Modulation of striatal cyclic nucleotide phosphodiesterase by calmodulin: Regulation by opiate and dopamine receptor activation

ivuur"pl,"rn,llrol/Jq?, vol.IX. pp x59toX64 Pergamon Press Ltd1979. Printed I"GreatBritam

MODULATION OF STRIATAL CYCLIC NUCLEOTIDE PHOSPHODIESTERASE BY CALMODULIN: REGULATION BY OPIATE AND DOPAMINE RECEPTOR ACTIVATION I. HANBAUER,J. Section

GIMBLE, K. SANKARAN

and

R. GERARD

on Biochemical Pharmacology. National Heart, Lung, and Blood National Institutes of Health, Bethesda, MD 20014, U.S.A. (Accepted

15 April

Institute,

!979)

Summary-Incubation of striatal slices with morphine (10m6 M) increased the cytosolic content of calmodulin. The double reciprocal plot of CAMP-phosphodiesterase (PDE) activitv. vs CAMP concentrations which appears to be biphasic with an apparent low and high K,-form in control slices was changed to a monophasic one with a low K,-form. The changes in the apparent K, for CAMP elicited by morphine (5 x lo-’ M) could be blocked by haloperidol (IO-’ M) and naltrexone (lo-’ M). In slices prepared from deafferented caudate nuclei, morphine (5 x lo-’ M) did not cause a change in the biphasic double reciprocal plot, but incubation of such slices with dopamine (2 x lo-’ M) still caused the appearance of a low &,-form of phosphodiesterase. Gel filtration (G-150) of soluble extracts indicated that a significantly greater proportion of the calmodulin was found in the fraction containing PDE activity in the morphine-treated slices than in control slices. Similar changes in PDE activity and calmodulin did not occur in cerebellar slices. These findings suggest that opiate receptors may be located on dopaminergic neurons, and that opiates may influence the regulation of PDE by calmodulin via post-synaptic dopamine receptors. Changes in PDE mediated by the increased availability of calmodulin can be used as an index of stimulation of dopamine receptors and may be related to the development of subsensitive receptor responses.

Several lines of independent evidence have shown that the activation of opiate receptors selectively increases the turnover-rate of dopamine in striatum (Clouet and Ratner, 1970; Costa Carenzi, Guidotti and Revuelta, 1973; Carenzi, Guidotti, Revuelta and Costa, 1975; Bloom Dewey, Harris and Brosing, 1976) and nucleus accumbens (Carenzi et al., 1975). The reduction in the number of striatal opiate receptors associated with the selective destruction of dopaminergic axons elicited by intranigral injection of 6-OH-dopamine (Schwartz, Pollard, Llorens. Malfrey, Gros, Pradelles and Dray, 1978), indicates that axoaxonic synapses may exist between striatal enkephalinergic interneurons and the long afferent axons projecting from dopaminergic neurons located in the substantia nigra (Pollard, Llorens-Cortes and Schwartz, 1977). Electrophysiological studies on the effect of opiates on nigral neurons provided evidence for an increased firing rate (Iwatsubo and Clbuet, 1977). but the molecular mechanisms participating in this event are yet unresolved. In the absence of this information, it is difficult to predict whether the activation of opiate receptors on dopaminergic terminals increases dopamine metabolism and release or only increases intraneuronal metabolism of dopamine. An increase in the release of dopamine following activation of opiate receptors can be inferred from various short-term effects of morphine which appear to be mediated by

Key words: tors, morphine,

striatal dopamine receptors, calmodulin, PDE.

opiate

activation of dopamine receptors (Costa, Cheney, Racagni and Zsilla, 1975). However, biochemical evidence in support of such an inference is lacking. It is known that a persistent activation of dopamine receptors causes an increase in the amount of calmodulin present in the supernatant fraction of striatal homogenates (Hanbauer, Gimble and Lovenberg, 1979). In order to investigate the regulatory interaction of opiate receptors on dopamine axons, the effect of morphine on calmodulin content and the kinetic properties of phosphodiesterase (PDE) was examined. The present report indicates that, in striatal slices, morphine increased the amount of calmodulin in the soluble fraction through a release of dopamine and thus acts as an indirect agonist of dopamine receptors. METHODS

