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Keywords:

  • adenylate cyclase;
  • D2 dopamine receptor;
  • heterologous sensitization;
  • neuronal cell line;
  • protein kinase A;
  • superactivation

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Production and maintenance of cell line
  6. Cyclic AMP accumulation assay
  7. Quantification of cyclic AMP
  8. Saturation binding assays
  9. Immunodetection
  10. Adenylate cyclase isoform identification
  11. Results
  12. Inhibition of adenylate cyclase in CAD-D2L cells
  13. Sensitization of cyclic AMP accumulation in CAD-D2L cells
  14. Effect of PKA activators on heterologous sensitization
  15. Effects of PDE inhibition on cyclic AMP accumulation
  16. Effects of modulators of PKA activity on PKA subunit expression
  17. Analysis of adenylate cyclase isoform expression pattern
  18. Discussion
  19. Acknowledgements
  20. References

Persistent activation of Gαi/o-coupled receptors results in a paradoxical enhancement of subsequent drug-stimulated adenylate cyclase activity. The exact mechanism of this up-regulation in the cyclic AMP signaling pathway, known as heterologous sensitization, remains undefined. The present study was designed to investigate the involvement of cyclic AMP-dependent protein kinase in D2L receptor–mediated sensitization in a neuronal cellular environment. The current studies were conducted in the Cath.a differentiated (CAD) cell line transfected stably with the D2L dopamine receptor (CAD-D2L). Long-term 18 h treatment with the D2 receptor agonist, quinpirole, resulted in a two-fold enhancement of forskolin-stimulated cyclic AMP accumulation. Similarly, long-term treatment with the PKA inhibitors, H89 or Rp-8Br-cAMP, also enhanced adenylate cyclase activity. In contrast, long-term activation of protein kinase A (PKA) by forskolin, isobutylmethylxanthine (IBMX), or dibutyryl cyclic AMP caused a significant reduction in subsequent forskolin-stimulated cyclic AMP accumulation and reduced both quinpirole- and H89-induced heterologous sensitization. The effects of PKA inhibitors and activators did not involve changes in PKA subunit expression. RT-PCR analysis of adenylate cyclase isoform expression patterns revealed the expression of mRNA for ACVI and ACIX in CAD-D2L cells. The ability of ACVI to be negatively regulated by PKA is consistent with the observation that inhibition of PKA results in heterologous sensitization of adenylate cyclase activity in CAD-D2L cells.

Abbreviations used
ACI–IX

type-I–IX adenylate cyclase

CBS

calf bovine serum

cyclic AMP

cyclic adenosine monophosphate

CAD

Cath.a differentiated cells

DTT

dithiothreitol

EBSS

Earle's balanced salt solution

ECF

enhanced chemifluorescence

HEK293

human embryonic kidney cells

IBMX

isobutylmethylxanthine

PDE

phosphodiesterase

PKA

protein kinase A

PMSF

phenylmethylsulfonyl fluoride

PVDF

polyvinylidene difluoride

SDS–PAGE

sodium dodecyl sulfate–polyacrylamide gel electrophoresis

TCA

trichloroacetic acid.

Prolonged activation of Gαi/o-coupled receptors induces an enhanced response to subsequent drug-stimulated cyclic AMP accumulation in several cellular models (Sharma et al. 1975; Thomas and Hoffman 1987; Watts and Neve 1996; Schoffelmeer et al. 1997; Rhee et al. 2000). This phenomenon, termed superactivation or heterologous sensitization of adenylate cyclase, occurs following persistent activation of a number of Gαi/o-coupled receptors including µ opioid receptors, D2 dopamine receptors, and CB1 cannabinoid receptors (Sharma et al. 1975; Watts and Neve 1996; Avidor-Reiss et al. 1997; Rhee et al. 2000). Enhanced responsiveness of the cyclic AMP signaling pathway has been proposed to be a neuroadaptive mechanism in response to chronic inhibitory neurotransmission and may play a role in the tolerance, dependence, and withdrawal commonly seen with various drugs of abuse (Sharma et al. 1975; Thomas and Hoffman 1987; Nestler and Aghajanian 1997; Tzavara et al. 2000).

