SHORT COMMUNICATION
Ontogenetic studies of cCMPdependent protein kinase in rat cerebellum (Receired 6 September 1978. Accepted 10 October 1978)
THERAT cerebellum is characterized by its high content of cyclic 3'.5'-guanosine monophosphate (cGMP). The cerebellar concentration of this nucleotide is about ten times higher than that of any other brain area examined so far (STEINERet a/., 1972: MAO & GUIDOTTI, 1974). Particularly high cGMP-concentrations were found in the cerebellar cortex (BIGGIO& GuroorTr. 1976; BIGGIOer ul., 1977; RUBIN& FERRENDELLI, 1977), where this nucleotide probably is preferentially located in Purkinje cells (MAO er ul., 1975). Upon destruction of the climbing fibers (one of the main excitatory inputs to the Purkinje cells) with 3-acetylpyridine both the Purkinje cells and the cerebellar & GUIDOTTI. 1976). PharcGMP level are affected (BIGGIO macological manipulations of the cerebellar GABAergic function change the firing rate of Purkinje cells (BISTIet a/., 1971; CURTISet a/.. 1970) and the cGMP content in the cerebellar cortex (MAOet ul., 1974~.b ; BIGGIOet a / . , 1977). These lines of evidence suggest that cGMP participates as second messenger in the regulation of the cerebellar Purkinje cell activity. Among the enzyme systems affected by cGMP are protein kinases which are stimulated by this nucleotide (GOLDBERG & HADDOX.1977). Relatively high levels of cGMP-dependent protein kinase (EC 2.7.1.37; cGPK) activities have been found in mammalian tissues with high concentrations of cGMP (Kuo et u'l., 1976b) in& SOLD, 1972: cluding the cerebella from rat (HOFFMAN LINCOLN et ol., 1976). and cow (TAKAIet ul.. 1975). and & SANO.1975; GILLet ul., 1976; other tissues (NAKAZAWA KUOe t ul.. 1976~:SHoii et ul., 1977). Changes in cGPK activity during development occur in lung. heart, brain and cerebellum of guinea pig (Kuo. 1975; Kuo er a/., 1976a). The aim of our study was to analyze the changes in cGPK activity during the ontogenesis of the rat cerebellum and to relate these changes to the temporal sequence of differentiation of the cerebellar neuronal components and to the concomitant changes in the cGMP content of that brain area. In rodents the cerebellar cGMP content is very low at birth and increases sharply during the first postnatal et (11.. 1972; SPANOet ul.. 1975). month (STEINER EXPERIMENTAL PROCEDURES Assax o f c C P K tictiriry. cGPK activity was defined as the difference between the incorporation of 3zPO;3 from
Abbreviations u s e d : CAMP, cyclic AMP. cGMP. cyclic Y.5'-guanosine monophosphate; cGPK. cyclic GMPdependent protein kinase: DEAE, diethylaminoethyl: KEM. buffer containing 25 mM-K-phosphate pH 7.5. I mM-EDTA and 50 mhc-mercaptoethanol.
[;-32P]-ATP into histones in the presence and absence of cGMP. One unit of cGPK activity was defined as the incorporation of 1 pmol 3 2 P O Q 3 in IOmin at 30 C. The specific activity of cGPK was expressed as enzyme units per mg protein charged onto thc DE column (see Preparation of cGPK). Unless otherwise specified, the assay of cGPK has initiated by the addition of 1 4 0 ~ 1of a mix containing 140 mM-Na acetate, 70 mM-Mg acetate, 5 mwaminophql(spec. act. line, 20pg histone IIA. 7 phc-[;.-"P]ATP P pH. 5-10 x lo' c.p.m. per pmol). and 0.7 ~ M - c G M (final 8) to lop1 of the 'peak eluate' (containing 10-40jcg protein and up to 10 units of cGPK). The reaction was carried out at 3 0 C for 10min and was terminated by pipetting 50p1 of the incubation mixture on to Whatman 3 MM filter paper strips (2.5 x 35cm), presiously soaked a i t h 'precipitating solution' (5:; wjv TCA. 0.25"" w I Na2W0,. 2 H 2 0 . 204 v/v H,SO, conc.; Krjo & GREI:NGARD. 1972) and dried before use. The strips were washed 3 times for 20 min with 31 'precipitating solution'. This procedurr typically leaves 0.0157" of the input radioactivity as blank while retaining 94":, of the phosphorylated histones on the filter. The assay in absence of cGMP was performed in the same way except that no cyclIc nucleotide \+as added. All assays were run in duplicate. Preparation o f c G P K . The animals were decapitated and the cerebella homogenized with 4 b o l of KEM (25 m w K phosphate pH 7.5, 1 mM-EDTA. 50 mM-mercaptoethanol). The homogenates were spun for 2Omin at 2O.OOOg. All operations were performed at 0 4-C. The sample (containing 0.2-1 mg protein and 40-250 units of cGPK) of each of the supernatants was placed onto a DE.52 cellulose column ( 3 0 0 ~ 1bed vol. approx. 8 mm high and 5.5 mm i.d.) equilibrated with KEM. The column was washed Kith 150p1 KEM. followed by 150pI KEM-0.175 M - N ~ C IThe . cGPK was then eluted with 300pl KEM 0.175w-NaCI. This fraction is referred to as the 'peak eluate'. The recotery of partially purified cGPK through this procedure uas consistently better than W,. Only trace amounts of cCPK-dependent activity could be detected in the fractions before or after the 'peak eluate'. The cGPK activity in the 'peak eluate'. as well as i n the supernatant before DE52 column. remained constant for at least one week when kept at 0-4 C. but nas lost upon freezing. A detailed analysis of this method m i l l be & GLYDOTTI. i n preparation). described elsewhere (BANDLE Assay c f c G M P binding activity. This determination was done as described by MURAD P I a / . (1971) except that KEM was used instead of 50 mM-Na-acetate pH 4 for the incubation. Other dererminutions. The degree of cGMP degradation
