Brain Research, 177 (1979) 301-309 © Elsevier/North-Holland BiomedicalPress

301

BIOCHEMICAL CHARACTERIZATION OF POSTSYNAPTICALLY LOCALIZED CYCLIC NUCLEOTIDE PHOSPHODIESTERASE

MARJORIE A. ARIANO and M. MICHAEL APPLEMAN Molecular Biology, Ahmanson Center for Biological Research, University of Southern California, Los Angeles, Calif. 90007 (U.S.A.)

(Accepted February 22nd, 1979)

SUMMARY This study demonstrates the postsynaptic localization of one of the isozymes of cyclic nucleotide phosphodiesterase (PDE) activity at asymmetrical, axospinous terminals in the rat corpus striatum and neocortex. Characterization of this enzymatic activity demonstrates that the PDE form surviving aldehyde fixation for electron cytochemistry can be considered to preferentially hydrolyze cyclic 3'5'-guanosine monophosphate, and it requires calcium and a heat-stable calcium-dependent regulator protein (CDR) for full hydrolytic activity. Ion exchange chromatographic analysis of extracts of corresponding unfixed brain regions demonstrates that only one enzyme activity peak exhibits similar aldehyde resistance and calcium and regulator protein activatibility. INTRODUCTION The cyclic nucleotides have been hypothesized to mediate effects of central nervous system neurotransmitters via their role as second messengersS,18,20,~4. Cyclic nucleotide phosphodiesterase (PDE) (EC 3.1.4.17), the enzyme activity capable of degrading these second messengers, exists in multiple forms capable ofhydrolyzing cyclic 3',5'-adenosine monophosphate (cyclic AMP), cyclic 3',5'-guanosine monophosphate (cyclic GMP), or cyclic 3',5'-cytidine monophosphateS,7,17, 27. The different PDE forms have different substrate specificities and kinetic properties, and appear to be differentially regulated. DEAE-cellulose chromatography resolves discrete PDE fractions from liver23,27, heart 2, and brain homogenates 28. In order of their elution from a chromatography column by a salt gradient, the first active fraction (D-I) preferentially hydrolyzes cyclic GMP when examined at a low substrate concentration. This enzyme activity, which has also been referred to as a low affinity cyclic AMP PDE, requires calcium and a calcium-dependent regulator protein (CDR) for full

302 activity16, zg. The second active DEAE fraction (D-2) is a cyclic GMP-stimulated cyclic AMP PDE 3,9, while the third (D-3) has greater cyclic AMP hydrolyzing activity 3. Neither the second nor the third PDE fractions are activated by calcium and CDR. Cyclic nucleotide phosphodiesterase activity may be cytochemically visualized by the precipitation of lead phosphate, formed by the sequential actions of the enzyme and 5'-nucleotidase on the cyclic nucleotide lo. This cytochemical method has been used to determine the specific site of PDE activity within various brain regions1,4,10, photoreceptor outer segments 22, at the neuromuscular junction 21, and in the renal medullalL Location of a phosphodiesterase activity juxtaposed to the postsynaptic side of a synaptic terminal allows the enzyme to regulate elevated cyclic nucleotide levels produced in response to the binding of first messenger neurotransmitters and implies a role for cyclic nucleotides in synaptic function. Cytochemical localization of PDE is considered specific when 3',5'-cyclic nucleotide must be used as a substrate, endogenous 5'-nucleotidase does not contribute to the formation of extraneous reaction product, and the PDE activity can be inhibited by one of the methyl xanthines 1,4. Although PDE activity has been anatomically localized within the central nervous system, there are no reports as to the identity of the enzyme form representing this activity. The present work was initiated to identify the specific form of the cytochemically visualized PDE through the investigation of activators, resistance to aldehyde fixation, and enzyme kinetic properties. MATERIALS AND METHODS