Although the precise mechanisms of cellular heterologous sensitization remain unknown, the development of sensitization is prevented by pertussis toxin pretreatment and also by sequestering released βγ subunits (Avidor-Reiss et al. 1996; Thomas and Hoffman 1996; Watts and Neve 1996; Watts et al. 1998; Rhee et al. 2000; Rubenzik et al. 2001). Heterologous sensitization has been associated with changes in the expression and localization of Gαi/o proteins(Van Vliet et al. 1993; Watts et al. 1999; Bayewitch et al. 2000). These observations are consistent with a Gαi/o/βγ-dependent mechanism for the development of heterologous sensitization. On the other hand, many isoforms of adenylate cyclase show differential sensitization, which may be dependent upon the cellular model and method of adenylate cyclase stimulation employed (Thomas and Hoffman 1996; Watts and Neve 1996; Avidor-Reiss et al. 1997; Nevo et al. 1998; Rhee et al. 2000). Additional observations suggest that the distal mechanisms of heterologous sensitization may in part be determined by the distinct regulatory properties of individual adenylate cyclase isoforms (Taussig and Zimmerman 1998; Cumbay and Watts 2001). Thus, the particular expression patterns of adenylate cyclase isoforms among cell lines and brain regions may impart differences in adenylate cyclase responsiveness leading to differential mechanisms of sensitization (McDermott and Sharp 1995; Lane-Ladd et al. 1997; Chern 2000; Cumbay and Watts 2001).

The unique mechanisms for heterologous sensitization of adenylate cyclase as a result of expression patterns and cellular model diversity form the basis of the present study. Specifically, we wanted to examine the potential mechanism of heterologous sensitization in a novel neuronal cell line that was derived from the central nervous system. Therefore, the present studies were executed in CAD cells, a variant of the Cath.a cell line, which was originally derived from a neuronal brain tumor (Suri et al. 1993). These clonally derived cells have a neuronal phenotype and are easily differentiated to develop processes and varicosities upon the removal of serum (Qi et al. 1997). CAD cells were transfected with the dopamine D2L receptor and were used as a model cell line to study drug-induced sensitization of adenylate cyclase. Long-term (18 h) treatment with the D2 receptor agonist, quinpirole, resulted in a heterologous sensitization of subsequent forskolin-stimulated cyclic AMP accumulation in CAD-D2L cells. Additional studies revealed that direct inhibition of PKA resulted in a similar enhancement of adenylate cyclase activity, suggesting that long-term inhibition of PKA induces heterologous sensitization in CAD-D2L cells. Furthermore, concomitant pretreatment with activators of PKA attenuated drug-induced heterologous sensitization, and, when used alone, PKA activators resulted in a subsequent desensitization of adenylate cyclase activity. Analysis of PKA subunit expression following pretreatment with both inhibitors and activators of PKA suggested that changes in subunit expression were not involved in sensitization. RT-PCR analysis confirmed the presence of mRNA corresponding to ACVI and ACIX in CAD cells. Taken together, the data described herein support a potential role for PKA in heterologous sensitization following long-term activation of dopamine D2L receptors in CAD cells.

Materials

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Production and maintenance of cell line
  6. Cyclic AMP accumulation assay
  7. Quantification of cyclic AMP
  8. Saturation binding assays
  9. Immunodetection
  10. Adenylate cyclase isoform identification
  11. Results
  12. Inhibition of adenylate cyclase in CAD-D2L cells
  13. Sensitization of cyclic AMP accumulation in CAD-D2L cells
  14. Effect of PKA activators on heterologous sensitization
  15. Effects of PDE inhibition on cyclic AMP accumulation
  16. Effects of modulators of PKA activity on PKA subunit expression
  17. Analysis of adenylate cyclase isoform expression pattern
  18. Discussion
  19. Acknowledgements
  20. References

[3H]Cyclic AMP (25 Ci/mmol) and [3H]spiperone (101 Ci/mmol) were purchased from NEN Life Science Products (Boston, MA, USA). Rabbit polyclonal PKA antibodies (anti-α-catalytic, β-catalytic, RIIα-regulatory, and RIβ-regulatory) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). H89 and Rp-8Br-cyclic AMP were purchased from Calbiochem (La Jolla, CA, USA). Quinpirole, spiperone, forskolin, dibutyryl cyclic AMP, isobutylmethylxanthine, pertussis toxin, growth media, and most other reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

Production and maintenance of cell line

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Production and maintenance of cell line
  6. Cyclic AMP accumulation assay
  7. Quantification of cyclic AMP
  8. Saturation binding assays
  9. Immunodetection
  10. Adenylate cyclase isoform identification
  11. Results
  12. Inhibition of adenylate cyclase in CAD-D2L cells
  13. Sensitization of cyclic AMP accumulation in CAD-D2L cells
  14. Effect of PKA activators on heterologous sensitization
  15. Effects of PDE inhibition on cyclic AMP accumulation
  16. Effects of modulators of PKA activity on PKA subunit expression
  17. Analysis of adenylate cyclase isoform expression pattern
  18. Discussion
  19. Acknowledgements
  20. References

