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Research ArticleCELLULAR AND MOLECULAR

Heterologous Sensitization of Recombinant Adenylate Cyclases by Activation of D2 Dopamine Receptors

Medhane G. Cumbay and Val J. Watts
Journal of Pharmacology and Experimental Therapeutics June 2001, 297 (3) 1201-1209;

Abstract

Persistent activation of Gαi/o-coupled receptors results in an enhanced responsiveness of drug-stimulated adenylate cyclase activity through an unknown mechanism. This agonist-induced heterologous sensitization of drug-stimulated cyclic AMP accumulation has been proposed to be a mechanism by which cells adapt to prolonged Gαi/o activation. Heterologous sensitization was examined in human embryonic kidney 293 cells stably expressing D2Ldopamine receptors in combination with recombinant isoforms of adenylate cyclase. The ability of each isoform to be differentially regulated by G protein subunits and other signaling intermediates allowed us to identify potential mechanisms that are involved in heterologous sensitization of adenylate cyclase. We now report that both short- and long-term activation of D2L dopamine receptors resulted in a marked degree of sensitization of ACI, ACII, ACV, and ACIX, but not ACVIII. The effects of agonist treatment on ACI, ACII, and ACVIII appeared to be dependent upon the ability of these adenylate cyclase isoforms to synergistically respond to selective activators in the presence of activated Gαs. Sensitization of ACV was characterized by enhanced cyclic AMP accumulation following Gαs or forskolin stimulation. Furthermore, agonist pretreatment enhanced the basal levels of cyclic AMP accumulation in ACV/D2L cells, an effect that was not observed with the other adenylate cyclase isoforms. ACIX, which has no known activators other than Gαs, showed robust agonist-induced sensitization of isoproterenol-stimulated cyclic AMP accumulation. In summary, heterologous sensitization appeared to be related to the ability of each adenylate cyclase isoform to be modulated by Gαs.

Acute activation of Gαi/o-coupled receptors inhibits drug-stimulated cyclic AMP accumulation. In contrast, prolonged activation of Gαi/o-coupled receptors results in an enhanced responsiveness of forskolin- and Gαs-stimulated adenylate cyclase [E.C. 4.6.1.1] activity through an unknown mechanism (Thomas and Hoffman, 1987; Ammer and Schulz, 1996; Watts and Neve, 1996; Rhee et al., 2000). This agonist-induced heterologous sensitization of drug-stimulated cyclic AMP accumulation has been proposed to be a mechanism by which cells adapt to prolonged inhibition of cyclic AMP synthesis and may be a cellular model of drug tolerance and dependence (Sharma et al., 1975;Ammer and Schulz, 1996; Watts and Neve, 1996; Nestler and Aghajanian, 1997). Heterologous sensitization of adenylate cyclase is caused by the persistent activation of a number of Gαi/o-coupled receptors, including μ-opioid, D2 and D4 dopamine, A3 adenosine, α2-adrenergic, M4muscarinic, and CB1 cannabinoid receptors (Sharma et al., 1975; Jones and Bylund, 1988; Ammer and Schulz, 1996; Thomas and Hoffman, 1996; Watts and Neve, 1996; Avidor-Reiss et al., 1997;Palmer et al., 1997; Schoffelmeer et al., 1997; Nevo et al., 1998;Watts et al., 1999; Rhee et al., 2000). In addition, studies have revealed that heterologous sensitization occurs in a number of cellular systems, including transient expression systems, stably transfected cultured cells, and primary cultured neurons (Watts and Neve, 1996;Schoffelmeer et al., 1997; Watts et al., 1998; Rhee et al., 2000; V. J. Watts, J. G. Lisinicchia, and M. A. Beazely, unpublished observations). Together these observations suggest that heterologous sensitization is a common phenomenon, and that understanding the mechanisms may reveal information about G protein-coupled receptor signaling in a variety of cell types.

