Induction of cyclic AMP and cyclic GMP 3′:5′-cyclic nucleotide phosphodiesterase activities in neuroblastoma lines under differentiating conditions

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Pergamon

Int[ J[ Devl Neuroscience\ Vol[ 04\ No[ 2\ pp[ 298Ð208\ 0886 Copyright Þ 0886 ISDN[ Published by Elsevier Science Ltd Printed in Great Britain[ All rights reserved 9625Ð4637:86 ,06[99¦9[99

PII] S9625Ð4637"86#99997Ð6

INDUCTION OF CYCLIC AMP AND CYCLIC GMP 2?]4?!CYCLIC NUCLEOTIDE PHOSPHODIESTERASE ACTIVITIES IN NEUROBLASTOMA LINES UNDER DIFFERENTIATING CONDITIONS MAURO GIORGI\ DANIELA GIORDANO\$ CRISTIANA CANIGLIA\$ STEFANO BIAGIONI$ and GABRIELLA AUGUSTI!TOCCO$% Dipartimento di Biologia di Base e Applicata\ Universita dell|Aquila\ L|Aquila\ Italy^ $Dipartimento di Biologia Cellulare e dello Sviluppo\ Universita di Roma {{La Sapienza||\ Roma\ Italy "Received 01 September 0885^ revised 13 January 0886^ accepted 29 January 0886# Abstract*It is now widely accepted that cyclic nucleotide phosphodiesterases "PDEs# play fundamental roles in signal transduction pathways^ they show a remarkable molecular complexity\ di}erent tissue distribution and complex regulatory mechanisms[ Here we report PDE isoforms expression in two dibutyryl cyclic AMP di}erentiated murine cell lines] the hybrid neuroblastomaÐglioma 097CC04 and the parental neuroblastoma N07TG1[ They di}er in the ability to establish functional synapses\ a feature present only in the former[ Ionic exchange chromatography elution pro_les of N07TG1 and 097CC04 undi}erentiated cell extracts show two main peaks of activity[ The _rst one hydrolyzes cyclic GMP and is speci_cally inhibited by Zaprinast\ thus representing a member of the PDE4 family[ The second peak hydrolyzes cyclic AMP and is signi_cantly inhibited by rolipram\ as all the PDE3 family members[ The induction of di}erentiation by dibutyryl cyclic AMP in both clonal lines results in an increase of PDE activities only after 2 hr of treatment\ suggesting that protein neosynthesis is involved[ Interestingly in both clones\ besides the increase in cyclic AMP hydrolyzing speci_c activity "2[0!fold in 097CC04 and 1[4!fold in N07TG1#\ we also observed an increase in cyclic GMP hydrolyzing activity "0[6!fold in 097CC04 and 3[2!fold in N07TG1#[ While the induction of PDE3\ previously reported also in other cellular systems\ could be considered as a feedback response to the higher cyclic AMP levels\ this is not true for the isoform that hydrolyzes cyclic GMP[ These data suggest that the induction of PDE isoforms in neuroblastoma cells could be related to the activation of neuronal di}erentiative pathway[ Þ 0886 ISDN Key words] phosphodiesterase\ PDE3\ PDE4\ neuroblastoma\ neuroblastoma!glioma\ neuronal di}er! entiation[

Cyclic nucleotide phosphodiesterases "PDEs# play a key role in signal transduction pathways using cyclic AMP and cyclic GMP as second messengers[ PDEs are a complex group of molecules^ at least seven families "PDE0 to PDE6# have been described that di}er in molecular size\ charge\ a.nity for substrates\ speci_c modulators "in some cases belonging to other transduction pathways# and pharmacological inhibitors[1\03\34 More than one gene has been identi_ed for each family\ except PDE4 and PDE6\ and enzyme heterogeneity can be further increased by alternative splicing of transcripts[ Because of their remarkable molecular complexity\ their di}erent tissue distribution and the existence of many regulatory mechanisms\ it is now widely accepted that PDEs play integrative and regulatory roles in signal transduction pathways[0\1 However only in a few cases has a correlation between expression of a single PDE isoform and a speci_c cellular function been demonstrated^ this is the case for PDE5 and visual signal transduction24 and for PDE2 and the antilipolytic e}ect of insulin[14 Regulation and integration of di}erent intracellular signals are particularly important for modu! lation of the electrical and metabolic activity in the nervous system\33 where high levels of PDE activity and many of the known isoforms are present[34 Calcium!calmodulin dependent PDE is the predominant isoform in the nervous system and appears widely distributed in di}erent brain areas[22 The expression of other PDE isoforms appears restricted to speci_c structures\ as is the case for PDE5 in the retina and PDE1 in the limbic system[ The localization of the various isoforms in speci_c compartments1 suggests an association of their activity with speci_c neuronal functions[ To investigate neuronal functions\ neuroblastoma clones have been widely used[19\36 They rep!

