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Elimination of GTP biphasic regulation of synaptosomal adenylate cyclase by manganese and solubilization.

Effects of divalent cations and solubilization with Lubrol-PX were studied on guanine nucleotide regulation of synaptosomal adenylate cyclase activity of the rat caudate nucleus. In the presence of Mg2+, both GTP and Gpp(NH)p exerted biphasic actions on the membrane-bound adenylate cyclase activity. The K0.5 value for the GTP stimulation of the cyclase was 47 nM, and the value for the GTP inhibition was 4.5 microM. One hundred microM dopamine selectively enhanced the stimulatory phase of the GTP action, whereas 10 microM morphine selectively enhanced the inhibitory phase of the GTP action. When Mg2+ was replaced by Mn2+, the inhibition of the membrane-bound adenylate cyclase by these nucleotides and morphine was completely abolished; but the catalytic activity of adenylate cyclase was not impaired. These results suggest that the inhibitory action of GTP is responsible for the morphine inhibition of synaptosomal adenylate cyclase. Lubrol-solubilized adenylate cyclase prefered Mn2+ to Mg2+ for its activity. The stimulation of adenylate cyclase by either GTP or Gpp (NH)p was eliminated in the Sepharose 6B-fractionated solubilized preparation in the presence of either Mg2+ or Mn2+. Ten mM NaF also failed to activate the fractionated adenylate cyclase. In the fractionated solubilized preparation, GTP and Gpp(NH)p failed to inhibit adenylate cyclase. These results indicate that GTP and Gpp(NH)p are unable to inhibit the resolved catalytic unit of the synaptosomal adenylate cyclase.

Membrane-bound adenylate cyclase is stimulated or inhibited by hormones. It has been recently shown that GTP plays a crucial role in mediating the actions of both stimu latory and inhibitory hormones on adenylate cyclase activity (1,2). Hormone-stimulable adenylate cyclase systems consist of at least three separate components: a hormone receptor, a guanine nucleotide regulatory protein (G protein), and a catalytic unit of adenylate cyclase (1)(2)(3)(4)(5). The catalytic unit separated from the G protein is active with Mn2+-ATP, but it is inactive with the physio logical substrate Mg2+-ATP (6). The G protein is essential to constitute the Mg2+ ATP-dependent activity of adenylate cyclase (6) and responsible for the activation of adenylate cyclase by guanine nucleotides and F (3, 7, 8). Stimulatory hormones increase the rate of GTP activation of adenylate cyclase (9, 10). In addition to the G protein-mediated stimulatory action of GTP, an inhibitory action of GTP on adenylate cyclase activity has been also demonstrated in a variety of tissues (11-15). In tissues in which GTP exerts both the stimulatory and the inhibitory action on adenylate cyclase activity, lower GTP concentrations (<0.1-1 /LM) stimulate and higher GTP concentrations (?0.1-1 uM) inhibit the cyclase (13-15). Hormones and neurotransmitters such as a-adrenergic agonists and adenosine inhibit adenylate cyclase in these tissues only in the presence of high GTP concentrations (13, 15-17). Cooper et al. (14) have shown that the inhibitory phase of the GTP biphasic action is linked to adenosine inhibition of rat adipocyte adenylate cyclase. These lines of evidence suggest that the inhibitory action of GTP is generally implicated in the action of inhibitory hormones on adenylate cyclase activity. However, the mechanism by which GTP and hormones inhibit adenylate cyclase has not yet been fully elucidated. It has been suggested that GTP inhibits adenylate cyclase through a component distinct from both the catalytic unit and the stimulatory G protein (18,19). In tissues in which GTP inhibits adenylate cyclase, however, it has not been investigated whether GTP directly inhibits the resolved catalytic unit or not.
