Peptides. Vol. 10, pp. 681-685. © Pergamon Press plc, 1989. Printed in the U.S.A. 0196-9781/89 $3.00 + .00

Neuropeptide-Dopamine Interactions. IV. Effect of Thyrotropin-Releasing Hormone

on Striatal Dopaminergic Neurons

H I R O M A S A I K E G A M I , S A L L Y A. S P A H N A N D C H A N D A N P R A S A D 1

Section o f Endocrinology, Department o f Medicine Louisiana State University Medical Center, New Orleans, LA 70112

R e c e i v e d 1 1 O c t o b e r 1988

IKEGAMI, H., S. A. SPAHN AND C. PRASAD. Neuropeptide-dopamine interactions. IV. Effect ofthyrotropin-releasing hormone on striatal dopaminergic neurons. PEPTIDES 10(3) 681~585, 1989.--Many biologic effects of TRH seem to be mediated via a dopaminergic mechanism. The present study examined the effects of chronic TRH administration on the properties of nigrostriatal dopaminergic neurons. Ten days, continuous subcutaneous TRH administration via an osmotic minipump led to a significant rise in striatal level of 3,4-dihydroxyphenylacetic acid, but not of homovanillic acid or dopamine. These treatments also did not elicit any significant changes in the maximal binding capacity (B,lax) or affinity (K o) of either D~- or D2-dopamine receptor. By contrast, TRH administration led to a significant increase in both B~,ax and K D of striatal mazindol binding. This effect of TRH, however, was not observed in in vitro studies. In conclusion, these data suggest that in vivo administration of TRH may modulate dopaminergic activities by altering, directly or indirectly, dopamine release and reuptake.

Thyrotropin-releasing hormone Striatum 3,4-Dihydroxyphenylacetic acid D~-dopamine receptor D2-dopamine receptor Mazindol binding sites

Homovanillic acid Dopamine

TRH is widely distributed in both the mammalian and nonmam- malian central nervous system (CNS), including the striatum, an area richly innervated with dopaminergic neurons (16,17). Exog- enous TRH has been shown to evoke a variety of CNS-related biologic activities independent of its endocrine function (14, 16, 17, 20, 27). Examples of some of the TRH-mediated effects that evidently share a common dopaminergic mechanism include: 1) potentiation of the behavioral effects of L-DOPA in pargyline- treated mice (14), 2) induction of rotational behavior in apomor- phine-treated mice (20), and 3) activation of tyrosine hydroxylase, the rate-limiting enzyme in dopamine (DA) synthesis (27).

Although studies on the interaction between TRH and various elements of striatal dopaminergic neurons suggest a definite modulatory role for this peptide, the data from different laborato- ries often seem contradictory [see (17) for review]. This apparent contradiction may have resulted from the differences in the experimental design and data analyses. Variables include TRH dosage, treatment route and duration, and the method(s) used to determine receptor properties. Furthermore, in most of the studies all elements of the dopaminergic neurons had not been evaluated simultaneously.

The present investigation was initiated to study the effects of low-dose continuous in vivo TRH administration on the levels of DA and its metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC)

and homovanillic acid (HVA). In addition, we also examined by Scatchard analysis, both ex vivo and in vitro, the effects of TRH on the properties of D l- and D2-DA receptors, and DA up- take sites.

DRUGS

[N-methyl-3H]-SCH23390 (70 Ci/mmol), [benzene ring-3H] - domperidone (45 Ci/mmol), and [4'-3H]-mazindol (15 Ci/mmol) were purchased from New England Nuclear, Boston, MA. TRH, haloperidol, DA, DOPAC, HVA, epinine, and all other chemicals were products of either Sigma Chemical Co. or Aldrich Chemi- cals. SCH 23390 and mazindol were gifts from Schering Corpo- ration and Sandoz, Inc., respectively.

METHOD

Male Sprague-Dawley rats (250-300 g) were purchased from Holtzmann Co., and housed in a temperature- and light-controlled (22°C, 12-hr light) room with free access to Purina Chow and water. All experiments were initiated at least 4 days after arrival of the rats in our animal care facility.

Each rat underwent dorsal subcutaneous implantation of an Alzet osmotic minipump (model 2001) filled (240 txl) with TRH

IRequests for reprints should be addressed to Dr. Chandan Prasad, Department of Medicine, LSUMC, 1542 Tulane Avenue, New Orleans, LA 70112.