CAD cells were stably transfected with the D2L dopamine receptor (CAD-D2L) using pcDNA3-D2L with Lipofectamine transfection reagent from Life Technologies (Grand Island, NY, USA) according to the manufacturer's protocol. Clones were isolated by selection with G418 (600 µg/mL) and were screened for D2 receptor expression using [3H]spiperone saturation binding isotherms as described below. Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum, 5% calf bovine serum (CBS), 50 U/mL penicillin, 50 µg/mL streptomycin, and 300 µg/mL G418. Cells were grown in a humidified incubator in the presence of 10% CO2 at 37°C.

Cyclic AMP accumulation assay

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Production and maintenance of cell line
  6. Cyclic AMP accumulation assay
  7. Quantification of cyclic AMP
  8. Saturation binding assays
  9. Immunodetection
  10. Adenylate cyclase isoform identification
  11. Results
  12. Inhibition of adenylate cyclase in CAD-D2L cells
  13. Sensitization of cyclic AMP accumulation in CAD-D2L cells
  14. Effect of PKA activators on heterologous sensitization
  15. Effects of PDE inhibition on cyclic AMP accumulation
  16. Effects of modulators of PKA activity on PKA subunit expression
  17. Analysis of adenylate cyclase isoform expression pattern
  18. Discussion
  19. Acknowledgements
  20. References

Cells were seeded at concentrations between 50 000 and 100 000 cells/well in 48-well cluster plates. For acute (15 min) experiments, cells were preincubated in 200 µL Earle's balanced salt solution (EBSS) assay buffer (EBSS containing 2% calf bovine serum, 0.02% ascorbic acid, and 15 mm Na+-HEPES) for 10 min at 37°C and placed on ice. Quinpirole (10 µm) in the absence or presence of spiperone (1 µm) was added to wells prior to the addition of forskolin (10 µm). Incubations were carried out for 15 min at 37°C, and the assay buffer was decanted. The culture plates were placed on ice and cells were lysed with 100 µL 3% trichloroacetic acid (TCA). The 48-well plates were stored at 4°C for at least 1 h before quantification. For long-term sensitization experiments, cells were preincubated for 18 h in the presence of drugs at 37°C in a humidified incubator in the presence of 10% CO2. For pertussis toxin treatment experiments, cells were initially treated with pertussis toxin (300 ng/mL) for 2 h, followed by a 18-h incubation with 1 µm quinpirole in the presence of pertussis toxin (final concentration of 150 ng/mL). Following drug pretreatment, the cells were washed three times for 3–4 min with 200 µL EBSS assay buffer, placed on ice, after which forskolin was added. Spiperone (1 µm) was added to the stimulation assay buffer to block activation of D2L receptors by residual agonist (Watts and Neve 1996). The cells were then incubated for 15 min at 37°C. The medium was removed, and the cells were lysed with 3% TCA. The 48-well plates were stored at 4°C until quantification of cyclic AMP was carried out.

Quantification of cyclic AMP

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Production and maintenance of cell line
  6. Cyclic AMP accumulation assay
  7. Quantification of cyclic AMP
  8. Saturation binding assays
  9. Immunodetection
  10. Adenylate cyclase isoform identification
  11. Results
  12. Inhibition of adenylate cyclase in CAD-D2L cells
  13. Sensitization of cyclic AMP accumulation in CAD-D2L cells
  14. Effect of PKA activators on heterologous sensitization
  15. Effects of PDE inhibition on cyclic AMP accumulation
  16. Effects of modulators of PKA activity on PKA subunit expression
  17. Analysis of adenylate cyclase isoform expression pattern
  18. Discussion
  19. Acknowledgements
  20. References

