Cyclo(Leu-Gly) has opposite effects on D-2 dopamine receptors in different brain areas

Peptides. Vol. 5, pp. 7-10. 1984. ~' Ankho International Inc. Printed in the U.S.A.

0196-9781/84 $3.00 + .00

Cyclo(Leu-Gly) has Opposite Effects on D-2 Dopamine Receptors in Different Brain Areas JOHN M. LEE,* RONALD F. RITZMANN *j AND JEREMY Z. FIELDSt

*Alcohol and Drug Abuse Research and Training Program, Department o f Physiology and Biophysics University o f Illinois at Chicago, Health Sciences Center, P.O. Box 6998, Chicago, IL 60680 and tDepartment o f Pharmacology, University o f Health Sciences, The Chicago Medical School 3333 North Green Bay Road, North Chicago, IL 60064 R e c e i v e d 17 O c t o b e r 1983 LEE, J. M., R. F. RITZMANN AND J. Z. FIELDS. Cyclo(Leu-Gly)has opposite effects on D-2 dopamine receptors in different brain areas. PEPTIDES 5(I ) 7-10, 1984.---Cyclo(Leu-Gly) (cLG), a diketeopiprazine analog of Pro-Leu-GlyNH~ (MIF), affects a number of physiological and behavioral responses to the endogenous neurotransmitter, doparnine (DA). In the present series of experiments, the effect of in viw~administration of cLG (8 mg/kg) was investigated five days following subcutaneous administration. It was found that cLG administration caused a supersensitive behavioral response, measured by increased stereotypic sniffing, to the DA agonist, apomorphine (APO). At the same time, an increase was found in the affinity for dopamine (DA), as measured by dopamine inhibition of aH-spiroperidol binding to D-2 DA receptors in striatum (nigro-striatal DA tract). In contrast, the same peptide treatment caused a subsensitive physiological response to APO-induced hypothermia, concomitant with a decrease in affinity for dopamine, as measured by DA inhibition of aH-spiroperidol binding to D-2 DA receptors in hypothalamus (incerto-hypothalamic DA tract). These results suggest that a single neuromodulatory agent, the peptide cLG, can elicit diametrically opposite effects on D-2 DA receptors and on the corresponding physiological endpoints in two different brain areas. Cyclo(Leu-Gly)

D-2 dopamine receptors

Hypothermia

PRO-Leu-Gly-NH,., (MIF), the C-terminal fragment of oxytocin, and its structural analog, cyclo(Leu-Gly) (cLG), have been shown to alter both dopamine-mediated behaviors and physiological responses. F o r example, in 1974, an effect of M I F on brain dopamine (DA) systems was demonstrated in an in vivo animal model. Plotnikoff and Kastin [14] showed that M I F was active in the L-DOPA potentiation test, which is a measure of increased dopaminergic synaptic activity. Drugs which potentiate L - D O P A are often active as anti-Parkinsonian agents. In addition, M I F administered unilaterally by intracerebroventricular injection produced marked rotational behavior similar to that seen for the DA agonist APO [6]. Other hypothalamic regulatory peptides, such as thyroid releasing hormone (TRH), somatostatin and leutinizing hormone releasing hormone (LHRH) did not produce such rotation. Both these effects of M I F were seen even in hypophysectomized animals, indicating that inhibition of Melanocyte Stimulatory Hormone (MSH) release from the pituitary, a classical hormonal action of MIF, is not involved. Moreover. recent reports have suggested the existence of specific, high-affinity binding sites for M I F in the striatum and in other areas innervated by DA-containing neurons [5].

~Requests for reprints should be addressed to Ronaid F. Ritzmann.