EM cytochemistry. Sprague-Dawley rats, 250-350 g, were anesthetized with sodium pentobarbital and vascularly perfused with cold 2 ~ EM-grade glutaraldehyde (Polysciences), 2 ~ paraformaldehyde in 0.05 M sodium cacodylate buffer containing 0.25 M sucrose at pH 7.4. Following dissection of the neostriatum or the neocortex, 100 #m slices were cut on a tissue chopper and incubated at 37 °C in a phosphodiesterase assay media containing 1 mM cyclic AMP or cyclic G M P (Sigma), 2 mM lead nitrate, and snake venom as a source of 5'-nucleotidase (Crotalus atrox, Sigma, 10 mg/ml) in a 60 mM Tris.maleate buffer containing 5 mM magnesium chloride and 0.25 M sucrose at pH 7.4. The reaction was stopped by the addition of l ~ osmium tetroxide in cacodylate buffer, the tissue sections were dehydrated in a graded acetone series, and embedded in Araldite 502. Thin sections were cut on a Sorval MT-2B ultramicrotome and examined, unstained, with a JEM 7 electron microscope. The following controls were performed to verify the specificity of the cytochemical localization: 5 mM isobutyl-methylxanthine (Aldrich) was added to inhibit PDE activity; cyclic G M P without additives or total omission of substrate yielded no reaction product formation; snake venom was omitted without reaction product formed; or the tissue was heated to 100 °C for 5 min prior to cyclic G M P PDE cytochemical assay, with no subsequent lead phosphate produced. Enzyme preparation. Brains were removed from 150-250 g Sprague-Dawley rats killed by decapitation. The neostriata or cerebral cortices were dissected out and the

303 tissue was homogenized in 10 vols. of chromatography buffer (0.05 M sodium acetate, 5 mM magnesium chloride, and 5 mM 2-mercaptoethanol, pH 6.4), with a Brinkmann Polytron at maximum speed, followed by sonication for 30 sec at a setting of 100 mW with a Braun Biosonic III. All procedures were performed at 4 °C. The homogenate was centrifuged at 30,000 × g for 20 mirt and a 5 ml aliquot of the supernatant was chromatographed on a 1 × 15 cm column of DEAE-cellulose (Bio-Rad), previously equilibrated with buffer containing either 1 mM CaC12 or 1 mM EGTA. The column was washed with the same buffer and eluted with an 80 ml linear gradient of 0.05 M-1.0 M sodium acetate. Fractions were collected from the point of sample application and assayed for PDE activity and calcium-sensitive protein regulator. P D E assay. Tissues were assayed for PDE activity according to the isotopic method of Russell, et al. 2a, using [3H]cyclic AMP (spec. act. 46.5 Ci/mmol) and [all]cyclic G M P (spec. act. 10.2 Ci/mmol) from New England Nuclear. Batch separation of products with the anion exchange resin, AG1-X-2 equilibrated with 50 ethanol, was the final step of the assay procedure. Product recoveries were calculated using [3H]adenosine or [aH]guanosine standards. One unit of PDE activity is defined as the amount of enzyme which hydrolyzes 1 nmol of cyclic nucleotide/min at 30 °C in the 0.4 ml assay vol. Under the conditions of assay (less than 30 % hydrolysis), activities were proportional to the amount of enzyme. CDR preparation. Calcium-dependent regulator was prepared by heating whole brain homogenates at 100 °C for 5 min, followed by sonication for 15 sec at a setting of 100 mW (Biosonic III), and centrifugation at 30,000 × g for 20 min. The supernatant was acid precipitated (pH 4) for 1 h at 0 °C, then dialyzed 18 h against 10 vols. of 5 mM Tris.HC1, pH 7.4 at 4 °C. CDR content of boiled samples was assayed by measuring the increase in hydrolysis of micromolar cyclic GMP by a standard PDE preparation which produced 5 ~ hydrolysis in the absence of the regulator. The quantity of CDR used to determine the activation of a PDE fraction is the amount which yields a 50 % increase in cyclic GMP hydrolysis under these conditions2L Protein was measured by the method of Lowry et al. 19. RESULTS Cyclic nucleotide phosphodiesterase activity, visualized as a lead phosphate precipitate, is localized postsynaptically at asymmetrical, axospinous terminals in rat neocortex and corpus striatum following incubation with millimolar concentrations of substrate (Figs. 1 and 2). Visualization of cyclic GMP PDE activity requires the addition of calcium and a heat-stable calcium-dependent regulator in the incubation medium (Fig. 3), while cyclic AMP PDE activity is demonstrable without additions. These results led us to further characterize the isozymes present in homogenates of these brain regions prior to fixation. DEAE cellulose chromatography of these preparations in the presence of calcium indicates the existence of a number of active fractions (Fig. 4A). Under standard assay conditions (10 -6 M substrate), cyclic GMP hydrolysis was significantly greater than cyclic AMP hydrolysis. Assays of the enzyme fractions with excess CDR increased the activity of D-l, but had no effect or even

304

Fig. 1. Cytochemical localization of phosphodiesterase activity in unstained rat neocortex. * Denotes a reacting dendrite following incubation of the tissue with 1 mM cyclic AMP. x 44,000.

