The activation and inhibition of adenyl cyclase from the brain of the madagascar cockroach (gromphadorhina portentosa)

The activation and inhibition of adenyl cyclase from the brain of the madagascar cockroach (gromphadorhina portentosa)

Comp. Biochem. Physiol., 1972, Vol. 43B,pp. 209 to 215. Pergamon Press. Printed in Great Britain THE ACTIVATION AND I N H I B I T I O N OF ADENYL CYC...

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Comp. Biochem. Physiol., 1972, Vol. 43B,pp. 209 to 215. Pergamon Press. Printed in Great Britain

THE ACTIVATION AND I N H I B I T I O N OF ADENYL CYCLASE FROM THE BRAIN OF T H E MADAGASCAR COCKROACH (GROMPHADORHINA PORTENTOSA)* A R N O L D S. ROJAKOVICK and R A L P H B. M A R C H Division of Insect Toxicology and Physiology, Department of Entomology, University of California, Riverside, California 92502 (Received 22 December 1971)

Abstract--1. The enzyme adenyl cyclase has been demonstrated for the first

time in insect nerve tissue. 2. The assay employed is linear with time for 20 min. A plot of activity against protein concentration reveals a non-linear response very similar to that previously reported for rat pineal gland adenyl cyclase. 3. Known mammalian adenyl cyclase activators, norepinephrine and epinephrine, significantly stimulate insect adenyl cyclase; however, isoproterenol is not effective. Fluoride ion is a very potent stimulator. 4. Ecdysterone and 4-aminobutryic acid both appear to inhibit cockroach brain adenyl cyclase. INTRODUCTION SINCE its discovery by Sutherland & Rall (1957) and Rallet al. (1957), the production of Y,5'-cyclic adenosine monophosphate (cAMP) from adenosine-5'-triphosphate (ATP) by adenyl cyclase has been extensively studied in mammalian tissues (Greengard & Costa, 1970). The level of cAMP in a cell at any given time is a result of its production by adenyl cyclase and its hydrolysis by phosphodiesterase to 5'-adenosine monophosphate (AMP). These enzymes have been shown to be present in nearly all mammalian tissues, nerve tissue containing the highest levels (Butcher & Sutherland, 1962; Suthedand et al., 1962). In comparison, relatively little work on these enzymes has been reported for invertebrates. Hence, it seemed logical to investigate insect nerve tissue for adenyl cyclase activity and to study the effects of certain known mammalian adenyl cyclase activators, as well as other compounds, upon its activity. MATERIALS AND METHODS Adenosine-8-14C-5"-triphosphate (50mc/mM) was purchased from International Chemical and Nuclear Corp. L-Norepinephrine bitartrate, L-epinephrine bitartrate, nL-isoproterenol HCI and glucagon were obtained from Sigma Chemical Co. Ecdysterone * This investigation was supported by N.I.H. Training Grant ES 47 from the National Institute of Environmental Health Sciences. 209

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ARNOLD S. ROJAKOVICKAND RALPH B. MARCH

was purchased from Schwartz-Mann Biochemical Co. All solutions were made in 5 × 10 -2 M Tris-(hydroxymethyl) aminomethane-HCl (Tris) buffer, pH 7"4. Statistical significance was assessed by the method of Dunnett (1955).

Obtaining the enzyme Adult Madagascar cockroaches (Gromphadorhina portentosa) of both sexes were decapitated and the head capsules were cut open and fastened in a Petri dish by softening the wax base with a hot glass rod. The brain, including the circumesophageal connectives but not the subesophageal ganglion was then excised with the aid of a dissecting microscope. The excised brains were homogenized in a Duall glass homogenizer in 5 × 10 -2 M Tris buffer, p H 7-4 at 0°C. Final tissue concentration was approximately 22 mg (wet wt.)/ml. Protein concentration was determined by the method of Lowry et al. (1951), using bovine serum albumin as a standard. Assay of the enzyme Adenyl cyclase activity was monitored by quantitating the conversion of 14C-ATP into I~C-cAMP. Assays were carried out in small laboratory-fabricated glass bulbs which were discarded after each experiment. Each incubation contained A T P (1"2 x 10 -3 M) including 0"5/zc ofATP-8-14C, MgC12"6H30 (5 x 10 -3 M), caffeine (3 × 10 -3 M) and approximately 100/zg of brain homogenate protein, plus any activator/inhibitor being studied, in a total volume of 100 td, with 5 x 10 -3 M Tris buffer, p H 7-4. Preineubation time of the enzyme with the activator/inhibitor under study was 5 min unless otherwise indicated. The reaction was initiated by the addition of substrate and was carried out for 10 min at 30°C in an agitating water-bath. Termination of the reaction was achieved by placing the incubation vessel in boiling water (92°C) for a period of 3 min. Previously boiled homogenate was used to obtain blank values. The incubation was then cooled and centrifuged to remove denatured protein. The cAMP formed was separated from A T P and other reaction products by the descending paper chromatographic system described and verified by Rabinowitz et al. (1965). Five-/zl aliquots of the centrifuged incubations were applied to Whatman 1 M M filter paper and the chromatogram was developed for 20 hr. After drying at room temperature, the positions of the nucleotides were observed under u.v. light. Marker cAMP was used to verify the location of the separated 14C-cAMP. ATP, ADP and A M P remain near the origin while cAMP migrates about 10 cm. Caffeine migrates with the solvent front. The location of cAMP and other products was also verified by autoradiography. The marker cAMP identified spots were cut out and counted in a scintillation cocktail described by Chase & Rabinowitz (1962) in a Packard Tri-Carb liquid scintillation spectrometer. RESULTS AND DISCUSSION