%To whom correspondence should be addressed[ 298

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resent a useful model because of their cellular homogeneity and their ability to express di}erent speci_c neuronal properties\ which furthermore can be modulated by varying the culture conditions[ We have previously described the pattern of PDE isoforms in the neuroblastoma cell line N07TG1[17 Here we report the characterization of cyclic AMP and cyclic GMP PDE activities in the hybrid cell line neuroblastomaÐglioma 097CC04 "derived from somatic hybridization of neuroblastoma N07TG1 and glioma C5!BU!029# that expresses preferentially parental neuroblastoma features[ The two lines di}er in their ability to complete neuronal di}erentiation after appropriate treatment^ in fact both clones display a morphological {{di}erentiated|| phenotype after treatment with dibutyryl cyclic AMP "dbcAMP#\ however\ only the hybrid clone is capable of establishing functional synapses with myotubes in culture[7 We have then compared the expression of PDE activities in both clones under di}erentiating conditions "in the presence of dbcAMP#\ as an attempt to establish a possible relationship between speci_c PDE isoforms and cellular events occurring in the maturation of neuronal cells[ 0[ EXPERIMENTAL PROCEDURES 0[0[ Materials ð2HŁCyclic AMP "sp[ act[ 17 Ci mmol−0# and ð2HŁcyclic GMP "sp[ act[ 05[7 Ci mmol−0# were obtained from Amersham "Buckinghamshire\ U[K[#[ RO 19!0613 "3!ð2!butoxy!3!methoxybenzylŁ! 1!imidazolidinone# was a gift from Ho}man La Roche "Basel\ Switzerland#^ rolipram "ZK 51600\ 3!ð2?!cyclopentyloxy!3?!methoxyphenylŁ!1 pyrrolidone# from Shering "Berlin\ Germany# and zap! rinast "M and B 11837\ 1!o!propoxyphenyl!7!azapurin!5!one# from Rho¼ne!Poulenc Rorer "Dagenham\ U[K[#[ All other reagents were of analytical grade and purchased from Sigma Chemical Co[ "St Louis\ MO\ U[S[A[#[ Culture media and sera were obtained from Flow "Ayrshire\ U[K[#[ 0[1[ Cell cultures Murine neuroblastoma N07TG1 and hybrid 097CC04 cell lines were cultured in Dulbecco|s modi_ed Eagle|s medium "DMEM# containing penicillin "4999 IU ml−0# and streptomycin "4 mg ml−0#\ supplemented with 09) fetal calf serum "FCS# and maintained in a humidi_ed atmo! sphere of 09) CO1 at 26>C[ Hypoxantine\ aminopterin and thymidine at a _nal concentration of 9[0 mM\ 09 mM and 05 mM respectively were added to the medium of 097CC04 cultures to avoid growth of revertants[ Cells were plated in 59 or 89 mm culture dishes "Falcon\ Becton Dickinson Europe\ France#[ After 13 hr the medium was replaced with either DMEM supplemented with 09) FCS "control cultures# or supplemented with 0) FCS and 4 mM dbcAMP and renewed after an additional 37 hr[ At _xed times after dbcAMP addition both control and treated cells were washed twice with phosphate bu}er saline "PBS# and stored at −79>C[ 0[2[ Enzyme extract Cells were collected by scraping in homogenization bu}er "19 mM TrisÐHCl pH 6[1\ 9[1 mM EGTA\ 4 mM MgCl1\ 0 mM phenylmethylsulfonyl~uoride "PMSF#\ 4 mM b!mercaptoethanol\ 09 mg ml−0 leupeptin\ 4 mg ml−0 bestatin\ 09 mg ml−0 pepstatin A\ 9[0) Triton X!099# and homo! genized at 3>C in a glassÐte~on homogenizer with 19 strokes[ The homogenates were centrifuged at 099 999 g for 34 min at 3>C and the supernatants used for ion exchange chromatography[ Alter! natively\ in the time course experiments after dbcAMP treatment\ homogenates were centrifuged in a microfuge "Biofuge A\ Haereus\ Postfach\ Germany# at 09 999 g for 0 hr at 3>C and the super! natants used for PDE enzyme assay[ In both cases ×89) of homogenate PDE activity was recovered in the supernatants[ 0[3[ DEAE chromatography The extracts from 097CC04 and N07TG1 cells grown in absence or presence of dbcAMP were diluted 0]1 with 49 mM sodium acetate pH 5[4\ 9[1 mM EGTA\ 4 mM b!mercaptoethanol\ 9[0 mM PMSF\ 4 mM NaF\ 9[94) Triton X!099 "bu}er A# and loaded onto a 4 ml diethylaminoethyl "DEAE# cellulose "Whatman\ Maidstone\ U[K[# column pre!equilibrated with the same bu}er[ After loading the column was washed with _ve bed volumes of bu}er A and PDE activity was eluted with