Although the precise molecular mechanisms for heterologous sensitization remain unknown, recent studies have provided evidence that sensitization is influenced by the signal transduction machinery within the cellular environment (Thomas and Hoffman, 1996; Watts and Neve, 1996; Watts et al., 1998; Rhee et al., 2000). Specifically, evidence suggests that agonist-induced sensitization may be influenced by the complement of adenylate cyclase isoforms present within the cell (McDermott and Sharp, 1995; Watts and Neve, 1996; Avidor-Reiss et al., 1997; Varga et al., 1998). There are at least nine distinct molecular forms of adenylate cyclase (ACI-ACIX) that have been partially characterized and each isoform appears to have distinct regulatory requirements with the exception that each isoform is stimulated by Gαs, (for review, see Taussig and Zimmerman, 1998). Types I, III, and VIII are stimulated by calcium/calmodulin and have differential sensitivity to modulation by αi/o (Nevo et al., 1998, V. J. Watts and M. G. Cumbay, unpublished observations). Additionally, ACI shows synergistic activation by Gαs and Ca2+, whereas ACVIII does not (Cali et al., 1994;Wayman et al., 1994; Nielsen et al., 1996). Types V and VI are robustly stimulated by Gαs and forskolin, whereas their activity is inhibited by calcium and Gαi(Taussig et al., 1994; Chabardes et al., 1999). The family of adenylate cyclases with the most members is represented by ACII, ACIV, and ACVII, which are characterized by their ability to be conditionally stimulated by G protein βγ subunits as well as their apparent lack of direct modulation by Gαi/o (Taussig and Zimmerman, 1998). In addition, ACII and ACVII are stimulated by activators of protein kinase C such as PMA (Watts and Neve, 1997; Taussig and Zimmerman, 1998). Finally, ACIX is stimulated by Gαs but is not potently stimulated by forskolin (Antoni et al., 1998; Hacker et al., 1998).

The regulatory properties described above and recent observations that the recombinant isoforms of adenylate cyclase show differential sensitization (Thomas and Hoffman, 1996; Watts and Neve, 1996;Avidor-Reiss et al., 1997; Nevo et al., 1998; Rhee et al., 2000) provide the basis for present studies. We have constructed HEK293 cells stably expressing D2L dopamine receptors in combination with ACI, ACII, ACV, ACVIII, or ACIX in an effort to identify potential mechanisms for heterologous sensitization of adenylate cyclase. In an effort to control for sensitization of endogenous adenylate cyclase activity in HEK293 cells, we have characterized and used conditions that show selective activation of recombinant adenylate cyclase isoforms. We now report that both short- and long-term activation of D2L dopamine receptors resulted in a marked degree of sensitization of ACI, ACII, ACV, and ACIX, but not ACVIII. This time-dependent heterologous sensitization appeared to be influenced by the regulatory properties of each adenylate cyclase isoform. In particular, sensitization appeared to be related to the ability of adenylate cyclase isoforms to be activated by Gαs, supporting a hypothesis that heterologous sensitization involves Gαs.

Experimental Procedures

Materials.

[3H]Cyclic AMP was purchased from PerkinElmer Life Science Products (Boston, MA). Forskolin, (−)-quinpirole, and spiperone were purchased from RBI/Sigma (Natick, MA). PMA and A23187 were purchased from Calbiochem (La Jolla, CA). Fetal clone serum and bovine calf serum were purchased from Hyclone (Logan, UT). Most other reagents were purchased from Sigma (St. Louis, MO).

Cell Culture.

HEK293 cells expressing ACI, ACII, ACV, and ACVIII were obtained from Dr. Daniel Storm (University of Washington, Seattle, WA). Each cell line was transfected with pcDNA1-D2L as described previously (Watts and Neve, 1996), creating the cell lines ACI/D2L, ACII/D2L, ACV/D2L, and ACVIII/D2L. HEK293 cells were transfected with pcDNA3-ACIX using lipofectAMINE (Life Technologies, Grand Island, NY) according to the manufacture's instructions. Clones were isolated by selection with G418 (600 μg/ml) and were screened for isoproterenol-stimulated cyclic AMP accumulation as described below. The ACIX cell line was then transfected with pcDNA1-D2L (10 μg) and pBabe Puro (1 μg) using lipofectAMINE and clones were isolated following selection with puromycin (2 μg/ml) and G418 (600 μg/ml). All cells were maintained in culture medium consisting of Dulbecco's modified Eagle's medium supplemented with 5% fetal clone serum, 5% bovine calf serum, 0.05 U/ml penicillin, and 50 μg/ml streptomycin. The medium used to maintain HEK-ACI/D2L, ACII/D2L, ACV/D2L, and ACVIII/D2L cells was supplemented with hygromycin (460 U/ml) and puromycin (2 μg/ml). The medium used for the ACIX/D2L cell line was supplemented with G418 (300 μg/ml) and puromycin (2 μg/ml). Cells were propagated in a humidified incubator in the presence of 10% CO2.

Cyclic AMP Accumulation Assay.

