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J. Biol. Chem., Vol. 277, Issue 14, 11859-11865, April 5, 2002

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Zinc Inhibition of cAMP Signaling^*

Claudette Klein^§, Roger K. Sunahara^¶, Tracie Y. Hudson, Tomasz Heyduk, and Allyn C. Howlett

From the  Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri 63104, the ^¶ Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109, and the  Neuroscience and Drug Abuse Research Program, J. L. Chambers Research Institute, North Carolina Central University, Durham, North Carolina 27707

Received for publication, September 12, 2001, and in revised form, January 11, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Zn²⁺ is required as either a catalytic or structural component for a large number of enzymes and thus contributes to a variety of important biological processes. We report here that low micromolar concentrations of Zn²⁺ inhibited hormone- or forskolin-stimulated cAMP production in N18TG2 neuroblastoma cells. Similarly, low concentrations inhibited hormone- and forskolin-stimulated adenylyl cyclase (AC) activity in membrane preparations and did so primarily by altering the V_max of the enzyme. Zn²⁺ also inhibited recombinant isoforms, indicating that this reflects a direct interaction with the enzyme. The IC₅₀ for Zn²⁺ inhibition was ~1-2 µM with a Hill coefficient of 1.33. The dose-response curve for Zn²⁺ inhibition was identical for AC1, AC5, and AC6 as well as for the C441R mutant of AC5 whose defect appears to be in one of the catalytic metal binding sites. However, AC2 displayed a distinct dose-response curve. These data in combination with the findings that Zn²⁺ inhibition was not competitive with Mg²⁺ or Mg²⁺/ATP suggest that the inhibitory Zn²⁺ binding site is distinct from the metal binding sites involved in catalysis. The prestimulated enzyme was found to be less susceptible to Zn²⁺ inhibition, suggesting that the ability of Zn²⁺ to inhibit AC could be significantly influenced by the coincidence timing of the input signals to the enzyme.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In general, Zn²⁺ is required as either a catalytic or structural component for a large number of enzymes and thus contributes to a wide variety of important biological processes including gene expression, replication, hormonal storage and release, neurotransmission, and memory. Zn²⁺ is also critical for the structural integrity of cells, influencing membrane stability and cytoskeletal organization (reviewed in Refs. 1-3). In this light, it is not surprising that dietary Zn²⁺ deprivation is lethal in mice and in man has been associated with a variety of abnormalities related to growth, sexual maturation, and wound healing (4). The complexity of Zn²⁺ actions is further appreciated by the fact that the consequences of excess Zn²⁺ are also severe being associated with a number of neurodegenerative disorders including Parkinson's and Alzheimer's disease and epilepsy (1-3). Toward understanding the paradoxical functions of Zn²⁺, attention has focused on defining the critical candidates that mediate its effects and whether they can reflect the physiological, pharmacological, or toxicological effects of the metal.

In the brain, Zn²⁺ along with iron is the most concentrated metal (1-4). Significant levels of chelatable histochemically reactive Zn²⁺ are present in a subset of glutamatergic neurons in which Zn²⁺ appears to be localized to synaptic vesicles (5-7). These Zn²⁺-containing neurons are primarily located in the hippocampus (mossy fibers), striatum, and neocortex. The concentrations of Zn²⁺ within these vesicles have been estimated to be as high as millimolar levels (3). Neuronal firing results in the release of both glutamate and Zn²⁺ into the synaptic cleft (4-7). Intense firing can result in Zn²⁺ concentrations of several hundred micromolars (4). This estimate is based upon the accumulation of Zn²⁺ in the perfusate of hippocampal slices. Thus, the actual localized concentrations of the metal may be significantly greater. The fate of neuronally released Zn²⁺ is not totally clear. It is likely that some of the metal is taken back up by the presynaptic neurons and reconcentrated into vesicles (8). The movement of Zn²⁺ from presynaptic to postsynaptic cell has been documented. In general, this translocation is associated with the neurotoxic effects of Zn²⁺ (4). For example, excessive firing of mossy fibers of the hippocampus leads to selective Zn²⁺ uptake and the destruction of the CA1 neurons that are innervated by these bundles. However, the fact that Zn²⁺ translocation from presynaptic to postsynaptic cells occurs allows for the possibility that this metal is also used in the central nervous system as a transcellular messenger (1-3).

The immediate targets of intracellular Zn²⁺ are unclear as are the physiological consequences of its actions on specific targets. In neuronal cells, targets such as glyceraldehyde 3'-phosphate dehydrogenase, NAD-catabolizing activities such as poly(ADP-ribose) synthetase, or the production of reactive oxygen species have been proposed as mediators of Zn²⁺ neurotoxicity (for review, see Ref. 9 and references therein). However, Zn²⁺ deprivation is also cytotoxic, and there are numerous examples where culturing cells in Zn²⁺-deficient medium results in cell death, whereas higher external concentrations of Zn²⁺ appear as antiapoptotic (for review, see Ref. 10 and references therein). In that case, Ca²⁺/Mg²⁺-dependent endonucleases, caspases, Bcl2/Bax ratios, and cytoskeletal components are some of the proposed targets that Zn²⁺ modulates with a protective outcome.

