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Towards a specific histochemical localization of adenylate cyclase in the rat hippocampus I. Methodical aspects
Acta histochem. 69, 171-175 (1981)
Division of Cell Biology and R egulation, Biosciences Section, The Karl Marx University, Leipzig, ODR (Director: Prof. Dr. sc. nat. G. SCHUSTER) Institute of Anatomy a nd Biology, Medical Academy Magdeburg, Magdeburg, GDR (Dircc:tol': Prof. Dr. se. med A. DaRN)
Towards a specific histochemical localization of adenyl ate cyclase in the rat hippocampus I. Methodical aspects!,2) By GERD POEGGEL and HANS-GERT BERKSTEIK (Reeeived November 5, 1980)
Summary The adequate histo chemical demonstration of adenyla t e cyclase still invol ves seriom methodical problems (low basal activity, fixation procedure, heavy m et al ions as capture agents etc.). ,.ye h a ve tried to revise fixation a pproach and composition ot incuba tion medium in order to specify the lllode of demonstmiion of the enzyme. The influence of strontium a nd b a rium (instead of lead) as well as dithiothreitole a nd sodium fluoride on the survive of adenylate cyclase activity was studied using a radiochemi cal model system.
Introduction 4 main reasons are responsible for the difficulties, which a histochemist must overcome, if he wants to localize adenylate cyclase (EC 4.6.1.1) specifically in 8itu (especially in the CNS): 1. The enzymatic activity may be very low in the tissue of interest. 2. Adenosine triphosphate, the natural substrate of the enzyme, is also converted by other ferments during incubation (as recently shown by gel staining after microdisc electrophoresis [POEGGEL et aJ. 1980]). :3. The heavy metal ion, which is used to capture and precipitate the inorganic phosphate (largely lead) is capable of catalyzing a nonenzymatic substrate-splitting on one hand, and of inhibiting the enzymatic activity, on t.he other (LEMAY and J ARETT 1975, KEMPEN et al. 1978). 4. The fixation may also depress parts of the adenylate cyclase activity.
1) '.I'his resear'ch was supported by the "Ministerium fUr Ho ch - und Fachschulwesen del' DDR". 2) Parts of the data wel'e presented at the III. SymposiUIll on Neurotransmission 1979, Helsinki.
172
G. POEGGEL and H .·G. BERNSTEIN
Bearing in mind the importance of adenylate cyclase as cAMP-generating enzyme for many metabolic processes, there were efforts to improve the assay conditions for adenylate cyclase (introduction of a synthetic, more specific substrate - adenylyl-imidodiphosphate - by YOUNT et al. [1970], testing of other heavy metal candidates for their ability to form an insoluble reaction product with PPi without considerable loss in enzymatic activity: Barium, KEMPEN et al. [1978]). The present report deals with our experience in modifying the incubation conditions for adenylate cyclase to get a more specific reaction as investigated in a radiochemical model system.
Material and methods Male young albino mts were stuili nd . The animals were kill ed under mild anaesthesia . Some of them were perfused intracttrdially prior to decapitation (formaldehyde, 3 %, 5 min; glutaraldehyde, 2 %,5 min). The brains of
+ T riton X 100 (0.01 % ) AI + 10 mM N a F AI + 10 mM NaF + 1 m1l1 dithiothreitole (DTT) AI + 10 mM N aF + 1 mM DTT + 1 mM Pb2+ AI + 10 mM NaF + 1 mM DTT + 1.5 mM Pb2+ AI + 10 rnM N flF + 10- 5 M guanylyl imidodiphosphl\te (G'MP-PNP, purch ased from Boehringer Mannheim, FRG) AI + 10 mM N aF + 1 mM DTT + 20 mM Sr2+ AI + 1 mM DTT + 20 mM S['2+ An + 10 mM NaF + 1 mM DTT + 20 mM Sr2+ An + 10 mM NaF + 1 rnM DTT = 8 mM B a
No. 1. AI No. No. No. No. No.
2. 3. 4. 5. 6.
No.7. No.8. No.9. No. 10.