Nucleotide phosphodiesterase activities in neuroblastoma lines

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39 ml of a linear 9[94Ð0 M sodium acetate gradient in bu}er A[ Fractions of 0[9 ml were collected and assayed for PDE activity[ Activity recovery from DEAEÐcellulose chromatography in several experiments ranged between 74 and 84)[ Fractions corresponding to the peaks of enzyme activity were pooled and concentrated with centricon 09 "Amicon\ Danvers\ MA\ U[S[A[# in a refrigerated centrifuge[ The samples were stored in 49) glycerol at −19>C and then used for further analyses[ 0[4[ PDE activity assay PDE activity was measured with the two step method described by Thompson and Appleman\46 in 59 mM Hepes pH 6[1 assay bu}er containing 9[0 mM EGTA\ 4 mM MgCl1\ 9[4 mg ml−0 bovine serum albumin "BSA#\ 29 mg ml−0 soybean trypsin inhibitor with a substrate concentration of 0 mM[ 0[5[ Immunoblot analysis Sodium dodecyl sulfate "SDS#Ðpolyacrylamide gel electrophoresis was performed on 7) slab gels according to Laemmli25 and the proteins transferred to nitrocellulose membranes according to Towbin et al[48 The blots were incubated overnight at 3>C with an IgG anti calcium!calmodulin dependent PDE "0]0999# raised in rabbit against bovine brain antigen[ A secondary anti!rabbit IgG alkaline phosphatase conjugated antibody was used to reveal the immunocomplexes[ Puri_ed bovine brain Ca1¦!CaM PDE was used as standard[ 0[6[ Other procedures Aliquots of cellular extracts were used for determination of lactate dehydrogenase "LDH# activity according to the Vassault50 procedure[ Choline acetyltransferase "ChAT# activity was measured according to the method of Fonnum[10 Protein concentration was determined as described by Bradford\3 using BSA as standard[

1[ RESULTS 1[0[ Characterization of 097CC04 PDE isoforms Extraction of cyclic nucleotide PDE activity from neuroblastomaÐglioma 097CC04 cells was dependent on the presence of Triton X!099\ as previously described for N07TG1[17 More than 89) of both cyclic AMP and cyclic GMP PDE activity was recovered in the presence of 9[0) Triton X! 099[ The speci_c activity of the enzyme extracted under these conditions was respectively 9[1 nmol of cyclic AMP hydrolyzed min−0 per mg protein and 9[97 nmol of cyclic GMP hydrolyzed min−0 per mg protein[ The DEAEÐcellulose elution pro_le ðFig[ 0"A#Ł shows the presence in 097CC04 cells of two peaks of activity\ eluting respectively at 149 mM "peak 0# and 479 mM "peak 1# sodium acetate[ Recovery of enzyme activity in the eluate was 74Ð84) in all experiments[ Fractions corresponding to peaks 0 and 1 were pooled in order to study the kinetic and biochemical properties of the isoforms[ The activity present in both peaks was neither stimulated nor inhibited by cyclic GMP in the con! centration range of 9[0Ð09 mM\ it was also found to be independent of calcium!calmodulin[ Calcium! calmodulin dependent activity was also absent in the cell extract^ this was further con_rmed by immunoblot analysis\ using a puri_ed polyclonal antibody against bovine brain PDE0 isoform "data not shown#[ For a further characterization of the isolated isoenzymes\ their response to speci_c inhibitors was evaluated[ Zaprinast exhibited a high inhibitory e}ect on cyclic GMP PDE activity present in peak 0\ with a IC49 of 9[4 mM ðFig[ 1"A#Ł\ whereas the activity eluted in peak 1 is signi_cantly inhibited by rolipram with an IC49 of 9[5 mM ðFig[ 1"B#Ł[ A lower inhibition was observed in the presence of RO 19!0613 "IC492[1 mM^ data not shown#[ These data altogether suggest that the isoform present in peak 0 belongs to the PDE4 family\ while the isoform present in peak 1 belongs to PDE3 family[ 1[1[ Induction of PDE activity in N07TG1 and 097CC04 by dbcAMP A general property of many neuroblastoma clones is the ability to respond to various culture conditions\ displaying a di}erentiated phenotype\ as shown by growth of neurites and high levels