Cells were seeded at densities between 100,000 and 150,000 cells/well in 48-well cluster plates and grown to confluence. Cells were pretreated for 2 or 18 h in culture medium, in the presence or absence of the D2 agonist quinpirole (1 μM). Where indicated, some pretreatments were performed in the presence of the D2 antagonist spiperone (1 μM). Following pretreatment, the cells were washed three times for 3 to 4 min with 200 μl/well of assay buffer (Earle's balanced salt solution containing 0.02% ascorbic acid and 2% bovine calf serum). The cells were then placed on ice and the indicated drugs were added in the presence of spiperone (1 μM) to prevent activation of D2Lreceptors by residual quinpirole (Watts and Neve, 1996). The cells were then incubated in a 37°C water bath for 15 min. Following the incubation, the stimulation media was decanted and the reaction was terminated with 100 μl/well of ice-cold 3% trichloroacetic acid. The 48-well cluster plates were stored at 4°C for up to 1 week prior to analysis.

Quantification of Cyclic AMP.

Cyclic AMP accumulation was quantified using a competitive binding assay adapted fromNordstedt and Fredholm (1990) with minor modifications (Watts and Neve, 1996). Duplicate samples of the cell lysate (15 μl) were added to reaction tubes. [3H]Cyclic AMP (∼1 nM final concentration) and cyclic AMP binding protein (ca. 150 μg) were diluted in cyclic AMP assay buffer (100 mM Tris/HCl, pH, 7.4, 100 mM NaCl, 5 mM EDTA) and then added to each well for a total volume of 550 μl. The tubes were incubated on ice for 2 h and were harvested by filtration (Packard Unifilter GF/C) using a 96-well Packard Filtermate Cell harvester (Meriden, CT). The filters were allowed to dry and Microscint O scintillation fluid was added. Radioactivity on the filters was determined using a Packard TopCount scintillation/luminescence detector. Cyclic AMP concentrations in each sample were estimated in duplicate from a standard curve ranging from 0.1 to 300 pmol of cyclic AMP per assay.

Results

Regulation of Endogenous Adenylate Cyclase in HEK-D2LCells.

Acutely, D2 agonists inhibit cyclic AMP accumulation in HEK293 cells expressing D2Lreceptors, whereas short-term (2 h) pretreatment with the same drugs results in a heterologous sensitization of forskolin (10 μM)-stimulated cyclic AMP accumulation (Fig.1; Watts and Neve, 1996). Long-term (18 h) activation of D2 dopamine receptors also sensitized forskolin (10 μM)-stimulated cyclic AMP accumulation (Fig.1). The apparent magnitude of agonist-induced heterologous sensitization in HEK-D2L cells was dependent upon the concentration of forskolin that was used to subsequently stimulate cyclic AMP accumulation (Table 1; Watts and Neve, 1996). For example, agonist treatment of HEK-D2L cells followed by stimulation with 10 μM forskolin caused a marked increase in cyclic AMP accumulation that was 7-fold greater than that observed in vehicle-treated cells (Table1; Watts and Neve, 1996). On the other hand, sensitization studies using forskolin at concentrations of 100 nM revealed no increase in cyclic AMP accumulation compared with basal levels and no enhanced response in agonist-treated cells (Table 1). Agonist treatment also failed to induce significant sensitization of adenylate cyclase activity in HEK-D2L cells following activation of the endogenous β-adrenergic receptor by isoproterenol (Table 1). Additionally, agonist treatment did not alter basal levels of cyclic AMP accumulation in HEK-D2L cells (Fig. 1).

Figure 1
Figure 1

Effect of agonist pretreatment on cyclic AMP accumulation in HEK-D2L cells. Cells were pretreated for 2 h (A) or 18 h (B) with vehicle or quinpirole (1 μM). Following agonist pretreatment, cells were washed extensively and cyclic AMP accumulation was determined in the absence (basal) or presence of 10 μM forskolin (FSK) for 15 min. Data shown are mean ± S.E. of seven to nine independent experiments. *p < 0.05 compared with vehicle-treated cells (Student's paired t test).

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Table 1

Effects of agonist treatment on cyclic AMP accumulation in HEK-D2L cells

In an effort to further characterize heterologous sensitization of endogenously expressed adenylate cyclase in HEK-D2L cells, we examined drug-stimulated cyclic AMP accumulation using isoform-selective adenylate cyclase activators. Short- and long-term exposure of HEK-D2L cells to quinpirole did not alter the cyclic AMP response to A23187, indicating that the endogenous adenylate cyclases in HEK-D2Lcells are not activated by Ca2+ (Table 1; data not shown for 2-h treatment). Similarly, cyclic AMP accumulation in HEK-D2L cells was not altered by the phorbol ester PMA in vehicle- or agonist-treated cells (Table 1). Together, these data show that agents that selectively activate ACI and ACVIII (A23187) or ACII (PMA) do not stimulate cyclic AMP accumulation in HEK-D2L cells under the conditions tested here. Although the exact complement of adenylate cyclases expressed in HEK cells is not known, these studies suggest that HEK-D2L cells are a useful system to examine heterologous sensitization of recombinant isoforms of adenylate cyclase that respond to the selective activators used in this study.