Male Sprague-Dawley rats (Zivic Miller, PA; 150 g body weight) were decapitated, their brains were removed rapidly and the caudate nuclei dissected. Slices of caudate nuclei (230 pm thick) were prepared with a Sorvall tissue sectioner (TC-2). The slices were preincubated in Krebs-Ringer solution pH 7.4 which was supplemented with 10 mM dextrose and ascorbic acid (0.2 mg/ml) and saturated with 95”/, O2 + 5% COz. After the addition of various drugs, as reported in Results, the incubation was continued for 30min. At the end of the incubation, the dishes containing the slices were removed from the incubation medium and they were prepared for various types of measurements. In some rats, unilateral transection of nigra-

recep-

859

I. HANRAIJI-R, J.

X60

GIMRLE. K. SANKARAN

striatal axons was performed 3 weeks before the experiments using stereotactic coordinates of KGnig and Klippel (1970). Chromatographic supernatant

separation

of striatal

of calmodulin

and PDE

in

homogenates

At the end of the incubation, striatal slices were homogenized in 0.05 M Tris pH 7.4 (glass-teflon homogenizer) and centrifuged at 3.5 x IO“ g,, for 30 min. The supernatant fraction was chromatographed on a Sephadex G-150 column (73 x 1Scm) equilibrated with the 0.05 M Tris buffer pH 7.4. The column was eluted with the same buffer. and 2.8 ml fractions were collected and analyzed for calmodulin and PDE activities. Assay

of calmodulin

CAMP-PDE

activity

by activation

of purijied

(high K,,-form)

Aliquots from the fractions eluted from the Sephadex G-150’column were heated for 1 min at 90°C and added to a reaction mixture containing 0.02 mM CAMP (0.02 &i 3H-cAMP), 0.1 mM Ca*+, 1 mM Mg*+, 0.6 mM dithiothreitol, 32 mM Tris buffer pH 7.4 and purified PDE which is free of calmodulin (1Opg protein) (Uzunov and Weiss, 1972). From each fraction, duplicate samples were also assayed in the absence of 0.1 mM Ca*+. but in the presence of 0.1 mM EGTA. The mixtures were incubated for 5 min at 37°C and the reaction was terminated by placing the test tubes in a 90°C water bath for 45 sec. The 5’-AMP formed during this incubation period was converted stoichiometrically to adenosine by further incubation for 30min at 37’C in the presence of 6Opg snake venom (Ophiophagus hannah, Sigma, St Louis, MO). The nucleoside was purified with aluminum oxide columns (0.5 x 3.5 cm) as described by Filburn and Karn (1973). From the difference in PDE activities found in the absence or presence of Ca’ +, the units of calmodulin present in each fraction were calculated. One unit is defined as the amount of calmodulin that elicits a two-fold increase in the activity of a standard amount of purified PDE. Kinetic

analysis

of cAMP-PDE

activir!

The activity of PDE was measured in aliquots of each fraction eluted from the Sephadex G-150 column. The various fractions containing PDE activity were combined and the kinetic properties of PDE were studied. The apparent V,,,, and K, of PDE were measured using CAMP as substrate. Samples were incubated in presence of 1 mM Mg*‘, 0.6 mM dithiothreitol 32 mM Tris buffer pH 7.4, 0.02 mM Ca*+ and varying concentrations of CAMP ranging from 3 to 3OOpM. The samples were incubated for 5 min at 37°C and the reaction was terminated by placing the test tubes in a 90°C water bath for 45 sec. Double reciprocal plots of CAMP concentration vs the initial velocity of PDE were drawn to estimate the apparent V,,, and K, of PDE.

Statistical

and R.