The reaction was initiated b y the addition of protein (50 to 100 f.lg). Incubation was c-arried out in a water bath at 37 °C for 15 min. Under the conditions tested , the kinetics of the react ion remained linear. Incubation procedure was terminated by the addition of 0.5 n HCI. The mixture was then boiled to denaturato protein. After cooling 400 pI 1 M imidazole buffer were added and the final mixturc w ~.s applied to a 0.5 X 8 cm column pack ed with neutral Al 20 a and eluted with 4 ml 0.1 M imidazole buffer ( pH = 7.1). The eluate was collected direct ly into a scintillation vial and measured. Using this approach over 90 % of the applied cyclic AMP were recovered. The
Towards a specific histochemical localization
173
protein content of the samples was estimated according to LOWRY and co-workers (1951)_ Human serum albumin served as standard protein_ Data were treated for statistics by a nonparametric U-test.
Results The basal activity of adenylate cyclase in rat hippocampus was established to be 577 ± 68 nKatfkg (system AI) and 625 ± 113 nKatfkg (system An). The influences of the revised media on the enzymatic activity are shown in the table. The addition of sodium fluoride increases the activity of adenylate cyclase significantly. Dithiothreitole, in combination with NaF, causes a nearly threefold enhancement of cAMP formation. Lead ions are capable of reducing adenylate cyclase activity even in the millimolar range. A slight increase in cAMP formation after the addition of 1.5 mM Pb2+ instead of ImM is obviously due to a spontaneous (nonenzymatic) substrate-splitting. The presence of G'MP-PNP, a wellknown analogue of GTP, also causes an enhancement of substrate conversion by adenylate cyclase. Of special intercst are the data obtained with Sr2+ and Ba2+ instead of Pb 2+. Although their concentrations in the incubation media reached the 8-fold (Ba2+) or even 20-fold (Sr2+) those of lead ions, their inhibiting influence on the enzyme was considerably low. Concerning thc effect of fixation it was found that after perfusion with formaldehyde, a decrease of adenylate cyclase activity to a level of 44 % of basal activity took place. Surprisingly, pretreatment with glutaraldehyde did not cause an inhibition of the enzymatic activity. The use of suberinacid dimethyl diimidoester, however, enhanced the formation of cyclic AMP up to 128 %.
Table 1. Influence of various modifications of incubation conditions for adenyl ate cyclase (tissue fixed with glutaraldehyde) Test systcm No.
2
Effect on activity (expressed as % of basal activity)
Significance [P] (n = 3)
127 130
> 0.05 (not significant)
3 4 5 6 7 8
267
9
60 83
10
29 47 (!) 136
75 62
< < < < < < < < <
0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03
174
G. POEGGEL a nd H.-G. BERNSTEIN
Discussion Our data permit the following concluRions: 1. The basal activity of adenyla t e cyelase In the rat hippo campus is low. This finding is coin cident with observations made by others (BIGL, personal eommunication). For histo ehemical purposes an enzyme, a cting at a low rate during its normal state, is extremely unfavourable, since it must be expected that hi stoehemical procedures cau se an additional loss in activity. 2. Incubation media , revised for the captur'e ion (strontium or bar ium instead of lead) should guarantee a higher survival ch an ce for enzymatically intact a d enylate cyelase molecules, thus increasing the form a tion rate of visible r eadion produet dming histoche mical in euba tion procedure. While a histochemical m e thod for stL'ontium precipitation of Pi a lready exists (ERXST 19720. and b), no histochemical data are yet availa ble concerning PPi precipitation. The r eplacement of lead b y bar ium does not yield a n electro n-dense reaction product (KEMPEN et a l. 1978). We are now a bout to study the possible u sefulness of b arium and strontium under "real" in situ conditions as substitutes for lead. 3. 'rhe addition of DTT and/or guanylyl imidodiphosphate, in combination with sodium fluoride, should help to increase the basal activity of adenylate cyclase in situ. 4. From the fixatives t ested, suberinacid dimethyl diimidoest er seems to be the " substance of choice". It must, however, be emphasized that glutaraldehyde , a fixative, much easier ava ila ble and widely in use, also gives suffi cient results in our t est system.
