Signal transduction pathways involved in presynaptic receptor-mediated inhibition of dopamine release in rat striatum

Neurochem. Int. Vol. 20, Suppl., pp, 85S-88S, 1992 Printed in Great Britain. All fights reserved

0197-0186/92 $5.00+0.00 Copyright © 1992 Pergamon Press plc

SIGNAL T R A N S D U C T I O N PATHWAYS INVOLVED IN PRESYNAPTIC RECEPTOR-MEDIATED INHIBITION OF DOPAMINE RELEASE IN RAT STRIATUM NANCY R. ZAHNISER,WAYNE A. CASSand FRANK A. FITZPATRICK Department of Pharmacology, University of Colorado, Health Sciences Center, Denver, CO 80262, U.S.A.

D-2 dopamine (DA) autoreceptors inhibit evoked release of DA in the rat striatum. The mechanisms underlying this presynaptic modulation of DA release are not known. Possible mechanisms include inhibition of adenylate cyclase activity, inhibition of phosphoinositide hydrolysis, production of arachidonic acid metabolites, activation of a K ÷ conductance or inhibition of a Ca ++ conductance. While striatal D-2 DA receptors can inhibit adcnylate cyclase, this action does not appear to be related to D-2 DA receptor modulation of release (Memo et al., 1986; Bowyer and Weiner, 1989). It has also been suggested that striatal D-2 DA receptors inhibit phosphoinositidc hydrolysis; however, more recent evidence has not supported this contention (KcUy et al., 1988). Metabolites of arachidonic acid, specifically lipoxygcnase mctabolitcs, have been suggested to mediate presynaptic inhibition produced by FMRFamide receptors in Aplysia sensory neurons (Piomelli et al., 1987; Buttner et al., 1989), but the role for these novel second messengers in the action of D-2 DA receptors has never been investigated. Electrophysiological studies indicate that impulse-regulating somatodendritic D-2 DA autoreceptors in substantia nigra are coupled via a guanine nuclcotidc regulatory protein (Innis and Aghajanian, 1987) to K + channels (Lacey et al., 1987). Activation of these D-2 DA receptors leads to an outward K + current that hyperpolarizes the membrane and decreases firing rate. The observations that the K + channel blockers tetraethylammonium (TEA) or 4-aminopyridine (4-AP) partially block D-2 DA receptor modulation of Ca ++-evoked 3H-DA release from striatal synaptosomes suggest that presynaptic release-modulating D-2 DA receptors may be linked to K + channels (Bowyer and Weiner, 1989). Postsynaptic D-2 DA receptors on striatal neurons also appear to be coupled to K + channels because the receptor activity can be blocked by nanomolar concentrations of quinine (Freedman and Weight, 1989). In addition to D-2 DA autoreceptors, striatal DA release is also inhibited by activation of several

other receptors, including AI adenosine receptors (Lupica et al., 1990). These observations led us to investigate the possibilities that arachidonic acid metabolites and/or K + channels mediate D-2 DA and A1 adenosine receptor modulation of striatal DA release. For release experiments, striatal slices (400 g m thick) were prepared from male Sprague-Dawley rats (170-300 g; Sasco Animal Laboratories), allowed to recover for 1 hr and then superfused at a rate of 1.0 ml/ min for 1 hr with aerated, warmed (34°C) Krebs' buffer. The buffer contained either 10 g M nomifensine or GBR 12909 to inhibit the synaptic DA transporter and to obtain detectable levels of DA. After the 1 hr superfusion period, 17 l-ml samples associated with each of two electrical stimulation periods (unipolar square-wave stimulations, 1 Hz for 1 min, 2 ms pulse duration, 18-22 mA; separated by 1 hr) were collected on ice. Drugs were included in the superfusion buffer 30 rain before the second stimulation. DA levels in the superfusion samples were determined using highperformance liquid chromatography coupled with electrochemical detection (Gerhardt et al., 1989). Stimulation-evoked overflow was calculated by subtracting spontaneous release from total release and summing all positive values (designated as S1 and $2, respectively). The method used to measure release from striatal slices preloaded with 3H-DA was similar to that described for the endogenous DA and has been described in detail previously (Dwoskin and Zahniser, 1986). Radioligand binding studies were used to determine whether or not these drugs interacted with D-2 DA receptors. 3H-Spiperone binding to rat striatal membranes was measured as described previously (Zahniser and Dubocovich, 1983), except that 20 mM Hepes buffer (pH 7.5) containing 154 mM NaCI was used instead of Krebs' buffer. The final concentration of 3H-spiperone used was 120 pM for competition studies and 5-500 pM for saturation studies. Nonspecific binding was defined with 10 # M S85S

