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Adenyl cyclase in the haemocytes of the American cockroach
J. Inset Physiol., 1975, Vol. 21, pp. 221 to 229. Pmganwn Ress. J%intedin &cut Brituin
ADENYL CYCLASE IN THE HAEMOCYTES AMERICAN COCKROACH ROBERT D. VANDENBERG and RICHARD
OF THE
R. MILLS
Department of Biology, Virginia Commonwealth University, Richmond, Virginia 23220, U.S.A. (Reekwed lOJune 1974) Abstract-Incubation of 14C-adenosinetriphosphate with lysed haemocytes produces W-cyclic 3’,5’-adenosinemonophosphate (CAMP). The addition of phosphodiesterase to similar reaction mixtures results in the conversion of CAMP to 5’-adenosinemonophosphate. Differential centrifugation of the haemocyte preparation revealed that adenyl cyclase activity occurred in both membrane bound and sohible fractions, although the evidence suggests that the enzyme activity was originally particulate. The signikance of these data is discwsed in regard to the sclerotization process and the mode of action of bursicon. INTRODUCTION
THE SCLEROTIZATION or tanning of insect cuticle is thought to involve the crosslinking of proteins by diquinones into a hard insoluble matrix. This relatively impermeable exoskeIeton provides the means to resist desiccation and aids in protection from other environmental hazards. The diquinones used in the sclerotization process arise via the oxidation of diphenols by phenoloxidase. The alternate oxidation and reduction theoretically produces the binding of protein-free amine side chains to the ring (PRYOR, 1940). A number of diphenols have been implicated in the tanning process (BRUNET,1967) but it is universally accepted that N-acetyldopamine is the major cuticle component, as originally proposed by KARLsON and SEKERIS(1962). Sclerotization is also under the influence of a hormone. COTTRELL(1962) and FRAENKJJLand HSIAO (1962) demonstrated the presence of a substance that was necessary for cuticle hardening and darkening. This substance was later deemed to be a hormone and was appropriately named bursicon by FRAENKE L and HSIAO (1965). In the American cockroach, bursicon is released by the terminal abdominal ganglion (MILLS et al., 1965) or, more precisely, by neurohaemal organs located posterior to the last 3 or 4 ganglia (MILLS, unpublished). The mode of action of the hormone has not been completely elucidated although it has been postulated that bursicon acts by increasing tyrosine uptake by haemocytes (MILLS and WHITEHEAD, 1970). Using a more sophisticated approach, POST (1972) confirmed these observations and recently it has been shown that dibutryl cyclic AMP apparently mimics the action of bursicon (VON KNORRE et al., 1972; SEZLIGMAN and DOY, 1972; VANDENBERG and MILLS, 1974). 221
222
ROBERT
D.
VANDENBERGAND RICHARD R.
MILLS
The goal of the present work has been to assay the haemocytes for the presence of adenyl cyclase. The results show that the enzyme activity is indeed present and supports the contention that CAMP may act as a second messenger in bursiconmediated membrane transport enhancement (VANDENBERGand MILLS, 1974). MATERIALS AND METHODS American cockroaches, Periplaneta awicann, were held in screen cages as previously described (MILLS, 1966) and animals (sixth-to-last instar) were removed while in the process of shedding their old cuticle. Haemolymph was obtained by placing a precalibrated capillary tube adjacent to a severed appendage (MILLS et al., 1966). The collected blood was immediately frozen by packing in dry ice and acetone, The frozen haemolymph preparations were diluted with an equal volume of 0.05 M MgSO, and subsequently frozen and thawed three times. The reaction mixture was made up according to the specifications of ROSENand ROSEN(1968) with minor modifications. The complete reaction mixture (modified) contained: 0.02 ml 0.1 M NaF, 0.02 ml 0.2 M dithioerythritol, 0.02 ml 30.0 mM MgSO,, 0.02 ml O-1 M theophylline, 0.08 ml 0.15 M Tris buffer pH 8.01 at 37”C, 0.05 ml ‘*C-ATP (Amersham/Searle 1 mCi/mmol), and 0.100 ml of the haemolymph preparation. The controls contained the complete reaction mixture except the enzyme was previously boiled. All reactants were made up using Tris buffer as a solvent. The reactants were dispensed into Corex centrifuge tubes using a Hamilton microlitre syringe. The assay used for cyclic AMP formation was a modification of the original assay as described by HIRATA and HAYAISHI(1967). The initial incubation time was 20 min at 37°C. The reaction