Experimental perturbation of an adenylate cyclase system is one of the useful approaches to investigate the roles of components of the cyclase system in regulation of the enzyme activity. It has been recently shown that Mn2+ impairs the GTP-dependent hormonal regulation of adenylate cyclase activity in frog erythrocytes (20), human platelets (19), and hamster adipocytes (21). The components of the adenylate cyclase system can be physically separated by solubilization of membranes with detergent and successive chromatography (22, 23). We demonstrated previously that opioids inhibit the syn aptosomal adenylate cyclase of the rat caudate nucleus through a GTP-dependent process (24). In the present study, we have investigated effects of Mn2+ and solubilization with Lubrol-PX on the regulation of the synaptosomal adenylate cyclase activity in an attempt to elucidate the mechanism by which GTP and morphine inhibit the enzyme. The present results suggest that the inhibitory action of GTP on the adenylate cyclase activity is responsible for the morphine inhibition of the cyclase. In addition, we show that GTP no longer inhibited the resolved catalytic unit of the synaptosomal adenylate cyclase.

Enzyme preparations:
The crude syn aptosomal fraction (8-12 mg protein) of rat caudate nucleus prepared as described pre viously (24) was lysed with 10 ml of 5 mM Tris-HCI (pH 7.4) for 30 min at 0°C. The crude synaptosomal lysates were precipitated by centrifugation at 1 5,000xg for 30 min (4°C). The lysates were suspended in ice cold 1 mM EGTA, 5 mM Tris-HCI (pH 7.4) to yield a protein concentration of 0.8-1.0 mg/ml. This suspension was stored at -40'C until use.
Adenylate cyclase assay: To avoid the complicating effects of the ATP-regenerating system on adenylate cyclase activity (25), the modified method (24) of Clement Cormier et al. (26) was used. The total 500 ul of standard assay mixture contained 50 el of enzyme preparation, 0.12 mM ATP, 1.2 mM 3-isobutyl-1 -methylxanthine, 50 mM Tris HCI (pH 7.4), and 4.8 mM MgC12 or MnCI2, unless otherwise indicated. The reaction was initiated by the addition of ATP. After the incubation for 5 min at 37'C, unless otherwise indicated, the reaction was terminated by boiling for 3 min. Cyclic AMP formed was assayed by the method of Tovey et al. (27) as described previously (24). The compounds including nucleotides employed in experi ments did not significantly affect the cyclic AMP assay. Adenylate cyclase activity was linear with time for at least 10 min. Each experiment was performed in triplicate with results differing by less than 5% and repeated at least twice using different enzyme pre parations. Data shown are the means of triplicate determinations of one representative experiment, unless otherwise indicated. The Student's t-test was used for the statistical analysis.
Protein concentration was determined by the method of Bradford (28) using bovine gamma globulin as a standard.
Chemicals: Dopamine HCI was purchased from Wako Pure Chemical Ind. Morphine HCI was purchased from Takeda Chemicals. ATP (A 2383) and GTP were from Sigma Chemicals. Gpp(NH)p was obtained from P-L Biochemcals Inc. Lubrol-PX was from Nakarai Chemicals. Other chemicals were all of reagent grade.

Membrane-bound
adenylate cyclase: Figure 1 shows that adenylate cyclase in the crude synaptosomal lysates was active in the presence of either Mg2+ or Mn2+. Since relatively high activities were obtained in the presence of either 4.8 mM MgCl2 or MnCI2, we used this concentration of the divalent cations exclusively in the following experi ments.
In the presence of 4.8 mM MgC12, both GTP and Gpp(NH)p showed biphasic effects on adenylate cyclase activity in the lysates (Fig. 2). The K0,5 value for the GTP stimu lation of the cyclase was 47 nM, and the value for the GTR inhibition was 4.5 uM. The effects of dopamine and morphine, which modulate adenylate cyclase activity through their receptors (24, 29), on the biphasic actions of these guanine nucleotides were studied. As shown in Fig. 2, 100 flM dopamine enhanced the stimulatory actions of these nucleotides. The K0.5 value for the GTP stimulation of adenylate cyclase was not altered, whereas the maximal stimulation by GTP was increased from 183±10% (mean± S.E.M., the number of separate experiments (n)=4) to 233±13% (n=4, P<0.05) by the addition of dopamine. Similar results were obtained with Gpp(NH)p as a substituent of GTP (Fig. 2). Dopamine did not affect the inhibitory actions of these nucleotides on the adenylate cyclase activity. Ten ,uM mor  phine enhanced the inhibitory action of GTP by reducing the K0 .5 value for the GTP inhibition from 4.5 jiM to 2.7 jiM and by increasing the maximal GTP inhibition from 29*2% (n=4) to 39+3% (n=4) (P<0.05). The Gpp(NH)p inhibition of adenylate cyclase was also enhanced slightly by morphine (Fig.  2). Morphine did not alter the stimulatory actions of these nucleotides. Thus, dopamine selectively enhanced the stimulatory actions of these guanine nucleotides, whereas mor phine selectively enhanced their inhibitory actions.