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sulpiride. To determine effects on agonist interactions, competition curves were constructed using +H-spiperone and the selective D-2 DA receptor agonist N 0437 (0.3 nM-10 /~M). ~H-DA uptake into striatal synaptosomes was measured in Krebs' buffer containing 0.1 mM pargyline for 2.5 min at 37°£?. For competition curves 10 nM 3H-DA was used, and for saturation curves the same concentration of 'H-DA was used in the absence or presence of unlabeled DA (10 n M - 10 pM). Nonspecific binding was defined with 10/1M GBR 12909. The assay using )H-mazindol to measure recognition binding sites associated with the DA transporter was similar to that described by Javitch et al. (1984). Stimulation-evoked overflow of endogenous DA was 76 + 2.7 pg DA/mg tissue. In controls, the response to the second stimulation was nearly identical to that of the first ($2/S1 ratio = 0.97 + 0.02). A maximally effective concentration of either N-0437 (I 0 nM) or adenosine (50 pM) decreased DA overflow by approximately 30%. Increasing the concentration of either drug did not significantly increase the percent of inhibition, suggesting that 30% represents the maximal inhibition under these conditions. A similar maximal effect was found with 30 nM pergolide, another selective D-2 DA receptor agonist. This 30% maximal inhibition is in contrast to studies that looked at Nq)437 inhibition of ~H-DA release, where the maximal effect is as great as 90% inhibition with 100 nM N-0437 (Van der Weide et al., 1988; unpublished observations). However, despite the differences in assay conditions and results, in both cases N-0437 is a potent agonist at these presynaptic D-2 DA receptors. When perfused together, the effects of I 0 nM N-0437 and 50/~M adenosine were additive. We first investigated the possible role of arachidonic acid metabotites in presynaptic inhibition in the striatum by determining the effect of inhibitors of key enzymes in the arachidonic acid cascade. The effect of arachidonic acid itself was also determined. Neither D-2 DA nor A i adenosine receptor modulation of endogenous DA overflow evoked from rat striatal slices by electrical field stimulation was affected by the phospholipase A2 inhibitor p-bromophenacyl bromide (BPB; 10 ,uM), by the cyclooxygenase inhibitor aspirin (100 ktM) or by arachidonic acid (30/.t M). BPB did, however, increase spontaneous outflow of DA from the slices in a dose-dependent manner whereas aspirin and arachidonic acid had no effect. Likewise, in slices prelabeled with 3H-DA, exposure to BPB, U 73122 (a phospholipase A2 inhibitor; I0 ~ M) and nordihydroguaiaretic (NDGA; a lipoxygenase inhibitor; 30 HM) significantlyincreased spontaneous outflow of

tritium while the cyclooxygenasc inhibitor~ aspirin and indomethacin (INDO, 30 ltM) had no effect. Thc increased outflow is likely due to the ability of BPB, U 73122, and NDGA to block the DA transporter. In low micromolar concentrations, BPB, U 73122 and NDGA, but not aspirin and INDO, blocked uptake of ~H-DA into striatal synaptosomes and inhibited binding of 3H-mazindol to a site associated with the DA transporter. Kinetic analyses indicated that the blockade produced by BPB was noncompetitive whereas that produced by NDGA was uncompetitive. in contrast to their potent interactions with the DA transporter, only one of these compounds, U 73122, affected ligand interactions (both agonist and antagonist) with the D-2 DA receptor. Taken together, these results indicate that arachidonic acid metabolites do not mediate the actions of presynaptic releasemodulating D-2 DA and A1 adenosine receptors on striatal DA release. However, certain of these inhibitors of arachidonic acid metabolism are relatively potent DA uptake blockers. Therefore, caution should be employed when using BPB, U-73122 and NDGA to study mechanisms involved in DA release because these compounds may increase overflow and appear to antagonize the action of presynaptic receptors. ro ~