was terminated by placing the tubes in a boiling water-bath for 3 min. After chilling in ice and sequentially adding 0.05 ml 8% &SO, and 7.2% Ba(OH),, the tubes were centrifuged in a Beckman Model J-21 refrigerated centrifuge. The resultant heavy white precipitate was sedimented by centrifugation. The supematant was removed and was reacted again with &SO, and Ba(OH),. After centrifugation, the final supernatant was removed and transferred to clean Corex tubes. This supematant was freeze-dried using a Virtis Model No. lo-010 automatic freeze-dryer until the volume was reduced to 40 ,ul. The freeze-dried supernatant was subsequently chromatographed on Whatman No. 1 paper for 20 hr in an ammonium acetate-ethanol 3 : 7 v/v solvent system. After development, the paper was removed from the chamber, air-dried, and cut into strips for scanning on a Packard 7201 Badiochromatogram Strip Scanner. The control value of 10 to 17 counts/min above background was subtracted from each experimental result. R, values were calculated for each peak recorded by the strip scanner. RESULTS Preliminary experiments were designed to assay for the presence of adenyl cyclase in the membranes of the haemocytes. Accordingly, the enzyme assay was
ADENYL
CYCLASJ3IN THIt HAEMO(syTB8
OF THS AMERICAN
COCKROACH
223
conducted using the procedures described in Materials and Methods and the products were chromatographed on paper. Fig. 1 illustrates the results obtained. t t h
-
.t
.E + ::
_ _
0
s
-Origin
8
:I
CAMP
FIG. 1. Radioactive “C-ATP was incubated (see Materials and Methods) with the blood cell preparation for 20min at 37°C. The products were extracted and chromatographed on Whatman No. 1 paper using an ammonium acetate-ethanol 3 : 7 v/v solvent system. After development, the strips were air dried and analysed on a Packard 7201 radiopaperchromatogram strip scanner. The R, value for authentic CAMP was found to be O-63 which corresponds to the peak marked CAMP in the figure. Authentic cyclic AMP has a R, value of 0.63 which corresponds with the peak denoted as CAMP in Fig. 1. From these data, it appeared that the 14C-ATP had been converted into W-cyclic 3’,5’ AMP, In a series of more detailed experiments to prove that adenyl cyclase was present, the reaction was conducted over a series of progressively lengthened time intervals. From Fig. 2 it can be seen that the levels of 14C-CAMP rise significantly during each sequential progression in time of incubation. In addition, a plot of enzymatic activity (change in radioactivity per mg protein) against time indicates that the reaction follows zero-order kinetics. The reaction products were also chromatographed in other solvent systems (MILLS and LAKE, 1971) and the major radioactive peak always corresponded to the R, of authentic cyclic 3’,5’ AMP. These data strongly suggested that adenyl cyclase was a component of haemocyte membranes and that cyclic 3’,5’ AMP may be a natural endogenous product. Due to the limited amount of product it was not feasible to subject the suspected CAMP to spectral analysis. Another series of experiments was designed to confirm CAMP identity. During the in r&o formation of CAMP, a phosphodiesterase exists which catalyses the hydrolytic cleavage of the cyclic nucleotide to produce 5’ AMP. This reaction was inhibited by the addition of theophylline in the previous experiments in order to facilitate the build-up of cyclic 3’,5’ AMP. In the following studies, the theophylline was deleted and phosphodiesterase (snake venom and/or bovine spleen phosphodiesterase from PL B&hem.) was added to the reaction mixture. It was surmised that if 5’ AMP was a product, this would tend to support the contention that CAMP had been produced.
224
Ronmrr D. VANDENBERG ANDRnxmm R. MILLS
Time,
min
R,=0.63
FIG. 2. A composite diagram depicting six different incubation times ranging from 20 to 120 min. As shown, the concentration of CAMP rises proportionally as the time of incubation increases. The R, values of authentic, ATP, ADP, 5’ AMP and CAMP (O-63) correspond to the area denoted. Incubation and analysis were conducted as described in Fig. 1.
5’AMP
CAMP
Origin
I 0
I
!
I
0
I
6
I
I
t
12 Distance,
I
I
I
I
I
18 cm
FIG. 3. Radioactive %-ATP was incubated (see Materials and Methods) with the blood ceII preparation with the deletion of theophylline, and the addition of 1 unit of phosphodiesterase. As shown in the figure, some of the CAMP has been converted to 5’ AMP by the addition of the enzyme.