In light of the importance of cAMP as a second messenger, we have examined whether Zn²⁺ could elicit its effects by altering adenylyl cyclase (AC)¹ activity. Biochemical studies of mutant ACs and recent crystallographic studies of a truncated soluble form of AC have indicated that the enzyme possesses two catalytic metal binding sites (11, 12). X-ray crystallography studies distinguished these two sites by their preferential occupancy by either Zn²⁺ or Mn²⁺. Although both sites are presumed to be occupied by Mg²⁺ in vivo (12), these observations allow for the possibility that Zn²⁺ could influence cAMP production particularly in the brain where Zn²⁺ concentrations are significant. In the studies reported here, we have examined the effects of Zn²⁺ on cAMP signaling in N18TG2 neuroblastoma cells. We have determined that low micromolar concentrations of Zn²⁺ inhibit hormone- and forskolin-stimulated cAMP accumulation directly by inhibiting AC. Furthermore, we have characterized the potent inhibitory effects of Zn²⁺ on AC in both isolated N18TG2 membranes and membranes isolated from Sf9 cells expressing recombinant isoforms.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture Conditions-- N18TG2 cells were grown in Dulbecco's modified Eagle's/Ham's F-12 (1:1) medium containing 10% heat-inactivated donor bovine calf serum and penicillin/streptomycin. The medium was changed 16-24 h prior to experimentation, and cells were confluent at the time of experimentation. Cells were dissociated from flasks by gentle trituration with phosphate-buffered saline containing 0.6 mM EDTA and resuspended to 2 × 10⁶ cells/ml in Gey's balanced salt solution without calcium but containing 0.1 mg/ml fatty acid-free bovine serum albumin, 10 mM Na-Hepes, pH 7.4, and the phosphodiesterase (PDE) inhibitor Ro20-1724 at 0.1 mM. Cells were incubated in that buffer in a shaking 37 °C water bath for increasing time periods during which different concentrations of Zn²⁺ were added.

cAMP Assay-- To determine cAMP levels in intact N18TG2 cells, we treated cells with or without stimulatory agents for a maximum of 4 min, a time within the linear range of stimulation, and then lysed them by boiling in sodium acetate, pH 4.5 (13). cAMP levels in the soluble fraction were measured using a modified Gilman assay (14). All assay points were taken in triplicate and assayed in triplicate. Triplicates generally agreed within 5% (13). Protein determinations were done according to Bradford (15).

AC Assay-- Membrane-bound AC activity was assayed by incubating N18TG2 and Hi5 membranes for 20 min or Sf9 membranes for 10 min at 30 °C in a HEPES-buffered mixture containing Mg²⁺, ATP, an ATP-regenerating system, cAMP, and PDE inhibitors (0.1 mM Ro20-1724 or isobutylmethylxanthine, respectively) and monitoring the conversion of [-³²P]ATP to [³²P]cAMP (16-18). Unless indicated otherwise, ATP and MgCl₂ were added at 0.5 and 10 mM, respectively, and EDTA was not added to the reaction mixture. When PGE₁-stimulated AC activity in N18TG2 membranes was monitored, 10⁴ M GTP was added. All samples were assayed in triplicate in agreement generally within 5%. The data are presented as the mean ± S.E. Enzyme activity is proportional to the concentration of membranes and the time of incubation. Unless indicated otherwise, when Zn²⁺ was added, it was present in the reaction mixture at the start of the incubation. The concentrations of Zn²⁺ added do not interfere with the energy-regenerating system. Because AC activity in Sf9 membranes is not dependent upon that system, it could be eliminated without altering the dose-response curves for Zn²⁺ inhibition. It is also noted that the concentrations of Zn²⁺ were sufficiently low as not to alter the concentration of Mg-ATP available as the substrate. This finding was evaluated using the free WinmaxC software developed by C. Patton at Stanford (Palo Alto, CA).