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Fig[ 0[ DEAEÐcellulose elution pro_les of PDE activities extracted from 097CC04 and N07TG1 {{di}er! entiated|| and control cells[ DEAEÐcellulose chromatographic elution pro_les of cellular extracts from control cells "A\ 097CC04^ B\ N07TG1 and from cells treated with dbcAMP for 4 days "C\ 097CC04^ D\ N07TG1#[ See Section 0 for details of the procedure[ PDE activity was assayed with 0 mM cyclic GMP ",# and with 0 mM cyclic AMP "R# as substrates[ Each reported elution pro_le is representative of at least three experiments[

of neurospeci_c proteins[19 This is the case also for N07TG1 and 097CC04 cells\ in which mor! phological di}erentiation "Fig[ 2#\ together with a block of proliferation can be induced by dbcAMP[ For a better assessment of the di}erentiating action of dbcAMP on 097CC04 cells ChAT and LDH activities were assayed[ As shown in Table 0 after 4 days of treatment\ an increase of the former and a decrease of the latter was observed\ thus con_rming that dbcAMP is capable of enhancing not only morphological di}erentiation but also to induce the expression of enzymes involved in the production of the neurotransmitter\ and down regulation of LDH\ as previously reported[36 Next we analyzed the time course of PDE activities from a few hours to 5 days after dbcAMP administration to both cell lines[ 097CC04 and N07TG1 cells behave di}erently in response to dbcAMP[ In the hybrid clone\ an increase of cyclic AMP hydrolyzing activity "PDE3# is already evident 2 hr after dbcAMP addition ðFig[ 3"A#Ł while induction of cyclic GMP hydrolizing activity "PDE4# requires a longer time "5 hr# ðFig[ 3"C#Ł^ conversely in N07TG1 cells PDE4 activity increases earlier than PDE3 ðFig[ 3"D and B#Ł[ However\ induction of PDE activities in all cases has never been observed earlier than 2 hr treatment "data not shown#[ In 097CC04 cells both isoenzymes Table 0[ ChAT and LDH activity expressed in 097CC04 cells {{di}erentiated|| with dbcAMP Enzymatic activity ChAT "09−2 fmol Ach min−0 per cell# LDH "09−6 unit per cell#

Ctrl

dbcAMP

Activity increase "fold#

6[529[1 3[829[0

19[521[2 9[829[94

1[6 9[07

Note] Ctrl\ control cultures^ dbcAMP\ culture treated with 4 mM dbcAMP for 4 days[ Data are expressed as means2S[E[ "n2#[

Nucleotide phosphodiesterase activities in neuroblastoma lines

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Fig[ 1[ Inhibition curves of 097CC04 PDE isoforms by speci_c PDE4 and PDE3 inhibitors[ "A# Inhibition by zaprinast "M and B 11837# of cyclic GMP PDE eluted in peak 0 ",# from 097CC04 control cells "IC49 9[4 mM#[ "B# Inhibition by rolipram "ZK 51600# of cyclic AMP PDE eluted in peak 1 from 097CC04 control cells "# "IC49 9[5 mM# and from cells treated for 4 days with dbcAMP "Ž# "IC49 2[1 mM#[ Inhibition by rolipram of cyclic AMP PDE eluted in peak 0 from 097CC04 treated cells "R#\ at 099 mM rolipram only 29) of activity was inhibited[ PDE activity was assayed with 0 mM cyclic nucleotides as substrates[ Calculated E[ S[ was in all cases ³4) of the reported values[