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Flc. I . Rat cerebellar cyclic GMP-dependent protein kinase, 13H]cGMP binding activity and cerebellar and ['HlcGMP binding wet weight during ontogenesis. The specific activities of cGPK (U--m) ( O - - - O ) (upper part of the figure) and the wet weight of the cerebellum (lower part of the figure) were determined from rats of the ages indicated. For each age 4 7 animals were used and the result expressed as the mean ~ s . E . M .For methods see Experimental Procedures.
in the assay of cGPK was measured by substituting cGMP with ['HlcGMP (1.5 x lo5 c.p.m.) for cGMP in reaction mixes. The C3H]cGMP was isolated according to MAO& GuiDom (1974) either before or after the incubation (10 min/30"C) and the recoveries were compared. [3zP]Phosphohistones were prepared in an assay for cGPK using a slight modification of the method of MAENO et al. (1975). Adult rat cerebella served as the enzyme source. After an incubation for 60min at 30°C the reaction mixture was chromatographed through a Sephadex (3-25 column (0.55 x 5 cm). The column was eluted with KEM in 100pI portions and the phosphohistones isolated in the excluded fractions. Wherever this preparation was used, the radioactive phosphohistones were measured as the material that was insoluble in 'precipitating solution' and was retained on Millipore HAWP filters. c G M P was measured with the radioimmunoassay as described by HARPER& BRWKER(1975). Protein concentrations were determined using the method of BRADFORD (1976) with bovine serum albumin as standard. RESULTS Appearance of cGPK dtiring the ontogenesis of rat cerebellum
Figure I shows c G M P binding, cGPK activity and cerebellar weight in Sprague-Dawley rats during the first 50 days postpartum. The cGPK activity is very low during the first 10 days after birth. It increases during the following 15 days, whereafter the adult level is reached. This development is paralleled by an increase in cGMP-binding activity and is preceded by a rapid increase of the cerebellar wet weight. When the (1-5 units) samples extracted from 0, 10, 20 and 30 day-old rats were challenged with the specific inhibitor (Type I) of CAMP-dependent protein kinase (SZMICIELSKI ef a/., 1977), no significant reduction
in the cGMP stimulated histone-phosphorylation could be observed. The same amount of inhibitor reduced the activity of I100 units of purified beef heart CAMP-dependent protein kinase, (EC 2.7.1.37) to 12.9?, of the control activity. cGMP degradation was the same in the samples from 0 to 30 day old rats and in adult controls. When complete reaction mixes containing the 'peak eluate' and C3H]cGMP were incubated for IOmin at 30'C, more than 82% of the radioactive label was recovered as cGMP in all assays. Absence of inhibitory or modulatory thermostable factors in the 'peak eluate' was indicated by recombination experiments. When 10 pI of heat-treated (2 min in boiling water bath) 'peak eluate' from 0, 10 or 30 day-old rats were incubated with 10 pl untreated 'peak eluate' no inhibition or stimulation of cGMP-dependent histone phosphorylation was observed. In addition a high activity of phosphoprotein-phosphatase does not seem to exist in the 'peak eluate' prepared from cerebella in the early developmental stages, since 907; of [32P]-labelled phosphohistones were recovered when they were added to a standard assay together with material from 3 day-old rats. The C3H]cGMP binding activity in the 'peak eluate' appeared specific for cGMP. The addition of 200-fold excess of non-radioactive cGMP reduced the amount of ['HlcGMP bound to 9% whereas only 39% of the bound C3H]cGMP could be replaced by the same molar excess of CAMP. DISCUSSION
To study changes of cGPK during cerebellar postnatal development in rat we have used a method for purification and measurement of cGPK which allowed the simultaneous processing of a large number of samples. The cerebellar cGPK preparation obtained with this method contained negligible amounts of c G M P phosphodiesterase. phosphoprotein-phosphatase or heat-stable protein kinase
I
I
:,
0
I (
(3)
contact w i t h p a r a l l e l f i b r e s (2)
5
(2)
_I
5
A 9 e
10
develoD
15
20
{contact t o Purkinje c e l l s (2)
(Days p o s t n a t a l )