681

682 IKEGAMI, SPAHN AND PRASAD

solution (8 mg/ml) or vehicle (saline) under light ether anesthesia. Ten days after pump implants, rats were killed by decapitation, the striata dissected from the brain as described by Glowinski and Iversen (30), and stored frozen at - 80°C until assayed. The daily intake of TRH was calculated to be 0.51 kO.02 mg/kg body weight.

Striatal tissue from one rat was taken up in I ml 0.1 M perchloric acid containing 100 nM epinine (PCA-epinine solution) and homogenized with a Polytron (setting 6, 3 x 5 set). A 10 pl aliquot was stored frozen for protein assay (6), the remainder was centrifuged at 12,000 x g for 10 min, and the supematant was used for catecholamine assay after 10 times dilution in PCA-epinine. Catecholamine levels were determined by high pressure liquid chromatography (HPLC) coupled with electrochemical detection system. Reverse-phase HPLC was performed on a Sphersorb S30DS2 column (3 p., 5 cm x 4.6 mm i.d., Little Champ, Regis) using a mobile phase of 0.15 M monochloroacetic acid containing 2 mM EDTA and 25-30 mgll sodium octylsulphate. The pH of the mobile phase was adjusted to between 3.0 and 3.05 with sodium hydroxide. The applied potential for the electrochemical detector (Bio Analytical Systems, West Lafayette, IN) was +0.75 V relative to an Ag/AgCl reference electrode. The sensitivity of the method, as determined by injection of authentic DA and its metabolites, was 0.2 pmol for DA and DOPAC, and 0.4 pmol for HVA.

To prepare for the receptor binding assays, rat striata were homogenized in 20 volumes of ice-cold buffer A (50 mM Tris-HCl, pH 7.4) using Polytron (setting 6, 1 x 20 set), and homogenate filtered through three layers of moist cheesecloth. The filtrate was centrifuged at 48,000 x g for 10 min at 4”C, the pellet resuspended in the original volume of buffer A, and recentrifuged. The final pellet was resuspended in about one-half of the original volume (34 mg protein/ml) of buffer B (50 mM Tris-HCl, 5 mM KCl, 2 mM CaCl,, 1 mM MgCl,, pH 7.4), and used for receptor binding. All other reagents used in receptor assay were prepared in buffer B.

Mazindol binding sites were measured as described elsewhere with only minor modification (5). However, D,- and D,-DA receptor assays (7,15) were substantially modified to facilitate simultaneous processing of a large number of samples. These changes are described in detail under Results. Briefly, the condi- tions for receptor binding assays were as follows: The reaction mixture for D,-DA receptor (0.25 ml) containing 124 pM to 2.75 nM 13H]-SCH 23390 (70 Ci/mmol) and 3545 pg membrane protein was incubated at 37°C for 30 min. The nonspecific [3H]-SCH 23390 binding was defined as that displaced by 1 p,M SCH 23390. For D,-DA receptor, the reaction mixture containing 119 pM to 2.5 nM [3H]-domperidone (45 Ci/mmol) and loo-125 kg membrane protein in a total volume of 1 .O ml was incubated at 37°C for 30 min. The nonspecific [3H]-domperidone binding was defined as that displaced by 5 pM haloperidol. Mazindol binding sites were measured by incubating the reaction mixture on ice for 2 hours, and it contained 3-30 nM [3H]-mazindol (15 Ci/mmol), 500 n&I NaCl, and 50-100 pg membrane protein in a total volume of 0.25 ml. The nonspecific binding was defined by 10 pM mazindol. All binding assays were stopped by adding 4.0 ml of cold buffer A followed by rapid filtration under vacuum through Whatman GF/B glass fiber filters. The filters were washed twice with 4.0 ml of ice-cold buffer A, placed in a minivial containing 1 ml of ethyleneglycol monoethylether, vortexed, and then radio- activity counted in 2.5 ml of scintillation fluid (Ready Gel, Beckman). The specific binding data were analyzed by Scatchard analysis (22) to calculate the K, and B,,, of the receptors.