thereafter continue to increase reaching a maximum value at day 4 of treatment ðFig[ 3"A and C#Ł[ However the increase in activity is signi_cantly higher for PDE3 than for PDE4 "as reported in Table 1#[ As for 097CC04 clone administration of dbcAMP to N07TG1 cells also results in a signi_cant induction of the two PDE isoforms reaching their maximum at days 4Ð5 of treatment ðFig[ 3"B and D#Ł^ however\ in this case induction is more remarkable for PDE4 than PDE3 "Table 1#[ Since dbcAMP also brings about a two to three!fold increase of the total protein content\ the higher speci_c activity of both PDE isoforms in the presence of dbcAMP demonstrates a speci_c regulatory response of these enzymes[ 1[2[ Characterization of PDE isoforms expressed in both clonal lines after dbcAMP treatment In order to establish whether dbcAMP would also induce a modi_cation in the pattern of expressed isoforms\ cell extracts of dbcAMP treated cells were run on DEAEÐcellulose chromatography[ The elution pro_les of 097CC04 and N07TG1 cells treated for 4 days with 4 mM dbcAMP are shown in Fig[ 0"C and D#\ respectively[ In both clones two main peaks are present^ they elute at the same saline concentration and show the same substrate speci_city than in control cells[ As for control cells the activity of each peak is independent of cyclic GMP\ calcium and calmodulin^ these data together with the response to speci_c inhibitors show that the higher PDE activity induced by dbcAMP treatment is due to the isoforms present in control cells[ Moreover\ the chromatographic analysis con_rms that the induction is preferential for the PDE3 isoform in 097CC04 clone ðFig[ 0"C#Ł and for PDE4 isoform in N07TG1 clone ðFig[ 0"D#Ł[ However in both clones in the presence of dbcAMP a small peak of cyclic AMP PDE activity coelutes with PDE4[ This activity shows a poor rolipram inhibition ðFig[ 1"B#Ł indicating that it does not belong to the PDE3 family^ the degree of rolipram inhibition is similar to that exhibited by the recently described PDE6 isoform\30 that displays high a.nity for cyclic AMP[ Inhibition studies of PDE activity from di}erentiated and undi}erentiated 097CC04 cells provide interesting observations[ In fact under the two culture conditions PDE4 shows the same rate of

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Fig[ 2[ Morphological appearance of control and dbcAMP treated 097CC04 neuroblastomaÐglioma hybrid cells[ Micrographs "59×# of control "A# and dbcAMP treated "B# cells grown as described in Section 0[ The {{di}erentiated|| cells were cultured in 4 mM dbcAMP for 4 days[

inhibition by zaprinast "data not shown#\ while rolipram inhibition of PDE3 shows a signi_cant change of the curve slope ðFig[ 1"B#Ł[ IC49 for PDE3 from di}erentiated cells is 2[1 mM while for undi}erentiated cells it is 9[5 mM[ 2[ DISCUSSION We have compared the expression of PDE isoforms during dbcAMP induced di}erentiation of two neuronal cell lines] neuroblastomaÐglioma hybrid 097CC04 and its parental neuroblastoma N07TG1[ Since the pattern of PDE isoforms in the parental cell line N07TG1 is already known\17 we have _rst characterized PDE activity in the hybrid 097CC04 cells[ DEAEÐcellulose chromatography has revealed two peaks of PDE activities\ showing distinct substrate speci_city^ both enzymes are neither stimulated by cyclic GMP or calcium!calmodulin nor inhibited by cyclic GMP[ The lack of any e}ect on enzyme activity by cyclic GMP and calmodulin\ indicates that PDE0\ 1 and 2 isoenzymes\ if present\ are expressed at very low levels which escape detection under standard assay conditions[ Cyclic GMP hydrolytic activity eluting at the lowest ionic strength ðpeak 0 in Fig[ 0"A#Ł is strongly inhibited by Zaprinast^13\15 thus it can be designated as a member of the {{cyclic GMP binding!cyclic GMP speci_c PDE|| family "PDE4#\ characterized in rat and bovine lung11\12\45 and in rat platelets[04\20 PDE4 isoenzymes in hybrid and parental clone N07TG1 show some distinctive properties as indicated by a di}erent degree of Zaprinast inhibition[17 PDE activity eluting in peak 1 speci_cally hydrolyzes cyclic AMP^ this characteristic together with its inhibition properties allows to classify it as a {{low Km RO 19 0613 inhibited form|| PDE3[01\03 This PDE isoform is expressed in several tissues08\16\39^ however\ its presence in the brain is worthy of

Nucleotide phosphodiesterase activities in neuroblastoma lines

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Fig[ 3[ Time course of PDE activities in {{di}erentiated|| and control cells[ Cells "5×093# were plated into 59 mm culture dishes and treated with 4 mM dbcAMP[ At the indicated time\ cells were collected and enzyme activity measured as discussed in Section 0[ cAMP "A and B# and cGMP "C and D# PDE activity of 097CC04 "A and C# and N07TG1 "B and D# clones[ Empty bars represent enzymatic activity of control cells\ full bars those of treated cells[

