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

THE ACTIVATION AND INHIBITION OF ADENYL CYCLASE FROM THE BRAIN OF THE MADAGASCAR COCKROACH (GROMPHADORHINA PORTENTOSA)*

ARNOLD S. ROJAKOVICK and RALPH B. MARCH

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 epi- nephrine, 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 produc- tion of Y,5'-cyclic adenosine monophosphate (cAMP) from adenosine-5'-tri- phosphate (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

210 ARNOLD S. ROJAKOVICK AND 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 de-

capitated 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, pH 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 ATP (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, pH 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 ATP 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 MM filter paper and the chromatogram was developed for 20 hr. After drying at room temper- ature, 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 AMP 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.

R E S U L T S AND D I S C U S S I O N

Consideration of the assay

Caffeine was added to the react ion mixture to prevent the b reakdown of c A M P by inhibi t ing phosphodiesterase. However , c h r o m a t o g r a p h y of s tandards, to- gether with au toradiography, indicated that this inhibi t ion was not complete since A M P was detected. T h e possibili ty o f incomplete blocking of phosphodies terase by methylxanth ines has also been suggested by H e p p et al. (1970).

F igure 1 demonst ra tes tha t the p roduc t ion of cAMp-14C f rom ATP-14C by cockroach bra in homogena tes is linear wi th t ime for 20 min. T h e non- l inear effects of increasing amoun t s of cockroach brain tissue u p o n catalytic activity bo th in the presence and absence of 1 m M N a F are shown in Fig. 2. Weiss & Costa (1968) repor ted similar results wi th identical prote in concentra t ions for rat pineal

A C T I V A T I O N ' A N D I N H I B I T I O N OF A D E N Y L CYCLASE I N MADAGASCAR COCKROACH 211

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FIG. 1. Activity of cockroach brain adenyl cyclase in producing cyclic 3",5"-AMP as a function of time.

80

70

~ 6o

.~ 3 0

u

2o

Io

o

. /~ - ( - NoF

50 I00 150 Enzyme, Fgm protein

FIG. 2. Effect of 1"0 m M NaF on the activity of cockroach brain adenyl cyclase in producing cyclic 3",5"-AMP as a function o f enzyme concentration. Each incu- bation vessel contained between 0.058 and 0"145 mg of brain protein. Each value

represents the mean of four experiments; brackets indicate S.E.

2 1 2 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 mM 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 mM (Fig. 3). Maximal stimulation is observed at 3 mM which is considerably lower

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FIo. 3. Effect of varying concentrations of NaF upon activity of cockroach brain adenyl cyclase.

than has been reported for various mammalian adenyl cyclases (Costa & Green- gard, 1969; Weiss, 1969; Hepp et al., 1970; Ramachandran, 1971). At concen- trations greater than 3 mM, cockroach adenyl cyclase also differs in that marked autoinhibition is observed.

Stimulation by catecholamines Norepinephrine and epinephrine stimulate cockroach brain adenyl cyclase

(Figs. 4 and 5). Maximal stimulation by both is achieved at a concentration of 500 /~M. Isoproterenol, over the same concentration range, had no effect. All three compounds have been shown to stimulate rat pineal gland adenyl cyclase at slightly lower concentrations than those reported here (Weiss & Costa, 1968; Weiss, 1969).

ACTIVATION AND INHIBITION OF ADENYL CYCLASE IN MADAGASCAR COCKROACH 213

E 450

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~ 350

300

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i00 500 i 0 0 0 2500 500(9

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FIG. 4. Effect of norepinephrine on adenyl cyclase activity of cockroach brain. Each value indicates the mean of four experiments; brackets indicate S.E. At a

concentration of 500/zM, Dunnett's test indicates P < 0"01.

c 600

~ 550

o_ 450

i 400

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FIG. 5. Effect of epinephrine on adenyl cyclase activity of cockroach brain. Each value indicates the mean of four experiments; brackets indicate S.E. At a concen-

tration of 500/zM, Dunnett's test indicates P < 0"01.

214 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 isopro- terenol, 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) impli- cated 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-amino- butyric 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 Cyclic 3',5'-AMP formed P value

(p mol/mg of protein per min) None 294 + 16 Ecdysterone (10 -4 M) 246 + 35 < 0"05 4-Aminobutyric acid (10 -4 M) 270 + 12 > 0'05

*Each value represents the mean of four experiments + S.E.

significant. Preincubation periods of 30 min were required or the reported dif- ferences 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 appli- cations.

ACTIVATION AND INHIBITION OF ADENYL CYCLASE IN MADAGASCAR COCKROACH 215

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Key Word Index--Adenyl cyclase, activation, inhibition; ATP; cyclic AMP ; cockroach nerve tissue; norepinephrine; epinephrine; NaF; isoproterenol; ecdysterone; 4-amino- butyric acid; glucagon; C-romphadorhina portentosa.