Cyclic AMP was quantified using a competitive binding assay with minor modifications (Watts and Neve 1996). Duplicate samples of the cell lysate (15 µL) were added to separate reaction tubes. [3H]Cyclic AMP (1 nm final concentration) was added to each assay tube, followed by cyclic AMP-binding protein (∼100 µg of crude adrenal extract in 500 µL of cyclic AMP buffer consisting of 100 mm Tris/HCl, pH 7.4, 100 mm NaCl, and 5 mm EDTA). The reaction tubes were incubated on ice for 2 h, and the samples were then harvested by filtration (Whatman GF/C filters) using a 96-well Packard Filtermate cell harvester. Filters were allowed to dry, and 40 µL of Packard Microscint 0 scintillation fluid was added to each sample well. Radioactivity on the filters was determined using a Packard TopCount scintillation counter. Cyclic AMP concentrations in each sample were determined in duplicate from a standard curve ranging from 0.1 to 100 pmol cyclic AMP. Dose–response curves for cyclic AMP accumulation were analyzed by non-linear regression using the program GraphPad Prism (San Diego, CA, USA). All values for cyclic AMP accumulation are expressed as pmol cyclic AMP per well unless otherwise indicated.

Saturation binding assays

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Production and maintenance of cell line
  6. Cyclic AMP accumulation assay
  7. Quantification of cyclic AMP
  8. Saturation binding assays
  9. Immunodetection
  10. Adenylate cyclase isoform identification
  11. Results
  12. Inhibition of adenylate cyclase in CAD-D2L cells
  13. Sensitization of cyclic AMP accumulation in CAD-D2L cells
  14. Effect of PKA activators on heterologous sensitization
  15. Effects of PDE inhibition on cyclic AMP accumulation
  16. Effects of modulators of PKA activity on PKA subunit expression
  17. Analysis of adenylate cyclase isoform expression pattern
  18. Discussion
  19. Acknowledgements
  20. References

Briefly, cells in 10 cm culture dishes were lysed with ice-cold hypotonic buffer (1 mm Na+-HEPES, pH 7.4, 2 mm EDTA). After 10–15 min, the cells were scraped from the plate and centrifuged at 30 000 g for 20 min. The resulting crude membrane fraction was resuspended in Tris-buffered saline using a Brinkmann Polytron homogenizer at setting 6 for 5 s and was subsequently used for radioligand binding assays. The binding of [3H]spiperone was assessed essentially as described (Watts and Neve 1996). Aliquots of the membrane preparation (5–20 µg of protein) were added to duplicate assay tubes containing the following (final concentrations): 50 mm Tris-HCl, pH 7.4 with 155 mm NaCl (Tris-buffered saline), 0.001% bovine serum albumin, and [3H]spiperone (5–200 pm). (+)-Butaclamol (5 µm) was used to define non-specific binding. Incubations were carried out at 37°C for 45 min, in a volume of 1.0 mL, and terminated by filtration as described above for the cyclic AMP binding assay. Data for saturation binding were analyzed by non-linear regression using the computer program, GraphPad Prism (San Diego, CA, USA) to determine Bmax and KD values. Saturation analysis of [3H]spiperone binding determined that the density of binding sites in membranes prepared from CAD cells expressing the D2L receptor (CAD-D2L) was 206 ± 56 fmol/mg of protein with a KD for [3H]spiperone of 22 ± 4 pm (n = 5).

Immunodetection

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Production and maintenance of cell line
  6. Cyclic AMP accumulation assay
  7. Quantification of cyclic AMP
  8. Saturation binding assays
  9. Immunodetection
  10. Adenylate cyclase isoform identification
  11. Results
  12. Inhibition of adenylate cyclase in CAD-D2L cells
  13. Sensitization of cyclic AMP accumulation in CAD-D2L cells
  14. Effect of PKA activators on heterologous sensitization
  15. Effects of PDE inhibition on cyclic AMP accumulation
  16. Effects of modulators of PKA activity on PKA subunit expression
  17. Analysis of adenylate cyclase isoform expression pattern
  18. Discussion
  19. Acknowledgements
  20. References