SHERARD

analysis

The apparent K, values of PDE in the absence or presence of morphine were determined by double reciprocal plots of several individual experiments and the results of each experimental group was expressed as the mean + SE. Where appropriate, the Student’s t-test was used to determine the statistical significance of the difference between means. RESULTS EfSect of morphine

on the apparent

K, of CAMP-PDE

Incubation of striatal slices with morphine decreased the K, of PDE for CAMP. Figure 1 shows the double reciprocal plot of enzyme activity versus CAMP concentrations for PDE present in the supernatant of homogenates from striatal slices incubated in the presence or absence of 10m6 M morphine. While the kinetic profile of the PDE present in the supernatant from striatal slices incubated without morphine appears to be multiphasic (top panel), the double reciprocal plot of the supernatant from homogenates from striatal slices incubated in the presence of morphine appears to be monophasic (center panel). Assuming that the multiphasic relationship is resolved in only two components shown in the double reciprocal plot in Figure 1 (top). the apparent K, values

COllNOl

.I--:i 30

I/V

20 10

-1

12 3 l/SInmolI. 3’5’sAMP

0

10%

4

5

Morphine

~:3-:--:--/,_ -1

0

1 2 3 I/S (nmoll 3’5.CAMP

4

5

1

2&M 30

IIV

t

201

M -1

Dopamine

0

1 2 3 I/S blmoll-’ 3’5’-cAMP

4

5

Fig. 1. Comparison of kinetic properties of CAMP phosphodiesterase in the supernatant fraction of striatal homogenates. Striatal slices were incubated in Krebs-Ringer solution pH 7.4 for 60 min. Thereafter 1O-6 M morphine or 2 x to-’ M dopamine were added and the incubation continued for 20 min. The apparent K, values were determined from Lineweaver-Burke plots as shown.

Opiate

Table 1. Increase

in the calmodulin content morphine in striatal slices

receptors elicited

by

Minutes Preincubation 90 15 60

Morphine (5 x lo-’ M) 0 1s 30

Calmodulin Units*/fig Protein 0.78 + 0.04(4) 0.99 * 0.10 (4) 1.20 + 0.11* (4)

Number of experiments in parenthesis. activation of purified PDE. t P < 0.01.

* 1 unit = 2-fold

of PDE can be calculated as 8 and lOOtiM CAMP. The addition of morphine (10e6 M) to the incubation medium of striatal slices changed the kinetic profile of PDE. The double reciprocal plot became monophasic with an apparent &-value for CAMP of 31 PM, which was below the high &,-form of PDE shown in the absence of morphine. Figure 1 (bottom) also shows that the changes in the kinetic profile of PDE elicited by morphine resembled those obtained after 30min incubation of striatal slices with dopamine (2 x lo-’ M). Effect of morphine on the calmodulin content and its association with PDE The results reported in Table 1 show that the soluble calmodulin content was increased in the homogenates from slices incubated for 30 min with morphine (5 x lo-’ M). The data also show that a 15 min incubation with the same dose of morphine was insufficient to increase the calmodulin content. The supernatant prepared from the homogenate of striatal slices incubated with morphine (lO-‘j M) was chromatographed on Sephadex G-150. In Figure 2 are shown the chromatographic elution profiles of calmodulin and PDE present in homogenates of striatal slices incubated in the presence or absence of morphine. In both preparations, the protein-containing PDE activity eluted with proteins in the high molecular weight range. As expected, the bulk of calmodulin, which has a smaller molecular weight than PDE, eluted several fractions after PDE (Fig. 2). The volume of buffer that eluted calmodulin was similar to that required to elute proteins with a molecular weight of approximately 20,000. Such an estimation of the molecular weight of calmodulin was in fact close to the estimation reported by Lin, Liu and Cheung (1974); Stevens, Walsh, Ho, Teo and Wang (1976) and Klee (1977). In slices incubated without morphine only a small proportion of the soluble calmodulin eluted together with PDE. In contrast, in slices incubated with morphine (10s6 M) the proportion of calmodulin eluted in the fractions containing PDE activity was much greater (Fig. 2). Not only was the amount of calmodulin increased, but also the characteristics of the PDE peak that is present in the striatal slices incubated with morphine differed from that present in striatal slices incubated in the absence of morphine. In the