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Fig. 1. Comparison of potencies of K ~ channel blockers to inhibit the modulation of evoked DA overflow from rat striatal slices produced by D-2 DA receptor activation. The inhibition produced by 10 nM N-0437 in control slices was defined as the maximal D-2 DA receptor-mediated modulation. Shown are mean values for N = 3-4. Next, we investigated the role of K ÷ channels in the presynaptic inhibitory actions of D-2 DA and AI adenosine receptors in the striatum by determining the effects of three K + channel lockers - quinine, 4-AP and TEA. In contrast to the results with the inhibitors of arachidonic acid metabolism, addition of quinine (100 nM), 4-AP (3 pM) or TEA (1 mM) to the superfusion buffer did not significantly affect spontaneous DA

Dopamine 90 release. By itself, quinine (100 nM) significantly increased evoked DA overflow by 17%. This increase is similar to that caused by a 2 n M concentration of the selective D-2 DA receptor antagonist raelopride, a concentration that completely blocks the effect of 10 nM N--0437. When quinine was coperfused with N 0437, it blocked the inhibition caused by N--0437 in a dose-dependent manner (Figure 1). The IC50 value for quinine was 6 nM, and a concentration of 100 nM quinine completely blocked the effect of N-0437. This potent dose-effect relationship is similar to that reported by Freedman and Weight (1989) for the ability of quinine to inhibit D-2 receptor-activated K ÷ currents in acutely dissociated striatal neurons. Relatively low concentrations of 4-AP (3/a M) or TEA (1 mM) also completely antagonized the inhibitory effect of N--0437 on evoked DA overflow (Figure 1). In contrast, the inhibitory modulation produced by adenosine, measured in the presence of raclopride to eliminate any D-2 DA receptor effects, was not affected by any of the K ÷ channel blockers (quinine, 1 /aM; 4-AP, 30 /aM; TEA, 1 mM). In addition, the results of the binding experiments confirmed that none of the K ÷ channel blockers, in the concentrations used in the present experiments, were direct-acting D-2 DA receptor antagonists. Quinine (100 nM), 4-AP (3/aM) or TEA (1 mM) did not affect either total or nonspecific binding of 3H-spiperone to rat striatal membranes, and the ability of N-0437 to inhibit binding also was the same in the absence and presence of the K ÷ channel blockers. These results with the K ÷ channel blockers, taken together with our observation that the releasemodulating effects of N--0437 and adenosine are additive, suggest that D-2 DA and A1 adenosine receptors inhibit DA release in the striatum by different mechanisms. Inhibition ofstriatal DA release by D-2 DA autoreceptors involves a K ÷ channel that is inhibited by low concentrations of quinine and that is also sensitive to 4-AP and TEA. Quinine is approximately two orders of magnitude more potent than 4-AP and five orders of magnitude more potent than TEA. In contrast, none of these K ÷ channel blockers affect AI adenosine receptor-mediated modulation. It appears that low doses of quinine can block the effects of both presynaptic (our data) and postsynaptic (Freedman and Weight, 1989) D-2 DA receptor activation in the striatum. Further experiments will be necessary to determine the identity of the specific K ÷ channel involved in the action of release-modulating D-2 DA autoreceptors. The role of Ca ÷+ channels in the action of both 1)--2 DA and AI adenosine receptor also needs to be

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explored. Nonetheless, the present results indicate, for the first time, that a quinine-sensitive K ÷ channel appears to mediate the actions of release-modulating D-2 DA receptors in the brain.

Acknowledgements---This work was supported by USPHS Grants NS26851 and GM41026 and Training Grant AA07464. The authors thank Mr. Gaynor Larson for excellent technical assistance. REFERENCES

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