OF THE AMERICAN COCKROACH
ADBNYLCYCLASEINTHEIiAEMOCYTES
225
As depicted in Fig. 3, it can be seen that incubation of W-ATP with haemocyte membrane fractions (in the presence of 1 unit of phosphodiesterase)yields 5’ AMP Units
of
Phosphodiesterase
Control
ATP
ADP I
I
I 6
0
I
I
I 12
Distance,
I
I
11
11
16
cm
FIG. 4. The effect of phosphodiesterase on the conversion of CAMP to 5’ AMP. Phosphodiesterase (0, 1, 2, and 4 units) were incubated with the standard reaction mixture without theophylline. A progressively higher peak of 5’ AMP is found due to the increased concentration of the enzyme. ATP
Drlgin fi
0
6’
12 Distance,
0
6
12 Distance,
24
18 cm
I
I
18
24
cm
FIG. 5. The localization of adenyl q&se in extracted blood cell membranes. The extract was centrifuged at 20,000 g and the pellet used as the enzyme. Radioactive W-ATP was incubated with the enzyme and the products chromatographed without prior extraction. As shown, ATP is converted to CAMP and no reaction occurs if the enzyme is previously boiled.
226
ROBBRT D.
VANDENBERG ANDRICHARD R. MILLS
as a product. The presence of CAMP suggests that insufficient phosphodiesterase was present to keep pace with CAMP formation via adenyl cyclase. However, when the phosphodiesterase concentration was increased (Fig. 4), it can be seen that a vast rise in 5’ AMP occurred. To further substantiate the presence of the adenyl cyclase, a boiled enzyme control was incubated with r4C-ATP. The complete absence of either 14C cyclic 3’,5’ AMP or 5’ AMP as a product is offered as further proof of haemocyte adenyl cyclase activity. The above data indicate that adenyl cyclase is present in cockroach haemocytes, but offer no proof as to its cytological location. The following study was designed to determine if all of the enzymatic activity was localized in the particulate fraction. After freezing and thawing the blood cell pellet three times, the preparation was centrifuged at 20,000 g for 15 min. The supernatant and pellet were subsequently incubated with the standard reaction mixture for 2 hr and the analysis conducted as denoted in Materials and Methods. As expected, the pellet showed considerable adenyl cyclase activity (Fig. 5), which agrees with all work to date (ROSEN and ROSEN, 1968). Unexpectedly, the supernatant also exhibited enzyme activity (Fig. 6). The enzyme has not been solubilized in previous work and it is possible that the insect system may be different from the mammalian system.
DISCUSSION The results of this study demonstrate that 14C-ATP can be converted to a labelled compound which is chromatographically identical to authentic CAMP. The addition of exogenous nucleotide phosphodiesterase catalyses the conversion of the labelled compound to 5’ AMP. Co-chromatography of authentic CAMP with the labelled compound confirms its identity as CAMP. These data indicate that CAMP is indeed the labelled reaction product and in turn suggest the presence of an adenyl cyclase system within, or located on, the haemocytes. The presence of the enzyme in cockroach haemocytes supports the contention that CAMP may be instrumental in increasing membrane permeability. It has previously been established that CAMP serves this function in mammalian systems (POSTERNACK et aE., 1962) and it is recognized that fluid secretion is enhanced in both insect salivary glands and Malpighian tubules by CAMP (BERRIDGEand PATEL, 1969; MADDRELLet al., 1972). Previous investigators have demonstrated the stimulatory effects of bursicon on tyrosine uptake by blood cells (MILLS and WHITEHEAD,1970; POST, 1972). It has also been postulated that activation of an adenyl cyclase system may be via a bursicon receptor site located on the haemocyte membrane (VANDENBERGand MILLS, 1974). In addition to these findings it is known that diphenolic glucosides bind to blood proteins which are in turn incorporated into the cuticle (KOEPPE and MILLS, 1972; KOEPPEet al., 1974). Cyclic AMP also has the potential to stimulate receptor sites on epithelial cells which may well mediate the uptake of these molecules. Thus it would seem that CAMP could stimulate the transport of several different
AIMNYL CTCLASEIN THE HAEMOCTTBS OF THE AMERICANCOCKROACH
cm
Distance,
FIG. 6. Production of CAMP by soluble adenyl cyclase. The extract was centrifuged at 20,000 g and the eupernatant used as the enzyme. Radioactive WATP was incubated with the enzyme and the analysis was as described in Fig. 5. ATP is converted to CAMP as shown with a corresponding increase in ADP. It is assumed that a soluble ATPase is also present in the enzyme preparation.