Samples for immunodetection were prepared as described by Boundy et al. (1998) with minor modifications. Cells grown in six-well cluster plates were pretreated with drugs for 18 h. Following drug treatment, the cells were placed on ice and 0.5 mL of ice cold buffer (10 mm HEPES, 0.4 m NaCl, 5 mm MgCl2, 0.5 mm EDTA, 0.1 mm EGTA and 1% NP-40) containing 20% glycerol, 1 mm dithiothreitol (DTT), 1 mm Na3VO4, and 0.15 mm phenylmethylsulfonyl fluoride (PMSF) was added to each well. Cells were scraped and transferred to polystyrene tubes, sonicated for 4 min (power setting 6, 50% duty cycle), and then incubated on ice for 30 min. Samples were centrifuged at 1000 g for 5 min, and the supernatant was retained for analysis. Total protein concentrations in the supernatant were determined using Pierce bicinchonic acid (BCA) protein assay (Rockford, IL, USA). Protein samples were equalized by dilution and resolved by sodium dodecyl sulfate–polyacrylamide gelelectrophoresis (SDS-PAGE) using a 4–15% polyacrylamide gradient gel and electro-transferred to Bio-Rad polyvinylidene difluoride (PVDF) membranes (Hercules, CA, USA). Nonspecific antibody binding was blocked by incubating membranes overnight in 5% non fat dried milk at 4°C. The membranes were washed with Tris-buffered saline and incubated with PKA subunit specific antibodies for 3 h. The PVDF membranes were washed and immunodetection was accomplished using Amersham Life Sciences enhanced chemifluorescence (ECF) western blotting kit (Buckinghamshire, UK) according to the manufacturer's instructions. Briefly, membranes were incubated with secondary antibody (fluorescein-linked, anti-rabbit Ig) for 1 h, washed and then incubated with tertiary antibody (anti-fluorescein, alkaline phosphatase conjugate) for 1 h. Membranes were again washed, exposed to ECF substrate for 10 min, dried at room temperature (25°C) for 20 min, and then scanned using Molecular Dynamics Storm Imaging System (Sunnyvale, CA, USA).

Adenylate cyclase isoform identification

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Production and maintenance of cell line
  6. Cyclic AMP accumulation assay
  7. Quantification of cyclic AMP
  8. Saturation binding assays
  9. Immunodetection
  10. Adenylate cyclase isoform identification
  11. Results
  12. Inhibition of adenylate cyclase in CAD-D2L cells
  13. Sensitization of cyclic AMP accumulation in CAD-D2L cells
  14. Effect of PKA activators on heterologous sensitization
  15. Effects of PDE inhibition on cyclic AMP accumulation
  16. Effects of modulators of PKA activity on PKA subunit expression
  17. Analysis of adenylate cyclase isoform expression pattern
  18. Discussion
  19. Acknowledgements
  20. References

Molecular analysis of the adenylate cyclase isoform expression pattern in CAD-D2L cells was completed as described by Varga et al. (1998) with minor modifications. Cells were grown in a 150-mm culture dish, and mRNA was isolated from total RNA using the MessageMaker kit according to manufacturer instructions (Life Technologies, Rockville, MD, USA). The SuperScript One-Step RT-PCR system was used according to manufacturer instructions (Life Technologies, Rockville, MD, USA). The reaction mixture contained 200 ng mRNA, 100 ng sense primer, 100 ng antisense primer, 1.0 µL RT/Taq mix, and 25 µL of 2X reaction buffer in a total volume of 50 µL. First strand synthesis was completed at 50°C for 30 min. The cDNA was denatured at 94°C for 2 min, followed by 35 amplification cycles as follows: 95°C for 30 s (denaturation), 55°C for 1 min (annealing), and 72°C for 1 min (extension). The primer sequences used were as follows: 5′-CGGCAGCTCGAGAA(A/G)AT(A/C/T)AA(A/G)ACIAT(A/C/T)GG-3′ (sense) and 5′-CCGGACTCGAGAC(A/G)TTIACIGTITTICCCCA-3′ (antisense). These degenerate primers correspond to two conserved regions in the second catalytic domain (CIIa) and are separated by approximately 300 bp in all adenylate cyclase isoforms. The underlined portions of the primers introduced XhoI restriction sites used for subsequent insertion into the single XhoI cloning site of pBS SK(–). The final 300 bp PCR products were isolated from a 2% agarose gel and digested with XhoI. The products were then ligated into the XhoI site of the pBS SK(–) vector using standard molecular techniques with T4 DNA ligase. The constructs were transformed into DH5α competent bacteria and positive clones were identified by restriction analysis using XhoI. Positive clones were subsequently sent to the University of Wisconsin DNA sequencing laboratories (Madison, WI, USA) for sequence analysis. Sequences were then individually aligned with known adenylate cyclase cDNA sequences using InforMax Vector NTI (Bethesda, MD, USA).