Student’s t-test was used for comparison between two indepen- dent groups. To compare several treated groups with a control

Tdd

60

40

20

0

Time (min)

FIG. 1. Time course of binding of [3H]-SCH 23390 (1 S9 nM) and [3H]-domperidone (1.14 nM) to D,- and D,-dopamine receptors, respec- tively.

group, the data were subjected to Kruskal-Wallis analysis of variance followed by the Mann-Whitney U-test.

RESULTS

Optimal Conditions for Dopamine Receptor Assay

The specific binding of 13H]-SCH 23390 to D,-DA receptor and [3H]-domperidone to D,-DA receptor was linear for the first 15 min at 37°C (Fig. 1). The binding increased slowly on further incubation, reaching saturation by 30 min (Fig. 1). At saturation the nonspecific binding was 11% and 36% of the total binding for D,- and Da-DA receptors, respectively.

Bound and free ligands are generally separated immediately after temination of the binding reaction. Because the period needed to achieve saturation of the binding reaction was relatively short (30 min), processing a large number of samples in a short time became difficult. To overcome this problem, the following experiment was designed. Binding reactions were terminated by adding 4.0 ml of cold buffer A, and the tubes were kept on ice. At different times (between 0.5 and 180 min) after stopping the reaction, bound and free ligands were separated by filtration through GF/B filters, as described under the Method section. The statistical analysis of data presented in Fig. 2 shows that the specific binding did not change significantly when the binding reaction mixture was stored on ice for up to 180 min after termination (D,-binding: H-value = 4.5 1, p = 0.6070; D,-binding: H-value=5.38, p=O.4955).

Chronic TRH Increases Striatal DOPAC Levels

The data presented in Fig. 3 show that ten days, continuous subcutaneous administration of TRH (0.5120.02 mg/kg body weight/day) resulted in a significant increase (67%, p<O.O05) in the levels of DOPAC (control: 20.2? 1.6 n = 21; treated: 33.7 *

TRH AND DOPAMINERGIC NEURONS 683

1000

,,-,'--=- ~ 500 .'-' ~ ,..._ ° ~ [3HI - Domperidone (D~)

o 36 6b Time Elapsed after Termination of Reaction (rain)

FIG. 2. Reaction mixtures for [3H]-SCH23390 (1.74 nM) and [3H]- domperidone (1.38 nM) bindings can be stored on ice for up to 3 hours after termination of binding.

2.6, n = 11), but not of DA (control: 47.3---4.8, n = 2 1 ; treated: 50.1_+4.6, n = 11)or HVA (control: 7.38---0.77, n = 2 1 ; treated: 10.21-+2.6, n = 11). A significant (p<0.005) decrease in DA/ DOPAC ratio (control: 2.45 --- 0.22, n = 21; treated: 1.28 - 0.06, n = 11) appropriately reflects the changes observed in DA and DOPAC levels.

TRH Does not Modulate DA Receptors

To examine whether elevations in striatal DOPAC levels resulted from interactions between TRH and dopaminergic recep- tors, the following two experiments were conducted. The effects of TRH on the binding of [3H]-SCH 23390 to D~-receptor, and [3H]-domperidone to D2-receptor was examined by adding in- creasing amounts of TRH in the binding assay. The data in Table 1 show that in vitro addition of up to 10 txM TRH does not affect either K D or Bma x of the binding of [3H]-SCH23390 or [3H]- domperidone. Furthermore, the data in Table 2 also show that 10 days, continuous subcutaneous administration of TRH (0.51 -+ 0.02 mg/kg/day) also did not affect the K D or Bma x of either ligand.

TRH and Striatal Mazindol Binding Sites

Inasmuch as a DA reuptake system has been suggested (21) to

~ 60

8 ~ 50 E

'~ 40 E

,,r 30 m o

20

0

~ control (n=21) ~ TRH-treated (n=11)

* p<O.O05

, 1

DA DOPAC HVA DA DOPAC

o o D

2

n O

FIG. 3. Effect of 10 days of continuous subcutaneous TRH infusion on striatal levels of DA, DOPAC, HVA, and DA/DOPAC ratio.