Hormones
Amino
acids
Proteins
FIG. 7. Diagrammatic representation of probable mechanisms involved in the mediation of membrane transport by cyclic AMP. It is suggested that adenyl cyclase may be activated by various hormonea and the cyclase in turn produces CAMP. The cAMP enhancea membrane transport and induces certain enzyme (i.e. tyrosine hydroxykne) synthesis. These procerser, couldtakeplaceinmany other cells (fat body, gut, etc.) as well as the blood celk
227
228
ROBERTD. VANDBNBBRG AND RICHARD R. MILLS
moieties as indicated by the circles in Fig. 7. Both small and large molecular weight components are involved and their translocation could be enhanced by CAMP. In addition, a number of ‘activators’ which are involved in the tanning process may have the potential to elicit a response. The diagrammatic cell shown in Fig. 7 need not represent any particular cell but illustrates processes that take place in epidermal, fat body, and blood cells. Acktwwledgements-We would like to thank DAVID Loux and NANCY TAYLOR for technical assistance, and Dr. JUDY BONDfor helpful discussions, This work was supported by grant No. GB-31376 from the National Science Foundation. REFERENCES BERRIDGEM. J. and PATELN. G. (1968) Insect salivary glands: stimulation of fluid secretion by 5-hydroxytryptamine and adenosine -3’,5’-monophosphate. Science, Wash. 162, 462-463. BRUNETP. C. J. (1967) Sclerotins. Endeuwour 26, 68-74. COT-L C. B. (1962) General observations on the imaginal ecdyais of blowflies. Trans. R. ent. Sot. Lend. 114, 317-333. FRAENKELG. and HSIAOC. (1962) Tanning in the adult fly: a new function of neurosecretion in the brain. Science, Wash, 141, 1057-1058. FRAENKELG. and HSIAO C. (1965) Buraicon, a hormone which mediates tanning of the cuticle in the adult fly and other insects. J. Insect Physiol. 11, 513-556. HIRATAM. and HAYAISHI0. (1967) Adenyl cyclase of Brewibucterium liquefaciens. Biochim. biophys. Actu 149, l-l 1. KARLSON P. and SBKERISC. E. (1962) Zum Tryosinstoffwechsel der Insekten-IX. Kontrolle des Tyrosinstotfwechsels durch Ecdyaon. Biochim. biophys. Acta 63, 489-495. KOEPPBJ. K. and MILU R. R. (1972) Hormonal control of tanning by the American cockroach: probable bursicon mediated translocation of protein-bound phenols. 3. Insect
Physiol. 18,465-469. KOEPPE J. K., M~us R. R., and BRUNETT. C. J. (1974) Cuticle sclerotization by the American cockroach: high molecular weight carriers of phenolic betaglucosidea. Biochim. biophys. Acta. In press. VON KNORREV. E., GERSCH M., and KUSCH T. (1972) Zur Frage der Beeinflussung des Tanning-Phanomens durch zyklisches 3’,5’-AMP. ZooLJb. Physiol. 76,434-440. MADDRELLS. H. P., PILCHERD. E. M., and GARDINWB. 0. C. (1971) Pharmacology of the Malpighian tubules of Rhodnius and Carasius: the structure-activity relationship of tryptamine analogues and the role of cyclic AMP. _7. exp. Biol. 54,779-804. MILLS R. R, (1966) A cockroach cage designed for the simple collection of ootheca. r. econ.
Ent. 59,490. MILLS R. R., GREENSLADE F. C., and COUCHE. F. (1966) Studies on vitellogeneais in the American cockroach. y. Insect Physiol. 13, 1539-1546. MILU R. R., GUERRAA. A., and MATHURR. B. (1965) Studies on the hormonal control of tanning in the American cockroach-I. Release of an activation factor from the terminal abdominal ganglion. 3. Insect Physiol. 11, 1047-1053. MILLS R. R. and LAKE C. R. (1971) Metabolism of tyrosine to p-hydroxyphenylpropionic acid and to p-hydroxyphenylacetic acid by the haemolymph of the American cockroach. Insect Biochem. 1,264-270. MILU R. R. and WHITBHEADD. L. (1970) Hormonal control of tanning in the American cockroach: changes in blood cell permeability. y. Insect Physiol. 16,331-340. POST L. C. (1972) Bursicon: its effect on tyrosine permeation into insect haemocytea. Biochim. biophys. Acta 290,424-428.
ADENYL
CYCLASE IN THR HAEMOCYTES
OF THB AMRRICAN COCKROACH
229
P~STEWACK T. H., SUTHERLAND E. W., and HENION W. F. (1962) Derivatives of cyclic-3’-, S-adenosine monophosphate. Bbchim. biehys. Actu 65,558-560. PRYOR M. G. M. (1940) On the hardening of the ootheca of Bkatta orientahk Proc. R. Sot. (B) 128,378-393. ROSBN0. A. and ROSBNS. M. (1968) The effect of catecholamines on the adenyl cyclase of frog and tadpole hemolysates. Biochem. biwhys. Res. Commun. 31, 82-91. SELICMAN I. M. and DOY F. A. (1972) Studies on cyclic AMP mediation of hormonally induced cytolysis of the alary hypodermal cells and of hormonally controlled DOPA synthesis in Lucillia cup&a. IsraelJ. ent. 7,129-142. VANDBNBBRO R. D. and MILU R. R. (1974) Hormonal control of tanning by the American cockroach: cyclic AMP as a probable intermediate. J. Insect Physiol. 20,623-627.