particular attention\ since this isoform is an homologue of the Drosophila dunce mutant\ which causes de_ciencies in associative learning and memory[07 Association of this isoform with brain function is also suggested in mammals by the reported potent antidepressant action of its speci_c inhibitor rolipram[52 Several PDE3 genes "PDE3 A\B\C\D# from rat and human tissue have been cloned on the basis of their homology to the dunce gene^ 06\26\42 the heterogeneity of this family is further increased by isogene splice variants\31 and by the presence of di}erent promoters for each gene[40 The irregular shape of peak 1 "Fig[ 0# may be due to the presence of several members of the family\ eluting in a very close range of ionic strength[ The 097CC04 pattern of PDE activity appears quite similar to that described in the parental neuroblastoma N07TG1 and di}ers considerably from that of the parental glioma\ which expresses as major component PDE0 isoenzyme^4 this observation con_rms that the hybrid line shows preferentially features of the neuronal parental cell line[ The absence of PDE0 in studied neuro! blastoma is not surprising^ in fact although calmodulin!dependent isoform is widely expressed in the central nervous system "CNS# it is not ubiquitously distributed in all neurons^27 moreover it has not been found in sympathetic neurons of the superior cervical ganglia\18 suggesting that this activity may be not expressed in the peripheral nervous system "PNS#[ As already described\ in neuroblastoma cell lines treatments increasing cyclic AMP levels result in the progression of the di}erentiation pathway as shown by the inhibition of cell division and neurite extension\ the increase of cell size and total protein content and the enhanced expression of speci_c neuronal enzyme such as ChAT[36 Induction of PDE3 following increased cyclic AMP levels has been reported in _broblasts treated with PGE or dbcAMP28\05\ in linfoblastoid cells\2 in human monocytes\47 in astrocytoma59 and glioma cells treated with noradrenalin and isoproterenol[5\35\37\38\43 Furthermore Sertoli cells in response to FSH show a 099!fold increase of their cAMP!speci_c PDE activity^8\09\02\43\44\51 this increase has been related to lower responsiveness to gonadotropin of these cells in the mature stage[00\41 The higher PDE activity reported in dbcAMP treated cell "Fig[ 3#\ may be dependent on enzyme cAMP!dependent phosphorylation as reported in myoblasts23 and thyroid cells[49 This does not seem to be the case in the neuroblastoma system\ since the increase of enzyme

C 9[19029[90 9[04529[91

T 9[51029[91 9[27829[92

cAMP 2[0 1[4

Activity increase "fold# C 9[97929[90 9[03229[90

T 9[02229[93 9[51129[92

cGMP 0[55 3[24

Activity increase "fold#

PDE speci_c activity "nmol substrates hydrolyzed min−0 mg−0#

C T 9[93129[990 9[30929[992 9[90029[990 9[3129[991

cAMP

8[7 2[7

Activity increase "fold#

C 9[9129[993 9[91029[993

T 9[0129[995 9[1329[90

cGMP

5 00[3

Activity increase "fold#

PDE activity per cell "fmol substrates hydrolyzed min−0:cell#

Note] C\ control cultures^ T\ cultures treated with 4 mM dbcAMP for 4 days[ Data are expressed as means2S[E[ "n5#[