(2)
3
25
f i n a l contact t o Purkinje c e l l dendrites (2)
temporary synapses w i t h P u r k i n j e c e l l body (2
a c t i v a t e d by mossy f i b r e
i n number ( 3 )
{contact t o Purkinje c e l l s (1)
e a r l i e s t signs of a c t i v i t y (2)
[increase
inhibitory activity (2)
I
$inhibitory activity (2)
a c t i v a t e d b y mossy f i b r e ( 2 )
i n c r e a s e i n number ( 3 )
2 7
I
30
f i n a l c o n t a c t w i t h c l i m b i n g f i b r e s t h r o u g h synapses on d e n d r i t e s ( 2 ,
L e m P o r a r v svnapses on c e l l body w i t h c l i m b i n q f i b r e s (
formation of apical d e n d r i t i c t r e e ( 4 )
c o n t a c t w i t h p a r a l l e l f i b r e s (1)
i n c r e a s e i n number
number! ( 3 )
Increase( i n
(1)
~
n monolayer rranoement - +
Flci 2. Postnatal development of cerebellar components of the rat. The wavy line indicates the approximate age of the rat at the inception o f the various events. The information represented was collected from the work of ( I ) WOODWARI) cr d.(1969). (2) SHIMONO ct d.(1976). (3) ALTMAN(1969). and (4) B I K K Y& BKAIXI-Y(1976). The development of cerebellar cGPK i s taken from Fig. I.
cGMP-dependent D r o t e i n kinase
Mossy f i b r e s
Parallel fibres
Climbing f i b r e s
Granule c e l l s
Stellate cells
Basket c e l l s
Golgi c e l l s
Purkinje c e l l s
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modulators that could interfere with the cGMP-stimulated histone phosphorylation. Our cGPK determination is based upon the difference of histone-phosphorylation in presence vs that in absence of cGMP. Thus, cyclic nucleotide-independent protein kinases would not interfere with the results. We have tested the possibility that in the cGPK preparation there are cGMP-independent catalytic subunits (as proposed by SHOJIet al., 1977). However when the 'peak eluate' fraction was applyed to Sephadex (3-100 and G-150 & GUIDOTTI, in preparation), we could columns (BANDLE not detect protein kinase- or cGMP-binding activities associated with proteins of molecular weights below 150,000. This observation is in agreement with the report of LINCOLN er al. (1976 and GILLC t a / . (1976). These authors. using highly purified cGPK preparations from bovine and pig lung, could not detect catalytic and regulatory subunits of cGMP-dependent protein kinases. The possibility that CAMP-dependent protein kinases could interfere in our cGPK-assay was also discounted because the inhibitor (Type I) of this kinase left the cGMP stimulated histonephosphorylation unaltered. In addition, the CAMP-dependent protein kinase is eluted from this column with a much & GUIDOTTI,in higher salt concentration (SZMIGIELSKI press). The cGPK is expected to exhibit cGMP binding activity (LINCOLN et al., 1976; TAKAIet a/., 1975). Measurement of cGMP receptor density using C3H]cGMP confirmed the specificity of cGPK assay method because the increase of cGPK activity was consistently paralleled by an increase of ['HIcGMP binding (see Fig. I). Our ontogenetic study revealed that cGPK activity is very low in newborn rats. It starts to increase 10 days later and rises in the following 15 days to its adult level. These data agree with a report of Kuo et al. (1976a) showing that in crude cerebellar homogenates of guinea pig the level 3f cGMP-dependent phosphorylation is lower in newborn ( 5 day-old) than in adult (300 day-old) animals. Guanylate cyclase (EC 4.6.1.2). which is responsible for the synthesis of cGMP. shows a very different pattern of activity during the development of rat cerebellum (KUMAKURA, 1977; SPANOet a/.. 1975). After the sixth day post partum there is a sharp increase in specific activity of this enzyme reaching a peak at day 10, then the activity decreases within the next 10 days to the adult level. Cerebellar cGMP-concentrations are low at birth and only start to increase after the 12th postnatal day (SPANOet al., 1975; BANDLE, unpublished); thus the change in cGPK-activity precedes this development. All these components. which may be involved in the chain of reaction that focuses around cGMP, reach their adult concentrations or activities at the time when the cerebellum becomes fully functional in the coordination of muscle movements. Figure 2 shows some developmental events during the ontogenesis of the rat cerebellum. The Purkinje cells reach their final number and location before post-'natal day 3. well before we could detect a rapid increase of cGPK activity. However, at this time the Purkinje cells have not yet developed dendrites. The formation of the apical dendritic tree of the Purkinje cells takes place between the & BRADLEY, 1976). 10th and the 30th postnatal day (BERRY
Present address: F. Hoffmann-La Roche Co. AG, Grenzacher-strasse 124, 4002 Basel, Switzerland. To whom correspondence should be addressed.