ADENYL CYCLASE IN THE HAEMOCYTES AMERICAN COCKROACH ROBERT D. VANDENBERG and RICHARD
OF THE
R. MILLS
Department of Biology, Virginia Commonwealth University, Richmond, Virginia 23220, U.S.A. (Reekwed lOJune 1974) Abstract-Incubation of 14C-adenosinetriphosphate with lysed haemocytes produces W-cyclic 3’,5’-adenosinemonophosphate (CAMP). The addition of phosphodiesterase to similar reaction mixtures results in the conversion of CAMP to 5’-adenosinemonophosphate. Differential centrifugation of the haemocyte preparation revealed that adenyl cyclase activity occurred in both membrane bound and sohible fractions, although the evidence suggests that the enzyme activity was originally particulate. The signikance of these data is discwsed in regard to the sclerotization process and the mode of action of bursicon. INTRODUCTION
THE SCLEROTIZATION or tanning of insect cuticle is thought to involve the crosslinking of proteins by diquinones into a hard insoluble matrix. This relatively impermeable exoskeIeton provides the means to resist desiccation and aids in protection from other environmental hazards. The diquinones used in the sclerotization process arise via the oxidation of diphenols by phenoloxidase. The alternate oxidation and reduction theoretically produces the binding of protein-free amine side chains to the ring (PRYOR, 1940). A number of diphenols have been implicated in the tanning process (BRUNET,1967) but it is universally accepted that N-acetyldopamine is the major cuticle component, as originally proposed by KARLsON and SEKERIS(1962). Sclerotization is also under the influence of a hormone. COTTRELL(1962) and FRAENKJJLand HSIAO (1962) demonstrated the presence of a substance that was necessary for cuticle hardening and darkening. This substance was later deemed to be a hormone and was appropriately named bursicon by FRAENKE L and HSIAO (1965). In the American cockroach, bursicon is released by the terminal abdominal ganglion (MILLS et al., 1965) or, more precisely, by neurohaemal organs located posterior to the last 3 or 4 ganglia (MILLS, unpublished). The mode of action of the hormone has not been completely elucidated although it has been postulated that bursicon acts by increasing tyrosine uptake by haemocytes (MILLS and WHITEHEAD, 1970). Using a more sophisticated approach, POST (1972) confirmed these observations and recently it has been shown that dibutryl cyclic AMP apparently mimics the action of bursicon (VON KNORRE et al., 1972; SEZLIGMAN and DOY, 1972; VANDENBERG and MILLS, 1974). 221
222
ROBERT
D.
VANDENBERGAND RICHARD R.
MILLS
The goal of the present work has been to assay the haemocytes for the presence of adenyl cyclase. The results show that the enzyme activity is indeed present and supports the contention that CAMP may act as a second messenger in bursiconmediated membrane transport enhancement (VANDENBERGand MILLS, 1974). MATERIALS AND METHODS American cockroaches, Periplaneta awicann, were held in screen cages as previously described (MILLS, 1966) and animals (sixth-to-last instar) were removed while in the process of shedding their old cuticle. Haemolymph was obtained by placing a precalibrated capillary tube adjacent to a severed appendage (MILLS et al., 1966). The collected blood was immediately frozen by packing in dry ice and acetone, The frozen haemolymph preparations were diluted with an equal volume of 0.05 M MgSO, and subsequently frozen and thawed three times. The reaction mixture was made up according to the specifications of ROSENand ROSEN(1968) with minor modifications. The complete reaction mixture (modified) contained: 0.02 ml 0.1 M NaF, 0.02 ml 0.2 M dithioerythritol, 0.02 ml 30.0 mM MgSO,, 0.02 ml O-1 M theophylline, 0.08 ml 0.15 M Tris buffer pH 8.01 at 37”C, 0.05 ml ‘*C-ATP (Amersham/Searle 1 mCi/mmol), and 0.100 ml of the haemolymph preparation. The controls contained the complete reaction mixture except the enzyme was previously boiled. All reactants were made up using Tris buffer as a solvent. The reactants were dispensed into Corex centrifuge tubes using a Hamilton microlitre syringe. The assay used for cyclic AMP formation was a modification of the original assay as described by HIRATA and HAYAISHI(1967). The initial incubation time was 20 min at 37°C. The reaction was terminated by placing the tubes in a boiling water-bath for 3 min. After chilling in ice and