Effects of Short- and Long-Term D2 Receptor Activation on ACI and ACVIII.

Type I or VIII adenylate cyclase was stably transfected in combination with the dopamine D2Lreceptor in HEK cells. Stimulation of either ACI/D2L cells and ACVIII/D2L cells with the calcium ionophoreA23187 markedly increased cyclic AMP accumulation (Figs.2 and3). Exposure of ACI/D2L cells to quinpirole enhancedA23187-stimulated cyclic AMP accumulation compared with vehicle-treated cells by 3- and 5-fold following short- and long-term agonist exposure, respectively (Fig. 2). Heterologous sensitization of ACI was prevented when quinpirole treatments were carried out in the presence of the D2 antagonist spiperone (Fig. 2). In contrast to the robust effects of agonist treatment on ACI activity, short-term agonist exposure on ACVIII/D2L cells did not result in significant heterologous sensitization of A23187-stimulated cyclic AMP accumulation (Fig. 3A). Furthermore, 18 h of agonist exposure produced only a modest degree of heterologous sensitization of ACVIII (1.3-fold) compared with vehicle-treated cells (Fig. 3B).

Figure 2
Figure 2

Agonist-induced heterologous sensitization ofA23187-stimulated cyclic AMP accumulation in ACI/D2L cells. Cells were pretreated for 2 h (A) or 18 h (B) with vehicle or quinpirole (1 μM). Following agonist pretreatment, cells were washed extensively and cyclic AMP accumulation was determined in the absence (basal) or presence of 10 μM A23187 for 15 min. Where indicated some pretreatments were completed in the presence of 1 μM spiperone (+Spip). Data shown are mean ± S.E. of seven to eight independent experiments. *p < 0.05 compared with vehicle-treated cells (Student's paired t test).

Figure 3
Figure 3

Agonist-induced heterologous sensitization ofA23187-stimulated cyclic AMP accumulation in ACVIII/D2Lcells. Cells were pretreated for 2 h (A) or 18 h (B) with vehicle or quinpirole (1 μM). Following agonist pretreatment, cells were washed extensively and cyclic AMP accumulation was determined in the absence (basal) or presence of 10 μM A23187 for 15 min. Where indicated some pretreatments were completed in the presence of 1 μM spiperone (+Spip). Data shown are mean ± S.E. of seven to eight independent experiments. *p < 0.05 compared with vehicle-treated cells (Student's paired t test).

Additional studies examined the ability of ACVIII to be synergistically activated by Gαs and Ca2+under the conditions used in Fig. 3. Values for drug-stimulated cyclic AMP accumulation levels in ACVIII/D2L cells were 4.4 ± 1.0 pmol/well (n = 7) in the presence of 1 μM isoproterenol (Gαs) and 7.3 ± 1.8 pmol/well (n = 7) in presence of 10 μM A23187(Ca2+). Simultaneous stimulation of ACVIII/D2L cells by both agents (isoproterenol and A23187) elevated cyclic AMP levels to 11.1 ± 2.1 pmol/well (n = 7), indicating that the activation of ACVIII by Gαs and Ca2+ is additive and not synergistic.

A23187-stimulated cyclic AMP accumulation appeared to be greater in ACI/D2L cells compared with ACVIII/D2L cells; however, the actual fold increase compared with basal levels of cyclic AMP accumulation was similar in both cell lines (Figs. 2 and 3). Thus, in the absence of prior D2 receptor activation, both cell lines are equally responsive to Ca2+. These results suggest that differences in Ca2+ responsiveness do not explain the lack of sensitization of ACVIII compared with ACI. On the other hand, differences in Ca2+ responsiveness as well as adenylate cyclase expression levels could be masked in the absence of phosphodiesterase inhibitors because of differences in cyclic AMP degradation. Thus, the observed differences between ACI and ACVIII were further explored in sensitization and cyclic AMP accumulation studies that were completed in the presence of the phosphodiesterase inhibitor IBMX. Similar to the studies described above, A23187-stimulated cyclic AMP accumulation was similar in ACI/D2L and ACVIII/D2Lcells following vehicle treatment (Fig.4A). Short-term sensitization studies completed in the presence of IBMX found that agonist treatment resulted in sensitization of only ACI and not ACVIII (Fig. 4A). The ability of cholera toxin-stimulated Gαs to stimulate both ACI and ACVIII in the presence and absence of A23187 was also examined in the presence of IBMX. The activation of Gαs(via cholera toxin) appeared to synergistically enhanceA23187-stimulated ACI activity, whereas, the effects of concurrent Gαs activation and A23187 on the activity of ACVIII appeared to be only additive (Fig. 4, B and C).