During this time the two major afferent inputs, the climbing and the parallel fibers, establish their final synaptic contacts to the Purkinje cell dendrites. The period during which cGPK appears and increases in activity coincides with that of the formation of the synapses between the Purkinje cells and the inhibitory interneurons (see Fig. 2). Laboratory of Preclinical Pharmacology, National Institute of Mental Health, Saint Elizabeth's Hospital, Washington, DC 20032, U.S.A.
E. BANDLE' A. G u i ~ o r r r *
REFERENCES
ALTMANJ. (1969) DNA metabolism and cell proliferation, in Handbook of Neurochemistry (LAJTHAA,, ed.), Vol. 11, pp. 137-182. Plenum Press, New York. BERRYM. & BRADLEY P. (1976) The growth of the dendritic trees of Purkinje cells in the cerebellum of the rat. Brain Res. 112, 1-35. A. (1976) Climbing fibers activation BIGGIOG . & GUIDOTTI and 3'5'-cyclic guanosine monophosphate (cGMP) content in cortex and deep nuclei of cerebellum. Brain Res. 107, 365-373. BICCIOG., GUIDOTTI A. & COSTAE. (1977) On the mechanism of the decrease in cerebellar cyclic G M P content elicited by opiate receptor agonists. Nauny1-Schmiedebergs Archs Pharmac. 296, 117-121. BISTIS., IOSIFG . & STRATA P. (1971) Suppression of inhibii o n in the cerebellar cortex by picrotoxin and bicuculline. Brain Res. 28, 591-593. BRADFORD A. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248-254. CURTISD. R., DUCCANA. W., FELIXD. & JOHNSTON G . A. R. (1970) GABA, bicuculline and central inhibition. Nature, Lond. 226, 1222-1224. GILLG . N., HOLDYK. E., WALTONG. M. & KANSTEIN C. G . (1976) Purification and characterization of 3'5'cyclic GMP-dependent protein kinase. Proc. natn. Acad. Sci., U.S.A. 73, 3918-3922. N. D. & HADDOXM. K. (1977) Cyclic G M P GOLDBERC metabolism and involvement in biological regulation. A . Rev. Biochem. 46, 823896. G . (1975) Femtomole sensitive HARPERJ. F. & BROOKER radioimmunoassay for cyclic AMP and cyclic G M P after 2 ' 0 acetylation. J . Cycl. Nucl. Res. 1. 207-218. HOFFMAN F. & SOLDG . (1972) A protein kinase activity from rat cerebellum stimulated by guanosine 3'5'-mono1 W phosphate. Biochem. biophys. Res. Commun. 49, 1 1107. KUMAKURA K. (1977) Studies on regulatory mechanisms of guanylate cyclase. Thesis, Sophia University, Tokyo. Kuo J. F. (1975) Changes in relative levels of guanosine 3'5'-dependent and adenosine 3'5'-monophosphatedependent protein kinase in lung, heart and brain of developing guinea pig. Proc. natn. Acad. Sci., U.S.A. 72, 22562259. P. (1972) An assay method for Kuo J. F. & GREENGARD cyclic AMP and cyclic G M P based upon their abilities to activate cyclic AMP-dependent and cyclic GMPdependent protein kinases. Adr. Cycl. Nucl. Res. 2, 41-50.