sequentially adding 0.05 ml 8% &SO, and 7.2% Ba(OH),, the tubes were centrifuged in a Beckman Model J-21 refrigerated centrifuge. The resultant heavy white precipitate was sedimented by centrifugation. The supematant was removed and was reacted again with &SO, and Ba(OH),. After centrifugation, the final supernatant was removed and transferred to clean Corex tubes. This supematant was freeze-dried using a Virtis Model No. lo-010 automatic freeze-dryer until the volume was reduced to 40 ,ul. The freeze-dried supernatant was subsequently chromatographed on Whatman No. 1 paper for 20 hr in an ammonium acetate-ethanol 3 : 7 v/v solvent system. After development, the paper was removed from the chamber, air-dried, and cut into strips for scanning on a Packard 7201 Badiochromatogram Strip Scanner. The control value of 10 to 17 counts/min above background was subtracted from each experimental result. R, values were calculated for each peak recorded by the strip scanner. RESULTS Preliminary experiments were designed to assay for the presence of adenyl cyclase in the membranes of the haemocytes. Accordingly, the enzyme assay was
ADENYL
CYCLASJ3IN THIt HAEMO(syTB8
OF THS AMERICAN
COCKROACH
223
conducted using the procedures described in Materials and Methods and the products were chromatographed on paper. Fig. 1 illustrates the results obtained. t t h
-
.t
.E + ::
_ _
0
s
-Origin
8
:I
CAMP
FIG. 1. Radioactive “C-ATP was incubated (see Materials and Methods) with the blood cell preparation for 20min at 37°C. The products were extracted and chromatographed on Whatman No. 1 paper using an ammonium acetate-ethanol 3 : 7 v/v solvent system. After development, the strips were air dried and analysed on a Packard 7201 radiopaperchromatogram strip scanner. The R, value for authentic CAMP was found to be O-63 which corresponds to the peak marked CAMP in the figure. Authentic cyclic AMP has a R, value of 0.63 which corresponds with the peak denoted as CAMP in Fig. 1. From these data, it appeared that the 14C-ATP had been converted into W-cyclic 3’,5’ AMP, In a series of more detailed experiments to prove that adenyl cyclase was present, the reaction was conducted over a series of progressively lengthened time intervals. From Fig. 2 it can be seen that the levels of 14C-CAMP rise significantly during each sequential progression in time of incubation. In addition, a plot of enzymatic activity (change in radioactivity per mg protein) against time indicates that the reaction follows zero-order kinetics. The reaction products were also chromatographed in other solvent systems (MILLS and LAKE, 1971) and the major radioactive peak always corresponded to the R, of authentic cyclic 3’,5’ AMP. These data strongly suggested that adenyl cyclase was a component of haemocyte membranes and that cyclic 3’,5’ AMP may be a natural endogenous product. Due to the limited amount of product it was not feasible to subject the suspected CAMP to spectral analysis. Another series of experiments was designed to confirm CAMP identity. During the in r&o formation of CAMP, a phosphodiesterase exists which catalyses the hydrolytic cleavage of the cyclic nucleotide to produce 5’ AMP. This reaction was inhibited by the addition of theophylline in the previous experiments in order to facilitate the build-up of cyclic 3’,5’ AMP. In the following studies, the theophylline was deleted and phosphodiesterase (snake venom and/or bovine spleen phosphodiesterase from PL B&hem.) was added to the reaction mixture. It was surmised that if 5’ AMP was a product, this would tend to support the contention that CAMP had been produced.
224
Ronmrr D. VANDENBERG ANDRnxmm R. MILLS
Time,
min
R,=0.63
FIG. 2. A composite diagram depicting six different incubation times ranging from 20 to 120 min. As shown, the concentration of CAMP rises proportionally as the time of incubation increases. The R, values of authentic, ATP, ADP, 5’ AMP and CAMP (O-63) correspond to the area denoted. Incubation and analysis were conducted as described in Fig. 1.
5’AMP
CAMP
Origin
I 0
I
!
I
0
I
6
I
I
t
12 Distance,
I
I
I
I
I
18 cm
FIG. 3. Radioactive %-ATP was incubated (see Materials and Methods) with the blood ceII preparation with the deletion of theophylline, and the addition of 1 unit of phosphodiesterase. As shown in the figure, some of the CAMP has been converted to 5’ AMP by the addition of the enzyme.