Materials-- [-³²P]ATP and [³H]cAMP were purchased from ICN and PerkinElmer Life Sciences, respectively. Tissue culture medium was purchased from Biowhittaker (Walkersville, MD). Heat-inactivated donor bovine calf serum was from JHR Biosciences (Lenexa, KS). All other reagents were from Sigma.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Zn²⁺ Inhibits cAMP Accumulation in N18TG2 Neuroblastoma Cells-- N18TG2 cells were incubated in the absence or presence of 300 µM Zn²⁺ for 2 h, and cAMP accumulation in response to forskolin and PGE₁ stimulation was monitored. As seen in Fig. 1, the incubation of cells with Zn²⁺ resulted in a dramatic attenuation of cAMP accumulation in response to both stimuli. It is unlikely that the low levels of cAMP reflect a stimulation of a PDE by Zn²⁺, because cells were preincubated throughout the experiment with PDE inhibitors. Moreover, Zn²⁺ had no effect on nonstimulated (basal) cAMP levels, also arguing against a Zn²⁺ effect on PDE activity. Because forskolin binds to and directly activates AC, the inhibition of cAMP accumulation would not likely reflect a primary effect on hormone receptors or G_s. When cells were preincubated with the heavy metal ionophore, pyrithione, a greater inhibition by Zn²⁺ was observed, suggesting that internalization of Zn²⁺ is necessary for the inhibition of cAMP production (Fig. 1).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   The effect of Zn2+ on cAMP accumulation in N18TG2 cells. Cells were preincubated for 2 h in the absence (open bars) or presence (solid bars) of 300 µM ZnCl2 or ZnCl2 plus 0.5 µM pyrithione. Basal forskolin-stimulated (104 M) or PGE1-stimulated (105 M) cAMP levels were determined as described under "Experimental Procedures." The data are representative of two experiments.

The extracellular levels of Zn²⁺ necessary to observe this inhibition were examined (Fig. 2). Little effect on forskolin-stimulated cAMP accumulation was seen when cells were preincubated with 1 or 10 µM ZnCl₂ for 2 h. Preincubation with 25 µM ZnCl₂ resulted in an approximate 25-30% attenuation of the forskolin response, whereas 150 µM resulted in an approximate 60-70% inhibition. When cells were incubated with Zn²⁺ and 5 µM pyrithione, concentrations of Zn²⁺ as low as 10 µM could significantly inhibit cellular cAMP accumulation in response to forskolin, and complete inhibition was observed at ~25 µM ZnCl₂. Thus, the relatively high extracellular concentrations of Zn²⁺ appear necessary to allow for the accumulation of sufficient intracellular levels. This is not unusual. For example, high concentrations of Zn²⁺ will suppress apoptosis in model cell culture systems, but concentrations approaching serum levels of Zn²⁺ (15-25 µM) will do so if pyrithione is present to facilitate uptake (19). In the experiment shown in this figure, pyrithione alone had a slight inhibitory action on forskolin-stimulated cAMP production. This effect was inconsistent and relatively minor such as not to alter the interpretation of the data.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Concentration-dependent effects of Zn2+. N18TG2 cells were preincubated with the indicated concentrations of ZnCl2 for 2 h (solid bars) at which time the levels of forskolin-stimulated (104 M) cAMP levels were determined. Parallel preincubations were performed in the presence of ZnCl2 plus 0.5 µM pyrithione (hatched bars). The levels of cAMP are reported as the percentage found in forskolin-treated cells that had not been preincubated with Zn2+ or pyrithione (open bar). Basal and forskolin-stimulated cAMP levels in control cells were ~65 and 625 pmol/mg, respectively. The data are representative of three experiments.

Fig. 3 indicates that when cells were preincubated with 100 µM ZnCl₂, a time period of more than 60 min was necessary to observe significant inhibition of forskolin-stimulated cAMP accumulation. The extent of inhibition increased with time and appeared maximum after 120 min. However, when pyrithione was present, the inhibition could be observed even after 30 min of incubation. In that case, maximum inhibition seemed to require between 60 and 90 min of incubation. The time dependence of the effects of Zn²⁺ is consistent with an uptake process that appears to be a limiting factor in eliciting the inhibition of cAMP accumulation.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   The effects of Zn2+ are time-dependent. N18TG2 cells were preincubated without additions (open bars) with 300 µM ZnCl2 (solid bars) or with 300 µM ZnCl2 plus 0.5 µM pyrithione (hatched bars) for the indicated times. Forskolin-stimulated (104 M) cAMP levels were then determined. 100% represents the levels achieved upon forskolin stimulation of cells preincubated in the absence of any additions. Basal levels were ~12% of those seen upon forskolin stimulation. The data are representative of two experiments.

It is generally accepted that the transfer of metals across the plasma membranes involves metal complexes that influence the efficiency with which the metal is delivered or transferred (8, 21-23). Thus, we evaluated the inhibition of cAMP accumulation by Zn²⁺ when the metal was presented as ZnCl₂ or as a complex of zinc ascorbate. As shown in Fig. 4, Zn²⁺ became a more potent inhibitor of forskolin-stimulated cAMP accumulation when ascorbate was present. In that case, significant inhibition was observed with 25 µM Zn²⁺ and appeared almost maximum with 75 µM. In contrast, zinc citrate and ZnSO₄ behaved in a fashion similar to ZnCl₂ (data not shown).