When 4.8 mM MgCl2 was replaced by the same concentration of MnCl2, the adenylate cyclase activity in the lysates increased by approximately 200% (Fig. 1). Figure 3    for the inhibition of adenylate cyclase mentioned above.
Lubrol-solubilized adenylate cyclase: To examine whether guanine nucleotides directly inhibit the catalytic unit or not, we studied the effects of GTP and Gpp(NH)p on the activity of Lubrol-solubilized adenylate cyclase. Lubrol-PX, when it was added to the lysate suspension, inhibited adenylate cyclase by 60% in the presence of 4.8 mM MgCl2; but it activated the cyclase three fold in the presence of 4.8 mM MnCl2. The 40% of Mgt+-dependent and the 80% of M n2+-dependent activities were recovered as the solubilized adenylate cyclase. As shown in Fig. 4, the Lubrol-solubilized adenylate cyclase showed very low activities in the presence of Mgt', but it was still active in the presence of Mn2+. The Lubrol-solubilized adenylate cyclase was applied on the Sepharose 6B column. Figure 5 shows the elution profile of adenylate cyclase activity from the column. The Mn 21 -dependent activity eluted as a single peak. The pooled peak fractions showed negligible adenylate cyclase activity in the presence of Mgt+. Both GTP and Gpp(NH)p failed to activate adenylate cyclase in the pooled peak fractions in the presence of either 4.8 mM MgCl2 or MnCI2 (Fig. 6). Since the stimulation of adenylate cyclase by Gpp(NH)p is quasi irreversible, the extent of the Gpp(NH)p stimulation depends on the duration of incubation. Even after the preincubation at 23°C for 2 hr, however, Gpp(NH)p (10 nM 100 ,uM) did not show any effect on the adenylate cyclase activity in the pooled peak fractions in the presence of either Mg2+ or Mn2+ (data not shown). The activation of adenylate cyclase by 10 mM F was also abolished in the pooled peak fractions (Table   1). Thus, the adenylate cyclase activity eluted from the column was dependent on Mn2+ and completely insensitive to the activation by either guanine nucleotides or F-. As shown in Fig. 6, both GTP and Gpp Fig. 5. Gel filtration of Lubrol-solubilized adenylate cyclase. Three ml of the Lubrol solubilized adenylate cyclase of rat caudate nucleus crude synaptosomal lysates was applied on a Sepharose 6B column (1.5 x 55 cm). The column was equilibrated and eluted at a flow rate of 9 ml/hr as described in the text. Each 50 UI aliquot of the 0.8 ml fractions was assayed for adenylate cyclase at 37 °C for 10 min in the pres ence of 4.8 mM MnC12 and other reagents as described in the text. VO and Vt indicate the void and the total volume of the column, respectively. The indicated fractions were pooled. Table  1.  (NH)p could no longer inhibit adenylate cyclase in the pooled peak fractions in the presence of either Mg2+ or Mn2+.

