If it were a peptide, chymotrypsin would be expected to inactivate it but be without an effect should it be a monophenolic amine. In order to ensure that monophenolic amines, if present, would be carried through the experimental procedures, octopamine was incubated with chymotrypsin prior to assay, in the same manner as the corpora cardiaca extracts. In this way the possibility that monophenolic amines would be precipitated with the enzyme or otherwise altered or destroyed was eliminated. On completion of the enzymatic digestion each of the samples and controls was assayed for activity by determining its glycogenolytic effect in the nerve-cord of the intact cockroach. The results are shown in Table 11, conclusively demonstrating that octopamine is not identical with the active factor contained in the corpus cardiacum. DISCUSSION This report confirms the observation by Hart and Steele (1969) that epinephrine has no effect on nerve-cord phosphorylase activity in vitro and at the same time extends these observations to show that its precursors, dopamine and norepinephrine, are similarly without effect. Although a number of authors (Ostlund, 1954; Von Euler, 1961; Frontali and Haggendal, 1969) claim to have demonstrated dopamine and norepinephrine in insect nervous tissues, our observations would seem to preclude a role for them as regulators of glycogen metabolism. The activation of nerve-cord phosphorylase by the monophenolic amines is in contrast to the effect of the catecholamines (Table 1). An examination of the structure of these compounds would suggest that a single hydroxyl group on the ring in the para position is required for activity. The addition of a second hydroxyl group in the meta position results in complete loss of activity. There is now good evidence to suggest that activation of nerve cord phosphorylase by extracts of the corpus cardiacum and monophenolic amines occurs as a result of an accumulation of cyclic adenosine 3',5'-monophosp hate (Hart and Steele, 1973 ; Robertson and Steele, 1972) . These results are important because they appear to be the first report of hormonal or biogenic amine-mediated activation of phosphorylase in a nervous tissue. It is noteworthy that catecholamines, while increasing levels of cyclic AMP in mammalian nervous tissue, do not alter phosphorylase activity (Kakiuchi and Rail, I968b ). The significance of nerve-cord phosphorylase activation, either by the corpus cardiacure factor or monophenolic amines, is uncertain. It may be important to remember that insect nervous tissue contains an order of magnitude more glycogen than does mammalian nerve tissue (Treherne, 1965). Wigglesworth (196o) showed that the prominent food reserve in cockroach nerve-cord is glycogen found in the perineurium cells. At present it is uncertain how the regulation of phosphorylase and the degradation of glycogen is related to the trophic function of these cells. The activation of nerve-cord phosphorylase by the monophenolic amines and the resulting glycogenolysis is most interesting in view of the observations by Carlson (i968a , b) on the firefly light organ. The monophenolic amines were the most active in eliciting a response from the light organ of all adrenergic compounds tested. Smalley (1965) has suggested that the transmitter involved in activation of the light organ controls glycogen metabolism in the end-cells, which is in
58
ROBERTSON AND STEELE
Insect Biochem.
agreement with the observation of Smith (1963) that the tracheal end-cells contain large deposits of glycogen. Neither tyramine nor its derivatives have been demonstrated in insect nervous tissue. However, whole insect homogenates have been shown to contain tyramine (Sekeris and Karlson, i962 ). Furthermore, Whitehead (1969) has shown that haemocytes can decarboxylate tyrosine to produce tyramine, while cockroach haemolymph can catalyse the [3-hydroxylation of tyramine to yield octopamine (Lake, Mills, and Brunet, 197o ). In our studies we have obtained evidence to show that cockroach nerve-cord can [3-hydroxylate tyramine (Robertson and Steele, 1973). This is not surprising, since Frontali and Haggendal (1969) have shown that cockroach nerve-cord contains dopamine and norepinephrine, and the same enzyme is known to 13-hydroxylate tyramine as well as dopamine (Kaufman and Friedman, 1965). T h e recent demonstration by Barker, Molinoff, and Kravitz (i972) and Molinoff and Axelrod (i972) that lobster nerve-cord contains relatively large amounts of octopamine is particularly interesting in the light of our observations. In the absence of an unequivocal demonstration of tyramine or its derivatives in insect nerve tissue the physiological significance of the observations reported here must remain in doubt. At the present time various nervous tissues are being analysed for their content of tyramine and derivatives so that, hopefully, a rationale may be provided for the observations made in the present study.