Consideration of the assay Caffeine was a d d e d to the reaction m i x t u r e to prevent the b r e a k d o w n o f c A M P b y inhibiting phosphodiesterase. H o w e v e r , c h r o m a t o g r a p h y of standards, together with a u t o r a d i o g r a p h y , indicated that this inhibition was not complete since A M P was detected. T h e possibility o f incomplete blocking o f phosphodiesterase b y m e t h y l x a n t h i n e s has also b e e n suggested b y H e p p et al. (1970). F i g u r e 1 d e m o n s t r a t e s t h a t the p r o d u c t i o n of c A M p - 1 4 C f r o m A T P - 1 4 C b y cockroach brain h o m o g e n a t e s is linear with time for 20 min. T h e non-linear effects of increasing a m o u n t s of cockroach brain tissue u p o n catalytic activity b o t h in the presence and absence of 1 m M N a F are s h o w n in Fig. 2. Weiss & Costa (1968) r e p o r t e d similar results with identical protein concentrations for rat pineal

ACTIVATION'AND INHIBITION

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211

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ARNOLD S. ROJAKOVICK AND RALPH B . MARCH

gland adenyl cyclase. With a crude mitochondrial fracton of mouse brain as described by Krishna et al. (1968), we observed perfect linearity with protein concentrations ranging from 150 to 900/zg/incubation. At a protein concentration of 730/,g/incubation this assay is linear with time for 25 min. Stimulation by fluoride ion It is also evident in Fig. 2 that 1 m M NaF stimulated adenyl cyclase activity roughly two- to fivefold. The stimulation of adenyl cyclase by fluoride ion was first observed by Rall & Sutherland (1958) for the mammalian liver enzyme and has since been shown for adenyl cyclase from many sources. Cockroach brain adenyl cyclase is stimulated by fluoride ion over a concentration range of 0.5-20 m M (Fig. 3). Maximal stimulation is observed at 3 mM which is considerably lower E

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ACTIVATION AND INHIBITION OF ADENYL CYCLASE IN MADAGASCAR COCKROACH

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ARNOLD S. ROJAKOVICK AND RALPH B. MARCH

The greater concentrations of epinephrine and norepinephrine required for the stimulation of cockroach adenyl cyclase, plus the failure of stimulation by isoproterenol, suggest a difference in the nature of the active sites of mammalian and cockroach adenyl cyclases. Glucagon has also been demonstrated to have a stimulatory effect on liver adenyl cyclase (Hepp et al., 1970). This protein showed no effect on cockroach nerve adenyl cyclase over a concentration range of 0.1-20/zg/ml.

Inhibition by ecdysterone and 4-aminobutyric acid The prothoracic glands of insects produce a steroid hormone responsible for moulting called ecdysone (Wigglesworth, 1970). Because of the effects of a number of mammalian hormones, albeit not steroid in structure, on adenyl cyclases from different tissues the effects of ecdysterone, a hydroxylated derivative of ecdysone, on cockroach brain adenyl cyclase was examined. Other investigators have also considered the interactions of insect hormones and cAMP. Leenders et al. (1970) observed an increase in the levels of cAMP with extracts of salivary glands of Drosophila hydei incubated with ecdysterone. Berridge et al. (1968, 1970) implicated cAMP, as well as 5-hydroxytryptamine, in control of fluid secretion by salivary glands of the blowfly, Calliphora erythrocephala. Recently, Gilbert et al. (1971) reported that ecdysone injected into diapausing pupae increased the level of cAMP and that juvenile hormone tended to inhibit this process. Both 4-aminobutyric acid and ecdysterone appear to weakly inhibit cockroach brain adenyl cyclase activity (Table 1); however, only the effect of the latter is statistically TABLE 1--COMPARISON OF THE EFFECT OF ECDYSTERONE AND 4-AMINOBUTYRIC ACID ON THE ACTIVITY OF COCKROACH BRAIN ADENYL CYCLASE*

Additions None Ecdysterone (10 -4 M) 4-Aminobutyric acid (10 -4 M)

Cyclic 3',5'-AMP formed

P value

(p mol/mg of protein per min) 294 + 16 246 + 35 270 + 12

< 0"05 > 0'05

*Each value represents the mean of four experiments + S.E. significant. Preincubation periods of 30 min were required or the reported differences were not observed. Additional research is necessary to assess the meaning of these results. Further studies are currently underway to determine the effects of insect hormones and various insecticides upon the catalytic activity of adenyl cyclase in nervous as well as other insect tissues.