MIF

Stereotypy

Another indication that M I F and c L G act at or modify dopaminergic systems is the demonstration that both peptides block the development o f dopaminergic supersensitivity. Production o f physiological hypersensitivity to DA in the nigro-striatal and incerto-hypothalamic DA systems is welldocumented [1, 2, 7, 16, 19]. Cyclo(Leu-Gly) blocked the development of, but not the expression of, DA supersensitivity produced by chronic opiate administration [15], chronic neuroleptic administration [1], and 6-hydroxydopamine (6-OHDA) lesions [16]. The molecular mechanism(s) by which the various DAmediated physiological responses are affected by M I F and/or cLG have not been clearly elucidated. Studies of the abovenoted animal models of dopaminergic supersensitivity have yielded clues about possible molecular mechanisms of peptide action. In the neuroleptic model, for example, the behavioral hypersensitivity to DA that follows chronic haloperidol administration is thought to be due to an increase in the number of postsynaptic D-2 DA receptors. It has been shown [3] that M I F and c L G reverse the increase in the number of striatal D-2 DA receptors which occurs as a result of this chronic neuroleptic administration. One possibility then, is that c L G interacts specifically with the DA receptor corn-

8

LEE, RITZMANN AND FIELDS

plex. This ability of a neuropeptide to act at the receptor level is similar to the findings of Murphy and Schuster [ 12], who have shown differential effects of the neuropeptide, cholecystokinin, on DA binding to its receptor in the striatum and olfactory tubercle after both in vivo and in vitro treatments. Previously, we have shown that the ability of MIF and cLG to block the physiological supersensitivity to DA in the striatum (APO-induced stereotypic sniffing) and the hypothalamus (APO-induced hypothermia) appears to be working through alterations of D-2 DA receptors [17]. However, in these previous studies [17], which only examined striatal receptors, we showed that neither changes in receptor number, nor changes in affinity for antagonist (3H-spiroperidol) binding to D-2 DA receptors, correlates strongly with the time course for the behavioral changes seen. Therefore, in the present study, we determined agonist interactions with the D-2 DA receptors by monitoring changes in the ability of DA to inhibit 3H-spiroperidol binding. These assays revealed the existence of multiple receptor subtypes which have differential affinities for DA, designated D-2-HI and D-2-LO. We found that there was a differential effect of cLG on the striatai and hypothalamic DA systems, and that the changes in agonist-receptor interaction at the D-2-HI receptor in each area could account for the physiological changes observed. Moreover, the data presented have suggested that D-2-HI DA receptors in the hypothaiamus, although they are biochemically similar to D-2-HI DA receptors in the striatum, are regulated by cLG in a different way. METHOD

Male Wistar rats (Charles River, MA) (175 to 225 g) were injected subcutaneously with 8 mg/kg of cLG or vehicle on Day I. On Day 5, the animals from each group were divided into two sub-groups. In one sub-group, the animals were injected (IP) with either 0.5 mg/kg or 4 mg/kg APO and monitored for stereotypy or hypothermia, respectively. The second sub-group was sacrificed with striatum and hypothaiamus dissected, and analyzed for DA inhibition of 3Hspiroperidol (0.075 nM) binding to D-2 DA receptors in the respective areas (see below). In the present study, the data are expressed as the frequency of animals showing at least the presence of continuous sniffing during the 30-minute period following an injection of APO [20]. Hypothermia is expressed as the change in body temperature (°C) between pre- and 30-minute postAPO injections. Special care was taken to ensure that the binding assays were carried out using only a low concentration of aHspiroperidol (0.075 nM), thus avoiding binding to a lower affinity aH-spiroperidol binding site (designated "R-LO") at :'H-spiroperidol concentrations greater than 0.3 nM[ 13]. It is possible that this site (R-LO) is non-dopaminergic, since binding changes at this site in the striatum do not correlate with striatally mediated behavioral changes (unpublished). Moreover, the site may be serotonergic, since co-incubation with maiansern can eliminate :'H-spiroperidol binding to this site [20]. In the striatum, the curves for DA inhibition were shallow (n=0.70) and the data fit better (,o<0.05) to a twosite model, F(2,5)=36.3, p<0.05, using the computer curve-fitting program, LIGAND [11]. The Kt values for DA were 81.3 nM for the higher affinity of the two sub-sites (designated D-2-HI), and 26,000 nM for the lower affinity site tD-2-LO}.

