Journal of NeurochemistryLippincott—Raven Publishers, Philadelphia© 1996 International Society for Neurochemistry

Diminished Noradrenergic Stimulation Reduces the Activityof Rolipram-Sensitive, High-Affinity Cyclic AMP

Phosphodiesterase in Rat Cerebral Cortex

Ying Ye and James M. O’Donnell

Department of Pharmacology and Therapeutics, Louisiana State University Medical Center, Shreveport, Louisiana, U.S.A.

Abstract: The present study examined the in vivo regula-tion of rolipram-sensitive, high-affinity cyclic AMP phos-phodiesterase (PDE4) in rat cerebral cortex. The hydroly-sis of cyclic AMP, formed by stimulation of ~3-adrenergicreceptors, was measured in cerebral cortical slices. Hy-drolysis of cyclic AMP formed under these conditionswas inhibited by the PDE4-selective inhibitor rolipram butnot by selective inhibitors of other PDE families. Intraven-tricular infusion of 6-hydroxydopamine (6-OHDA; 200 /.tg)decreased the rate constant of cyclic AMP hydrolysis andincreased the cyclic AMP half-life 17 days, but not 1 or7 days, following the treatment. A reduction in norepi-nephrine (NE) content occurred first; the NE level wasreduced to 42, 24, and 6% of control at 1, 7, and 17 daysafter 6-OHDA infusion, respectively. This was followedby the development of supersensitivity of /~-adrenergicreceptor-linked adenylyl cyclase, which occurred 7 daysafter the infusion. The reduction in PDE4 activity occurredlast. When a higher dose of 6-OHDA (300 ,ug) was used,the reduction in the rate constantof cyclic AMP hydrolysisoccurred by 7 days; at this time NE contentwas depletedto 6% of control. Similar to 6-OHDA treatment, continu-ous blockade of ~3-adrenergicreceptors, produced bychronic propranolol infusion, decreased the rate constantof cyclic AMP hydrolysis. Therefore, the current resultsindicate that diminished stimulation of /3-adrenergic re-ceptors, either by loss of noradrenergic innervation orby receptor blockade, reduces the activity of PDE4. Thissuggests that PDE4 regulation may contribute in the ho-meostasis of the noradrenergic receptor—effector systemin the brain. Key Words: Phosphodiesterase—CyclicAMP hydrolysis—Noradrenergic system —6-Hydroxydo-pamine—Propranolol—Rolipram.J. Neurochem. 66, 1894—1902 (1996).

Phosphodiesterase (PDE), the enzyme that macti-yates cyclic nucleotide second messengers, is com-posed of multiple isozymes. These isozymes are classi-fied into seven families based on distinct molecularstructure, substrate preference, susceptibility to regula-tion and inhibition, and tissue distribution (Beavo eta!., 1994). Besides these multiple families of PDE,there is an additional level of complexity. Most fami-lies have subfamilies, i.e., subtypes, coded by distinct

isogenes, and each isogene may havemultiple productsdue to alternative splicing or different initiation sites.

In rat cerebral cortex, at least three families of PDEare present (Thompson and Appleman, 1970; Nichol-son et al., 1989): Ca2~/ca1modulin-stimulatedPDE1,cyclic GMP-stimulated PDE2, and rolipram-sensitive,high-affinity cyclic AMP PDE4. Recently, a novelform of PDE has been detected in rat cerebral cortex(Mukai et al., 1994), but the identity of this isozymeis yet to be resolved.

PDE4 is of particular interest in neuropsychophar-macology. It is the major isozyme hydrolyzing cyclicAMP formed by stimulation of ~3-adrenergicreceptor-linked adenylyl cyclase, even though PDE1 accountsfor most cyclic AMP hydrolytic activity in cell-freepreparations of rat brain (Whalin et al., 1989; Challissand Nicholson, 1990; Barber et al., 1992). Specificinhibitors of PDE4, such as rolipram, exhibit antide-pressant effects in both animal models predictive ofantidepressant activity (Wachtel and Schneider, 1986;O’Donnell, 1993) and in clinical trials (Fleischhackeret al., 1992). Unlike classical antidepressants, the ef-fects of rolipram appear to be mediated by inhibitingPDE4 rather than inhibiting monoamine oxidase ormonoamine reuptake (Kehr et al., 1985; Schmiechenet al., 1990; O’Donnell, 1993). The finding that theinhibition of PDE4 has potential therapeutic use in thetreatment of depression makes it important to obtaina better understanding of this enzyme.