View larger version (25K): [in this window] [in a new window]   Fig. 4.   The effect of ascorbate on Zn2+ inhibition. N18TG2 cells were preincubated for 2 h in the absence (open bar) or presence of the indicated concentrations of ZnCl2 (solid bars) and 1 mM ascorbate (hatched bars). Samples were then assayed for forskolin-stimulated (104 M) cAMP levels. 100% is the level attained in control cells. The data are representative of three experiments.

Zinc Inhibits AC Activity in N18TG2 Membranes-- Plasma membranes purified by sucrose gradient centrifugation from N18TG2 cells were assayed for hormone-stimulated AC activity in the presence of varied concentrations of ZnCl₂. As shown in Fig. 5A, micromolar concentrations of Zn²⁺ effectively inhibited AC activity such that at 60 µM ZnCl₂, little PGE₁-stimulated enzyme activity could be detected. The IC₅₀ for ZnCl₂ was ~8-9 µM. Similar results were obtained using ZnSO₄. Because the assay for AC is performed in the presence of a PDE inhibitor and PDE activity is monitored by the tracer [³H]cAMP present in the assay, it is clear that the decreased levels of cAMP do not reflect the activation of PDE by Zn²⁺. Low micromolar concentrations of Zn²⁺ also effectively inhibited forskolin-stimulated AC activity (Fig. 5B). However, in this case, significant forskolin-stimulated activity could still be observed when 60 µM ZnCl₂ was present. The data, analyzed as a simple two-site binding model, suggested that two classes of binding sites could be present, one with an apparent IC₅₀ of ~2-3 µM and another greater than 10-fold.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Inhibition of AC activity in N18TG2 membranes by Zn2+. Sucrose gradient-purified plasma membranes were assayed for PGE1-stimulated (A) and forskolin-stimulated (B) AC activity in the presence of the indicated concentrations of ZnCl2. The concentrations of activators were 105 and 104 M, respectively. Basal activity was 60 pmol/min/mg. The data are representative of three (A) and four (B) experiments. GraphPad Prism (GraphPad Software, Inc.) was used to fit the data to simple one-site (A) and two-site (B) binding models.

Kinetic Analyses of the Effects of Zn²⁺-- We first determined the effects of Zn²⁺ on forskolin-stimulated AC activity measured at increasing concentrations of substrate. The results obtained when AC activity in N18TG2 membranes was assayed in the presence or absence of 10 µM Zn²⁺ are depicted in Fig. 6A. An analysis of the rate of substrate utilization indicated that the K_m slightly increased from 0.23 ± 0.02 to 0.39 ± 0.09 mM in the presence of Zn²⁺. The V_max of the activity in N18TG2 membranes showed a 2-fold decrease in the presence of Zn²⁺ going from 645 ± 19 pmol/min/mg to 318 ± 25 pmol/min/mg. Current data indicate that the active site of AC contains two metal ion binding sites referred to as A and B, which can be selectively occupied by Zn²⁺ and Mn²⁺, respectively (12). To assess whether the inhibitory Zn²⁺ site is indeed the metal site A, we also assayed forskolin-stimulated activity using a constant ATP concentration and varying Mg²⁺ concentrations in the absence or presence of Zn²⁺. As depicted in Fig. 6B, the presence of 40 µM ZnCl₂ resulted in an approximate 4-fold decrease in V_max. The respective V_max values were 258 ± 11 pmol/min/mg and 73 ± 10 pmol/min/mg. K_m values were 0.6 ± 0.6 µM and 1.3 ± 0.12 µM when AC activity was measured in the absence and presence of 40 µM Zn²⁺, respectively. With 10 µM ZnCl₂, we observed an approximate 2-fold decrease in both the apparent K_m and V_max for the Mg²⁺ dependence of AC activity (data not shown). The sum of the data suggests that the major effect of Zn²⁺on AC activity is to reduce the rate of enzyme conversion of substrate to product.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   The effects of Zn2+ on the kinetic properties of the enzyme A, N18TG2 membranes were assayed for forskolin-stimulated (104 M) AC activity at increasing concentrations of ATP ranging from 0.005 to 2.0 M. MgCl2 was added at 10 mM. When present, ZnCl2 was added at a final concentration of 10 µM. The data are representative of two experiments. B, N18TG2 membranes were assayed for forskolin-stimulated (104 M) AC activity at increasing concentrations of Mg2+ ranging from 0.75 to 10.0 mM. ATP was added at 0.5 mM. When present, ZnCl2 was added at a final concentration of 40 µM. The data are representative of two experiments.