Short communication K i o J . F.. K L OW. N. & SHOJI M. (1976a) Regulation by cyclic G M P and stimulatory protein kinase modulator of cyclic GMP-dependent protein kinase from brain and other tissues. Adi.. Biocheni. Pswhophurmacol. 15. 379-389. Kr.0 J. F.. K i . o W. N.. SHOJI M.. DAVISC. W., SEERY L L Y T. E. (1976h) Purification and general properties of guanosine 3'5'-monophosphate-dependent protein Linase from guinea pig fetal lung. J . hiol. Chern. 251. 1759 1766. K r o W. N.. SHOJIM . & KLW J . F. ( 1 9 7 6 ~ )Localization of stimulator) modulator from rat brain and its specific effect o n guanosine 3':5'-monophosphate-dependent protein kinase from cerebellum and other tissues. Bio~~licrii. hiop/ij.\. R r s Cortiniir?i.70. 2 8 s 2 8 6 . LINCOL\T. M.. H A I I .C. L.. PARKC. R. & CORBINJ . D. (1976) Guanosine 3': 5'-cyclic monophosphate binding proteins in rat tissues. Proc. r i u r r i . .Ac(rd. Sci.. U.S.A. 73, 2559 2563. MATSO H.. UFDA 7.& G R F r S G A R l l P. (1975) Adenosine 3': 5'-monophosphate-dependent protein phosphatase acti\ity in synaptic membrane fractions. J . CFcl. Nucl. Res. I , 37 18. MAO C. C & ~ i ' I 1 X ) T T IA. (1974) Simultaneous isolation of adenosine 3'5'-cyclic monophosphate (CAMP) and Fuanosrnc 3'5'-cyclic monophosphate (cGMP) in small tissue samples. 4nulyr. Biochmi. 59, 63-68. A. & COSTAE. ( 1 9 7 4 ~The ) regulation MAOC. C.. GC.II)OTTI of cyclic gtianosine monophosphate in rat cerebellum: possible involtement of putative amino acid neurotransmitters Brtriti Rt,.\. 79, 510 514. M.40 C. C.. G ~ ' I I ) O T T A.I & COSTAE. (l974h) Interactions hetaeen ;.-aminohutqric acid and cyclic guanosine 3':5' monophosphate in rat cerebellum. Molec. Pharmac. 10, 736-745. MA^ C. C.. GL.IIIOTTI A. & LANDISS. (1975) Cyclic G M P : reduction of cerebellar concentrations in 'nervous' mutant mice. Brcriri R e s . 90, 335-339. MLRAII F.. MASGASIIXLOV. & VAUGHANM . (1971) A simple sensitive protein binding assay for guanosine 3':5' monophosphate. Proc. riutri. Acud. Sri.. U.S.A. 68. 736 739.
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NAKAZAWA K. & SANO M. (1975) Partial purification and properties of guanosine 3'5'-monophosphate-dependent protein kinase from pig lung. J . h i d Cliem. 250, 741 5-7419. RUBINE. H. & FERENDELLI J. A. (1977) Distribution and regulation of cyclic nucleotide levels in cerebellum iu viro. J . Neurocheni. 29, 43-51. SHIMONO T., NOSAKA S. & SASAKI K. (1976) Electrophysiological study on the postnatal development of neuronal mechanisms in the rat cerebellar cortex. Bruin Res. 108, 279-294. SHOJIM., PATRICK J. G.. TSEJ. & K u o J . F. (1977) Studies on the cyclic GMP-dependent protein kinase from bovine aorta. J . hiol. C h i . 252, 4347-4353. M. SPANOP. F., KUMAKURA K.. GOVONl s. & TRARVCCHI (1975) Post-natal development and regulation of cerebellar cyclic guanosine monophosphate system. Phcrrmcrc. Res. Commun. 7, 223-237. STEINERA.. FERENDELLI J. A. & KIPSIS D. M. (19721 Radioimmunoassay for cyclic nucleotides. 3. Effects of ischemia, changes during development and regional distribution of adenosine 3'5'-monophosphate and guanosine 3'5'-monophosphate in mouse brain. J . hiol. Cheni. 247, Il2l-ll24. SZMIGIELSKI A. & GLWOTTI A. Action of liarmaline and diazepam o n the cerebellar content of cyclic G M P and o n the activities of two endogenous inhibitors of protein kinase. Neurocbem. Res.. in press. SZMIGIELSKI A,. GUIDOTTIA. & COSTA E. (1977) Endogenous protein kinase inhibitors. purification. characterization and distribution in different tissues. J . hid. Cheoi. 252, 3848-3853. TAKAIY.. NISHIYAMA K.. YAMAMURAH. & NISHIZCKA Y. (1975) Guanosine 3': 5'-monophosphate-dependent protein kinase from bovine cerebellum. J . hid. Chem. 250, 469s-4695. WOODWARDD. J.. HOFFER8.J. & LAPHAML. W. (1969) Correlative survey of electrophysiological, neuropharmacological and histochemical aspects of cerebellar maturation in rat. in Neurohioloyj, of Crrehellur Erolurion c r r i d Derelopmenf (LLINASR.. ed.). pp. 725-741. AMA. Chicago.