Several studies using cultured cells have demon-strated that PDE4 is regulated in vitro. For instance,PDE4 activity is increased in immature granulosa andSertoli cells incubated with follicle-stimulating hor-mone (Conti et al., 1982, 1984), human monocytes

Resubmitted manuscript received November 6, 1995; acceptedDecember 8, 1995.

Address correspondence and reprint requests to Dr. Y. Ye at De-partment of Pharmacology and Therapeutics, Louisiana State Uni-versity Medical School, 1501 Kings Highway, Shreveport, LA71130, U.S.A.

Abbreviations used: EHNA, erythro-9- ( 2-hydroxy-3-nonyl) ade-nine; KRB, Krebs—Ringer-bicarbonate buffer; NE, norepinephrine;6-OHDA, 6-hydroxydopamine; PDE, phosphodiesterase.

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PHOSPHODIESTERASE AND NORADRENERGIC ACTIVITY 1895

(U937) stimulated with epinephrine or salbutamol(Barber et a!., 1992; Torphy et al., 1992), and FRTL-5 thyroid cells stimulated with thyroid-stimulating hor-mone (Sette et al., 1994a). This suggests that alter-ations in the rate of cyclic AMP synthesis may resultin a compensatory change in the activity of PDE4.

It is not clear whether PDE4 undergoes regulationin vivo. Acute or repeatedtreatment with desipramine,which should indirectly stimulate /3-adrenergic-lmnkedadeny!yl cyclase, was reported as not altering cyclicAMP PDE activity in the pineal gland (Moyer et al.,1980). By contrast, a single dose of the /3-adrenergicagonist isoproterenol has been shown to increase PDEactivity in the pineal gland 1 h following its administra-tion; this increased PDE activity is evidenced by anincrease in the Vmas for cyclic AMP hydrolysis (Ole-shansky and Neff, 1975). Neitherof these studies iden-tified the isozyme involved in the hydrolysis of cyclicAMP. Some studies have provided indirect evidencesuggesting that PDE4 is regulated in vivo (Schultz andSchmidt, 1986; Schoeffter and Stoclet, 1990; O’Don-nell, 1993). To date, no data have been reported thatdirectly address the issue of whether PDE4 undergoesregulation in the CNS, especially with altered norad-renergic activity. The present study directly addressedthis question by determining whether the activity ofPDE4 in rat cerebral cortex is decreased followingnoradrenergic lesioning and, if so, whether the de-creased activity is due to diminished stimulation of /3-adrenergic receptors.

MATERIALS AND METHODS

AnimalsMale Sprague—Dawley rats (weighing 225—300 g; Har-

lan, Indianapolis, IN, U.S.A.) were used for the experiments.The rats were housed in a temperature (22—24°C)- and light(6:00 a.m.—6:00 p.m.)-controlled room and were allo’vedfree access to food pellets and water. The studies reportedhere have been carried out in accordance with the Guideforthe Care and Use of Laboratory Animals as adopted andpromulgated by the National Institutes of Health.

Central noradrenergic lesionsNoradrenergic lesions were produced by intracerebroven-

tricular administration of 6-hydroxydopamine (6-OHDA)(Sporn et a!., 1977). Rats were pretreated with atropinemethylnitrate (10 mg/kg, i.p.), anesthetized with pentobar-bital (50 mg/kg, i.p.), and placed in a stereotaxic frame.Each lateral cerebral ventricle was infused with 6-OHDA(100 or 150 ~sg;see Results) dissolved in 10 pd of 0.2%ascorbic acid/0.9% NaC1. The infusions were made 0.5 mmposterior to bregma, ±1.6mm lateral from the midline, and3.2—3.4 mm ventral from the skull surface with the upperincisor bar set at 0 (O’Donnell et al., 1994). In experimentstesting the effects of6-OHDA 1 day followingits administra-tion, 6-OHDA was given via indwelling cannulae, whichwere implanted into both lateral cerebral ventricles 5—7 daysearlier; this was done to minimize direct effects of the surgi-cal trauma. The noradrenergic lesions were verifiedby deter-mination of norepinephrine (NE) levels using HPLC

(O’Donnell and Seiden, 1983; O’Donnell, 1993); proteincontent was determined by the method of Bradford (1976).

13-Adrenergic receptor blockadeChronic, central /1-adrenergic receptor blockade was

achieved by subcutaneous infusion of propranolol (Eison etal., 1988). Propranolol (30 mg/kg/day, 14 days) dissolvedin water was released from subcutaneously implanted os-motic pumps (model 2ML2; Aiza Corp., Palo Alto, CA,U.S.A.). After 14 days, the pumps were removed. The ratswere killed following a 24-h drug wash-out period, and cy-clic AMP hydrolysis was measured in slices of cerebral cor-tex. /3-Adrenergic receptor blockade was verified by the up-regulation of the receptors measured by saturation bindingof ‘25I-pindolol to membrane preparations of the cerebralcortices (O’Donnell and Frazer, 1985; O’Donnell, 1990)from the rats that receivedpropranolol following an identicaltreatment regimen.