Figure 4
Figure 4

Regulation of cyclic AMP accumulation in ACI/D2L and ACVIII/D2L cells in the presence of 500 μM IBMX. ACI/D2L, and ACVIII/D2L cells were pretreated for 2 h with vehicle or quinpirole (1 μM) (A). Following agonist pretreatment, cells were washed extensively and cyclic AMP accumulation was determined in the presence of 10 μMA23187 for 15 min. B and C, basal and A23187-stimulated cyclic AMP accumulation in cells following 2 h of pretreatment in culture medium in the absence (control) or presence of 200 ng/ml cholera toxin (+CTX). Data shown are mean ± S.E. of three to seven independent experiments. *p < 0.05 compared with vehicle-treated cells (Student's t test).

Effects of Short- and Long-Term D2 Receptor Activation on ACII.

HEK ACII/D2L cells were used to examine drug-stimulated cyclic AMP accumulation following pretreatment with quinpirole for 2 or 18 h. Quinpirole pretreatment resulted in marked sensitization of PMA-stimulated cyclic AMP accumulation compared with cells that were vehicle-treated (Fig.5). The magnitude of sensitization was time-dependent and was greater following long-term agonist exposure. Both short- and long-term quinpirole-induced sensitization were blocked when pretreatments were carried out in the presence of spiperone (Fig.5). In contrast to effects on PMA-stimulated cyclic AMP accumulation, agonist treatment of ACII/D2L cells did not alter cyclic AMP accumulation under basal, forskolin-stimulated, isoproterenol-stimulated, or A23187-stimulated conditions (Table2).

Figure 5
Figure 5

Agonist-induced heterologous sensitization of PMA-stimulated cyclic AMP accumulation in ACII/D2L cells. Cells were pretreated for 2 h (A) or 18 h (B) with vehicle or quinpirole (1 μM). Following agonist pretreatment, cells were washed extensively and cyclic AMP accumulation was determined in the absence (basal) or presence of 100 nM PMA for 15 min. Where indicated some pretreatments were completed in the presence of 1 μM spiperone (+Spip). Data shown are mean ± S.E. of seven to eight independent experiments. *p < 0.05 compared with vehicle-treated cells (Student's paired t test).

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Table 2

Effects of agonist treatment on ACII activity

Effects of Short- and Long-Term D2 Receptor Activation on ACV.

ACV/D2L cells were exposed to quinpirole for 2 or 18 h and basal and drug-stimulated cyclic AMP levels were examined. The basal activity of ACV was markedly enhanced (ca. 4-fold) following 2 h of quinpirole pretreatment compared with vehicle-pretreated cells (Fig. 6A). Extending agonist incubations to 18 h resulted in an even greater increase in ACV basal activity that was approximately 18-fold greater than vehicle-treated cells (Fig. 6B). Short- and long-term heterologous sensitization of basal levels of ACV-stimulated cyclic AMP accumulation was prevented when quinpirole treatments were carried out in the presence of spiperone (Fig. 6, A and B). The observed agonist-induced increases in basal cyclic AMP levels were specific to ACV/D2L cells and were not observed with the other cell types examined in the present study (Figs. 1-7).

Figure 6
Figure 6

Agonist-induced heterologous sensitization of basal and drug-stimulated ACV activity. ACV/D2L cells were pretreated for 2 or 18 h with vehicle or quinpirole (1 μM). Following agonist pretreatment, cells were washed extensively. Basal cyclic AMP accumulation was examined following 2 h (A) and 18 h (B) of agonist treatment. Where indicated some agonist pretreatments were completed in the presence of 1 μM spiperone (+Spip). Isoproterenol- (Iso, 1 μM) and forskolin (FSK, 100 nM)-stimulated cyclic AMP accumulation was also measured following 2 h (C) and 18 h (D) of agonist pretreatment. Data shown are mean ± S.E. of five to nine independent experiments. *p < 0.05 compared with vehicle-treated cells (Student's paired t test).

We identified conditions that could be used to activate ACV with relative selectivity compared with the endogenous adenylate cyclase in HEK-D2L cells. Stimulation of cyclic AMP accumulation in HEK-D2L cells by 1 μM isoproterenol and 100 nM forskolin was modest or absent, respectively (Table 1). In contrast, these agents had marked effects in ACV/D2L cells and the levels of cyclic AMP were increased from 4.1 ± 1.7 pmol/well (n = 9) under basal conditions to 42 ± 8.4 pmol/well (n = 9) in the presence of 1 μM isoproterenol and to 89 ± 26 pmol/well in the presence of 100 nM forskolin (n = 5). Sensitization studies further demonstrated that pretreatment of ACV/D2L cells with quinpirole for 2 or 18 h resulted in a significant increase in drug-stimulated cyclic AMP accumulation. The responses to 1 μM isoproterenol and 100 nM forskolin were increased by approximately 6-fold following 2 h of agonist treatment and 10-fold following 18 h of agonist treatment (Fig.6, C and D). Agonist treatment studies carried out in HEK-D2L cells revealed no significant sensitization of endogenous adenylate cyclase to 1 μM isoproterenol or 100 nM forskolin (Table 1).