Acknowledgement--We thank Barbara M. Beaver for consultation on statistical applications.

ACTIVATION AND INHIBITION OF ADENYL CYCLASE IN MADAGASCAR COCKROACH

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REFERENCES BERRIDOEM. J. (1970) The role of 5-hydroxytryptamine and cyclic A M P in the control of fluid secretion by isolated salivary glands, aT. exp. Biol. 53, 171-186. BERRIDGEM. J. & PAT~L N. G. (1968) Insect salivary glands: stimulation of fluid secretion by 5-hydroxytryptamine and adenosine-3", 5"-monophosphate. Science 162, 462-463. BUTCHERR. W. & SUTm~RLANDE. W. (1962) Adenosine 3',5'-phosphate inbiological materials. a~. biol. Chem. 237, 1244-1250. CHASE G. D. & RABINOWITZJ. L. (1962) Principles of Radioisotope Methodology, p. 203. Burgess, Minneapolis. COSTA E. & GREENCARDP. (Editors) (1969) Neurobiological significance of cyclic 3',5'adenosine monophosphate. In Advances in Biochemical Psychopharmacology, Vol. 1, pp. 131-164, Raven Press, New York. DUNNETT C. W. (1955) A multiple comparison procedure for comparing several treatments with a control, aT. Am. Stat. Ass. 50, 1096-1121. GILBERT L. I., APPLEBAUMS., GORELLT. A., SIDALLJ. B. & SIEN Y. C. (1971) Aspects of research on insect growth hormones. Bull. Wld Hlth Org. 44, 397-398. HEPP K. D., EDEK R. & WlELAND O. (1970) Hormone action on liver adenyl cyclase activity. Eur. jT. Biochem. 17, 171-177. KRISHNA G . , WEISS B. & BRODIE B. B. (1968) A simple, sensitive method for the assay of adenyl cyclase. J. Pharmaeol. exp. Ther. 163, 379-385. LEENDERS H. J., WULLEMS G. J. & BERENDES H. D. (1970) Competitive interaction of adenosine 3',5'-monophosphate on gene activation by ecdysone. Expl Cell Res. 63, 159-164. LOWRY O. H., ROSENBROUGHN. J., FARR A. L. & RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent. ~t. biol. Chem. 193, 265-275. RABINOWlTZ M., DESALLESL., MEISLER J. & LORAND L. (1965) Distribution of adenyl cyclase activity in rabbit skeletal muscle fractions. Biochim. biophys. Aeta 97, 29-36. RALL T. W. & SUTHERLANDE. W. (1958) Formation of a cyclic adenine ribonucleotide by tissue particles, ft. biol. Chem. 232, 1065-1076. RALL T. W., SUTHERLANDE. W. & BERTHETJ. (1957) The relationship of epinephrine and glucagon to liver phosphorylase. 3¢. biol. Chem. 224, 463-475. RAMACHANDRAN J. (1971) A new simple method for separation of adenosine 3",5'-cyclic monophosphate from other nucleotides and its use in the assay of adenyl cyclase. Analyt. Biochem. 43, 227-239. SUTHERLAND E. W. & RALL T. W. (1957) The properties of an adenine ribonucleotide produced with cellular particles, ATP, 1Vig~+, and epinephrine or glucagon. J. Am. Chem. Soc. 79, 3608. SUTHERLANDE. W., RALL T. W. & MENON T. (1962) Adenyl cyclase. ~t. biol. Chem. 237, 1220-1227. WEISS B. (1969) Similarities and differences in the norepinephrine and sodium fluoride sensitive adenyl cyclase system. ~t. Pharmacol. exp. Ther. 166, 330-338. WEISS B. & COSTA E. (1968) Selective stimulation of adenyl cyclase of rat pineal gland by pharmacologically active catecholamines. ~t. Pharmacol. exp. Ther. 161, 310-319. WIGGLESWORTHV. B. (1970) Insect Hormones. Freeman, San Francisco. Key Word Index--Adenyl cyclase, activation, inhibition; A T P ; cyclic AMP ; cockroach nerve tissue; norepinephrine; epinephrine; NaF; isoproterenol; ecdysterone; 4-aminobutyric acid; glucagon; C-romphadorhina portentosa.