Cyclic AMP hydrolysis assayCyclic AMP hydrolysis was measured in cerebral cortical

slices following activation of /3-adrenergic receptor-linkedadenylyl cyclase with isoproterenol (Whalin et al., 1989;Challiss and Nicholson, 1990). Rats were killed by decapita-tion, and cerebral cortices were dissected and chopped into0.3- X 0.3-mm slices using a Mcllwain tissue chopper. Theslices were suspended in oxygenated (95% 02, 5% C0

2)37°C Krebs—Ringer-bicarbonate buffer (KRB; 11.1 mMdextrose, 26.2 mM NaHCO3, 5 mM HEPES, 2.4 mMMgSO4, 0.4mM KH2PO4, 118 mM NaC1, 4.8 mM KC1, 1.3mM CaC12, and 0.01 mM Na2EDTA). The slices werewashed once and preincubated in oxygenated KRB at 37°Cfor 30 mm. After three additional washes, 1.5-mi aliquotsof the slices (containing ‘-~1.5—2.0mg of protein) werepipetted into individual vials and incubated for another 30mm. At the end of this incubation, 10 ~.tMisoproterenol wasadded to stimulate cyclic AMP accumulation. Five minuteslater, /3-adrenergic receptors were blocked by addition of100 jjM timolol, and cyclic AMP hydrolysis was allowed toproceed. The hydrolysis of cyclic AMP was terminated byaddition of ice-cold perchloric acid (final concentration,1.67%) at 1, 2, 3, 5, 10, or 15 mm following addition oftimoiol. The samples were sonified and then centrifuged at25,000 g for 15 mm at 4°C.The pellets were resuspendedin 1.0 M NaOH for protein content assay (Bradford, 1976).Cyclic AMP levels in the supernatant were determined byradioimmunoassay (O’Donnell, 1993). Whennecessary, ali-quots of the tissue slices were taken to measure NE concen-trations.

Identification of the families of PDE involved incyclic AMP hydrolysis

Family-specific PDE inhibitors were used to identify thetypes of PDE involved in hydrolyzing cyclic AMP formed bystimulation of /3-adrenergic receptor-linked adenylyl cyclase.Cerebral cortical slices were stimulated by 10 beM isoprotere-nol. Five minutes later, further stimulation was blocked byaddition of 100 p~Mtimolol; PDE inhibitors were also addedat this time. Cyclic AMP hydrolysis was allowed to proceedin the presence or absence of family-specific PDE inhibitors.Roliprarn, ranging from 0.01 to 10 ptM, was used as a PDE4-specific inhibitor. Fiuphenazine (10 1.tM), erythro-9-(2-hy-droxy-3-nonyl)adenine (EHNA; 10 1eM), milrinone (10~.tM),and zaprinast (10 ~M) were used as specific inhibitorsfor PDE1, PDE2, PDE3, andPDE5, respectively. The inhibi-

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1896 Y. YE AND J. M. O’DONNELL

tors were dissolved in dimethyl sulfoxide (final concentration,0.06%). The hydrolysis was terminated 3.5 mm later by addi-tion of ice-cold perchloric acid. The unhydrolyzed cyclicAMP was quantified by radioimmunoassay.

Data analysisRate constant (k) values of cyclic AMP hydrolysis were

obtained by fitting the data of the first phase of hydrolysisto a first-order kinetic equation, C = (C0 — C15 mm) e_kt+ C15 ,,.,~. The half-life (t~,2) values of cyclic AMP werecalculated by the equation t1,2 = ln2/k. Significant differ-ences between the control and treated groups were deter-mined using two-tailed Student’s t tests. The time depen-dency of the changes was tested by a two-way ANOVA(treatment by time). The correlations among the changes ofNE level, cyclic AMP formation, and rate constant of thehydrolysis were tested by an analysis of covariance.

Drugs and chemicalsCyclic AMP antiserum complex and ‘

251-pindolol werepurchased from DuPont/NEN (Boston, MA, U.S.A.), and2SI~cyclicAMP-tyrosine methyl ester was from ICN (Costa

Mesa, CA, U.S.A.). Rolipram was a gift from Schering AG(Berlin, Germany), and fiuphenazine was a gift from E. R.Squibb & Sons (Princeton, NJ, U.S.A.). Milrinone andzapri-nast were purchased from Calbiochem (La Jolla, CA,U.S.A.) and EHNA HC1 was from Research BiochemicalsInternational (Natick, MA, U.S.A.). All other drugs andchemicalswere obtained from Fisher Scientific (Dallas, TX,U.S.A.) or Sigma Chemical Co. (St. Louis, MO, U.S.A.).