Effects of Short- and Long-Term D2 Receptor Activation on ACIX.

Agents that selectively activate ACIX are not available, but HEK cells expressing ACIX show marked isoproterenol-stimulated cyclic AMP accumulation compared with HEK-D2Lcells (Fig. 7). Because HEK-D2L cells showed a trend toward developing heterologous sensitization to isoproterenol (Table 1), sensitization experiments with ACIX/D2L cells were completed simultaneously with HEK-D2L cells for direct comparison. Both ACIX/D2L and HEK-D2L cells were pretreated with quinpirole for 2 or 18 h and cyclic AMP levels were examined. Pretreatment with quinpirole resulted in sensitization of isoproterenol-stimulated ACIX activity following both 2 and 18 h of treatment. Both short- and long-term agonist-induced sensitization of ACIX was prevented by coincubation with spiperone (Fig. 7). In contrast to effects of agonist treatment in ACIX/D2L cells, quinpirole pretreatment failed to produce a significant degree (<2-fold) of sensitization to isoproterenol in HEK-D2L cells. Furthermore, basal levels of cyclic AMP accumulation in ACIX/D2L and HEK-D2L cells were not altered following short- or long-term agonist treatment (Table1; data not shown for ACIX).

Figure 7
Figure 7

Agonist-induced heterologous sensitization of isoproterenol-stimulated cyclic AMP accumulation in ACIX/D2L cells. Cells were pretreated for 2 h (A) or 18 h (B) with vehicle or quinpirole (1 μM). Following pretreatment, cells were washed extensively and cyclic AMP accumulation was stimulated with isoproterenol (1 μM) for 15 min. Where indicated, some pretreatments were completed in the presence of 1 μM spiperone (+Spip). Basal levels of cyclic AMP accumulation in vehicle-treated cells (18 h) were 3.0 ± 0.9 pmol/well for HEK-D2Lcells and 2.1 ± 0.4 pmol/well for ACIX/D2L cells (n = 6 for both cell lines). Data shown are mean ± S.E. of four to six independent experiments. *p < 0.05 compared with vehicle-treated cells (Student's paired t test).

Discussion

Sensitization of the cyclic AMP signaling pathway is a neuroadaptive response that may be involved in drug dependence, antidepressant drug therapy, and schizophrenia (Memo et al., 1983; Chen and Rasenick, 1995; Nestler and Aghajanian, 1997; Duman, 1998; Bohn et al., 2000). Mechanistic studies of sensitization in native tissues are difficult because individual brain regions routinely express multiple isoforms of adenylate cyclase. In the present study, we examined heterologous sensitization of select neuronal isoforms of adenylate cyclase by stably transfecting the D2 dopamine receptor with individual isoforms of adenylate cyclase in HEK293 cells. We used protocols or conditions that would allow us to selectively activate ACI, ACII, ACV, ACVIII, or ACIX to determine the isoform specificity and the potential mechanisms for agonist-induced heterologous sensitization of adenylate cyclase.

The present study suggests that isoform specificity for short- and long-term sensitization is similar and that agonist-induced heterologous sensitization is time-dependent for the isoforms of adenylate cyclase examined here. We found that a significant degree of sensitization occurred rapidly (within 2 h) and extending the agonist pretreatment to 18 h appeared to enhance the observed magnitude of sensitization for ACI, ACII, ACV, and ACIX. These observations are consistent with previous studies examining ACI and ACV (Watts and Neve, 1996; Avidor-Reiss et al., 1997; Nevo et al., 1998;Rhee et al., 2000). A study completed in HEK293 cells reported that short-term activation (30 min) of M2 muscarinic receptors sensitizes drug-stimulated ACVI activity, but not ACI or ACII activity (Thomas and Hoffman, 1996). These results may be explained by the short agonist treatment time and the fact that ACVI (and ACV) may show more rapid and robust sensitization compared with other adenylate cyclase isoforms.