Acknowledgements We are deptful to Prof. D r. sc. nat. H. LUPPA for stimulating discussions. We also wish to thank Doz. Dr. sc. med. K. J . HALBHUBER (Jena) for the gift of suberin acid dimethyl diimidoeste r. The courtesy of Prof. Dr. sc. nat. A. ERMISCH (Leipzig) in providing laboratory equipment is gratefully acknowledged.
References ERNST, S. A., Transport a denosine triphosphatase cytochemistry. 1. Biochemical characterization of a cytochemical m edium for the ultrastructural localization of ouabain-sensitive potassium d ependent phosphatase act ivity in the avian salt gland. J . Histochem. Cytochem. 20, 13 - 22 (1972). Tntnsport adenosine triphosphatase cytochemistry. II. Cytochemical localization of ouabain-sensitive phosphatase activity in the secretory epithelium of the avian salt gland. J. Histo chern. 20,23-28 (1972). HALBHvBER, K.-.T., FRoBER, H_, FEUERSTEIN, H., und GEYER, G., Immunologische und enzymatisehe Akti vit1tt nach Fixiemng mit Diimidoesterverbindungen. Mit 10 Abbildungen. Acta. Histochem. 66, 85-100 (1980 ). K EMPEN, H. J. M., DE PONT, J . •T. H. H. M., BONTING, S. L., and STADHOUDERS, A. M., The cytoche mical localiza tion of a d enylate cyclase: Fact, or artifact? J_ Histochem. Cytochem. 26. 298 - 312 (1978). LE MAY, A., and .JARETT, L., Pitfalls in the use of lead nitrate for the histochemical demonstration of adenylate cyclase activity. J . Cell BioI. 65, 39 - 50 (1975). LOWRY, O. H., HOSENBROUGH, N . •J., LEWIS, A., FARR, J., a nd RANDALL, A. L., Protein measurem ent with the folin phenol reagent ..J. BioI. Chern. 193,265 - 272 (1951). NAKAZAWA, K., SANO, M., and SAITO, T., Subcellular distribution and properties of guanyla te cyelase in rat cerebellum. Biochim. Biophys. Acta 444, 563 - 570 (1976).
Towards a specific histochemical localization
175
POEGGEL, G., BF
Division of Cell Biology and R egulation, Biosciences Section, The Karl Marx University, Leipzig, ODR (Director: Prof. Dr. sc. nat. G. SCHUSTER) Institute of Anatomy a nd Biology, Medical Academy Magdeburg, Magdeburg, GDR (Dircc:tol': Prof. Dr. se. med A. DaRN)
Towards a specific histochemical localization of adenyl ate cyclase in the rat hippocampus I. Methodical aspects!,2) By GERD POEGGEL and HANS-GERT BERKSTEIK (Reeeived November 5, 1980)
Summary The adequate histo chemical demonstration of adenyla t e cyclase still invol ves seriom methodical problems (low basal activity, fixation procedure, heavy m et al ions as capture agents etc.). ,.ye h a ve tried to revise fixation a pproach and composition ot incuba tion medium in order to specify the lllode of demonstmiion of the enzyme. The influence of strontium a nd b a rium (instead of lead) as well as dithiothreitole a nd sodium fluoride on the survive of adenylate cyclase activity was studied using a radiochemi cal model system.
Introduction 4 main reasons are responsible for the difficulties, which a histochemist must overcome, if he wants to localize adenylate cyclase (EC 4.6.1.1) specifically in 8itu (especially in the CNS): 1. The enzymatic activity may be very low in the tissue of interest. 2. Adenosine triphosphate, the natural substrate of the enzyme, is also converted by other ferments during incubation (as recently shown by gel staining after microdisc electrophoresis [POEGGEL et aJ. 1980]). :3. The heavy metal ion, which is used to capture and precipitate the inorganic phosphate (largely lead) is capable of catalyzing a nonenzymatic substrate-splitting on one hand, and of inhibiting the enzymatic activity, on t.he other (LEMAY and J ARETT 1975, KEMPEN et al. 1978). 4. The fixation may also depress parts of the adenylate cyclase activity.
1) '.I'his resear'ch was supported by the "Ministerium fUr Ho ch - und Fachschulwesen del' DDR". 2) Parts of the data wel'e presented at the III. SymposiUIll on Neurotransmission 1979, Helsinki.