RESULTS

Effects of family-specific PDE inhibitors on cyclicAMP hydrolysis

In cerebral cortical slices of control rats, cyclic AMPformed by stimulation of /3-adrenergic receptor-linkedadenylyl cyclase was hydrolyzed rapidly. The t

112 ofcyclic AMP was 2.10 ±0.21 mm; the rate constant ofhydrolysis was 0.35 ±0.04 min~(Fig. 1).

To determine the families of PDE that were involvedin this hydrolysis, cyclic AMP was hydrolyzed in theabsence or presence of different family-specific PDEinhibitors for 3.5 mm. The hydrolysis of cyclic AMPin cerebral cortical slices formedby isoproterenol stim-ulation was blocked by 1 or 10 1uM concentrationsof the PDE4-selective inhibitor rolipram (Fig. 2A; p<0.05 compared with the hydrolysis in the absenceof inhibitor) but not by 10 j.tM concentrations of flu-phenazine, EHNA, milrinone, or zaprinast (Fig. 2B),PDE1, 2, 3, and 5 selective inhibitors, respectively.

Effects of intraventricular infusion of 6-OHDA oncyclic AMP hydrolysis, NE content, andisoproterenol-stimulated cyclic AMP formation

Pretreatment with 6-OHDA (200 jig) produced atime-dependent reduction in the rate constant of cyclicAMP hydrolysis (p < 0.05, treatment by time interac-tion). The rate constant of cyclic AMP hydrolysis didnotshow any significant change 1 day or 7 days follow-ing intraventricular infusion of 6-OHDA (200 jig; Fig.3A). However, 17 days following the infusion, the rateconstant decreased by 38%, from the sham-lesioned

FIG. 1. Time course of cyclic AMP hydrolysis. Rat cerebral corti-cal slices were stimulated by 10 ~sMisoproterenol. Five minutesfollowing this stimulation, 100 pM timolol was added to stop fur-ther activation of /9-adrenergic receptor-linked adenylyl cyclase(0 mm), and cyclic AMP hydrolysis was allowed to proceed. Thehydrolysis of cyclic AMP was terminated at the indicated timesfollowing the blockade of /3-adrenergic receptors. Data are mean±SEM (bars) values for six separate experiments.

value of 0.34 ±0.03 to 0.21 ±0.02 min~(Fig. 3A;p < 0.01), and the t1,2 of cyclic AMP was increasedfrom 2.10 ±0.21 to 3.36 ±0.28 mm.

Basal cyclic AMP levels were comparable in cere-bral cortical slices from control and lesioned rats. Pre-treatment of 6-OHDA produced a time-dependentchange in isoproterenol-stimulated cyclic AMP forma-tion (p < 0.001, treatment by time interaction). Thecyclic AMP accumulation stimulated by 10 jiM isopro-terenol was not changed 1 day following 6-OHDA(200 jig) infusion but increased by about two- to three-fold from the control value 7 and 17 days followingthe infusion (Fig. 3C). By 10 mm following the initia-tion of hydrolysis, i.e., from the addition of timolol,the cyclic AMP levels no longer differed from basallevels in cerebral cortical slices from both control andlesioned rats.

To verify noradrenergic lesions, NE levels were de-termined in aliquots of cerebral cortical slices takenfrom the same rats. Intraventricular 6-OHDA (200 jig)infusion produced a time-dependent depletion (p<0.005, treatment by time interaction); NE contentin cerebral cortex was reduced to 42, 24, and 6% ofcontrol values 1, 7, and 17 days, respectively, afteradministration of 6-OHDA (Fig. 3B).

Overall, following intraventricular 6-OHDA (200jig) infusion, the reduction of NE level was the first tooccur, followed by the enhancement of isoproterenol-stimulated cyclic AMP formation and then the reduc-tion in the rate constant of cyclic AMP hydrolysis.The increase of isoproterenol-stimulated cyclic AMPformation appeared to be correlated with the depletionof NE as shown by an analysis of covariance (p<0.005). However, there was not a significant cone-

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PHOSPHODIESTERASE AND NORADRENERGIC ACTIVITY 1897

sion was time dependent or depended on the extent ofNE depletion, cyclic AMP hydrolysis was measuredin cerebral cortical slices from rats that had received300 jig of 6-OHDA by intracerebroventricular infusion(in contrast to the 200 jig used previously); this was