TABLE 1

EFFECT OF IN VITRO TRH ON [3H]-SCH 23390 AND [3H]-DOMPERIDONE BINDING TO RAT STRIATAL MEMBRANES

TRH (I~M)

[3H]-SCH 2 3 3 9 0 [3H]-Domperidone Binding (D]) Binding (D2)

KD Bmax KD Bmax

0 (control) 0.78 _ 0.03 1162 - 128 0.73 ± 0.07 1012 + 160 1.0 0.84 ± 0.02 1247 _+ 91 0.84 _± 0.07 1025 _-. 170 10.0 0.80 ± 0.07 1195 --- 112 0.88 - 0.06 953 - 166

K D =nM, Bma x = fmol/mg protein, p-value compare to control >0.05 in all cases.

Values are the means of three separate experiments, each performed in triplicate.

play an important role in the formation of DOPAC, we investi- gated the potential effects of TRH on mazindol binding sites. Although mazindol nonselectively inhibits both dopamine and norepinephrine uptake systems, its binding to striatal membranes largely reflects a labelling of DA uptake sites (5). The data in Table 3 show that in vitro addition of up to 10 txM TRH did not significantly affect either K D or Bma x of [3H]-mazindol binding sites in the rat striatum. A 10 days, continuous subcutaneous administration of TRH (0.51 ---0.02 mg/kg/day) to rats, however, profoundly increased the total number of mazindol binding sites

TABLE 2

EFFECT OF CHRONIC IN VIVO TRH ON [3H]-SCH 23390 AND [3H]-DOMPERIDONE BINDING TO RAT STRIATAL MEMBRANES

[3H]-SCH23390 [3H]-Domperidone Binding (Dl) Binding (D2)

KD Bmax KD Bmax

Control 0.92+--0.03(6) 984---65(6) 0.73---0.06(5) 785--+- 47(5) TRH 0.83 ± 0.11 (6) 863 --- 55(6) 0.90 ___ 0.37(5) 908 ___ 118(5)

K D = nM, Bma x = fmol/mg protein, p-value compare to control >0.05 in all cases.

Chronic subcutaneous administration of TRH (0.51 + 0.06 mg/kg/day) for 10 days was achieved using an Alzet minipump.

TABLE 3

EFFECT OF IN VITRO TRH 3 ON [ H]-MAZINDOL BINDING TO RAT STRIATAL MEMBRANES

[3H]-Mazindol Binding

TRH (IxM) K D B,,,, x

0 (control) 58.0 ~- 6.1 7.52 m 0.61 1.0 60.0 -4- 5.2 9.00 +__ 1.03 10.0 54.3 --- 6.3 8.09 _ 1.31

KD =nM, Bmax = pmol/mg protein, p-value compare to control >0.05 in all cases.

Values are the means of four separate experiments, each performed in triplicate.

684 IKEGAMI, SPAHN AND PRASAD

° _ _

o

E

E

E

12

0 0 Bmax K D

EZicon trol (n=8) I T R H - t r e a t e d (n=11

* p<O.01

60

4O ~ E

20

Mazindol Binding

FIG. 4. Subchronic, subcutaneous infusion of TRH increases the number, and decreases the affinity of striatal mazindol binding sites.

(Bmax), whereas the affinity (KD) of the binding sites decreased significantly (Fig. 4). The increase in the Bma x was about 136% (control: 4.81+_0.72; TRH-treated: 11.33_+1.93 pmol/mg pro- tein, p<0.01) , and the decrease in affinity was about 71% (con- trol KD=29.96+_6.32; TRH-treated KD=51.19+_6.09 nM, p<0.025).

DISCUSSION

The data presented here show that chronic TRH administration to rats causes profound alterations in the nigrostriatal dopaminer- gic system. These changes include elevations in levels of DOPAC, and the first time demonstration of upregulation of mazindol binding sites with no change in the properties of dopamine receptors. While peptides are generally thought to cross the blood-brain barrier (BBB) poorly, a recent study (29) suggests that the rate of transfer of TRH through the BBB is some three times higher than that of D-mannitol. The changes observed here, however, clearly suggest that pharmacologically relevant levels of TRH reach the striatum when chronically administered through subcutaneous route.

DOPAC, the major dopamine metabolite in the striatum, is formed by the oxidation of DA by the mitochondrial enzyme, monoamine oxidase. Although the relative contribution of intra- and extracellular pools of DA that serve as the source for DOPAC is a matter of debate (28), it has long been assumed that recently released and reuptaken DA is the main source of DOPAC (21). Thus, the agents that potentiate DA release and/or reuptake should elevate, whereas inhibitors of reuptake system should lower, the levels of DOPAC (10).