and striatal

861

PDE

supernatant from slices incubated with morphine, the kinetic profile of PDE in the combined fractions of the PDE peak eluted from Sephadex G-150 appeared to be monophasic and exhibited an apparent K, for CAMP of 30 PM. This monophasic profile could be reverted to a biphasic profile when the supernatant was chromatographed on a Sephadex G-150 column equilibrated and eluted with buffer containing 1 mM EGTA (results not shown). Evidence that the effects of morphine on striatal PDE are mediated by dopaminergic neurons (a) EfSect of receptor agonists. The data in Table 2 indicate that the shift in the kinetic properties of PDE elicited by morphine (5 x lo-’ M) could be prevented by incubation with antagonists of either dopamine or opiate receptors. Preincubation of striatal slices with haloperidol (lo-’ M) or naltrexone (lo-’ M) before the addition of morphine (5 x lo-’ M) prevented the shift of the kinetic profile elicited by morphine. The double reciprocal plot of PDE activity versus the CAMP concentrations, remained biphasic when morphine was added after preincubation with one of two inhibitors. (b) EfSect of hemitransection. The results in Table 3 show that brain hemitransection did not alter the kinetic profile of PDE. In addition, when striatal slices prepared from hemitransected brain were incubated with morphine (5 x lo-’ M), the double reciprocal plot of the PDE activity versus the CAMP concentrations present in the supernatant prepared from these

m

6

c z

FRACTION NUMBER

Fig. 2. Chromatographic studies on PDE and calmodulin in the supernatant fraction of caudate nucleus. Top: Sephadex G-150 column chromatogram of the supernatant fraction (3.1 x 104ga,) prepared from caudate nucleus slices after incubation in Krebs-Ringer solution pH 7.4 for 90 min. Bottom: Sephadex G-150 column chromatogram of the supernatant fraction (3.1 x 104g,,) prepared from caudate nucleus slices. The slices were preincubated for 60 min in Krebs-Ringer solution pH 7.4. After addition of 10e6 M morphine the incubation was continued for 30 min.

I. HANRAFFR. .I. GIMRLE. K. SANKARAN and R. SHERARD

X62

Table 2. Pattern

Addition ____

of phosphodiesterase isozymes in striatal and opiate-receptors Number of K,n-fOrms

to Incubation medium _____

None Morphine. 5 x 10 ’ M Morphine + Naltrexone, 10 ’ M Morphine + Haloperidol. lOmYM

slices: role of dopamine-

I(,

apparent values of PDE (/iM)

14 & 1.9: IO0 * I5 31 * 4.6 II: 83 76 15;

2 (5)

! (3 2(l) 2(l)

Slices were preincubated in Krebs-Ringer solution for 45 min. Naltrexone (IO ’ M), or haloperidol (10 ~’ M) were added and the preincubation continued for 15 min. After addition of morphine (5 x 10m7 M) the slices were incubated for 30 min and thereafter homogenized in 0.32 M sucrose. The apparent K, values were derived from double reciprocal plots of the initial velocity of PDE in the supernatant fraction vs the cAMP concentrations.

slices revealed

the

presence

of at

least

two

kinetic

PDE activity from the homogenate of striatal slices from hemitransected brain incubated with 2 x lo-’ M dopamine for 30 min appeared to exist as a single kinetic form with a K, for CAMP of 31 PM (Hanbauer et u/., 1979). (c) F&t of reserpine treutment. The PDE activity present in the homogenate of striatal slices prepared from striata of reserpine-treated rats and incubated with morphine (5 x IO-’ M) revealed biphasic kinetics. In contrast, the corresponding preparation obtained from slices incubated with apomorphine (lo-’ M) exhibited a monophasic profile (Table 4).

forms.

In contrast,

Specijicity striatal

of the dopamine

action

on the reyulation

oj

PDE

In order to ascertain that the release of calmodulin was a specific response triggered by morphine in tissues which contain dopamine receptors, the following experiments were performed: (1) cerebellar slices which are practically devoid of opiate (Hokfelt, Elde, Johansson, Terenius and Stein, 1977) or dopamine receptors (Ungerstedt, 1971) were incubated with either morphine or dopamine. The slices were homogenized and the PDE of the soluble fraction was studied. Table 5 shows that the double reciprocal plot of PDE activity versus CAMP concentrations was not modified by the incubation with dopamine or morphine. (2) Striatal slices were incubated for 30min with oxotremorine (10-h M) a muscarinic receptor

Table

3. Pattern

Addition None Morphine,

agonist, or isopreterenol (lo-’ M) a /i-adrenergic receptor agonist. The properties of soluble PDE did not differ from those untreated slices (results not shown).