FIG. 2. Effects of family-specific PDE inhibitors on the hydrolysisof cyclic AMP. Rat cerebral cortical slices were stimulated by10 p~Misoproterenol for 5 mm, and then further stimulation wasblocked by 100 ,uM timolol. Timolol was added together withdifferent PDE inhibitors. The hydrolysis was allowed to proceedin the absence (0 point) or presence of the inhibitors for 3.5 mm:basal levels of cyclic AMP (LI), levels of cyclic AMP formed byisoproterenol stimulation (•), unhydrolyzed cyclic AMP levelsfollowing a 3.5-mm hydrolysis in the absence (0 point) or pres-ence of rolipram (~),and unhydrolyzed cyclicAMP levels follow-ing a 3.5-mm hydrolysis in the absence (0 point) or presenceof family-specific inhibitors (LI). A: Different concentrations ofrolipram. B: Selective inhibitors of PDE1, 2, 3, and 5. *p < 0.05compared with value without PDE inhibitors. Data are mean±SEM (bars) values for three separate experiments.

lation between the changes in cyclic AMP hydrolysisand NE content or isoproterenol-stimulated cyclicAMP formation.

Effects of intraventricular infusion of a high doseof 6-OHDA on cyclic AMP hydrolysis, NEcontent, and isoproterenol-stimulated cyclic AMPformation

To determine whether the change of cyclic AMPhydrolysis observed 17 days following 6-OHDA infu-

FIG. 3. Effects of 6-OHDA treatment on the rate constant ofcyclicAMP hydrolysis, NE content, and isoproterenol-stimulatedcyclicAMP formation in rat cerebral cortex. A: Cyclic AMP hydro-lysis was measured in cerebral cortical slices (see Fig. 1) at theindicated times after the rats had received intracerebroventricu-lar infusion of either vehicle (LI) or 200 ~tgof 6-OHDA (U). Therate constant was obtained by fitting the levels of unhydrolyzedcyclic AMP (0, 1, 2, and 3 mm following addition of timolol) toa first-order kinetic equation. B: NE content (in ng/mg of protein)was measured in the aliquots of cerebral cortical slices from thesame rats. C: Cyclic AMP was formed by stimulating slices with10 sM isoproterenol for 5 mm. The values shown are isoprotere-nol-stimulated cyclicAMP levels from which the basal levels hadbeen subtracted. ~ < 0.01 compared with NE contents 1 dayor 7 days following 6-OHDA treatment; *p < 0.05, °°p< 0.01,

< 0.001 compared with corresponding values for sham-lesioned rats. Data are mean ± SEM (bars) values for four orfive separate experiments.

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1898 Y. YE AND J. M. O’DONNELL

formation was increased about threefold above the con-trol value (Fig. 4C).

Effects of continuous propranolol infusion oncyclic AMP hydrolysis and isoproterenol-stimulated cyclic AMP formation

To determine whether the change in cyclic AMPhydrolysis in 6-OHDA-treated rats was due to reducedstimulation of /3-adrenergic receptors caused by a le-sion-induced reduction in NE release, the hydrolysisassay was conducted using cerebral cortical slices fromrats subjected to chronic blockade of /3-adrenergic re-ceptors. This was accomplished by subcutaneous infu-sion of the /3-adrenergic antagonist propranolol.Chronic propranolol infusion (30 mg/kg/day, 14 days,s.c.) decreased the rate constant of cyclic AMP hydro-lysis from 0.35 ±0.04 min~(control) to 0.20 ±0.02min~(propranolol-treated) (Fig. 5A; p <0.01). Thet1,2 values for cyclic AMP hydrolysis were 2.10 ±0.21

FIG. 4. Effects of a high dose of 6-OHDA (300 ~sg)on the rateconstant of cyclic AMP hydrolysis, NE content, and isoprotere-nol-stimulated cyclicAMP formation in cerebral cortex. A: CyclicAMP hydrolysis was measured in cerebral cortical slices (seeFig. 1) from rats 7 days after they had received intracerebroven-tricular infusion of either vehicle (LI) or 300 1sg of 6-OHDA (•).The rate constant was obtained by fitting the levels of unhy-drolyzed cyclic AMP (0, 1, 2, and 3 mm following addition oftimolol) to a first-order kinetic equation. B: NE content (in ng/mg of protein) was measured in the aliquots of cerebral corticalslices. C: Cyclic AMP was formed by stimulating slices with 10jiM isoproterenol for 5 mm. The values shown are isoproterenol-stimulated cyclic AMP levels from which the basal levels hadbeen subtracted. *p < 0.05, °°p<0.01, °°°p< 0.001 in compar-ison with corresponding values for sham-lesioned rats. Data aremean ±SEM (bars) values for six separate experiments.