Recombinant AC Is Inhibited by Zn²⁺-- N18TG2 cells appear to express AC6 as their predominant activity (18). To determine whether AC itself is the target of Zn²⁺ inhibition, we examined recombinant AC6. We also examined the other member of this isoform family, AC5, which is expressed primarily in the striatum, a site of relatively high neuronal levels of Zn²⁺ (reviewed in Refs. 1-3 and 24-26). Low micromolar concentrations of Zn²⁺ effectively inhibited the forskolin-stimulated activity of both isoforms (Fig. 7A). The data were identical to those obtained when ZnSO₄ or ZnCl₂ plus ascorbate was evaluated (data not shown). The fact that Zn²⁺ inhibits the recombinant enzyme indicates that this reflects a direct interaction with the enzyme. The inhibition of forskolin-stimulated AC5 and AC6 activity was dramatic (100% at 10 µM) occurring over a narrow range of Zn²⁺ concentrations. An analysis of the data by Hill plot revealed a coefficient of 1.33, indicating that the inhibition was a cooperative process. The IC₅₀ for ZnCl₂ inhibition was ~1-2 µM, a value similar to that of the apparent higher affinity site seen when forskolin-stimulated AC activity in N18TG2 membranes was monitored. No evidence for the lower affinity site present in N18TG2 membranes was obtained in our experiments using recombinant enzyme. This would suggest that the apparent lower affinity site seen using N18TG2 membranes is not inherent to the AC enzyme but may reflect the participation of additional AC regulators present in those membranes. Because of the lower specific activity of the AC6 preparations, additional analyses were performed using AC5.

View larger version (14K): [in this window] [in a new window]   Fig. 7.   Zn2+ inhibition of recombinant ACs at different Mg2+ concentrations. A, Sf9 membranes expressing AC5 () and Hi5 membranes expressing AC6 () were assayed for forskolin-stimulated (104 M) AC activity in the presence of the indicated concentrations of ZnCl2. MgCl2 was added at 10 mM. The activity seen with 0.1 µM ZnCl2 was equivalent to that seen when no Zn2+ was added. The results are representative of three experiments. The data are presented as the percent of maximum activity as the specific activity of the AC6 preparation is ~15-fold lower than that of AC5. B, the dose-response curve for Zn2+ inhibition of AC5 was examined when activity was measured in the presence of 40 mM MgCl2. The data are representative of two experiments.

The Inhibitory Zn²⁺ Site Does Not Appear to Be the Catalytic Metal Binding Site-- The inability of Zn²⁺ to dramatically alter the K_m for Mg²⁺ or Mg²⁺/ATP would suggest that Zn²⁺ is not competitively inhibiting the binding of Mg²⁺ and, therefore, is not binding to metal site A. To further address this issue, we assessed the dose-response curve for Zn²⁺ inhibition of AC5 and of the activity in N18TG2 membranes using a range of Mg²⁺ concentrations varying from 3 to 40 mM. Concentrations of Mg²⁺ >50 mM were found to be partially inhibitory and were therefore not investigated. The reasons for this inhibition are currently unclear. The dose-response curve obtained when 40 mM MgCl₂ was added to the assay for recombinant AC5 is representative of the results and is shown in Fig. 7B. A comparison with the data in Fig. 7A indicates that the dose-response curve was unaffected by the increased concentration of Mg²⁺ present. Neither the IC₅₀ nor the cooperative nature of the inhibition by Zn²⁺ was altered. Reducing the Mg²⁺ concentration to 3 mM also did not change the enzyme sensitivity to Zn²⁺ (data not shown). Such findings are consistent with the premise that Zn²⁺ does not bind to a catalytic metal binding site unless Zn²⁺ binds to such a site in a manner that appears kinetically irreversible.

Several experiments were performed to address the reversible nature of Zn²⁺ inhibition. In the experiment shown in Fig. 8A, we monitored the time course of AC5 inhibition by 15 µM Zn²⁺. As shown, the rate of forskolin-stimulated enzyme activity in the presence of Zn²⁺ was reduced but linear over the time frame of the experiment, suggestive of a reversible reaction (27, 28). The reversibility of the inhibition was further evaluated by adding EDTA to the assay after the first 10 min of incubation with Zn²⁺. In that case, the rate of forskolin-stimulated cAMP synthesis was substantially recovered. The addition of EDTA to control samples did not alter the rate of cAMP formation. Similar observations were made when AC activity in N18TG2 membranes was monitored (data not shown). The data indicate that the inhibition of AC by Zn²⁺ is reversible.

