Assessment of the role of TRH in the release of [3H]-dopamine from rat nucleus accumbens-lateral septum slices

0361-9230393$6.00 + .oO Copyright0 1993 Pergamon Press Ltd.

BrainResemh Bulletin,Vol. 3 I, pp. 62 1-625, 1993 Printedin the USA. ~11rightsreserved.

Assessment of the Role of TRH in the Release of [3H]-Dopamine From Rat Nucleus A~~umbens-Lateral Septum Slices MILAGROS

Ml?NDEZ,*’

JULIO

MORAN,? SHERWIN WILK,$ AND JEAN-LOUIS CHARLI*’

PATRICIA

JOSEPH-BRAVO*

*Institute de Biotecnologia, Universidad Nacionai Authoma de ML;xico, Cuernavaca, Morelos 62271, hhkico ~~nstit~to de Fisiolog~aCelular, U~iversidadNaei~~alA~t~noma de hbhico, MCxico D.F., M&co ~~e~artme~t O~P~arrna~olog~, carat Sinai ~~~~1 o~~edici~e, New Yurk, NY IO029 Received

20 August

1992; Accepted

3 November

1992

MENDEZ, M., J. MORAN, S. WILK, P. JOSEPH-BRAVO AND J.-L. CHARLI. Assessment of the role of TRH in the w/em?

~~~3~3-d~~mine~r~mrat m&us ~~~~rn~e~s-~~ter~ septum &es. BRAlM RES BULL 31(5) 62 I-625, 1933.-We have studied f3H]-dopamine (i3H]-DA) release from rat nucleus accumbens lateral septum slices in response to various paradigms aimed at increasing endogenous or exogenous thyrotropin releasing hormone (TRH) concentrations in the extracellular space. High KC1 concentrations significantly enhanced [)H]-DA release by fourfold. TRH ( 10m4or 5 X 10e4 M) did not affect [3H]-DA release. The release of [‘HI-DA was not stimulated by TRH either in the presence of N-I-carboxy-2-phenylethyl (N’“benzyl>histidyl&raphthylamide, a specific pyroghitamyl peptidase II inhibitor, or that of specific inhibitors of prolyl endopeptidase and pyroghrtamyl peptidase 1. None of the peptidase inhibitors modified the f3H]-DA release by themselves. These results suggest that the TRH stimulation of [3H]-DA release in vitro observed in previous studies is not due to peptide inactivation but may be due to a nonspecific effect. TRH enhancement of DA release in nucleus accumbens in vivo may not be the result ofa direct effect ofTRH OR DA terminals. Thy~tropin releasing hormone ~ro~u~rnyi peptidase II

Dopamine

Nucleus accumbens

Peptidase

THYROTROPIN releasing hormone (TRH) is a neuroactive peptide present in many regions of the central nervous system (9). Several behavioral effects are produced by peripheral injections of TRH in rats, including increased locomotor activity (8,16). This effect is also obtained after injection of TRH directly in some brain areas, such as nucleus accumbens (8,17,23), which is densely innervated by TRHergic fibers and terminals (IO) and contains a high concentration of TRH receptors (24). Pha~a~lo~ca~ and bi~hemic~ evidence suggests that TRH produces some of these shalom effects by stimulating the turnover and release of dopamine (DA) in nucteus accumbens (7, I I, f 8,23). Several TRH anafogues, fess susceptible than TRH to peptidase action in vitro (5,6,X?), have a stronger effect than TRH (6,8,22). They enhance DA release in several brain regions, including nucleus accumbens and septum (I 7,22).

Lateral septum

Release

In vitro, TRH can be degraded by three peptidases: two of them, prolyl endopeptidase @X.3.4.21.26) (PE) and pyrogiutamyl peptidase I (E.C.3.4.19.3.) (PP I), are soluble intracellular nonspecific enzymes not involved in TRH catabolism in the extracellular fluid (14). The third enzyme, pyroglutamyl peptidase II (E.C.3.4.19.-) (PP II), is a specific ectoenzyme mainly present in brain, probabiy responsible for TRH inactivation once it is released into the extracellular space f2,3,26). One of the brain regions with highest activity is the nucleus accumbensIateraI septum (25). Evidence that endogeneous TRH effectively stimulates DA release in vivo has heen difhcuh to confirm because of the iack of specific antagonists for this peptide. We have recently shown that a specific inhibitor of PP 11, ~-i~rboxy-2-phenyIethy1 (N’“benzyl)-histidyl-&taphthyIamide (CPHNA), increases tbe

r Present addrew Departamento de Fisiologia, Fact&ad de Medicina, Universidad National Autdnoma de Mexico, Mexico D.F., Mexico. ’ Requests for reprints should be addressed to Dr. J. L. Charh, IBUNAM, A.P. 5 10-3, Cuemavaca, Mar. 6227 1, Mexico.

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M&VDEZ ET AL.

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Tima (min) FIG. 1. Time course of [3H]-DA release induced by KC1 and TRH from nucleus accumbens-lateral septum slices. Slices were loaded with [3H]-DA, perifused at a flow rate of 0.5 ml/min, and I ml fractions collected for radioactivity determination as described in the Method section. Arrows indicate the change of basal medium to medium containing 55 mM KC2 (A) or 100 pi’4 TRH (B). Results are expressed as the percentage of radioactivity released into medium per min from total radioactivity in the tissue. Vaiues are the mean + SEM of five (A) or seven (B) independent experiments. Asterisks indicate statistical significance from basai release at min 4 f***p < O.OOf; **p < 0.02; *p <: 0.05) using Student’s t-test analysis. Mean amount of ~ioactiv~ty present at start of perifusion before the 2%min washout period: 15409 cpm. Mean amount of radioactivity released 1 min before stimulus: 193 cpm.

recovery of TRH released from slices from different rat brain regions, including nucleus accumbens-lateral septum (3). To gain further insight into the physioiogical role of TRH in nucleus accumbens and test whether increased biological activity of TRH analogues is due to their resistance to peptidases, we determined if inhibition of PP II with CPHNA enhances, alone or in combination with TRH or specific inhibitors of PP I and PE, [3H]DA release from nucleus a~urn~n~late~ septum slices.

METHOD