Characterization of the two striatal binding sites indicates that the high affinity component (D-2-HI) has a biochemical profile that is consistent with a dopaminergic site. That is. the rank order of potency, from highest to lowest, was: DA. APO, Norepinephrine, isoproterenol and serotonin (data not shown). Similarly, in the hypothalamus. DA inhibition of 3H-spiroperidol binding indicates that two receptor subsites are present (p<0.01). We have also designated these sites as D-2-HI and D-2-LO, to agree with the terminology for the striatal binding sites. Again, biochemical characterization of the higher affinity sub-site (D-2-HI) is consistent with a dopaminergic receptor (same rank order as above). Characterization of the lower affinity site (D-2-LO) is less clear. RESULTS

Five days after a single injection (8 mg/kg) of cLG, SC, the striatally-mediated, APO-induced stereotypy was increased. In the cLG-treated group, 55% of the animals engaged in continuous sniffing compared to 22% for the control group (Table I). The peptide treatment caused, at the same time, a leftward shift in the curve for DA inhibition of 3H-spiroperidol binding. The Ki for DA binding to the D-2-HI site changed from 81.3 nM in controls to 1.0 nM for the cLG group (see Fig. 1 and Table 1). The change in the percentage of receptors in the D-2-HI state (control=2lY'~; cLG=43%) was also consistent with the direction of the behavioral changes. There was no significant change in the affinity (K,=26,000 nM) at the D-2-LO sites in the peptide treatment group. The number of D-2-LO sites actually decreased. On the other hand, five days after a single injection of cLG, the physiological response to DA in the hypothalamus, as measured by APO-induced (4.0 mg/kg) hypothermia, was decreased. In the peptide group, there was a temperature decrease of only 0.34_0.13°C, compared to a decrease of 0.66___0.12°C in the control animals (Table 1). DA inhibition of '~H-spiroperidol binding to D-2 DA receptors in hypothaiamus also revealed two sub-populations of receptors: D-2-HI and D-2-LO (Fig. 1 and Table I). The affinity of DA for D-2-HI site was 0.026 nM, which is about 3,000-fold higher affinity than for the corresponding striatal D-2-HI site. The hypothalamic D-2-HI site comprises 37% of D-2 binding, while D-2-LO (Kt=50,000 nM) accounts for the remainder (63%). Peptide treatment resulted in a rightward shift of the curve of at least five orders of magnitude for the D-2-HI site to a Kt of > ! ,000 riM. This decrease in affinity for DA at the D-2-HI site is consistent with the physiological subsensitivity seen in APO-induced hypothermia. Since it has been shown that MIF can act in vitro to alter the affinity of DA receptors for :'H-APO binding [4], we investigated the in vitro effect of cLG on D-2 binding. We could not find any such in vitro effect for cLG on DA inhibition of :'H-spiroperidoi binding in either the striatum or hypothalamus at any concentration of cLG between l0 -'z M to 10-~ M (data not shown). DISCUSSION

The present results suggest that the mechanism of action for cLG as a dopaminergic neuromodulator is the regulation of DA receptor binding sites and. consequently, of DAmediated physiological and behavioral responses. In addition. the data also indicate that cLG. a single neuromodulatory agent, can elicit diametrically opposite effects, not only

c L G E F F E C T S ON D-2 D O P A M I N E RECEPTORS

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FIG. 1. Dopamine inhibition of :'H-spiroperidol in (A) striatum and (B) hypothalamus. Y-axis represents percent bound of total :'Hspiroperidol (0.075 nM) to each tissue. X-axis is -log [dopamine]. l - - - - 1 , Vehicle: ~ - - F I , cyclo(Leu-Gly)-treated.

TABLE I IN VIVO EFFECT OF cLG ON D-2 DOPAMINE RECEPTORS AND PHYSIOLOGICAL ENDPOINTS IN STR1ATUM AND HYPOTHALAMUS

Striatum Treatment

n

D-2-HI (KI =nM)

n

APO-Induced Stereotypy

Veh cLG

3 3

81 ± 8.3 1.0 ± 0.2*

24 12

22% 55%*

Hypothalamus Treatment

n

D-2-HI (K~=nM)

n

APO-Induced Hypothermia(°C)

Veh cLG

3 3

0.026 _+ 0.017 >l.000*

l0 l0

-0.66 _+ 0.12 -0.34 ± 0.13"

D-2-HI affinities were determined in each brain region by DA inhibition of :~H-spiroperidol (0.075 nM) and analyzed using the computer curve-fitting program L1GAND (see test). Stereotypy was determined using 0.5 mg/kg APO and expressed as percent of animals showing stereotypic behavior. Significance was determined by Fischer Exact Probability Test (*p<0.05) [18]. Hypothermia was determined using 4.0 mg/kg APO and expressed as mean _ SEM. Significance was determined by two-way Student's t-test. *(,o<0.05).