View larger version (15K): [in this window] [in a new window]   Fig. 8.   A, a reversibility of Zn2+ inhibition. Forskolin-stimulated (104 M) AC5 activity was determined in the absence () or presence ( and ) of 10 µM ZnCl2. In the latter case, an aliquot was removed after the initial 7 min of incubation, and EDTA was added to achieve a final concentration of 0.1 mM EDTA (). The assay was continued, and cAMP production was monitored at the indicated time. The rates of enzyme activity were: control, 1.9 nmol/min/mg; Zn2+ before EDTA addition, 0.66 nmol/min/mg; Zn2+ after EDTA addition, 1.25 nmol/min/mg. The data are representative of three experiments. B, Zn2+ inhibition of recombinant AC5 at different Mn2+ concentrations. Sf9 membranes expressing AC5 were assayed for forskolin-stimulated (104 M) AC activity in the presence of the indicated concentrations of ZnCl2 and in the absence () or presence of 10 mM () or 1 mM MnCl2 (). MgCl2 was added at 5 mM. The normalized results are representative of two experiments.

To address whether the site of Zn²⁺ inhibition is located at the other metal site, the preferential binding site of Mn²⁺ (site B), we assessed the effects of Mn²⁺ on the efficacy of Zn²⁺ inhibition of AC5. The data depicted in Fig. 8B shows that 0.1, 1, and 10 mM Mn²⁺ had no effect on the IC₅₀ for Zn²⁺. Note that Mn²⁺ is a potent activator of AC activity, and thus higher maximal activities accompany increasing Mn²⁺ concentrations. The data support the premise that the site of Zn²⁺ inhibition is distinct from the catalytic metal binding sites.

Additional support for this premise was obtained in our studies of the C441R mutant of AC5 (11). This mutant AC5 displays a dramatic decrease in catalysis with only a slight change (2-fold) in the ability to bind substrate. The mutation is located at the residue adjacent to Asp-440, one of the critical aspartates that participate in coordinating metal binding (12). The close proximity of Cys-441 led the authors to suggest that a mutation in this residue results in a conformational change that disrupts the orientation of Asp-440 and thus the metal binding pocket (11). As shown in Fig. 9A, the C441R mutant behaves in a fashion identical to wild-type AC5 in response to ZnCl₂ inhibition of forskolin-stimulated activity (IC₅₀ of 1.8 µM). Thus, this mutation did not supplant the Zn²⁺ inhibition of AC5.

View larger version (13K): [in this window] [in a new window]   Fig. 9.   Zn2+ inhibition of different ACs. A, the C441R mutant AC5 was assayed for forskolin-stimulated (104 M) AC activity in the presence of the indicated concentrations of ZnCl2. The activity seen with 0.1 µM ZnCl2 was the same as when no Zn2+ was added. The data are representative of two experiments. Recombinant AC1 (B) and AC2 (C) were assayed for forskolin-stimulated (104 M) activity in the presence of the indicated concentrations of ZnCl2. In each case, the activity observed in the presence of 0.1 µM ZnCl2 was the same as that when no Zn2+ was added. The data are representative of three experiments.

The Effects of Zn²⁺ Are Isoform-specific-- To examine the effects of Zn²⁺ on other AC isoforms, we focused our attention on recombinant AC1 as representative of the AC1, AC3, and AC8 isoform family, and AC2 as representative of the AC2, AC4, and AC7 family (24-26). AC1 and AC2 are of particular interest, because like AC5 they are expressed in regions of the brain with relatively high vesicular levels of Zn²⁺ (1-3, 24-26). As illustrated in Fig. 9B, recombinant AC1 was effectively inhibited over a narrow range of Zn²⁺ concentrations, displaying an IC₅₀ of 1.4 µM, similar to that of AC5. The dose-response curve of AC2, however, was distinct from the other isoforms (Fig. 9C). AC2 was not inhibited by the low micromolar concentrations of ZnCl₂ that were effective against AC1 and AC5. It appeared that such concentrations were actually somewhat stimulatory to the enzyme. Only at concentrations of ZnCl₂ of >10 µM did we observe an inhibitory effect. This would suggest that the high affinity inhibitory site seen in AC1 and AC5 is different in AC2. Because the residues surrounding the metal binding sites are strictly conserved across all isoforms, these data would be consistent with the premise that Zn²⁺ binds to a site distinct from the catalytic metal binding sites.

The Effects of Zn²⁺ Are Attenuated When It Is Added to an Active Enzyme-- In the experiment shown in Fig. 10A, hormone-stimulated AC activity of N18TG2 membranes was inhibited ~60-70% when assayed in the presence of 10 or 40 µM ZnCl₂ (Fig. 10A, last two bars). However, if Zn²⁺ was added 5 min after hormone activation of AC was initiated, the inhibition was attenuated (being almost negligible with 10 µM ZnCl₂ and only ~20% with 40 µM ZnCl₂). Similarly, when Zn²⁺ was added after the assay of forskolin-stimulated AC had been initiated, the inhibition was also attenuated (Fig. 10B). The calculation of the possible chelation of Zn²⁺ by ATP in the presence of Mg²⁺ using WinmaxC indicated that minimal changes in the free concentration of Zn²⁺ would be expected. This finding rules out the possibility that ATP is simply chelating Zn²⁺. The data suggest that Zn²⁺ is a less potent inhibitor of AC activity when the enzyme has achieved an activated state.