the highest dose that did not produce lethality. The300 jig dose of 6-OHDA depleted NE to 6% of controlvalues 7 days after its administration (Fig. 4B); at thistime, the rate constant of cyclic AMP hydrolysis wasreduced to 0.20 ±0.04 min~from the control valueof0.45 ±0.10 mm_i (Fig. 4A;p <0.05). Seven daysafter lesioning, isoproterenol-stimulated cyclic AMP

FIG. 5. Effect of chronic propranolol infusion on the rate con-stant of cyclicAMP hydrolysis and isoproterenol-stimulated cy-clic AMP formation. A: Rate constant of cyclic AMP hydrolysis.Rats received propranolol (30 mg/kg/day for 14 days, s.c.) viasubcutaneously implanted osmotic pumps (U). Control rats un-derwent surgery but had no pumps implanted (LI). After 14days,the pumps were removed, and a 24-h drug wash-out periodwas allowed before measurement of cyclicAMP formation andhydrolysis. The rate constant was obtained by fitting the levelsof unhydrolyzed cyclicAMP (0, 1, 2, and 3 mm following additionof timolol) into a first-order kinetic equation. B: Cyclic AMP wasformed by stimulating slices with 10 jiM isoproterenol for 5 mm.The values shown are isoproterenol-stimulated cyclicAMP levelsfrom which the basal levels had been subtracted. ~*p < 0.01compared with control. Data are mean ±SEM (bars) values forsix separate experiments.

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TABLE 1. Effect of chronic, subcutaneous infusion ofpropranolol on binding of ‘

251-pindolol to (3-adrenergicreceptors on cerebral cortical membranes

Bmax (fmol/mg) K

0 (nM)

Control 116.4 ±13.9 0.21 ±0.06Propranolol 164.4 ±12.2’ 0.34 ±0.03

The rats received propranolol (30 mg/kg/day for 14 days, s.c.)similarly to those used for the cyclic AMP hydrolysis study (seeFig. 5). After 14 days, the pumps were removed, and a 24-h drugwash-out period was allowed before the rats were killed for measure-ment of ‘

25l-pindolol binding. Data are mean ±SEM values fromfour separate experiments.

p < 0.05 in comparison with control.

and 3.61 ±0.36 mm in control and propranolol-treatedrats, respectively. Isoproterenol-stimulated cyclicAMP formation was not increased significantly in pro-pranolol-treated rats (Fig. SB).

To verify that central /3-adrenergic receptors wereblocked by chronic, subcutaneous infusion of propran-olol, the density of /3-adrenergic receptors on cerebralcortical membranes was determined. Propranolol treat-ment increased the Bma. value for 1251-pindolol bindingfrom 116.4 ±13.9 to 164.4 ±12.2 fmol/mg of protein(Table 1; p < 0.05). Although propranolol treatmentdid not result in a significant change in the KD value,there was a tendency toward an elevated KD value fori201..pindolol binding in propranolol-treated rats (Ta-

ble 1).

DISCUSSION

The results of the present study showed that norad-renergic lesioningreduced PDE activity in rat cerebralcortex in a time-dependent manner. The hydrolysis ofcyclic AMP formed by stimulation of /3-adrenergicreceptors was blocked by the PDE4-selective inhibitorrolipram but not by selective inhibitors of other PDEfamilies (see below). Thus, the observed change incyclic AMP hydrolysis likely was due to a change inthe activity of PDE4.

The reduction in PDE4 activity was preceded bythe development of supersensitivity of /3-adrenergicreceptor-linked adenylyl cyclase. This suggests that thein vivo regulation of PDE4 was not as rapid as theregulation of /3-adrenergic receptor-linked adenylyl cy-clase. When a higher dose of 6-OHDA was used toproduce larger lesions more rapidly, the reduction inPDE4 activity occurredearlier. Thus, it appears that theextent of NE depletion might be more closely related tothe regulation of PDE4 activity than the time since 6-OHDA administration. However, analysis of covari-ance did not reveal a correlation between NE depletionor i soproterenol-stimulated cyclic AMP formation andPDE4 activity. It is likely that changes inPDE4 activityare delayed in time relative to the other two effects oflesioning.