The addition of dopamine receptor agonists to the incubation medium of striatal slices changes the double reciprocal plot of PDE activity vs CAMP concentration from a biphasic to a monophasic profile (Hanbauer et ul., 1979). Since a biphasic profile denotes a high degree of heterogeneity in the molecular forms of PDE present. it might be concluded that as a result of dopamine receptor stimulation this molecular heterogeneity is reduced. Previous experiments have shown that the change in kinetic profile of PDE is due to an increase in the calmodulin content present in the supernatant fraction of striatal slices incubated with dopamine (Hanbauer rt cl/., 1979). This increase in soluble calmodulin content could be due either a release of this protein from a membranebound pool or to a stimulation of calmodulin synthesis. A release process is probably operative because dopamine added to cell-free preparations of striatal synaptic membranes increases the calmodulin content of the medium (Revuelta, Uzunov and Costa, 1976). The modification of the kinetic properties of PDE is caused by an interaction of calmodulin and Ca2+

of phosphodiesterase isozymes in slices brain: lack of effect of morphine

10 incubation

5 x lo-’

medium

M

from

hemitransected

Number of K,-forms

K, apparent values of PDE (PM)

2 2

14 + 2.6: 112 f 8.0 15 + 0.1; 87 f 8.0

Hemisection of the brain was completed in rats 3 weeks before killing. Striatal slices were incubated in Krebs-Ringer solution with and without morphine. The slices were homogenized with 0.32 M sucrose and centrifuged for 30min at 3.5 x 104q,,. Double reciprocal plot of the initial velocity of PDE in the supernatant fraction versus the CAMP concentrations were drawn for each experiment. The Km-values arc expressed as mean t SEM of 3 such experiments.

863

Opiate receptors and striatal PDE Table 4. Molecular forms of phosphodiesterase in striatal slices from reserpine injected rats: effect of dopamine and opiate receptor agonists

Addition to incubation medium None Morphine, 5 x loo7 M Apomorphine, lo-’ M

Number of &-forms

K, apparent values of PDE (yM)

2 2

7.6 + 3.0; 55 * 5 7.4 + 2.5; 56 + 4 25 f 2

1

Rats were injected with reserpine (5 mg/kg, s.c.) 3.5 ht before killing. Striatal slices were preincubated in Krebs-Ringer solution for 45 min at 37°C. Morphine (5 x lo-’ M) or apomorphine (IO-’ M) were added and the incubation continued for 30min. The slices were homogenized in 0.32 M sucrose. The apparent K, values were derived from double reciprocal plots of the initial velocity of PDE in the supernatant fraction vs the CAMP concentrations. The K, values are expressed as mean + SE of 3 such experiments.

with a specific form of PDE

(Uzunov and Weiss, 1972; Lin et al., 1974). Calmodulin decreases the K,

for CAMP of the high &-form of PDE. Since the release of calmodulin increases the abundance of the apparent low &-form of PDE, it determines a higher degree of molecular uniformity in the PDE present in the supernatant fraction of striatal slices. The present work with Sephadex G-150 chromato~aphy showed that, in supernatant fractions of homogenates from striatal slices, the amount of calmodulin which comigrates with PDE increased following a 30 min incubation with dopamine receptor agonists. Since the increase in the amount of calmodulin that eluted with PDE could be prevented by equilibrating and eluting the Sephadex G-150 column with 1 mM EGTA, the interaction of PDE and calmodulin promoted by dopamine receptor stimulation is CaZ+dependent. In the present experiments, the increase in soluble calmodulin content and the change in the kinetic profile of striatal PDE have been used as an index of dopamine receptor activation. Using such an index, the present authors have attempted to resolve whether, in the striatum, opiate receptors regulate the release of dopamine from dopaminergic axon terminals. This verification was important because histological evidence indicates that in the striatum, axoaxonic synapses are very infrequent. In contrast, other experiments favor the interaction between opiate receptor activation and dopamine release by showing