Ontogenetic studies of cCMPdependent protein kinase in rat cerebellum (Receired 6 September 1978. Accepted 10 October 1978)
THERAT cerebellum is characterized by its high content of cyclic 3'.5'-guanosine monophosphate (cGMP). The cerebellar concentration of this nucleotide is about ten times higher than that of any other brain area examined so far (STEINERet a/., 1972: MAO & GUIDOTTI, 1974). Particularly high cGMP-concentrations were found in the cerebellar cortex (BIGGIO& GuroorTr. 1976; BIGGIOer ul., 1977; RUBIN& FERRENDELLI, 1977), where this nucleotide probably is preferentially located in Purkinje cells (MAO er ul., 1975). Upon destruction of the climbing fibers (one of the main excitatory inputs to the Purkinje cells) with 3-acetylpyridine both the Purkinje cells and the cerebellar & GUIDOTTI. 1976). PharcGMP level are affected (BIGGIO macological manipulations of the cerebellar GABAergic function change the firing rate of Purkinje cells (BISTIet a/., 1971; CURTISet a/.. 1970) and the cGMP content in the cerebellar cortex (MAOet ul., 1974~.b ; BIGGIOet a / . , 1977). These lines of evidence suggest that cGMP participates as second messenger in the regulation of the cerebellar Purkinje cell activity. Among the enzyme systems affected by cGMP are protein kinases which are stimulated by this nucleotide (GOLDBERG & HADDOX.1977). Relatively high levels of cGMP-dependent protein kinase (EC 2.7.1.37; cGPK) activities have been found in mammalian tissues with high concentrations of cGMP (Kuo et u'l., 1976b) in& SOLD, 1972: cluding the cerebella from rat (HOFFMAN LINCOLN et ol., 1976). and cow (TAKAIet ul.. 1975). and & SANO.1975; GILLet ul., 1976; other tissues (NAKAZAWA KUOe t ul.. 1976~:SHoii et ul., 1977). Changes in cGPK activity during development occur in lung. heart, brain and cerebellum of guinea pig (Kuo. 1975; Kuo er a/., 1976a). The aim of our study was to analyze the changes in cGPK activity during the ontogenesis of the rat cerebellum and to relate these changes to the temporal sequence of differentiation of the cerebellar neuronal components and to the concomitant changes in the cGMP content of that brain area. In rodents the cerebellar cGMP content is very low at birth and increases sharply during the first postnatal et (11.. 1972; SPANOet ul.. 1975). month (STEINER EXPERIMENTAL PROCEDURES Assax o f c C P K tictiriry. cGPK activity was defined as the difference between the incorporation of 3zPO;3 from
Abbreviations u s e d : CAMP, cyclic AMP. cGMP. cyclic Y.5'-guanosine monophosphate; cGPK. cyclic GMPdependent protein kinase: DEAE, diethylaminoethyl: KEM. buffer containing 25 mM-K-phosphate pH 7.5. I mM-EDTA and 50 mhc-mercaptoethanol.
[;-32P]-ATP into histones in the presence and absence of cGMP. One unit of cGPK activity was defined as the incorporation of 1 pmol 3 2 P O Q 3 in IOmin at 30 C. The specific activity of cGPK was expressed as enzyme units per mg protein charged onto thc DE column (see Preparation of cGPK). Unless otherwise specified, the assay of cGPK has initiated by the addition of 1 4 0 ~ 1of a mix containing 140 mM-Na acetate, 70 mM-Mg acetate, 5 mwaminophql(spec. act. line, 20pg histone IIA. 7 phc-[;.-"P]ATP P pH. 5-10 x lo' c.p.m. per pmol). and 0.7 ~ M - c G M (final 8) to lop1 of the 'peak eluate' (containing 10-40jcg protein and up to 10 units of cGPK). The reaction was carried out at 3 0 C for 10min and was terminated by pipetting 50p1 of the incubation mixture on to Whatman 3 MM filter paper strips (2.5 x 35cm), presiously soaked a i t h 'precipitating solution' (5:; wjv TCA. 0.25"" w I Na2W0,. 2 H 2 0 . 204 v/v H,SO, conc.; Krjo & GREI:NGARD. 1972) and dried before use. The strips were washed 3 times for 20 min with 31 'precipitating solution'. This procedurr typically leaves 0.0157" of the input radioactivity as blank while retaining 94":, of the phosphorylated histones on the filter. The assay in absence of cGMP was performed in the same way except that no cyclIc nucleotide \+as added. All assays were run in duplicate. Preparation o f c G P K . The animals were decapitated and the cerebella homogenized with 4 b o l of KEM (25 m w K phosphate pH 7.5, 1 mM-EDTA. 50 mM-mercaptoethanol). The homogenates were spun for 2Omin at 2O.OOOg. All operations were performed at 0 4-C. The sample (containing 0.2-1 mg protein and 40-250 units of cGPK) of each of the supernatants was placed onto a DE.52 cellulose column ( 3 0 0 ~ 1bed vol. approx. 8 mm high and 5.5 mm i.d.) equilibrated with KEM. The column was washed Kith 150p1 KEM. followed by 150pI KEM-0.175 M - N ~ C IThe . cGPK was then eluted with 300pl KEM 0.175w-NaCI. This fraction is referred to as the 'peak eluate'. The recotery of partially purified cGPK through this procedure uas consistently better than W,. Only trace amounts of cCPK-dependent activity could be detected in the fractions before or after the 'peak eluate'. The cGPK activity in the 'peak eluate'. as well as i n the supernatant before DE52 column. remained constant for at least one week when kept at 0-4 C. but nas lost upon freezing. A detailed analysis of this method m i l l be & GLYDOTTI. i n preparation). described elsewhere (BANDLE Assay c f c G M P binding activity. This determination was done as described by MURAD P I a / . (1971) except that KEM was used instead of 50 mM-Na-acetate pH 4 for the incubation. Other dererminutions. The degree of cGMP degradation
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Flc. I . Rat cerebellar cyclic GMP-dependent protein kinase, 13H]cGMP binding activity and cerebellar and ['HlcGMP binding wet weight during ontogenesis. The specific activities of cGPK (U--m) ( O - - - O ) (upper part of the figure) and the wet weight of the cerebellum (lower part of the figure) were determined from rats of the ages indicated. For each age 4 7 animals were used and the result expressed as the mean ~ s . E . M .For methods see Experimental Procedures.