Previous studies of recombinant isoforms of adenylate cyclase have suggested that sensitization is also influenced by the choice of agent or condition used to examine cyclic AMP accumulation (Thomas and Hoffman, 1996; Watts and Neve, 1996). Similar to our previous work, agonist pretreatment of ACII/D2L cells resulted in a marked degree of sensitization to PMA-stimulated cyclic AMP accumulation (Watts and Neve, 1996). We also found that agonist treatment failed to alter forskolin- and isoproterenol-stimulated cyclic AMP accumulation in ACII/D2L cells. The lack of ACII sensitization to these two agents appears to be consistent with other reports using nonselective activators such as forskolin, Gαs-coupled receptor agonists, or constitutively active Gαs (Thomas and Hoffman, 1996; Avidor-Reiss et al., 1997; Nevo et al., 1998; Rhee et al., 2000). In fact, agonist treatment of cells expressing ACII results in a significant decrease in stimulated cyclic AMP accumulation (Nevo et al., 1998; Rhee et al., 2000). The observed sensitization of ACII to protein kinase C activation is in contrast to one study that found that chronic μ-opioid receptor did not induce sensitization of phorbol ester-stimulated ACII activity (Avidor-Reiss et al., 1997). The reason for this discrepancy is unclear and may be due to differences in the Gαi/o-coupled receptors (D2 versus μ), the choice of cell lines (HEK293 versus COS 7), transfection methodology (stable versus transient), or other laboratory procedures (Watts and Neve, 1996; Avidor-Reiss et al., 1997). Specificity for sensitization of drug-stimulated cyclic AMP accumulation was also observed for other isoforms of adenylate cyclase. For example, long-term agonist treatment in ACI/D2L cells enhanced the cyclic AMP response toA23187 by 5-fold, whereas the response to 10 μM forskolin was enhanced only 2.3-fold (data not shown) compared with the 7-fold sensitization observed in HEK-D2L cells. Sensitization specificity was also observed for isoproterenol-stimulated ACIX activity compared with basal levels of cyclic AMP accumulation in ACIX/D2L cells.

In addition to the specificity described above, unique sensitization characteristics were observed upon examining sensitization of ACV-modulated cyclic AMP accumulation. Initial studies in ACV/D2L and HEK-D2L cells revealed sensitization of ACV when 1 μM isoproterenol or 100 nM forskolin was used to stimulate cyclic AMP accumulation in both cell lines. Moreover, ACV was the only isoform that exhibited sensitization of basal cyclic AMP accumulation. Opioid receptor stimulation also caused sensitization of the basal activity of ACV and the closely related ACVI (Avidor-Reiss et al., 1997). These observations may indicate that agonist pretreatment increases basal activity through a mechanism that is distinct to ACV and ACVI. Recently, a mutant ACV (F400Y) was shown to exhibit enhanced basal activity in the absence of drug stimulation (Zimmermann et al., 1999). Furthermore, the F400Y ACV mutant was also sensitized to activation by forskolin or Gαs (Zimmermann et al., 1999). Thus, persistent agonist treatment appears to result in a sensitized phenotype of ACV that shares a number of biochemical properties with the F400Y ACV mutant (Zimmermann et al., 1999).

Short- and long-term heterologous sensitization are blocked by pretreatment with pertussis toxin, implicating Gαi/o in heterologous sensitization (Watts and Neve, 1996; Watts et al., 1998, 1999; Rhee et al., 2000). There is also evidence that a decrease in the expression of Gαi may be involved in heterologous sensitization (Van Vliet et al., 1993; Reithmann and Werdan, 1995;Watts et al., 1999). More recently, a change in the detergent solubility of Gαi was correlated with the onset of heterologous sensitization (Bayewitch et al., 2000). The present results showing that ACVIII is weakly sensitized, combined with observations that ACVIII is only weakly inhibited by Gαi/o proteins, also support a strong role for Gαi/o proteins in sensitization (Nevo et al., 1998; V. J. Watts and M. G. Cumbay, unpublished observations). However, we also found marked sensitization of adenylate cyclases that are not directly modulated by Gαi/o, ACII and ACIX (Taussig and Zimmerman, 1998). Together, these observations suggest that there is an important role of Gαi/o proteins in sensitization, but this role appears to differ among recombinant isoforms of adenylate cyclase.

Receptor activation of Gαi/o proteins also promotes the release of βγ subunits, and studies have shown that sequestration of βγ subunits (via the coexpression of proteins that bind βγ subunits) prevents the development of heterologous sensitization (Avidor-Reiss et al., 1996; Thomas and Hoffman, 1996;Rhee et al., 2000). Because the individual isoforms are differentially regulated by βγ subunits, the mechanism for βγ-mediated sensitization would be predicted to differ among isoforms. For example, Gαs- and protein kinase C-stimulated ACII activity is potentiated by βγ subunits, whereas, the activity of ACI is inhibited by βγ subunits (Tang and Gilman, 1991; Watts and Neve, 1997; Taussig and Zimmerman, 1998), and several isoforms of adenylate cyclase are not modulated by βγ subunits (for review, seeTaussig and Zimmerman, 1998). The complexity of βγ-effects are also evident from studies that have shown that sequestration of βγ subunits can enhance the activity of ACV and also block agonist-induced sensitization of ACV (Bayewitch et al., 1998; Rhee et al., 2000). The observations described above suggest that unambiguously identifying the role of βγ subunits in sensitization will be difficult. Furthermore, the effects of βγ subunits will be dependent upon the recombinant isoforms under investigation.