172
G. POEGGEL and H .·G. BERNSTEIN
Bearing in mind the importance of adenylate cyclase as cAMP-generating enzyme for many metabolic processes, there were efforts to improve the assay conditions for adenylate cyclase (introduction of a synthetic, more specific substrate - adenylyl-imidodiphosphate - by YOUNT et al. [1970], testing of other heavy metal candidates for their ability to form an insoluble reaction product with PPi without considerable loss in enzymatic activity: Barium, KEMPEN et al. [1978]). The present report deals with our experience in modifying the incubation conditions for adenylate cyclase to get a more specific reaction as investigated in a radiochemical model system.
Material and methods Male young albino mts were stuili nd . The animals were kill ed under mild anaesthesia . Some of them were perfused intracttrdially prior to decapitation (formaldehyde, 3 %, 5 min; glutaraldehyde, 2 %,5 min). The brains of
+ T riton X 100 (0.01 % ) AI + 10 mM N a F AI + 10 mM NaF + 1 m1l1 dithiothreitole (DTT) AI + 10 mM N aF + 1 mM DTT + 1 mM Pb2+ AI + 10 mM NaF + 1 mM DTT + 1.5 mM Pb2+ AI + 10 rnM N flF + 10- 5 M guanylyl imidodiphosphl\te (G'MP-PNP, purch ased from Boehringer Mannheim, FRG) AI + 10 mM N aF + 1 mM DTT + 20 mM Sr2+ AI + 1 mM DTT + 20 mM S['2+ An + 10 mM NaF + 1 mM DTT + 20 mM Sr2+ An + 10 mM NaF + 1 rnM DTT = 8 mM B a
No. 1. AI No. No. No. No. No.
2. 3. 4. 5. 6.
No.7. No.8. No.9. No. 10.
The reaction was initiated b y the addition of protein (50 to 100 f.lg). Incubation was c-arried out in a water bath at 37 °C for 15 min. Under the conditions tested , the kinetics of the react ion remained linear. Incubation procedure was terminated by the addition of 0.5 n HCI. The mixture was then boiled to denaturato protein. After cooling 400 pI 1 M imidazole buffer were added and the final mixturc w ~.s applied to a 0.5 X 8 cm column pack ed with neutral Al 20 a and eluted with 4 ml 0.1 M imidazole buffer ( pH = 7.1). The eluate was collected direct ly into a scintillation vial and measured. Using this approach over 90 % of the applied cyclic AMP were recovered. The
Towards a specific histochemical localization
173
protein content of the samples was estimated according to LOWRY and co-workers (1951)_ Human serum albumin served as standard protein_ Data were treated for statistics by a nonparametric U-test.
Results The basal activity of adenylate cyclase in rat hippocampus was established to be 577 ± 68 nKatfkg (system AI) and 625 ± 113 nKatfkg (system An). The influences of the revised media on the enzymatic activity are shown in the table. The addition of sodium fluoride increases the activity of adenylate cyclase significantly. Dithiothreitole, in combination with NaF, causes a nearly threefold enhancement of cAMP formation. Lead ions are capable of reducing adenylate cyclase activity even in the millimolar range. A slight increase in cAMP formation after the addition of 1.5 mM Pb2+ instead of ImM is obviously due to a spontaneous (nonenzymatic) substrate-splitting. The presence of G'MP-PNP, a wellknown analogue of GTP, also causes an enhancement of substrate conversion by adenylate cyclase. Of special intercst are the data obtained with Sr2+ and Ba2+ instead of Pb 2+. Although their concentrations in the incubation media reached the 8-fold (Ba2+) or even 20-fold (Sr2+) those of lead ions, their inhibiting influence on the enzyme was considerably low. Concerning thc effect of fixation it was found that after perfusion with formaldehyde, a decrease of adenylate cyclase activity to a level of 44 % of basal activity took place. Surprisingly, pretreatment with glutaraldehyde did not cause an inhibition of the enzymatic activity. The use of suberinacid dimethyl diimidoester, however, enhanced the formation of cyclic AMP up to 128 %.