~~~~r~uis Male Wistar rats (150-200 g) fed ad lib a Purina Chow diet and maintained in a 12L: 12D period (07~-~9~ h) were used in all experiments. The specific peptidase inhibitors, pyroglutamyi diazomethyl ketone (PDMK), N-benzyioxy carbonyf prolyl prolinal (ZPP) and CPHNA were synthesized as previously

TRH-DA

INTERACTION

IN NUCLEUS

623

ACCUMBENS

Cl

Basal

-

KCI

m

TRH

30-

20-

70-

OFIG. 2. Effect of KC1 and TRH on [‘HI-DA release from nucleus accumbens-lateral septum slices in a serial incubation system (15). Slices were placed in a 5 ml plastic syringe that had been cut at the bottom and melted onto a nylon mesh (63 pm). They were transfered to a 2 ml glass vial containing KRB, loaded with [‘HI-DA, washed, incubated with basal medium for 10 min periods, and then transferred to a vial con~ning medium with 56 mM KC1 or ID0 pM TRH; incubation was continued for IO min. Results are expressed as in Fig. 1.Values represent the amount of radioactivity released during 10 min before (basal) or after (KC]; TRH) change of medium. Data are mean ? SEM of the number of independent experiments indicated above each bar. *Statistical significance from basal release (p c 0.001) by one-way ANOVA analysis. Mean amount of radioactivity present at start ofserial incubation: 88 12 cpm. Mean amount of radioactivity released during basal period (10 min): 55 I cpm.