The results discussed above suggest that, similar to their acute regulatory properties, the precise mechanisms of heterologous sensitization of adenylate cyclase may differ between individual isoforms (Thomas and Hoffman, 1996; Watts and Neve, 1996; Avidor-Reiss et al., 1997; Taussig and Zimmerman, 1998; Rhee et al., 2000). In spite of their differential regulation, all recombinant isoforms of adenylate cyclase are activated by Gαs and we have proposed that one mechanism of sensitization is enhanced Gαs activity or enhanced Gαs/adenylate cyclase interactions (Watts and Neve, 1996). We have shown that select recombinant isoforms of adenylate cyclase as well as the endogenous adenylate cyclases expressed in C6 glioma, HEK293, and NS20Y cells are sensitized following persistent D2 receptor activation (Watts and Neve, 1996; Watts et al., 1998). In addition, several other reports have provided support for a role of Gαsin heterologous sensitization of adenylate cyclase (Jones and Bylund, 1990; Chen and Rasenick, 1995; Ammer and Schulz, 1996; Watts and Neve, 1996; Ammer and Schulz, 1997). We now report that persistent activation of the D2 dopamine receptor results in marked sensitization of ACI, ACII, ACV, ACIX, but not ACVIII. Our present observations may be explained by the ability of these isoforms to be modulated by Gαs. Persistent agonist treatment induced a marked degree of heterologous sensitization ofA23187-stimulated ACI activity and PMA-stimulated ACII activity. Although the activation of ACI and ACII differs in many ways, both are synergistically activated by Gαs under our sensitization conditions, suggesting that they may share an overlapping mechanism of heterologous sensitization (Jacobowitz and Iyengar, 1994;Wayman et al., 1994; Watts and Neve, 1997; Taussig and Zimmerman, 1998). In contrast, ACVIII is not synergistically activated by Gαs in presence of A23187 and also shows very weak heterologous sensitization (Cali et al., 1994; Nielsen et al., 1996; present study). Thus, heterologous sensitization of ACI, ACII, and ACVIII may be dependent upon synergistic response to selective activators and Gαs. Sensitization studies of ACV also provided additional support for a role of Gαs in heterologous sensitization. Finally, ACIX, for which the only direct modulator appears to be Gαs, showed robust agonist-induced heterologous sensitization in ACIX/D2L cells (Hacker et al., 1998; Taussig and Zimmerman, 1998). The observations described above provide strong evidence that Gαs is required for sensitization of adenylate cyclase and that the magnitude of sensitization is dependent on the ability of Gαs to modulate each isoform.

The precise molecular mechanisms for heterologous sensitization of recombinant adenylate cyclase are unknown; however, the present study suggests a primary role for Gαs. We have also demonstrated that the specificity for sensitization is dependent upon the specific modulators or conditions that are used to examine cyclic AMP accumulation following agonist treatment.

Acknowledgments

We acknowledge Christopher Johnston and Dr. Kim Neve for careful reading of the manuscript. We also thank Dr. Daniel Storm for the cDNA for ACIX and HEK293 cells expressing ACI, ACII, ACV, and ACVIII.

Footnotes

  • Send reprint requests to: Val J. Watts, Ph.D., Purdue University, MCMP 1333, RHPH 224A, West Lafayette, IN 47907. E-mail: wattsv{at}pharmacy.purdue.edu

  • This work was supported by U.S. Public Health Service Grant MH60397, a National Alliance for Research on Schizophrenia and Depression Young Investigator Award, and Purdue University.

  • Abbreviations:
    ACI-IX
    type I-IX adenylate cyclase
    PMA
    phorbol-12-myristate-13-acetate
    HEK
    human embryonic kidney
    IBMX
    isobutylmethylxanthine
    • Received October 6, 2000.
    • Accepted March 7, 2001.

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Research ArticleCELLULAR AND MOLECULAR

Heterologous Sensitization of Recombinant Adenylate Cyclases by Activation of D2 Dopamine Receptors

Medhane G. Cumbay and Val J. Watts
Journal of Pharmacology and Experimental Therapeutics June 1, 2001, 297 (3) 1201-1209;
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