097CC04 N07TG1

Clones

Table 1[ Induction by dbcAMP of PDE actvity in 097CC04 and N07TG1 cells

205 M[ Giorgi et al[

Nucleotide phosphodiesterase activities in neuroblastoma lines

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activity requires at least 2 hr to become evident\ thus suggesting that transcriptional or translational regulation is involved[ The observed induction of cyclic GMP hydrolyzing activity "PDE4# by elevation of cyclic AMP level appears of particular interest[ In fact while induction of PDE3\ may be considered as a negative feedback response to increased cellular levels of cyclic AMP\ PDE4 induction represents an interesting mechanism of cross!talk between cyclic nucleotides\ as for PDE1 and PDE2[1 However\ in the case of PDE1 and 2\ cyclic GMP regulates cyclic AMP hydrolysis by an allosteric regulation of the enzyme activity^ in our case cyclic AMP regulation of cyclic GMP hydrolysis occurs via a more complex mechanism\ as indicated by the time interval required to observe this phenomenon[ The only evidence of a cyclic AMP!dependent stimulation of PDE4!cyclic GMP hydrolytic activity\ has been reported for the catalytic subunit of cyclic AMP!dependent protein kinases on partially puri_ed lung PDE[6 However\ this is a short term phenomenon while in our system the cyclic AMP! stimulated induction of PDE4 activity requires a few hours[ The present data do not allow to establish whether cyclic AMP directly enhances the expression of PDE4 or whether some other unknown regulatory factor is involved[ The induction of both PDE isoforms observed in neuroblastoma N07TG1 and 097CC04 lines could be part of the neuronal di}erentiation process\ as previously suggested for PDE3 in the di}erentiation of seminiferous tubule cells[00 Although the kinetic of PDE induction is very similar in both lines and chromatographic analysis has shown that both isoforms are induced\ the response of the two cell lines shows some diversity[ In fact the induction is more remarkable for PDE3 in 097CC04 cells and for PDE4 in N07TG1 cells "Fig[ 3#[ The di}erent response to dbcAMP observed in the two clones could be related to their di}erent ability to express speci_c neuronal properties\ and to the acquisition of 097CC04 cells to form functional synapses[ Furthermore as suggested by rolipram inhibition of PDE3\ di}erentiation could induces in 097CC04 cells a PDE3 subtype less sensitive to rolipram ðFig[ 1"B#Ł[ Expression of di}erent members of PDE3 family in relation to di}erentiation has been already reported in Sertoli cells[32 Finally PDE elution pro_les of both clones under di}erentiating condition show the presence of small amounts of cyclic AMP hydrolyzing activity ðFig[ 0"C and D^ Fig[ 1"B#Ł\ eluting at low ionic strength and not inhibited by rolipram which can possibly be ascribed to PDE6\30 an isoform characterized by its poor inhibition by rolipram and by a high a.nity for cyclic AMP "Km 9[1 mM#[ This observation appears particularly interesting^ in fact PDE6 has only been identi_ed by DNA cloning techniques\ except a possible identi_cation in T!lymphocyte cells21 and in superior cervical ganglion[18 Until now it has never been biochemically characterized[ If cyclic AMP activity present in peak 0 will be con_rmed as PDE6\ neuroblastoma cells may prove an interesting system for the biochemical characterization of this isoform and possibly for its isolation[ Acknowled`ements*We are grateful to La Roche for gifts of RO 19 0613\ Schering for rolipram\ Rho¼ne!Poulenc Rorer for zaprinast and Dr Randall Kincaid for antibody against the calmodulin!stimulated isoform[ This work was supported by M[ U[R[S[T[\ A[ I[R[C[ and C[ N[R[ grants[