Table 1. Influence of various modifications of incubation conditions for adenyl ate cyclase (tissue fixed with glutaraldehyde) Test systcm No.
2
Effect on activity (expressed as % of basal activity)
Significance [P] (n = 3)
127 130
> 0.05 (not significant)
3 4 5 6 7 8
267
9
60 83
10
29 47 (!) 136
75 62
< < < < < < < < <
0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03
174
G. POEGGEL a nd H.-G. BERNSTEIN
Discussion Our data permit the following concluRions: 1. The basal activity of adenyla t e cyelase In the rat hippo campus is low. This finding is coin cident with observations made by others (BIGL, personal eommunication). For histo ehemical purposes an enzyme, a cting at a low rate during its normal state, is extremely unfavourable, since it must be expected that hi stoehemical procedures cau se an additional loss in activity. 2. Incubation media , revised for the captur'e ion (strontium or bar ium instead of lead) should guarantee a higher survival ch an ce for enzymatically intact a d enylate cyelase molecules, thus increasing the form a tion rate of visible r eadion produet dming histoche mical in euba tion procedure. While a histochemical m e thod for stL'ontium precipitation of Pi a lready exists (ERXST 19720. and b), no histochemical data are yet availa ble concerning PPi precipitation. The r eplacement of lead b y bar ium does not yield a n electro n-dense reaction product (KEMPEN et a l. 1978). We are now a bout to study the possible u sefulness of b arium and strontium under "real" in situ conditions as substitutes for lead. 3. 'rhe addition of DTT and/or guanylyl imidodiphosphate, in combination with sodium fluoride, should help to increase the basal activity of adenylate cyclase in situ. 4. From the fixatives t ested, suberinacid dimethyl diimidoest er seems to be the " substance of choice". It must, however, be emphasized that glutaraldehyde , a fixative, much easier ava ila ble and widely in use, also gives suffi cient results in our t est system.
Acknowledgements We are deptful to Prof. D r. sc. nat. H. LUPPA for stimulating discussions. We also wish to thank Doz. Dr. sc. med. K. J . HALBHUBER (Jena) for the gift of suberin acid dimethyl diimidoeste r. The courtesy of Prof. Dr. sc. nat. A. ERMISCH (Leipzig) in providing laboratory equipment is gratefully acknowledged.
References ERNST, S. A., Transport a denosine triphosphatase cytochemistry. 1. Biochemical characterization of a cytochemical m edium for the ultrastructural localization of ouabain-sensitive potassium d ependent phosphatase act ivity in the avian salt gland. J . Histochem. Cytochem. 20, 13 - 22 (1972). Tntnsport adenosine triphosphatase cytochemistry. II. Cytochemical localization of ouabain-sensitive phosphatase activity in the secretory epithelium of the avian salt gland. J. Histo chern. 20,23-28 (1972). HALBHvBER, K.-.T., FRoBER, H_, FEUERSTEIN, H., und GEYER, G., Immunologische und enzymatisehe Akti vit1tt nach Fixiemng mit Diimidoesterverbindungen. Mit 10 Abbildungen. Acta. Histochem. 66, 85-100 (1980 ). K EMPEN, H. J. M., DE PONT, J . •T. H. H. M., BONTING, S. L., and STADHOUDERS, A. M., The cytoche mical localiza tion of a d enylate cyclase: Fact, or artifact? J_ Histochem. Cytochem. 26. 298 - 312 (1978). LE MAY, A., and .JARETT, L., Pitfalls in the use of lead nitrate for the histochemical demonstration of adenylate cyclase activity. J . Cell BioI. 65, 39 - 50 (1975). LOWRY, O. H., HOSENBROUGH, N . •J., LEWIS, A., FARR, J., a nd RANDALL, A. L., Protein measurem ent with the folin phenol reagent ..J. BioI. Chern. 193,265 - 272 (1951). NAKAZAWA, K., SANO, M., and SAITO, T., Subcellular distribution and properties of guanyla te cyelase in rat cerebellum. Biochim. Biophys. Acta 444, 563 - 570 (1976).
Towards a specific histochemical localization
175
POEGGEL, G., BF