that the number of opiate receptors present in the striatum is reduced by almost 40% following destruction of dopaminergic axons with 6-OH-dopamine (Schwartz et al., 1978). The present results indicate that in striatal slices a prolonged incubation with dopamine or opiate receptor agonists changes the double reciprocal plot of PDE activity vs CAMP concentrations from a biphasic into a monophasic profile. In addition, evidence is presented that the appearance of the monophasic profile of PDE activity versus CAMP concentrations elicited by morphine was inhibited by naltrexone or haloperidol. In contrast, analogous changes elicited by dopamine receptor agonists were inhibited by halo~rido1 but not by naltrexone. The inhibition of the response to morphine by haloperidol suggests that the functions of opiate and dopamine receptors are interrelated. Since the striaturn contains abundant innervation by enkephalinergic and dopaminergic neurons, whereas this innervation is absent in the cerebellum (Ungerstedt, 1971; Hokfelt et al., 1977; Hong, Yang and Costa, 1977) the lack of response of cerebellar PDE following incubation of cerebellar slices with dopamine or morphine supports the view that the changes in the kinetic properties of PDE elicited in the striatum are specific. Moreover the activation of muscarinic or fi-adrenergic receptors in the striatum fails to change the profile of the double reciprocal plot of PDE activity vs CAMP concentrations. These findings are not only in

Table 5. Pattern of phosphodiestera~ isozymes in slices of cerebellum: tack of effect of morphine Addition to incubation medium None Morphine, 5 x lo-’ M Dopamine, 2 x IO-’ M

Number of Km-forms

K, apparent values of PDE (PM)

2 2 2

7.8 f 1.8; 101 h 8 6.8 + 1.8; 98 + 6 8.0 i 2.0; 100 & 8

Slices were incubated in Krebs-Ringer solution saturated with O2 for 60min, 5 x lo-’ M morphine was added and the incubation continued for 30 min. The slices were homogenized in 0.32 M sucrose and centrifuged for 30min at 3.5 x lo4 9.“. Double reciprocal plot of the initial velocity of PDE in the supernatant fraction vs the CAMP concentrations were drawn from each experiment. The values of the &-forms are expressed as mean + SEM of such experiments.

864

1. HANBAUER,

J. GIMBLE, K. SANKARAN and R. SHERAK~

support of the specificity of the action of morphine, but they also suggest that the activation of adenylate cyclase may not always be associated with an increase of the soluble calmodulin content required for the activation of PDE. In fact, isoproterenol, similarly to dopamine, stimulates striatal adenylate cyclase (Premom, Tassin, Thierry and Bockaert. 1976) but fails to modify striatal PDE activity. Since the present data show that the activation of PDE by dopamine is inde~ndent from the availabihty of striatal opiate receptors, but the change of PDE activity caused by opiate receptor stimulation requires the availability of striatal dopamine receptors, it can be inferred that stimulation of opiate receptors modifies PDE by releasing dopamine. The proposed reIationship between opiate and dopamine receptors is supported by experiments showing that morphine fails to act after destruction of dopaminergic axons or after depletion of the dopamine content by reserpine. In conclusion, the present experiments confirm that, in the striatum, calmodulin functions in conjunction with dopamine receptors. The increase in the size of the soluble calmodulin pool is associated with a decrease in K, of PDE for CAMP. This suggests that calmodulin may not only function as a coupler of dopamine recognition sites with adenylate cyclase as proposed by others (Costa, Gnegy, Revueha and Uzunov, 1977), but it also is involved in the regulation of PDE activity. It appears, that by facilitating the coupling of recognition sites with adenylate cyclase, calmodulin increases the receptor response, whereas by activating PDE it reduces this response. The present experiments support the notion that in the striatum, enkephalinergic nerve terminals innervate dopaminergic axons and that the activation of these opiate receptors causes a release of dopamine.

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