The present study also revealed that chronic pro-pranolol infusion decreased PDE4 activity. The reduc-tion in the rate constant of cyclic AMP hydrolysiswas similar to that seen in lesioned rats. Subcutaneousinfusion of propranolol blocks central /3-adrenergic re-ceptors (Eison et al., 1988); this was verified by anincrease in the Bmax value for 251-pindolol binding tomembranes prepared from cerebral cortex in this study.The tendency for an increase in the K

0 value and thelack of a significant increase in isoproterenol-stimu-lated cyclic AMP formation inpropranolol-treated ratsmay be due to the presence of residual propranolol.Because chronic blockade of /3-adrenergic receptorsaffected cyclic AMP hydrolysis in a manner similar tonoradrenergic lesions, it is likely that lesion-inducedreduction in PDE4 activity is due to reduced stimula-tion of /3-adrenergic receptors by endogenous NE.

In rat cerebral cortex at least three families of PDEhave been identified: Ca

2~/ca1modulin-dependentPDEI, cyclic GMP-stimulated PDE2, and rolipram-sensitive, high-affinity cyclic AMP PDE4 (Nicholsonet al., 1989). Of the three, PDE assays in cell-freecortical preparations show that PDE I accounts formost of the activity hydrolyzing cyclic AMP. How-ever, in a synaptosomal preparation of the cortex, thePDE activities present are PDE2 and PDE4, with PDE2being of greater quantitative importance (Whalin etal., 1989; Barber et al., 1992). For a long time, theimportance of PDE4 in the CNS was not appreciateduntil another approach was taken that measures cyclicAMP decay following cessation of stimulation of ade-nylyl cyclase in intact cells (Barber and Butcher,1988). Applying this technique, together with the useof family-specific PDE inhibitors, Whalin et al. (1989)described the critical role of PDE4 in hydrolyzing cy-clic AMP associated with the noradrenergic system;these findings are further supported by the results ofChalliss and Nicholson (1990). The discrepancy be-tween the cell-free and cell-intact studies probably isdue to artifacts associated with the elimination of com-partmentalization and alterations in the process of iso-lation. The results of the current study are consistentwith the notion that PDE4 is the major isozyme hy-drolyzing cyclic AMP formed by stimulation of /3-adrenergic receptor-linked adenylyl cyclase. In corticalslices, the hydrolysis of cyclic AMP formed by ~3-adrenergic receptor stimulation was blocked by roli-pram (a PDE4 inhibitor) but not by fluphenazine (aPDE1 inhibitor), EHNA (a PDE2 inhibitor), milri-none (a PDE3 inhibitor), or zaprinast (a PDES inhibi-tor) at comparable concentrations. Therefore, it ap-pears that the reduction of PDE activity observed fol-lowing diminished noradrenergic activity is primarilydue to the reduction of PDE4 activity.

It has been shown that PDE4 also is involved inthe hydrolysis of histamine- and adenosine-stimulatedcyclic AMP accumulation (Stanley et al., 1989;Whalin et al., 1989). The present experimental designnot only measured PDE4 activity, but also provided

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the ability to focus on PDE4 activity that is associatedwith /3-adrenergic receptors, because cyclic AMP wasformed by isoproterenol stimulation. Thus, it was pos-sible to identify changes in PDE4 activity directly asso-ciated with altered stimulation of /3-adrenergic recep-tors in rat cerebral cortex.

The present results are in agreement with findingsfrom in vitro studies showing increased PDE4 activityresulting from increased concentrations of hormonesand neurotransmitters. PDE4 activity is increased incultured cells, such as immature granulosa and Sertolicells stimulated with follicle-stimulating hormone(Conti et al., 1982, 1984), human monocytes (U937)stimulated with epinephrine or salbutamol /3-adrener-gic receptor agonists (Barber et al., 1992; Torphy etal., 1992)], and FRTL-5 thyroid cells stimulated withthyroid-stimulating hormone (Sette et al., 1 994a).Early studies also showed that a low-Km cAMP PDEactivity is increased in astrocytoma cells stimulatedwith NE (Uzunov et al., 1973; Browning et al., 1976)and in fibroblasts stimulated with prostaglandmn E~(D’Armiento et al., 1972; Maganiello and Vaughan,1972); the specific families of PDE are not identified.

Two in vivo studies have been conducted to addressdirectly the regulation of PDE with different results(Oleshansky and Neff, 1975; Moyer et al., 1980).Oleshansky and Neff (1975) have shown that a singledose of isoproterenol increases the activity of a low-Km cyclic AMP PDE in the pineal gland, as evidencedby an increase in the Vmax for cyclic AMP hydrolysis.This effect is blocked by a /3-adrenergic antagonistand by a protein synthesis inhibitor, suggesting theinvolvement of enzyme up-regulation resulting fromstimulation of /3-adrenergic receptors. This observationsupports the findings of the present study. However,another study has shown that neither acute nor repeatedtreatment with desipramine, an antidepressant thatblocks NE reuptake and indirectly increases the stimu-lation of /3-adrenergic receptors, has any significanteffect on PDE activity in the pineal gland (Moyer etal., 1980). Neither of the studies (Oleshansky andNeff, 1975; Moyer et al., 1980) identified the isozymeinvolved in cyclic AMP hydrolysis.