Fig. 2. Cyclic GMP-PDE hydrolytic activity in corpus striatum, following assay with 1 mM cyclic GMP with the addition of 2 m M calcium and excess CDR in the incubation fluid. * Shows a reacting postsynaptic dendrite in the unstained thin section, x 40,400.

305

Fig. 3. Neostriatum following incubation of the 100 # m tissue slice with 1 m M cyclic G M P alone. Formation of reaction product, representative of phosphodiesterase activity, is not evident without the addition of modifiers. × 34,000.

t A.

cAMP •

• •

r A M P cGMP

cGMP o ' STANDARD o

a

ACTIVATED ASSAY

• •

o o

STANDARD ASSAY ACTIVATED ASSAY

GI % FIXATIVE

•

A

0,1% F I X A T I V E

ASSAY

6O

~

50 CDR

J

E

ttl I--

i

1.2

i.o

~_~o I--

:16 3.4

3

7

11

15

FRACTION

19

23

27

NUMBER

31

35

3

FRACTION n

15

19

25

2"r

31

38

NUMBER

Fig. 4. D E A E cellulose chromatography profiles of cortical phosphodiesterase activity. PDE activity is assayed with micromolar substrate in the standard assay; 20 # M CaCI2 and excess CDR are added in the activated assay; or with the fixative diluted to a final concentration of 0.1%. Solid bar indicates C D R activity. A: column equilibrated with 1 m M CaCI2 prior to sample application. B: column equilibrated with 1 m M E G T A prior to sample application.

306

reduced the activity of peaks eluted at a higher salt gradient. When enzyme fractions were exposed to aldehyde fixative conditions in vitro (see Methods), cyclic nucleotide hydrolysis decreased but was still significant in the D-1 peak. Addition of excess CDR and 20 #M calcium to the dilute fixative solution increased the hydrolytic activity of the D-1 peak, but had no effect on either D-2 or D-3 (Table I). DEAE chromatography in the presence of 1 mM EGTA yielded greatly reduced enzyme activity when compared to that with calcium (Fig. 4B). Enzyme activity was markedly stimulated upon addition of excess CDR and calcium chloride to the assay. Also in this case, the activity eluting at higher salt concentrations was rapidly inactivated upon addition of dilute fixative solutions, while the D-1 peak retained 10-20~ of its hydrolytic activity. The presence of CDR was determined on the chromatographically resolved enzyme fractions. The activator consistently eluted beyond the PDE activity, at quite high salt concentrations in both calcium- and EGTA-equilibrated chromatography columns (Fig. 4). A 6 0 - 8 0 ~ decrease in total phosphodiesterase activity occurred in equal weights of unfixed and fixed homogenates following use of the fixative. The surviving P D E activity in E M - p r e p a r e d tissues c o u l d be s t i m u l a t e d 2 0 - 3 0 ~ w i t h excess C D R

and m i c r o m o l a r c a l c i u m ( T a b l e I). K i n e t i c c o m p a r i s o n s o f h o m o g e n a t e s f r o m fresh and fixed b r a i n p r e p a r a t i o n s g a v e s u b s t r a t e affinities a n d r e a c t i o n velocities o f c o m p a r a b l e values, b u t were n o t c o n s i s t e n t enough to further distinguish which P D E i s o z y m e s u r v i v e d the aldehyde p r o c e s s i n g f o r E M c y t o c h e m i s t r y . TABLE I Characterization o f rat brain P D E fractions

DEAE resolved PDE activities from pooled rat neocortex and corpus striatum were assayed with micromolar cyclic AMP or cyclic GMP in the standard assay. Comparison is also made with liver isozymes prepared in the same manner. The activated assay included 20/~M CaC12 and excess CDR in addition to the substrate. Fixative-treated extracts were tested with 0.1 ~ EM fixative in the 0.4 ml assay volume. Data represent the average of at least 3 experiments with values expressed as nmol of enzyme/ml eluate/min.