View larger version (19K): [in this window] [in a new window]   Fig. 10.   The effects of Zn2+ on activated AC. PGE1-stimulated (105 M) (A) and forskolin-stimulated (104 M) (B) AC activity was measured in N18TG2 membranes in the absence (solid bars) or presence (last two bars in 20-min group) of 10 or 40 µM ZnCl2. Alternatively, Zn2+ was added to the assay of hormone- or forskolin-stimulated activity 5 min after the assay had been initiated (open and hatched bars). Cyclic AMP synthesis was determined after an additional 5 and 15 min, resulting in a total incubation time of 10 and 20 min, respectively. The data are representative of three experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have demonstrated that the incubation of neuroblastoma cells with Zn²⁺ attenuates their ability to synthesize cAMP in response to hormone or forskolin stimulation. This appears to reflect a reversible inhibition of AC and thus would be compatible with a homeostatic role for this regulation. This would also be consistent with the fact that little or no loss in cell viability (trypan blue exclusion) upon incubation with Zn²⁺ was observed during the time course of our experiments.² The concentrations of Zn²⁺ that are effective in attenuating the cell response to hormone or forskolin between 25 and 150 µM may be encountered in vivo (1-5, 19). However, the extracellular concentrations needed to inhibit cAMP accumulation in N18TG2 cells could be significantly reduced under conditions that facilitated cellular uptake of the metal. One condition was the use of the heavy metal ionophore, pyrithione, whereas another condition more apt to reflect a physiological relevant condition was the addition of ascorbate. It is not uncommon that the form of metal chelation influences the efficiency of metal transport (8, 21-23). Ascorbate is of particular interest, because it accumulates in the brain (being maintained at relatively high (1-2 mM) concentrations (29, 30)). It is further concentrated in synaptic vesicles of glutamatergic neurons and released into the extracellular space as a result of neuronal activity (29, 30). Therefore, ascorbate has the potential to ligate Zn²⁺, rendering the metal a more potent neuromodulator. It is also noted that we are probably manifesting the effects of a passive influx of Zn²⁺. Passive influx through the neuronal membrane has been demonstrated to occur when extracellular levels of Zn²⁺ are elevated, e.g. when Zn²⁺ is added to the cell culture medium or when Zn²⁺ is released from presynaptic vesicles (31). However, experiments using cultured neuronal cells have also demonstrated that Zn²⁺ uptake can be stimulated upon activation of voltage-gated Ca²⁺or NMDA channels, for example (1-3, 32). Thus, physiological conditions that stimulate Zn²⁺ uptake will potentially facilitate a more effective inhibition of AC.

That Zn²⁺ uptake is required to observe the inhibition of AC would indicate that inhibition does not result from the nonspecific binding of Zn²⁺ either to the plasma membrane or to the exofacial residues of the AC. That Zn²⁺ inhibition of AC1, AC5, and AC6 appears identical could argue that amino acid residues of the transmembrane domains, which show limited homology, are not involved but that Zn²⁺ binds to the cytosolic CI and/or CII regions, which exhibit considerable homology. This assumption is substantiated by the fact that a soluble AC construct composed of the CI region of AC5 and the CII region of AC2 is inhibited by low micromolar concentrations of Zn²⁺ (12) and that this reflects a decrease in V_max of the enzyme.³ It is of interest to note that the IC₅₀ of the soluble construct is ~15 µM, an intermediate value among that of the contributing isoforms. Although this value may reflect the loss of regions outside of the domains in question, it may also reflect the contribution of both subunits in defining the Zn²⁺ binding site.

In our attempts to determine the mechanism by which Zn²⁺ inhibits AC, we have made a number of observations to support the premise that it does not function at the catalytic metal binding sites. We wish to emphasize that our findings do not preclude Zn²⁺ binding to site A, a possibility indicated by crystallographic studies. Rather, the biochemical effects of Zn²⁺ binding to this "novel" inhibitory site may be of an affinity such that we are not in a position to observe enzymatically the effects of Zn²⁺ binding to alternative sites. We also note that Zn²⁺ does not generally inhibit two-metal ion-requiring enzymes as are ACs (34). Perhaps upon the disruption of the Zn²⁺ site that inhibits AC activity, we will observe that Zn²⁺ functions more typically, supporting or enhancing AC activity when it occupies a catalytic metal binding site. In that light, it is tempting to speculate that for AC2, which has a lower affinity Zn²⁺ inhibitory site (IC₅₀ = 25 µM), the stimulation of activity observed at the lower Zn²⁺ concentrations reflects the binding of Zn²⁺ to site A. Such questions are currently being explored.