The results of some studies have provided indirectevidence suggesting that PDE4 might be regulated invivo. O’Donnell (1993) has shown that the dose—re-sponse curve for a behavioral effect of rolipram isshifted to left in adult rats that were lesioned with6-OHDA as neonates. Schultz and Schmidt (1986)reported a similarenhanced sensitivity to rolipram afternoradrenergic lesions. One interpretation of such ob-servations is that PDE4 might be down-regulated inrats with noradrenergic lesions, and hence less inhibi-tor is required to achieve a similar level of PDE4 inhi-bition. Alternatively, it has been reported that phos-phorylation of a PDE4 subtype can increase its sensi-tivity to inhibition (Alvarez et al., 1995). Anotherindirect line of evidence comes from a study of agingrats (Schoeffter and Stoclet, 1990). The effects of iso-

proterenol on maximal cyclic AMP accumulation andrelaxation in aorta are decreased in aging rats. Nochange in adenylyl cyclase activity is detected to ac-count for the reduction, but the dose—response curvesfor rolipram and Ro 20-1724 to produce relaxationare shifted toward the right, suggesting a possible up-regulation of PDE4 in the aorta of aging rats. Theresults from these indirect studies are consistent withthe current findings.

Studies using cultured cells also have shown thatdifferent mechanisms could be involved in the regula-tion of PDE4. The increase of PDE4 activity in FRTL-5 thyroid cells shortly after thyroid-stimulating hor-mone addition is mediated by phosphorylation (Setteet al., 1994a). By contrast, the increase in PDE4 activ-ity in U937 cells following the prolonged exposure to/3-adrenergic agonist (Torphy et al., 1992) and imma-ture Sertoli cells to follicle-stimulating hormone(Swinnen et al., 1989) results from the up-regulationof the isozyme. In addition, it appears that differentsubfamilies of PDE4 and their variant forms are differ-entially expressed and regulated (Engels et al., 1994;Sette et al., 1994b; Alvarez et al., 1995). The presentdata showed a reduction in PDE4 activity with dimin-ished noradrenergic activity, but the subfamilies ofPDE4 involved and the mechanisms underlying thechange remain to be resolved. The slow nature of theregulation suggests the possibility of down-regulation.

There is evidence indicating that PDE4 is involvedin mood regulation. Inhibitors of PDE4 exhibit antide-pressant effects in behavioral tests predictive of antide-pressant activity (Wachtel, 1983; O’Donnell, 1993).In addition, clinical trials show that they are effectivein treating patients with major depression (Fleisch-hacker et al., 1992). However, unlike traditional anti-depressants, the effects produced by rolipram are notachieved by inhibiting monoamine oxidase or mono-amine reuptake (Kehr et al., 1985; Schneider et al.,1995). The possibility of direct stimulation of neuro-transmitter receptors can be excluded as well becauserolipram does not compete with various ligands thatbind to those receptors (Schneider et al., 1986). Al-though rolipram increases the turnover and release ofNE, the contribution of such an action to the antide-pressant-like effects of rolipram is doubted, becausethe blockade of dopaminergic or /.3-adrenergic recep-tors does not interfere with antidepressant-like effectsof rolipram (Wachtel and Schneider, 1986; O’Donnell,1993). The potency of PDE4 inhibitors for antagoniz-ing the effects of reserpine in mice correlates well withtheir potency for inhibiting the binding of [3H] -

rolipram in vivo (Schmiechen et al., 1990). Theseobservations suggest that the effects are mediated at apostreceptor level, most likely through the inhibitionof PDE4. The finding that the inhibition of PDE4 haspotential therapeutic use in the treatment of depressionraises questions about the role of the isozyme in de-pression. One of the hypotheses concerning the patho-physiology of depression posits an imbalance between

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PHOSPHODIESTERASE AND NORADRENERGIC ACTIVITY 1901

adenylyl cyclase/protemn kinase A systems and phos-pholipase C/protein kinase C systems (Wachtel,1988); PDE4 could be involved in such an imbalance.

Acknowledgment: This research was supported by grants(MH40694 and MH51175) and aResearch Scientist Devel-opment Award (MHO1231) from the National Institute forMental Health. The authors thank Sandra Frith, CharlesDempsey, and Geneva Meachum for excellent technical andsecretarial support.

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