Enzyme fraction*

D-1 cyclic AMP cyclic GMP D-2 cyclic AMP cyclic GMP D-3 cyclic AMP cyclic GMP Homogenates** cyclic AMP cyclic GMP

Untreated fractions

Fixative treated

Liver

Brain

Brain

Standard

Standard

Activated

Standard

Activated

0.5 7

13 28

19 45

7 12

8 13

2 1

4 4

2 7

4 2

3 4

1 0.2

7 4

4 2

4 4

4 3

62 41

105 73

146 89

17"** 27***

22*** 33***

* See text for characteristics of the D E A E resolved peaks, D-l, D-2, D-3. ** Expressed as enzyme/g of tissue/min. *** Homogenate of in vivo fixed tissue.

307 DISCUSSION This study demonstrates that the form of cyclic nucleotide phosphodiesterase activity which can be cytochemically visualized at postsynaptic sites in the rat corpus striatum and neocortex is one which preferentially hydrolyzes cyclic GMP rather than cyclic AMP at substrate concentrations in the physiological range. Other investigators have reported similar patterns of postsynaptic cyclic AMP hydrolytic activity at morphologically equivalent synapses1,4,10,21 but correlation of this anatomical localization with the different enzymatic properties known to exist for the multiple forms of phosphodiesterase activity17,2s had not been carried out. Utilization of glutaraldehyde and paraformaldehyde in preparative steps for cytochemistry appears to rapidly inactivate the two cyclic AMP hydrolyzing isozymes of PDE which are detected by DEAE cellulose chromatography in fresh tissue extracts. The requirement for added calcium and CDR for cytochemical visualization of reaction product in aldehyde fixed brain slices incubated with cyclic GMP, indicates that the surviving enzyme is the cyclic GMP-preferring form. This conclusion is further substantiated by the data using homogenates of fixed tissue which show a significant stimulation of cyclic nucleotide hydrolysis upon addition of these modifiers. It has been recently reported 15 that in brain, the highest activity of the cyclic GMP PDE as well as guanylate cyclase is located in the corpus striatum, suggesting an important function for the guanosine nucleotide in this area. The presence of calcium and CDR also increases the residual hydrolytic activity of D-I, the first enzyme peak resolved by DEAE chromatography, when it is exposed to dilute fixative solutions prior to assay (Table I). D-2 and D-3, the cyclic AMP PDE peaks, are effectively inhibited when assayed in the presence of dilute fixative, and the use of calcium and CDR modifiers does not overcome this inhibition. Use of an EGTA-equilibrated DEAE chromatography column results in a 78 ~ loss of cyclic GMP hydrolytic activity when compared to that from the Cag'+-containing column. The activation of the enzyme fraction from the chelator column upon addition of calcium and CDR is 1.6-fold greater than that from the Ca2+-containing column, suggesting that EGTA may chelate calcium or another metal essential to the enzyme for basal activity or stability independent of the CDR protein. Our results with the first PDE activity peak are in agreement with those of Uzunov and co-workers for their peak II on gel electrophoresis12, 80, in that both have a low affinity for cyclic AMP (Kin of 350 #M) and a high affinity for cyclic GMP (Kin of approximately 6/zM). Although addition of excess CDR to the PDE assay increases the affinity of this enzyme for cyclic AMP, it lowers the Km only to 80 #M, while the Km for cyclic GMP is an order of magnitude lower. The presence of CDR and Ca ~+ in the assay doubles the velocity of cyclic GMP hydrolysis at high substrate concentrations, and in the cytochemical analysis, leads to the formation of enough reaction product for visualization at the ultrastructural level. Though Gnegy and colleagues describe their peak II phosphodiesterase as a cyclic AMP PDE, we feel this enzyme is more aptly referred to as cyclic GMP PDE in view of this data. Greenberg et al. ~5, have studied the enzymatic regulation of cyclic GMP levels in