OF THE AMERICAN COCKROACH
ADBNYLCYCLASEINTHEIiAEMOCYTES
225
As depicted in Fig. 3, it can be seen that incubation of W-ATP with haemocyte membrane fractions (in the presence of 1 unit of phosphodiesterase)yields 5’ AMP Units
of
Phosphodiesterase
Control
ATP
ADP I
I
I 6
0
I
I
I 12
Distance,
I
I
11
11
16
cm
FIG. 4. The effect of phosphodiesterase on the conversion of CAMP to 5’ AMP. Phosphodiesterase (0, 1, 2, and 4 units) were incubated with the standard reaction mixture without theophylline. A progressively higher peak of 5’ AMP is found due to the increased concentration of the enzyme. ATP
Drlgin fi
0
6’
12 Distance,
0
6
12 Distance,
24
18 cm
I
I
18
24
cm
FIG. 5. The localization of adenyl q&se in extracted blood cell membranes. The extract was centrifuged at 20,000 g and the pellet used as the enzyme. Radioactive W-ATP was incubated with the enzyme and the products chromatographed without prior extraction. As shown, ATP is converted to CAMP and no reaction occurs if the enzyme is previously boiled.
226
ROBBRT D.
VANDENBERG ANDRICHARD R. MILLS
as a product. The presence of CAMP suggests that insufficient phosphodiesterase was present to keep pace with CAMP formation via adenyl cyclase. However, when the phosphodiesterase concentration was increased (Fig. 4), it can be seen that a vast rise in 5’ AMP occurred. To further substantiate the presence of the adenyl cyclase, a boiled enzyme control was incubated with r4C-ATP. The complete absence of either 14C cyclic 3’,5’ AMP or 5’ AMP as a product is offered as further proof of haemocyte adenyl cyclase activity. The above data indicate that adenyl cyclase is present in cockroach haemocytes, but offer no proof as to its cytological location. The following study was designed to determine if all of the enzymatic activity was localized in the particulate fraction. After freezing and thawing the blood cell pellet three times, the preparation was centrifuged at 20,000 g for 15 min. The supernatant and pellet were subsequently incubated with the standard reaction mixture for 2 hr and the analysis conducted as denoted in Materials and Methods. As expected, the pellet showed considerable adenyl cyclase activity (Fig. 5), which agrees with all work to date (ROSEN and ROSEN, 1968). Unexpectedly, the supernatant also exhibited enzyme activity (Fig. 6). The enzyme has not been solubilized in previous work and it is possible that the insect system may be different from the mammalian system.
DISCUSSION The results of this study demonstrate that 14C-ATP can be converted to a labelled compound which is chromatographically identical to authentic CAMP. The addition of exogenous nucleotide phosphodiesterase catalyses the conversion of the labelled compound to 5’ AMP. Co-chromatography of authentic CAMP with the labelled compound confirms its identity as CAMP. These data indicate that CAMP is indeed the labelled reaction product and in turn suggest the presence of an adenyl cyclase system within, or located on, the haemocytes. The presence of the enzyme in cockroach haemocytes supports the contention that CAMP may be instrumental in increasing membrane permeability. It has previously been established that CAMP serves this function in mammalian systems (POSTERNACK et aE., 1962) and it is recognized that fluid secretion is enhanced in both insect salivary glands and Malpighian tubules by CAMP (BERRIDGEand PATEL, 1969; MADDRELLet al., 1972). Previous investigators have demonstrated the stimulatory effects of bursicon on tyrosine uptake by blood cells (MILLS and WHITEHEAD,1970; POST, 1972). It has also been postulated that activation of an adenyl cyclase system may be via a bursicon receptor site located on the haemocyte membrane (VANDENBERGand MILLS, 1974). In addition to these findings it is known that diphenolic glucosides bind to blood proteins which are in turn incorporated into the cuticle (KOEPPE and MILLS, 1972; KOEPPEet al., 1974). Cyclic AMP also has the potential to stimulate receptor sites on epithelial cells which may well mediate the uptake of these molecules. Thus it would seem that CAMP could stimulate the transport of several different
AIMNYL CTCLASEIN THE HAEMOCTTBS OF THE AMERICANCOCKROACH
cm
Distance,
FIG. 6. Production of CAMP by soluble adenyl cyclase. The extract was centrifuged at 20,000 g and the eupernatant used as the enzyme. Radioactive WATP was incubated with the enzyme and the analysis was as described in Fig. 5. ATP is converted to CAMP as shown with a corresponding increase in ADP. It is assumed that a soluble ATPase is also present in the enzyme preparation.