Although the results of the studies on the effects of TRH on DA release from the striatum are conflicting, many studies clearly suggest a stimulation of DA release by TRH [see (17) for review]. Our observation of elevation in striatal DOPAC levels after chronic TRH (Fig. 3) is consistent with the DA-releasing activity of TRH. In fact, TRH and two of its analogs--MK-771 and DN-1417 -- have been shown to increase DOPAC content in the

striatum or cerebroventricular perfusate after acute and chronic peptide treatments (8,11). From recent evidence (28), including data showing that many DA uptake blockers do not decrease striatal DOPAC contents (4, 19, 26), it has been hypothesized that a major portion of DOPAC may derive from the intraneuronal pool of newly synthesized DA. In light of this hypothesis and known in vivo tyrosine hydroxylase activating (27) effects of a TRH analog (DN-1417), a second mechanism for TRH elevation of striatal DOPAC levels can be presented. Inasmuch as tyrosine hydroxy- lase is chronically activated, more and more of newly synthesized DA is converted to DOPAC, leading to elevation of its concen- tration.

In the striatum, two types of DA receptors exist whose stimulation affects cellular cAMP. Stimulation of the DA receptor called D t increases cAMP, whereas stimulation of the second type of DA receptor (O 2 type) reduces cAMP formation (24). It is possible that DA-like effects of TRH may be mediated through dopaminergic receptor(s). Results of several investigations (8, 13, 23), using [3H]-spiperone as ligand, suggest no significant effect of chronic TRH on the striatal Dz-receptor. However, in those investigations the receptor assays were performed at a single- ligand concentration that was close to or below the K D of D2-receptor; therefore, it is difficult to evaluate the true effect of TRH. In our receptor assays, using several ligand concentrations, we have determined that chronic TRH treatment does not alter K D or Bma x of [3H]-domperidone binding to the striatal membrane. Furthermore, the addition of up to 10 IxM TRH in D~- or D2-receptor assays did not significantly alter any of the binding parameters (K D or Bmax). In conclusion, increases in striatal DOPAC levels are not mediated through changes in striatal D l- or De-receptors.

The termination of neurotransmitter action at the synapse is regulated by removal of the neurotransmitter from the synaptic cleft. Therefore, one mechanism by which TRH could modulate dopaminergic activity is the reuptake of DA. Unfortunately, results of studies on potential modulation of striatal DA uptake by TRH are conflicting. Narumi et al. (9) reported a dose-dependent augmentation of [~4C]-DA uptake by rat striatal slices; however, Battaini et al. (1), using rat striatal Pz-fraction, could not demon- strate any significant effect of TRH on [3H]-DA uptake. A long-term stimulation by DA, released through the action of TRH, may possibly lead to an increase in mazindol binding sites. It must be remembered, however, that DA is a very weak inhibitor of 3H-mazindol binding by striatal membranes (5). The possibility that the apparent changes in mazindol binding sites after TRH treatment may not result from TRH, but from its metabolites cyclo(His-Pro) has been considered. Cyclo(His-Pro), a cyclic dipeptide partly derived from TRH metabolism (18), inhibits DA uptake by striatal synaptosomes (1). Two types of pyroglutamate aminopeptidases catalyze the conversion of TRH to cyclo(His- Pro): Type I (soluble, and exhibits broad substrate specificity) and Type II (membrane bound, concentrated at synaptic junction, and selective for TRH) (2, 12, 25). It is conceivable, therefore, that on chronic TRH treatment, TRH within synaptic cleft area is contin- uously being converted to cyclo(His-Pro), which then blocks DA reuptake; a constant blockade of DA uptake site leads to upregu- lation of [3H]-mazindol binding.

ACKNOWLEDGEMENTS

This research was supported in part by a grant from Scottish-Rite Schizophrenia Program, N.M.J., U.S.A., Smokeless Tobacco Research Council (STRC#0141-01), and NIH (DK37378). The superb secretarial assistance of Mrs. Ellen M. Brown, and editorial comments of Mr. Charles F. Chapman are gratefully acknowledged.

TRH AND D O P A M I N E R G I C NEURONS 685

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