308 r o d e n t b r a i n a n d have shown t h a t the corpus s t r i a t u m has the highest p r o p o r t i o n o f C D R - d e p e n d e n t P D E , followed by the b r a i n stem, a n d t h a t cyclic G M P P D E m a y be m o r e i m p o r t a n t t h a n guanylate cyclase in c o n t r o l l i n g the b a s a l cyclic G M P levels. The present investigation d e m o n s t r a t e s a p o s t s y n a p t i c l o c a t i o n for cyclic G M P h y d r o l y t i c activity in the n e o s t r i a t u m a n d n e o c o r t e x suggesting a role for the guanosine nucleotide in synaptic t r a n s m i s s i o n in these structures. The C D R r e q u i r e m e n t for full P D E h y d r o l y t i c activity focusses a t t e n t i o n on this protein, which also interacts with adenylate cyclase6,1a, 14,26 a n d strengthens the p r o p o s e d role o f the a c t i v a t o r in regulating synaptic levels o f the cyclic nucleotides. ACKNOWLEDGEMENTS This w o r k was s u p p o r t e d in p a r t by the K r o c F o u n d a t i o n a n d U S P H S G r a n t s A M 05925, A M 16367, A M 20978, a n d a n N I H Career D e v e l o p m e n t A w a r d to M.M.A. REFERENCES 1 Adinolfi, A. M. and Schmidt, S. Y., Cytochemical localization of cyclic nucleotide phosphodiesterase activity at developing synapses, Brain Research, 76 (1974) 21-31. 2 Appleman, M. M. and Terasaki, W. L., Regulation of cyclic nucleotide phosphodiesterase, ,4dvanc. Cyclic Nucleotide Res., 5 (1975) 153-162. 3 Appleman, M. M., Thompson, W. J. and Russell, T. R., Cyclic nucleotide phosphodiesterases, .4dvanc. Cyclic Nucleotide Res., 3 (1973) 65-98. 4 Ariano, M. A. and Adinolfi, A. M., Subcellular localization of cyclic nucleotide phosphodiesterase in the caudate nucleus, Exp. NeuroL, 55 (1977) 84-94. 5 Beavo, J. A., Hardman, J. G. and Sutherland, E., Hydrolysis of cyclic guanosine and adenosine 3',5'-monophosphate by rat and bovine tissues, J. biol. Chem., 246 (1970) 5649-5655. 6 Brostrom, C. O., Huang, Y.-C., Breckenridge, B. McL. and Wolff, D. J., Identification of a calciumbinding protein as a calcium-dependent regulator of brain adenylate cyclase, Proc. nat. Acad. Sci. (Wash.), 72 (1975) 64-68. 7 Cheng, Y.-C. and Bloch, A., Demonstration, in leukemia L-1210 cells, of a phosphodiesterase acting on 3',5'-cyclic CMP but not on 3',5'-cyclic AMP or 3',5'-cyclic GMP, J. biol. Chem., 253 (1978) 2522-2524. 8 Ferrendelli, J. A., Kinscherf, D. A. and Chang, G. M., Comparison of the effects of biogenic amines on cyclic GMP and cyclic AMP levels in mouse cerebellum in vitro, Brain Research, 84 (1975) 63-73. 9 Filburn, C. R., Colpo, F. and Sacktor, B., Regulation of cyclic nucleotide phosphodiesterases of cerebral cortex by Ca 2+ and cyclic GMP, J. Neurochem., 30 (1978) 337-346. 10 Florendo, N. T., Barrnett, R. J. and Greengard, P., Cyclic 3',5'-nucleotide phosphodiesterase: cytochemical localization in cerebral cortex, Science, 171 (1971) 745-747. 11 Florendo, N. T., Pitcock, J. A. and Muirhead, E. E., Cyclic 3',5'-nucleotide phosphodiesterase; cytocbemical localization in rat renomedullary interstitial cells. Phosphodiesterase in rat renal medulla, J. Histochem. Cytochem., 26 (1978) 441--451. 12 Gnegy, M. E., Costa, E. and Uzunov, P., Regulation of transsynaptically elicited increase of 3',5'cyclic AMP by endogenous phosphodiesterase activator, Proc. nat. ,4cad. Sci. (Wash.), 73 (1976) 352-355. 13 Gnegy, M. E., Uzunov, P. and Costa, E., Regulation of dopamine stimulation of striatal adenylate cyclase by an endogenous Ca~+-binding protein, Proc. nat. Acad. Sci. (Wash.), 73 (1976) 3887-3890. 14 Gnegy, M. E., Uzunov, P. and Costa, E., Participation of an endogenous Ca2+-binding protein activator in the development of drug-induced supersensitivity of striatal dopamine receptors, J. Pharmacol. exp. Ther., 202 (1977) 558-564. 15 Greenberg, L. H., Troyer, E., Ferrendelli, J. A. and Weiss, B., Enzymatic regulation of the concentration of cyclic GMP in mouse brain, Neuropharmacology, 17 (1978) 737-745.