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28[ Manganiello V[ and Vaughan M[ "0861# Prostaglandin E e}ects on adenosine 2?]4?!cyclic monophosphate concentration and phosphodiesterase activity in _broblasts[ Proceedin`s of the National Academy of Sciences of the U[S[A[ 58\ 158Ð 162[ 39[ Marchmont R[ J[\ Ayad S[ R[ and Houslay M[ D[ "0870# Puri_cation and properties of the insulin!stimulated cyclic AMP phosphodiesterase from rat liver plasma membranes[ Biochemistry Journal 084\ 534Ð541[ 30[ Michaeli T[\ Bloom T[ J[\ Martins T[\ Loughney K[\ Ferguson K[\ Riggs M[\ Rodgers L[\ Beavo J[ A[ and Wigler M[ "0882# Isolation and characterization of a previously undetected human cAMP phosphodiesterase by complementation of cAMP phosphodiesterase!de_cient Saccharomyces cerevisiae[ Journal of Biolo`ical Chemistry 157\ 01814Ð01821[ 31[ Monaco L[\ Vicini E[ and Conti M[ "0883# Structure of two rat genes coding for closely related rolipram!sensitive cAMP phosphodiesterases[ Journal of Biolo`ical Chemistry 158\ 236Ð246[ 32[ Morena A[\ Boitani C[\ De Grossi S[\ Stefanini M[ and Conti M[ "0884# Stage and cell!speci_c expression of the adenosine 2?\4?!monophosphate!phosphodiesterase genes in the rat seminiferous epithelium[ Endocrinolo`y 025\ 576Ð584[ 33[ Nestler\ E[ J[ and Greengard\ P[\ Protein phosphorylation and regulation of neuronal function[ In Basic Neurochemistry] Molecular\ Cellular and Medical Aspects\ 4th ed\ eds G[ J[ Siegel\ B[ W[ Agrano}\ R[ W[ Albers and P[ B[ Molino}[ Raven Press\ New York\ 0883\ pp[ 338Ð363[ 34[ Nicholson D[\ Challiss R[ A[ J[ and Shashid M[ "0880# Di}erential modulation of tissue function and therapeutic potential of selective inhibitors of cyclic nucleotide phosphodiesterase isoenzymes[ Trends in Pharmacolo`ical Sciences 01\ 08Ð16[ 35[ Onali P[\ Schwartz J[ P[\ Hanbauer I[ and Costa E[ "0870# Regulation by a b!adrenergic receptor of a Ca1¦!indipendent adenosine 2?\4!"cyclic# monophosphate phosphodiesterase in C5 glioma cells[ Biochimica et Biophysica Acta 564\ 174Ð 181[ 36[ Prasad K[ N[ "0864# Di}erentiation of neuroblastoma cells in culture[ Biolo`ical Reviews 49\ 018Ð154[ 37[ Schwartz J[ P[ and Passonneau J[ V[ "0863# Cyclic AMP!mediated induction of the cyclic AMP phosphodiesterase of C! 5 glioma cells[ Proceedin`s of the National Academy of Sciences of the U[S[A[ 60\ 2733Ð2737[ 38[ Schwartz J[ P[ and Onali P[ "0873# Beta!adrenergic receptor regulation of a cyclic AMP phosphodiesterase in C5 glioma cells[ Advances Cyclic Nucleotide Protein Phosphorylation Research 05\ 084Ð192[ 49[ Sette C[\ Iona S[ and Conti M[ "0883# The short!term activation of a rolipram!sensitive\ cAMP!speci_c phosphodiesterase by thyroid!stimulating hormone in thyroid FRTL!4 cells is mediated by a cAMP!dependent phosphorylation[ Journal of Biolo`ical Chemistry 158\ 8134Ð8141[ 40[ Sette C[\ Vicini E[ and Conti M[ "0883# The rat PDE2:IVd phosphodiesterase gene codes for multiple proteins di}er! entially activated by cAMP!dependent protein kinase[ Journal of Biolo`ical Chemistry 158\ 07160Ð07163[ 41[ Steinberger A[\ Hintz M[ and Heindel J[ J[ "0866# Changes in cyclic!AMP responses to FSH in isolated rat Sertoli cells during sexual maturation[ Biolo`y of Reproduction 08\ 455Ð461[ 42[ Swinnen J[ V[\ Joseph D[ R[ and Conti M[ "0878# Molecular cloning of rat homologues of the Drosophila melano`aster dunce cAMP phosphodiesterase] evidence for a family of genes[ Proceedin`s of the National Academy of Sciences of the U[S[A[ 75\ 4214Ð4218[ 43[ Swinnen J[ V[\ Joseph D[ R[ and Conti M[ "0878# The mRNA encoding a high!a.nity cAMP phosphodiesterase is regulated by hormones and cAMP[ Proceedin`s of the National Academy of Sciences of the U[S[A[ 75\ 7086Ð7190[ 44[ Swinnen J[ V[\ Tsikalas K[ E[ and Conti M[ "0880# Properties and hormonal regulation of two structurally related cAMP phosphodiesterases from the rat Sertoli cell[ Journal of Biolo`ical Chemistry 155\ 07269Ð07266[ 45[ Thomas M[ K[\ Francis S[ H[ and Corbin J[ D[ "0889# Characterization of a puri_ed bovine lung cGMP!binding cGMP phosphodiesterase[ Journal of Biolo`ical Chemistry 154\ 03853Ð03869[ 46[ Thompson W[ J[ and Appleman M[ M[ "0860# Multiple cyclic nucleotide phosphodiesterase activities from rat brain[ Biochemistry 09\ 200Ð205[ 47[ Thorphy T[ J[\ Zhow H[ L[ and Cieslinski L[ B[ "0881# Stimulation of b adrenoreceptors in a human monocyte cell line "U826# up!regulates cyclic AMP!speci_c phosphodiesterase activity[ Journal of Pharmacolo`y and Experimental Theraupeutics 152\ 0084Ð0194[ 48[ Towbin H[\ Staehelin T[ and Gordon J[ "0868# Electrophoretic transfer of proteins from polyacrylamide gel to nitro! cellulose sweets] procedure and some application[ Proceedin`s of the National Academy of Sciences of the U[S[A[ 65\ 3249Ð3243[ 59[ Uznov P[\ Shein H[ M[ and Weiss B[ "0862# Cyclic AMP phosphodiesterase in cloned astrocytoma cells] norepinephrine induces a speci_c enzyme form[ Science 079\ 293Ð295[ 50[ Vassault\ A[\ UV method with pyruvate and NADH[ In Methods in Enzymatic Analysis\ ed[ H[ U[ Bergmeier[ Verlag Chemie\ Weinheim!Basel\ 0872\ pp[ 008Ð025[ 51[ Verhoeven G[\ Cailleau J[ and De Moor P[ "0870# Hormonal control of phosphodiesterase activity in cultured rat Sertoli cells[ Molecular and Cellular Endocrinolo`y 13\ 30Ð40[ 52[ Wacthel H[ and Schneider H[ H[ "0875# Rolipram\ a novel antidepressant drug\ reverses the hypothermia and hypokinesia of monoamine!depleted mice by an action beyond postsynaptic monoamine receptors[ Neuropharmacolo`y 14\ 0008Ð 0015[