in the assay of cGPK was measured by substituting cGMP with ['HlcGMP (1.5 x lo5 c.p.m.) for cGMP in reaction mixes. The C3H]cGMP was isolated according to MAO& GuiDom (1974) either before or after the incubation (10 min/30"C) and the recoveries were compared. [3zP]Phosphohistones were prepared in an assay for cGPK using a slight modification of the method of MAENO et al. (1975). Adult rat cerebella served as the enzyme source. After an incubation for 60min at 30°C the reaction mixture was chromatographed through a Sephadex (3-25 column (0.55 x 5 cm). The column was eluted with KEM in 100pI portions and the phosphohistones isolated in the excluded fractions. Wherever this preparation was used, the radioactive phosphohistones were measured as the material that was insoluble in 'precipitating solution' and was retained on Millipore HAWP filters. c G M P was measured with the radioimmunoassay as described by HARPER& BRWKER(1975). Protein concentrations were determined using the method of BRADFORD (1976) with bovine serum albumin as standard. RESULTS Appearance of cGPK dtiring the ontogenesis of rat cerebellum
Figure I shows c G M P binding, cGPK activity and cerebellar weight in Sprague-Dawley rats during the first 50 days postpartum. The cGPK activity is very low during the first 10 days after birth. It increases during the following 15 days, whereafter the adult level is reached. This development is paralleled by an increase in cGMP-binding activity and is preceded by a rapid increase of the cerebellar wet weight. When the (1-5 units) samples extracted from 0, 10, 20 and 30 day-old rats were challenged with the specific inhibitor (Type I) of CAMP-dependent protein kinase (SZMICIELSKI ef a/., 1977), no significant reduction
in the cGMP stimulated histone-phosphorylation could be observed. The same amount of inhibitor reduced the activity of I100 units of purified beef heart CAMP-dependent protein kinase, (EC 2.7.1.37) to 12.9?, of the control activity. cGMP degradation was the same in the samples from 0 to 30 day old rats and in adult controls. When complete reaction mixes containing the 'peak eluate' and C3H]cGMP were incubated for IOmin at 30'C, more than 82% of the radioactive label was recovered as cGMP in all assays. Absence of inhibitory or modulatory thermostable factors in the 'peak eluate' was indicated by recombination experiments. When 10 pI of heat-treated (2 min in boiling water bath) 'peak eluate' from 0, 10 or 30 day-old rats were incubated with 10 pl untreated 'peak eluate' no inhibition or stimulation of cGMP-dependent histone phosphorylation was observed. In addition a high activity of phosphoprotein-phosphatase does not seem to exist in the 'peak eluate' prepared from cerebella in the early developmental stages, since 907; of [32P]-labelled phosphohistones were recovered when they were added to a standard assay together with material from 3 day-old rats. The C3H]cGMP binding activity in the 'peak eluate' appeared specific for cGMP. The addition of 200-fold excess of non-radioactive cGMP reduced the amount of ['HlcGMP bound to 9% whereas only 39% of the bound C3H]cGMP could be replaced by the same molar excess of CAMP. DISCUSSION
To study changes of cGPK during cerebellar postnatal development in rat we have used a method for purification and measurement of cGPK which allowed the simultaneous processing of a large number of samples. The cerebellar cGPK preparation obtained with this method contained negligible amounts of c G M P phosphodiesterase. phosphoprotein-phosphatase or heat-stable protein kinase
I
I
:,
0
I (
(3)
contact w i t h p a r a l l e l f i b r e s (2)
5
(2)
_I
5
A 9 e
10
develoD
15
20
{contact t o Purkinje c e l l s (2)
(Days p o s t n a t a l )
(2)
3
25
f i n a l contact t o Purkinje c e l l dendrites (2)
temporary synapses w i t h P u r k i n j e c e l l body (2
a c t i v a t e d by mossy f i b r e
i n number ( 3 )
{contact t o Purkinje c e l l s (1)
e a r l i e s t signs of a c t i v i t y (2)
[increase
inhibitory activity (2)
I
$inhibitory activity (2)
a c t i v a t e d b y mossy f i b r e ( 2 )
i n c r e a s e i n number ( 3 )
2 7
I
30
f i n a l c o n t a c t w i t h c l i m b i n g f i b r e s t h r o u g h synapses on d e n d r i t e s ( 2 ,
L e m P o r a r v svnapses on c e l l body w i t h c l i m b i n q f i b r e s (
formation of apical d e n d r i t i c t r e e ( 4 )
c o n t a c t w i t h p a r a l l e l f i b r e s (1)
i n c r e a s e i n number
number! ( 3 )
Increase( i n
(1)
~
n monolayer rranoement - +
Flci 2. Postnatal development of cerebellar components of the rat. The wavy line indicates the approximate age of the rat at the inception o f the various events. The information represented was collected from the work of ( I ) WOODWARI) cr d.(1969). (2) SHIMONO ct d.(1976). (3) ALTMAN(1969). and (4) B I K K Y& BKAIXI-Y(1976). The development of cerebellar cGPK i s taken from Fig. I.