DISCUSSION
GTP has been reported to exert not only a stimulatory but also an inhibitory (11, 12) or biphasic effect (14, 15, 30) on basal adenylate cyclase activity in other systems. Hydrolysis resistant Gpp(NH)p has been known to strongly activate adenylate cyclase. The inhibitory or biphasic action of Gpp(NH)p on adenylate cyclase activity, however, has been recently shown in human platelets (11, 31), rat fat cells (32), and Chinese hamster ovary cells (33). In this paper, we demon strated that both GTP and G pp (N H) p had the biphasic effect on the activity of the synaptosomal membrane-bound adenylate cyclase of the rat caudate nucleus in the presence of Mg2+. Morphine enhanced the Mg2+-dependent inhibitory actions of these guanine nucleotides, but it did not affect the stimulatory action of GTP in the presence of Mg2+ or Mn2+. When the GTP inhibition of the membrane-bound adenylate cyclase was abolished by Mn2+, morphine no longer inhibited adenylate cyclase. Since Mn2+ rather increases the binding of [3H]dihydromor phine to opioid receptors in the rat brain (34, 35), it is unlikely that Mn2+ eliminates the morphine inhibition of adenylate cyclase by disturbing the binding of morphine to the receptors. Therefore, it is conceivable that Mn2+ functionally uncouples the opioid inhibitable adenylate cyclase system in the synaptosomes. These results support our previous suggestions that the Mg2+ dependent inhibitory action of GTP on adenylate cyclase activity is responsible for the opioid inhibition of the synaptosomal enzyme and that opioids inhibit adenylate cyclase by enhancing the GTP inhibition of the cyclase (24). These suggestions are also supported by the observations in other systems that high inhibitory concentrations (>0.1-1 ,uM) of GTP are required for the inhibition of adenylate cyclase by a adrenergic agonists in neuroblastoma-glioma hybrid cells (NG 108-15 cells) (15) and human platelets (11) and by adenosine analogs in rat adipocytes (13, 14) and rat cerebral cortical membranes (16).
Biochemical and genetic studies are revealing the molecular basis of the GTP activation of adenylate cyclase, which involves the G protein (2). However, little is known about the mechanism of the GTP inhibition of adenylate cyclase. In the present study, we investigated whether GTP directly inhibits the catalytic unit or not. Judging from both the requirement of Mn2+ for the catalytic activity and the insensitivity to the activation by either guanine nucleotides or F (6, 22, 23), the active catalytic unit was completely separated from the G protein activity by Sepharose 6B gel filtration. The inhibitory actions of GTP and of Gpp(NH)p were abolished in the separated catalytic unit. These results provide evidence that a structure(s) distinct from the catalytic site of adenylate cyclase is involved in the GTP inhibition of the synaptosomal adenylate cyclase. From our results and the findings that the binding of opioid agonists to brain opioid receptors is also regulated by GTP (35, 36), it is conceivable that GTP both regulates the opioid receptor and inhibits the catalytic unit of adenylate cyclase through a component discrete from both the receptor and the catalytic unit.
An inhibitory guanine nucleotide regulatory component distinct from the stimulatory G protein has been proposed in rat adipocytes on the basis of differential sensitivities of the GTP stimulatory and inhibitory action to Mn2+ (14), organic mercurials (14), trypsin digestion (18), and radiation inactivation (37). Our present results also show that Mn2+ reduced much more the GTP inhibition than the GTP stimulation of the synaptosomal adenylate cyclase. However, since the mecha nisms underlying the differential modulations of the GTP stimulation and inhibition by these treatments are not known, these findings can not eliminate the possibility that GTP exerts both the stimulatory and in hibitory actions through a single population of G protein. One of the possible mechanisms mediating the GTP biphasic action through a single G protein is the hydrolysis of GTP to inactive or less active GDP by the GTPase associated with the G protein (38, 39). Assuming that high concentrations of GTP activate the GTPase leaving GDP bound to the G protein, the inactive or less active form of the G protein may accumulate in the presence of the high concentrations of GTP. This accumulation of the inactive or less active G protein may result in the inhibition of adenylate cyclase. The possibility that GTP and opioids inhibit adenylate cyclase by stimulating the GTPase has been recently suggested in NG 108-15 cells (40). How ever, since GTPase-resistant Gpp(NH)p also inhibited the synaptosomal adenylate cyclase, our results are inconsistent with the pos sibility mentioned above.
Elucidation of the mechanisms by which GTP exerts multiple regulatory actions on receptor adenylate cyclase systems is essential to the understanding of the regulation of adenylate cyclase activity by hormones or neurotransmitters. Further study is required to clarify more fully the molecular basis of the GTP biphasic action on adenylate cyclase activity. The Lubrol-solubilized adenylate cyclase presented in this report may provide a useful tool for the investigation of functions of components in adenylate cyclase systems, and it is a material which can be used for further purification of the catalytic unit of the cyclase.