This still leaves the question of how Zn²⁺ binding to this novel site leads to an attenuation of AC activity. The dose-response curve indicates that this is a cooperative process, reflecting either the binding of two (more than one) Zn²⁺ molecules or a Zn²⁺-induced protein-protein interaction. Although we can only speculate at this point, several observations could favor the latter interpretation. Based on current crystallographic data, the physical relationship of CI to CII is altered upon occupancy of the substrate binding site. Subsequent association of AC with G_s or forskolin is believed to allosterically influence the interaction to enhance the catalytic capacity of the enzyme (20, 33). Thus, there is precedence for the regulation of AC via changes in protein-protein interactions. We have also determined that a preassociation of AC with forskolin or G_s protects the enzyme from the inhibitory effects of Zn²⁺. We interpret such findings to suggest that both activators induce a conformational change(s) in the enzyme that minimizes the effects of Zn²⁺. This interpretation as opposed to one evoking steric hindrance of the Zn²⁺ binding site is favored, because forskolin and G_s bind to adjacent but distinct regions of AC and thus would obscure different residues of the enzyme (12, 33).

That the G_s·AC complex would display an altered sensitivity to Zn²⁺ could explain the biphasic dose-response curve for forskolin-stimulated AC activity in N18TG2 membranes (Fig. 5B) compared with that of the recombinant enzyme (Fig. 7A). It can be expected that a population of enzyme in N18TG2 membranes is associated in equilibrium with G and as such exhibits a decreased sensitivity to Zn²⁺. A greater population of enzyme would be associated with G_s when PGE₁-stimulated AC activity in N18TG2 membranes is monitored, thus explaining the higher IC₅₀ value for Zn²⁺ inhibition (Fig. 5A). Having shown that AC1, AC5, and AC6 display an IC₅₀ of ~1-2 µM while AC2 inhibition occurs with an IC₅₀ of ~20-25 µM, we could also account for the data obtained using N18TG2 membranes if a population of AC isoforms were present. Our Northern analysis of AC expression in N18TG2 cells did not detect the mRNA for AC2 (18), but we did not probe for the presence of the other members of this family. Although other isoforms may be present, they are not typically predominant in neuronal cells, and biochemical evidence for their presence in N18TG2 cells has not been reported. The aforementioned observations also suggest the coincidence timing requirements to effect an inhibition of AC by Zn²⁺. Cyclic AMP synthesis would be relatively refractory to an influx of Zn²⁺ should that occur subsequent to hormonal stimulation of the enzyme. It would also appear that the consequences of Zn²⁺ relative to cAMP synthesis will not only depend upon the timing of the input signals but will reflect the nature of the AC isoform predominant in a particular cell or tissue. In regions of the brain in which AC2 is expressed, the stimulation of cAMP synthesis could still occur in the presence of Zn²⁺ and may actually be enhanced under conditions that effectively limit cAMP synthesis supported by either AC1 or AC5.

In this study, we have described a novel regulation of AC by Zn²⁺, whereby the presence of this metal reversibly inhibits its activity. Such a regulation raises the possibility that in the regions of the brain like the hippocampus where Zn²⁺ is believed to function as either a neuromodulator or neurotoxic agent, it could do so in part by altering cAMP levels. In addition to defining a previously unappreciated mechanism of Zn²⁺ action, these experiments suggest a novel target for mediating the physiological effects of Zn²⁺ in the central nervous system as well as in other tissues in which Zn²⁺ concentrations may be significant.

	ACKNOWLEDGEMENTS

We thank Dr. A. G. Gilman (University of Texas Southwestern Medical Center) for support and generosity in providing us with numerous reagents and Dr. R. Taussig (University of Michigan Medical School) for providing us with Sf9 membranes expressing C441R. We also acknowledge the kind gift of Hi-5 membranes expressing recombinant AC-6 from Dr. R. Iyengar (Mt. Sinai School of Medicine) and the expert technical assistance of J. Collins.

	FOOTNOTES

^* This research was supported in part by National Institutes of Health Grants GM34497 (to A. G. G.), GM50514 (to T. H.), DA 03690, and K05-DA00182 (to A. H.) and by the American Heart Association (to C. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, St. Louis University School of Medicine, 1402 S. Grand Blvd., St. Louis, MO 63104. Tel.: 314-577-8155; Fax: 314-577-8156; E-mail: kleinc@slu.edu.

Published, JBC Papers in Press, January 22, 2002, DOI 10.1074/jbc.M108808200

² T. Y. Hudson, unpublished observations.

³ R. K. Sunahara, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: AC, adenylyl cyclase; PGE₁, prostaglandin E₁; G_s, subunit of G protein that stimulates adenylyl cyclase; PDE, phosphodiesterase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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