at the molecular level (receptor binding), but also on the corresponding physiological or behavioral endpoints associated with dopaminergic tracts in two different brain areas. The data also suggest that c L G alters binding of DA to the higher affinity sub-site (D-2-HI) of the D-2 DA receptor, and that changes in this, and only this receptor binding parameter. correlate with physiological and behavioral changes. It has been hypothesized that M I F and related peptides act initially through a specific peptide receptor. Recently, binding of tritiated peptides ( N - T y r - M I F and MIF) to putative M I F receptors have been reported, and the CNS distil-

bution of these binding sites appears to parallel dopaminergic innervation [5,9]. If, in fact, specific receptors for M I F do exist, then there are a number of mechanism(s) by which c L G can oppositely regulate striatal and hypothalamic DA receptors. One possibility is that the M I F receptor-effector complexes are different in the two brain areas. That is, there could exist M I F receptor sub-types, such as MIF-I and MIF-2 sub-populations. This situation is reminiscent of the division of many other neurotransmitter receptors into subpopulations, such as muscarinic and nicotinic for acetylcholine binding, alpha-adrenergic and beta-adrenergic for N E binding, and kappa, mu and delta sites for opiate binding. Thus, it is conceivable that each sub-type of M I F receptor might be coupled to a different second messenger. Another possibility for the differential effect o f c L G may stem from the fact that the DA receptors themselves are different in the striatum and hypothalamus. Therefore, it is possible that they are regulated differently by the same intermediate response elicited by the putative M I F / c L G receptor-effector complex. Evidence for the idea that the receptors are different is supported by the fact that the affinity of D-2-HI for DA is about 3,000-fold higher in the hypothalamus than in the striatum (see Fig. l and Table l). More specifically, the D-2 receptors in the two areas may have different receptor-effector complexes (i.e., adenylate cyclase versus an ion cannel) which they are coupled to, and are differentially affected by the activation of the M I F / c L G receptor. At this point, very little work has been done to define D-2 receptors in the hypothalamus, and further research may help delineate this present controversy. It would, of course, be of interest to know how both the striatal and hypothalamic D-2-HI site responds to other known receptor neuromodulators (i.e., GTP, sodium, etc.). Finally, there is a distinct possibility that the peptides could be acting on an additional non-dopaminergic neurotransmitter/neuromodulatory system that results in differential effects in the two areas. To investigate this possibility, we are presently looking at the effects of cLG on striatal cholinergic systems. We have been presuming that c L G works postsynaptically, based upon our earlier study demonstrating that c L G is active in blocking the behavioral supersensitivity to DA

l0

LEE, RITZMANN AND FIELDS

following presynaptic lesions by 6-OHDA [16]. Another possibility is that cLG could be acting presynaptically in one brain area, but postsynaptically in the other. However, the effects of MIF and cLG on presynaptic functions (i.e., on DA synthesis, release and reuptake) have been contradictory [8, 9, 10], and a number of these studies have used only a single peptide dose. Since the dose-response curve for the pharmacological actions of cLG and MIF are bell-shaped (inverted " U " ) , it may be appropriate to re-examine the peptide effects on presynaptic functions more thoroughly in each of the brain regions. The primary site of action of MIF/cLG, therefore, remains unclear. In summary, MIF and cLG differentially affect the biochemistry and physiological outputs of the nigro-striatal and incerto-hypothalamic DA systems. Specifically, peptide

treatment results in alterations, in opposite directions, in the two brain areas, in D-2 DA receptors at the recognition site for the neurotransmitter. However, these biochemical changes are in precisely the same direction as are the peptide-induced changes in physiological endpoints. What remains to be determined are the details of the molecular events, from recognition of MIF/cLG-type peptides at specific peptide receptors to neuromodulation of the D-2 DA receptors and their associated physiological endpoints.

ACKNOWLEDGEMENTS

The authors would like to thank Margaret Tyler for her excellent work in the preparation of this manuscript. This work was supported in part by BRSG Grants RR-5366 to J.Z.F. and 82508 to R.F.R.