(3,27,28). TRH (two different batches) was from Peninsula Laboratories, Belmont, CA; [3H]-dopamine (27.9 Ci/ mmol) and L-[2,3,4,S-[3H~-proline~-TRH ([3H]-pro-TRH) (102 ~i/mmol) from New England Nuclear Co., Boston, MA. Before use, each TRH lot was analyzed by thin layer chromatography (TLC) in CHQ: CHIIOH: NH40H (125:75:25) and by celtulose phosphate P8 1 paper (Whatman) chromatography in 1 M acetic acid. Only one spot with TRH Rf was detected in each case. TRH immunoreactivity of each lot was determined using a radioimmunoassay (I 5) and found identical to that of previous lots used in the laboratory. described

Release Experiments

13H]-DA uptake and release were performed essentially as described (11) with some modifications (20). Briefly, 250 pm wide nucleus accumbens-lateral septum slices (1.2 mg protein) were obtained in a McIIlwain tissue chopper (B~nkman), washed in Krebs Ringer Bicarbonate (KRB: 125 mM NaCl, 4.4 mM KCI, I.2 mM KH2P04, 1.3 mM MgSO,, 26 mM NaHC03, 10 mM glucose, 2.5 mM CaC12) pH 7.4, oxygenated with 95% 02/5% CO? at room temperature, preincubated at 37’C for 5 min, and prelabelled with 0.5 pCi/ml [3H]-DA (IO-’ M)in KRB for 20 min. Slices were recovered, washed with fresh KRB, and petifused at a flow rate of 0.5 ml/min in a Manostat multichannel pump (silicone tubing). Following a 22-min washout period, fractions of 1 ml were collected every min. The effect of a depolarizing stimulus (56 mM KCl), 100 PM or 500 PM TRH (two different batches) or TRH plus peptidase inhibitors (IO-’

M PDMK and ZPP, 10s4 M CPHNA) were studied on the efflux of radioactivity. TRH recovery (percentage of added TRH recovered from perifusion without tissue) was determined using radiochemicallly pure [3H]-pro-TRH (100 PM). Value was 93%. The high potassium medium was made by substituting 56 mM NaCl for an equimolar amount of KCI. Results are expressed as percentage rate constants (the amount of radioactivity released into the medium per min expressed as a percentage of total radioactivity in the tissue at the time of collection). In all experiments, pargyline (10 hM) and ascorbic acid (0.1 mg/ml) were added. For comparison, we also performed some 13H]-DA release experiments using a serial incubation technique developed in our laboratory ( 15) and brieBy described in legend for Fig. 2. ~hromatograph~c Analyses Serial Incubation Fiuids

ofRadiochemicals in Periifusion or

More than 80% of radioactivity released from slices loaded with L3H]-DA corresponded to its Rf in TLC analysis (n-butanol: water:acetic acid, 25: 10:4) (13); the remaining radioactivity was spread on the TLC plate and not associated with the Rf of any DA metabolite. RESULTS As reported (1 I), 56 mM KC1 increased almost fourfold (3HJDA release from perifused (Fig. 1A) or serially incubated nucleus accum~ns-lateral septum slices (Fig. 2). in contrast, TRH (100 PM) had no effect on basal [3H]-DA release (Figs. 1B and 2), nor potentiated the KC1 evoked release (not shown). A highe concentration of TRW (500 PM) was also ineffective (not shown). No response was observed whether experiments were performed at 1I, 14, or 18 h or using different batches of TRH (not shown). TRH degradation by peptidases present in the extracellular medium or at the cell surface was not the cause of unresponsiveness; CPHNA (IO“ M), a specific PPII inhibitor, alone or combined with specific inhibitors of PPI (PDMK. 10e5 M) and PE (ZPP, 10m5M), did not increase basal release of [3H]-DA from perifused nucleus a~um~ns-latex septum slices (Table I).

TABLE 1 EFFECTS OF TRH, 56 mM KCI, AND PEPTIDASE INHIBITORS ON [‘Hj-DOPAMINE RELEASE FROM NUCLEUS ACCUMBENS-LATERAL SEPTUM SLICES

Condition None KCI (56 mM) TRH (IOOpM) CPHNA PDMK t ZPP + CPHNA TRH (100 PM) + CPHNA TRH (100 PM) + PDMK + ZPP + CPHNA

[‘HI-DopamineRelearn (WControl) 100.0

349.0* 83.3 108.0 112.6 89.6 96. I

Slices were loaded and perifused as shown in Fig. 1A with or without KCl, TRH, or the peptidase inhibitors. Inhibitors concentrations: CPHNA, 10m4M; PDMK and ZPP, 10-s M. Values represent the percentage of [3H]-DA release, considering basal relase as 100%. Data are the mean of 2-6 independent experiments. SEM were always less than 10%. * Statistical significance from basal release (p < 0.001) by one-way ANOVA.