cGMP-dependent D r o t e i n kinase
Mossy f i b r e s
Parallel fibres
Climbing f i b r e s
Granule c e l l s
Stellate cells
Basket c e l l s
Golgi c e l l s
Purkinje c e l l s
1346
Short communication
modulators that could interfere with the cGMP-stimulated histone phosphorylation. Our cGPK determination is based upon the difference of histone-phosphorylation in presence vs that in absence of cGMP. Thus, cyclic nucleotide-independent protein kinases would not interfere with the results. We have tested the possibility that in the cGPK preparation there are cGMP-independent catalytic subunits (as proposed by SHOJIet al., 1977). However when the 'peak eluate' fraction was applyed to Sephadex (3-100 and G-150 & GUIDOTTI, in preparation), we could columns (BANDLE not detect protein kinase- or cGMP-binding activities associated with proteins of molecular weights below 150,000. This observation is in agreement with the report of LINCOLN er al. (1976 and GILLC t a / . (1976). These authors. using highly purified cGPK preparations from bovine and pig lung, could not detect catalytic and regulatory subunits of cGMP-dependent protein kinases. The possibility that CAMP-dependent protein kinases could interfere in our cGPK-assay was also discounted because the inhibitor (Type I) of this kinase left the cGMP stimulated histonephosphorylation unaltered. In addition, the CAMP-dependent protein kinase is eluted from this column with a much & GUIDOTTI,in higher salt concentration (SZMIGIELSKI press). The cGPK is expected to exhibit cGMP binding activity (LINCOLN et al., 1976; TAKAIet a/., 1975). Measurement of cGMP receptor density using C3H]cGMP confirmed the specificity of cGPK assay method because the increase of cGPK activity was consistently paralleled by an increase of ['HIcGMP binding (see Fig. I). Our ontogenetic study revealed that cGPK activity is very low in newborn rats. It starts to increase 10 days later and rises in the following 15 days to its adult level. These data agree with a report of Kuo et al. (1976a) showing that in crude cerebellar homogenates of guinea pig the level 3f cGMP-dependent phosphorylation is lower in newborn ( 5 day-old) than in adult (300 day-old) animals. Guanylate cyclase (EC 4.6.1.2). which is responsible for the synthesis of cGMP. shows a very different pattern of activity during the development of rat cerebellum (KUMAKURA, 1977; SPANOet a/.. 1975). After the sixth day post partum there is a sharp increase in specific activity of this enzyme reaching a peak at day 10, then the activity decreases within the next 10 days to the adult level. Cerebellar cGMP-concentrations are low at birth and only start to increase after the 12th postnatal day (SPANOet al., 1975; BANDLE, unpublished); thus the change in cGPK-activity precedes this development. All these components. which may be involved in the chain of reaction that focuses around cGMP, reach their adult concentrations or activities at the time when the cerebellum becomes fully functional in the coordination of muscle movements. Figure 2 shows some developmental events during the ontogenesis of the rat cerebellum. The Purkinje cells reach their final number and location before post-'natal day 3. well before we could detect a rapid increase of cGPK activity. However, at this time the Purkinje cells have not yet developed dendrites. The formation of the apical dendritic tree of the Purkinje cells takes place between the & BRADLEY, 1976). 10th and the 30th postnatal day (BERRY
Present address: F. Hoffmann-La Roche Co. AG, Grenzacher-strasse 124, 4002 Basel, Switzerland. To whom correspondence should be addressed.
During this time the two major afferent inputs, the climbing and the parallel fibers, establish their final synaptic contacts to the Purkinje cell dendrites. The period during which cGPK appears and increases in activity coincides with that of the formation of the synapses between the Purkinje cells and the inhibitory interneurons (see Fig. 2). Laboratory of Preclinical Pharmacology, National Institute of Mental Health, Saint Elizabeth's Hospital, Washington, DC 20032, U.S.A.
E. BANDLE' A. G u i ~ o r r r *
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