REFERENCES BARKER,D. L., MOLINOFF,P. B., and KRAVlXZ,E. A. (1972), ' Octopamine in the lobster nervous system', Nature, Lond., (New Biol.), 236, 61-62. CARLSON, A. D. (I968a), 'Effect of adrenergic drugs on the lantern of the larval Photuris firefly', J. exp. Biol., 48, 381-387. CARLSON, A. D. (I968b), 'Effect of drugs on luminescence in larval fireflies', ft. exp. Biol., 49, I95-I99. CARROLL, N. V., LONGLEY,R. W., and ROE, J. H. (1956), 'The determination of glycogen in liver and muscle by use of anthrone reagent', ~. biol. Chem., 220, 583-593. FISKE, C. H., and SUBBAROW,Y. (1925), 'The cholorimetric determination of phosphorus', ft. biol. Chem., 66, 375-4oo. FaONTALI, N., and HAGOENOAL, I. (1969), 'Noradrenaline and dopamine content in the brain of the cockroach, Periplaneta americana', Brain Res., 14, 540-542. HART, D. E., and STEELE,J. E. (I969), ' Inhibition of insect nerve cord phosphorylase activity by 5-hydroxytryptamine', Experientia, 25, 243. HART, D. E., and STEELE,J. E. (I973),ff. Insect Physiol., in the press. KAKIUCHI, S., and RALL, T. W. (I968a), ' The influence of chemical agents on the accumulation of adenosine 3',5' phosphate in slices of rabbit cerebellum', Mol. Pharmac., 4, 367-378. KAKIUCHI, S., and RALL, T. W. (1968b), ' Studies on adenosine 3',5' phosphate in rabbit cerebral cortex', Mol. Pharmac., 4, 379-388. KAUFMAN, S., and FRIEDMAN, S. (I965), 'Dopamine-13-oxidase', Pharmac. Rev., 17, 71-1oo. LAKE, C. R., MILLS R. R., and BRUNET,P. C. J. (i97o), ' ~-Hydroxylation of tyramine by cockroach haemolymph', Biochim. biophys. Acta, 215, 226-228. MOLINOFF, P. B., and AXELROD,J. (I972), ' Distribution and turnover of octopamine in tissues', J. Neurochem., 19, 157-163. OSTLUND, E. (I954), 'The distribution of catecholamines in lower animals and their effect on the heart', Acta physiol, scand., 3I, Suppl. I I2, 1-67. PRINGLE, J. W. S. (I938), 'Proprioception in insects. 1. A new type of mechanical receptor from the palps of the cockroach', ft. exp. Biol., 15~ lOI-113.
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RaLL, T. W., SUTHERLAND,E. W., and BERTHET, J. (x957), ' T h e relationship of epinephrine and glucagon to liver phosphorylase. IV. The effect of epinephrine and glucagon on the reactivation of phosphorylase in liver homogenate', )t. biol. Chem., 224, 463-475. ROBERTSON, H. A., and STEELE,J. E. (I972), 'Activation of insect nerve cord phosphorylase by octopamine and adenosine 3',5' monophosphate', ft. Neurochem., I9~ 16o3-16o6. ROBERTSON,H. A., and STEELE,J. E. (1973), unpublished work. SEKERIS, C. E., and KAaLSON, P. (I962), ' Z u m Tyrosinstoffwechsel der Insekten. VII. Der Katabolische abbau des Tyrosins und die Biogenese der Sklerotisierungssubstanz n-Acetyldopamin. Biochim. biophys. Acta, 62, Io3-I I3. SMALLEY, K. N. (I965), 'Adrenergic transmission in the light organ of the firefly, Photinus pyralis', Comp. Bioehem. Physiol., I6, 467-477. SMITH, D. S. (I963), ' T h e organization and innervation of the luminexent organ in a firefly Photinus pyralis', ft. cell. Biol., I6, 323-359. STEELE, J. E. (I963), ' T h e site of action of insect hyperglycaemic hormone', Gen. comp. Endocrin., 3, 46-52. STEELE, J. E. (i964), ' T h e activation of phosphorylase in an insect by adenosine 3',5'-phosphate and other agents. Am. Zool., 4, 328. STEELe, J. E. (i972), unpublished work. TRmmRNE, J. E. (1965), The neuroehemistry of Arthropods. Cambridge Monographs in Experimental Biology. No. 14 . London: Cambridge University Press. VAN HANDEL, E. (I965), 'Estimation of glycogen in small amounts of tissue', Analyt. Biochem., IX9 256-265. VON EULER, U. S. (I96I), 'Occurrence of catecholamines in acrania and invertebrates', Nature, Lond., 19o~ 17o-171. WHITEHEAD, D. L. (I969), ' N e w evidence for the control mechanism of sclerotization in insects', Nature, Lond., 224, 721-723. WIGGLESWORTH, V. B. (I96O), ' T h e nutrition of the central nervous system in the cockroach Periplaneta americana L. The role of the perineurium and glial cells in the mobilization of reserves',ff, exp. Biol., 37, 5oo--513.
Key Word Index: Catecholamines, glycogen phosphorylase, hyperglycaemic hormone, insect, octopamine, tyramine.