Hormones
Amino
acids
Proteins
FIG. 7. Diagrammatic representation of probable mechanisms involved in the mediation of membrane transport by cyclic AMP. It is suggested that adenyl cyclase may be activated by various hormonea and the cyclase in turn produces CAMP. The cAMP enhancea membrane transport and induces certain enzyme (i.e. tyrosine hydroxykne) synthesis. These procerser, couldtakeplaceinmany other cells (fat body, gut, etc.) as well as the blood celk
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ROBERTD. VANDBNBBRG AND RICHARD R. MILLS
moieties as indicated by the circles in Fig. 7. Both small and large molecular weight components are involved and their translocation could be enhanced by CAMP. In addition, a number of ‘activators’ which are involved in the tanning process may have the potential to elicit a response. The diagrammatic cell shown in Fig. 7 need not represent any particular cell but illustrates processes that take place in epidermal, fat body, and blood cells. Acktwwledgements-We would like to thank DAVID Loux and NANCY TAYLOR for technical assistance, and Dr. JUDY BONDfor helpful discussions, This work was supported by grant No. GB-31376 from the National Science Foundation. REFERENCES BERRIDGEM. J. and PATELN. G. (1968) Insect salivary glands: stimulation of fluid secretion by 5-hydroxytryptamine and adenosine -3’,5’-monophosphate. Science, Wash. 162, 462-463. BRUNETP. C. J. (1967) Sclerotins. Endeuwour 26, 68-74. COT-L C. B. (1962) General observations on the imaginal ecdyais of blowflies. Trans. R. ent. Sot. Lend. 114, 317-333. FRAENKELG. and HSIAOC. (1962) Tanning in the adult fly: a new function of neurosecretion in the brain. Science, Wash, 141, 1057-1058. FRAENKELG. and HSIAO C. (1965) Buraicon, a hormone which mediates tanning of the cuticle in the adult fly and other insects. J. Insect Physiol. 11, 513-556. HIRATAM. and HAYAISHI0. (1967) Adenyl cyclase of Brewibucterium liquefaciens. Biochim. biophys. Actu 149, l-l 1. KARLSON P. and SBKERISC. E. (1962) Zum Tryosinstoffwechsel der Insekten-IX. Kontrolle des Tyrosinstotfwechsels durch Ecdyaon. Biochim. biophys. Acta 63, 489-495. KOEPPBJ. K. and MILU R. R. (1972) Hormonal control of tanning by the American cockroach: probable bursicon mediated translocation of protein-bound phenols. 3. Insect
Physiol. 18,465-469. KOEPPE J. K., M~us R. R., and BRUNETT. C. J. (1974) Cuticle sclerotization by the American cockroach: high molecular weight carriers of phenolic betaglucosidea. Biochim. biophys. Acta. In press. VON KNORREV. E., GERSCH M., and KUSCH T. (1972) Zur Frage der Beeinflussung des Tanning-Phanomens durch zyklisches 3’,5’-AMP. ZooLJb. Physiol. 76,434-440. MADDRELLS. H. P., PILCHERD. E. M., and GARDINWB. 0. C. (1971) Pharmacology of the Malpighian tubules of Rhodnius and Carasius: the structure-activity relationship of tryptamine analogues and the role of cyclic AMP. _7. exp. Biol. 54,779-804. MILLS R. R, (1966) A cockroach cage designed for the simple collection of ootheca. r. econ.
Ent. 59,490. MILLS R. R., GREENSLADE F. C., and COUCHE. F. (1966) Studies on vitellogeneais in the American cockroach. y. Insect Physiol. 13, 1539-1546. MILU R. R., GUERRAA. A., and MATHURR. B. (1965) Studies on the hormonal control of tanning in the American cockroach-I. Release of an activation factor from the terminal abdominal ganglion. 3. Insect Physiol. 11, 1047-1053. MILLS R. R. and LAKE C. R. (1971) Metabolism of tyrosine to p-hydroxyphenylpropionic acid and to p-hydroxyphenylacetic acid by the haemolymph of the American cockroach. Insect Biochem. 1,264-270. MILU R. R. and WHITBHEADD. L. (1970) Hormonal control of tanning in the American cockroach: changes in blood cell permeability. y. Insect Physiol. 16,331-340. POST L. C. (1972) Bursicon: its effect on tyrosine permeation into insect haemocytea. Biochim. biophys. Acta 290,424-428.
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P~STEWACK T. H., SUTHERLAND E. W., and HENION W. F. (1962) Derivatives of cyclic-3’-, S-adenosine monophosphate. Bbchim. biehys. Actu 65,558-560. PRYOR M. G. M. (1940) On the hardening of the ootheca of Bkatta orientahk Proc. R. Sot. (B) 128,378-393. ROSBN0. A. and ROSBNS. M. (1968) The effect of catecholamines on the adenyl cyclase of frog and tadpole hemolysates. Biochem. biwhys. Res. Commun. 31, 82-91. SELICMAN I. M. and DOY F. A. (1972) Studies on cyclic AMP mediation of hormonally induced cytolysis of the alary hypodermal cells and of hormonally controlled DOPA synthesis in Lucillia cup&a. IsraelJ. ent. 7,129-142. VANDBNBBRO R. D. and MILU R. R. (1974) Hormonal control of tanning by the American cockroach: cyclic AMP as a probable intermediate. J. Insect Physiol. 20,623-627.