309 16 Kakiuchi, S. and Yamazaki, R., Calcium-dependent phosphodiesterase activity and its activating factor (PAF) from brain: studies on cyclic 3',5'-nucleotide phosphodiesterase, Biochim. biophys. Res. Commun., 41 (1970) 1104-1110. 17 Kakiuchi, S., Yamazaki, R. and Teshima, Y., Cyclic 3',5'-nucleotide phosphodiesterase. IV. Two enzymes with different properties from brain, Biochim. biophys. Res. Commun., 42 (1971) 968-974. 18 Kebabian, J'. W. and Greengard, P., Dopamine-sensitive adenyl cyclase: possible role in synaptic transmission, Science, 174 (1971) 1346-1349. 19 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 20 McAfee, D. A., Schorderet, M. and Greengard, P., Adenosine 3',5'-monophosphate in nervous tissue, increase associated with synaptic transmission, Science, 173 (1971) 1156-1158. 21 Pardos, G. J. and Lentz, T. L., Cytochemical localization of cyclic Y,5'-nucleotide phosphodiesterase activity in the neuromuscularjunction and skeletal muscle of the newt, Brain Research, 107 (1976) 355-364. 22 Robb, R. M., Histochemical evidence of cyclic nucleotide phosphodiesterase in photoreceptor outer segments, Invest. Ophthal. 13 (1974) 740-747. 23 Russell, T. R., Terasaki, W. L. and Appleman, M. M., Separate phosphodiesterases for the hydrolysis of cyclic adenosine 3',5'-monophosphate and cyclic guanosine 3',5'-monophosphate in rat liver, J. biol. Chem., 248 (1973) 1334-1340. 24 Siggins, G. R., Hoffer, B. J. and Bloom, F. E., Studies on norepinephrine-containingafferents to Purkinje cells of rat cerebellum. Iii. Evidence for mediation of norepinephrine effects by cyclic 3',5'-adenosine monophosphate, Brain Research, 25 (1971) 535-553. 25 Teo, T. S., Wang, T. H. and Wang, J. H., Purification and properties of the protein activator of bovine heart cyclic adenosine 3',5'-monophosphate phosphodiesterase, J. biol. Chem., 248 (1973) 588-595. 26 Teshima, Y. and Kakiuchi, S., Membrane-bound forms of Ca2÷-dependent protein modulator: Ca2÷-dependent and independent binding of modulator protein to the particulate fraction from brain, J. Cyclic Nucleotide Res., 4 (1978) 219-241. 27 Thompson, W. J. and Appleman, M. M., Characterization of cyclic nucleotide phosphodiesterase of rat tissues, J. biol. Chem., 246 (1971) 3145-3149. 28 Thompson, W. J. and Appleman, M. M., Multiple cyclic nucleotide phosphodiesterase activities from rat brain, Biochemistry, 10 (1971) 311-316. 29 Uzunov, P., Gnegy, M. E., Lehne, R., Revuelta, A. V. and Costa, E., A neurobiological role for a protein activator of cyclic nucleotide phosphodiesterase, Advanc. Biochem. Psychopharmacol. 15 (1976) 283-334. 30 Uzunov, P., Lehne, R., Revuelta, A. V., Gnegy, M. E. and Costa, E., A kinetic analysis of the cyclic nucleotide phosphodiesterase regulation by the endogenous protein activator. A study of rat brain and frog sympathetic chain, Biochim. biophys. Acta (Amst.), 422 (1976) 326-334. 31 Uzunov, P. and Weiss, B., Separation of multiple molecular forms of cyclic adenosine Y,5'-monophosphate phosphodiesterase in rat cerebellum by polyacrylamide gel electrophoresis, Biochim. biophys. Acta (Amst.), 284 (1972) 220-226.