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MENDEZ ET AL.

Similar results were obtained with TRH added in the presence of CPHNA or CPHNA C ZPP + PDMK (Table 1); this was also observed in the serial incubation system (not shown). DISCUSSlON TRH-induced enhancement of spontaneous locomotor activity has been explained by TRH activation of DA release in nucleus accumbens and septum (7,11,18,22,23), although some effects are inconsistent (4,22). In particular, some in vitro studies report a small but not significant stimulation of DA release at high concentrations ( 10m4M) of TRH (22). This weak action of TRH may be related to its low stability due to degradation by peptidases in bioIogica1 fluids or cell membranes (26) because some analogues resistant to enzymatic degradation (CC3509 and RX77368 at lo-’ M-10e3 M) increased DA release from some brain regions more efficiently than TRH itself (22). Our interest in the physiological relevance of PPII led us to study the consequence of its inactivation on the effect of TRH on DA release in nucleus accumbens-lateral septum slices because the contradicting reports mentioned could be due to TRH enzymatic inactivation. However, we did not find an increased release of [3H]-DA by TRH in contrast to previous reports (7.1 1) but in agreement with Sharp et al. (22) who found that only the TRH analogues were able to induce a significant [3H]-DA release. However, high doses of analogues were used in these studies. Furthermore, the TRH analogue ~-butyrola~one-~-car~nylhistidyl prolineamide (DN-1417) induces DA release more efficiently than TRH ( 16), despite being as efficiently hydrolyzed by PPII as TRH (12). Another analogue, N-[t(S)-4-oxo-Zazetidinyl] carbonyll-L-histidyl-L-prolineamide (YM14673), increases spontaneous locomotor activity 100X better than TRH if injected in the nucleus accumbens but does not bind to TRH receptors and is equally degraded by PPII as TRH is ( 19). The lack of effect of TRH was not due to TRH inactivation by soluble PE or PPI leaking out of slices; in the presence of their specific inhibitors (PDMK, ZPP) TRH did not increase 13H]-DA release. This agrees with previous studies showing these enzymes do not regulate TRH levels in the extracellular fluid

(14). The specific inhibitor of PPiI, CPHNA, did not modify [3H]-DA release with or without TRH although increasing (3X) the recovery of endogeneous TRH released from nucleus accumbens lateral septum slices at the concentration used in this study (3). These data suggest that the lack of TRH effect is not due to its inactivation by PPII. The high potency of the analogues mentioned above may not be justified by a different degradation susceptibility. The discrepancy remains open. but our negative results cannot be explained by any obvious methodological problem: a) two different batches of TRH were used, each with its expected Rf and immunoreactivity and, the different release methods used in this study adequately responded to a depolarizing KC1 stimulus similarly to other reports (11,22) and, b) because TRH effect on locomotor activity is dependent upon circadian rhythms ( I ) we tested TRH at various times during the cycle with no difference in response. Alternative explanations should be considered for either an indirect or a nonspecific effect mediating TRH-DA interactions. For example, TRH-like peptides are present in some regions of the rat brain (2 1), so activation of a TRH-like peptide receptor may induce DA release in the nucleus accumbens in vitro. The in vivo effects observed with TRH may be indirect on the DA terminals or else, modulating the effect of another neurotransmitter; the concentration of the latter not being adequate in the in vitro system. In conclusion, TRH inactivation by peptidases is not the event responsible for its lack of induction of [3H]-DA release in vitro. A reevaluation of TRH role on DA turnover in nucleus accumbens is thus necessary. ACKNOWLEDGEMENTS

This paper is dedicated to Ricardo Lopez-Escalera, who passed away on February 2 I, I99 I, and was of great technical help to this paper. The authors thank Herminia Pasantes-Morales for helpful discussion and the technical aid of E. Mata and S. Gonzalez. Supported in part by grants from Foundation Miguel Aleman, A.C. and the Third World Academy of Sciences.

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INTERACTION

IN NUCLEUS

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