REFERENCES 1. Bhargava, H. and R. F. Ritzmann. Inhibition of neuroleptic-

induced dopamine receptor supersensitivity by cyclo(Leu-Gly). Pharmacol Biochem Behav 13: 633-636, 1980. 2. Carlson, K. and J. Almasi. Time course of dopaminergic hypersensitivity following chronic narcotic treatment. Pharmacol Biochem Behav 11: 263-287, 1979. 3. Chiu, S., C. Paulose and R. Mishra. Neuroleptic drug-induced dopamine receptor supersensitivity: Antagonism by L-prolyi-L-leu-cyl-glycinamide. Science 214: 1261-1262, 1981. 4. Chiu, S., C. Paulose and R. Mishra. Effect of LprolyI-L-leucyi-glycinamide (PLG) on neuroleptic-induced catalepsy and dopamine/neuroleptic receptor binding. Peptides 2: 105--I i 1, 1981. 5. Chiu, S., Y. Wong, J. Ferrisi, R. Johnson and R. Mishra. Binding study of PLG, a novel anti-Parkinsonian agent in normal human brain. Pharmacol Res Commun 15: 41-51, 1983. 6. Cohn, M. and M. Cohn. Comparison of the regulation of rotational behavior by hypothalamic oligopeptides. Psychoneuroendocrinology 2: 197-202, 1977. 7. Cox, B., M. Ary and P. Lomax. Dopaminergic mechanism in withdrawal hypothermia in morphine-dependent rats. Life Sci 17: 41-51, 1975. 8. Friedman, E., J. Friedman and S. Gershon. Dopamine synthesis: Stimulation by a hypothalamic factor. Science lg2: 831-833, 1973. 9. Kostrzewa, R., M. Spirtes, T. Klara, C. Christensen, A. Kastin and T. John. Effects of PLG (MIF-I) on dopaminergic neurons. Pharmacol Biochem Behav 5: 125-127, 1976. 10. Kostrzewa, R., H. Fukushima, C. Harston, K. Perry, R. Fuller and A. Kastin. Striatal dopamine turnover and MIF. Brain Res Bull 4: 799-802, 1979,

11. Munson, P. and D. Rodbard. LIGAND: A versatile computerized approach for characterization of ligand binding systems. Anal Biochem 107: 220-239, 1980. 12. Murphy, R. and D. Schuster. Modulation of'~H-dopamine binding by cholecystokinin octapeptide (CCK-8). Peptides 3: 539543, 1982. 13. Pedigo, N., T. Reisine, J. Fields and A. Yamammura. :3Hspiroperidoi binding to two receptor sites in both the corpus striatum and frontal cortex of rat brain. J Pharmacol 50:451453, 1978. 14. Plotnikoff, N. and A. Kastin. Pharmacological studies with a tri-peptide L-proly-L-leucyl-glycinamide. Arch lnt Pharmacodyn Ther 211: 211-224, 1974. 15. Ritzmann, R. F., R. Waiter, H. Bhargava and L. Flexner. Blockade of narcotic-induced dopamine receptor supersensitivity by cyclo(Leu-Gly). Proc Natl Acad Sci USA 76: 5997-5998, 1979. 16. Ritzmann, R. F. and H. Bhargava. Effect of cyclo(Leu-Gly) on chemical denervation supersensitivity of dopamine receptors induced by intracerehroventricular injection of 6-hydroxydopamine in mice. Life Sci 27: 2075-2080, 1980. 17. Ritzmann, R. F., J. Lee and J. Z. Fields. Modification of morphine-induced changes in striatal :~H-spiroperidol binding and stereotyped behavior by cyclo(Leu-Gly). Life Sci 30: 1573-1580, 1982. 18. Siegai, S. Non-Parametric Statistics for Behavioral Science. New York: McGraw-Hill, 1956. 19. Understedt, U. Post-synaptic supersensitivity after 6-hydroxydopamine-induced degeneration of the nigro-striatai dopamine system. Acta Physiol Sc'and Suppl 367: 69-93, 1971. 20. Withy, R., R. Mayer and P. Strange. Use of :~H-spiperone for labelling dopaminergic and serotonergic receptors in bovine caudate. J Neuroc'hem 37: 1144-1154, 1981.