Inhibition of adenylate cyclase in CAD-D2L cells

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Production and maintenance of cell line
  6. Cyclic AMP accumulation assay
  7. Quantification of cyclic AMP
  8. Saturation binding assays
  9. Immunodetection
  10. Adenylate cyclase isoform identification
  11. Results
  12. Inhibition of adenylate cyclase in CAD-D2L cells
  13. Sensitization of cyclic AMP accumulation in CAD-D2L cells
  14. Effect of PKA activators on heterologous sensitization
  15. Effects of PDE inhibition on cyclic AMP accumulation
  16. Effects of modulators of PKA activity on PKA subunit expression
  17. Analysis of adenylate cyclase isoform expression pattern
  18. Discussion
  19. Acknowledgements
  20. References

We examined the ability of D2 agonists to inhibit forskolin-stimulated cyclic AMP accumulation CAD-D2L cells. Acutely, the D2 receptor agonist, quinpirole, markedly inhibited forskolin-stimulated cyclic AMP accumulation, and this inhibition was prevented by the D2 antagonist, spiperone (Fig. 1). To investigate the functional implications of cellular differentiation, we examined acute inhibition of adenylate cyclase activity following 3–4 days of serum starvation. Differentiation did not alter the magnitude of quinpirole-mediated inhibition of forskolin-stimulated cyclic AMP accumulation revealing 49 ± 6% (n = 6) inhibition in control CAD-D2L cells and 56 ± 8% inhibition (n = 5) in differentiated CAD-D2L cells (Fig. 1). Differentiation did not alter the basal levels of cyclic AMP accumulation, although forskolin stimulation of cyclic AMP accumulation was significantly enhanced in cells following growth in serum-free media (Fig. 1). This enhanced response to forskolin following differentiation was explored in greater detail because serum starvation slows cellular proliferation (Qi et al. 1997). Cell counting experiments revealed that the individual wells from differentiated cells contained nearly 30% (29 ± 3%, n = 3) fewer cells when compared with the wells of undifferentiated cells indicating that the increase in forskolin-stimulated cyclic AMP accumulation in differentiated cells is likely to be even greater if viewed on a per cell basis.

image

Figure 1. Effects of differentiation on D2L receptor-mediated inhibition of forskolin-stimulated cyclic AMP accumulation. CAD-D2L cells were grown in presence (control) or absence of serum (differentiated) for 3–4 days. Forskolin-stimulated cyclic AMP accumulation was measured in the absence or presence of 10 µm quinpirole (+ Quin) or 10 µm quinpirole and 1 µm spiperone (+ Quin + Spip) following a 15-min incubation. Basal levels of cyclic AMP accumulation were 2.6 ± 0.4 pmol/well in control cells and 3.4 ± 0.8 pmol/well in differentiated cells. Basal levels of cyclic AMP were not altered by any of the drug conditions. Data shown are the mean ± SEM of either five or six independent experiments, each assayed in duplicate. *p < 0.05 compared with vehicle (Dunnett's post-hoc repeated measures anova).

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Sensitization of cyclic AMP accumulation in CAD-D2L cells

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Production and maintenance of cell line
  6. Cyclic AMP accumulation assay
  7. Quantification of cyclic AMP
  8. Saturation binding assays
  9. Immunodetection
  10. Adenylate cyclase isoform identification
  11. Results
  12. Inhibition of adenylate cyclase in CAD-D2L cells
  13. Sensitization of cyclic AMP accumulation in CAD-D2L cells
  14. Effect of PKA activators on heterologous sensitization
  15. Effects of PDE inhibition on cyclic AMP accumulation
  16. Effects of modulators of PKA activity on PKA subunit expression
  17. Analysis of adenylate cyclase isoform expression pattern
  18. Discussion
  19. Acknowledgements
  20. References

Short-term (2 h) treatment of CAD-D2L cells with quinpirole did not alter subsequent forskolin-stimulated cyclic AMP accumulation (results not shown). In contrast, long-term (18 h) treatment of CAD-D2L cells with quinpirole resulted in a significant enhancement of subsequent forskolin-stimulated cyclic AMP accumulation (Fig. 2). This long-term heterologous sensitization was blocked in the presence of spiperone (Fig. 2) and also by pertussis toxin pretreatment. Specifically, experiments completed in the absence of pertussis toxin treatment revealed that long-term treatment with quinpirole enhanced forskolin-stimulated cyclic AMP accumulation from 19 ± 4 pmol/well in vehicle-treated cells to 33 ± 8 pmol/well in quinpirole-treated cells (p < 0.05, n = 3). This enhancement was blocked following pertussis toxin treatment (see methods) where forskolin-stimulated cyclic AMP accumulation was 10 ± 2 pmol/well in vehicle-treated cells and 10 ± 1 pmol/well in quinpirole-treated cells (n = 3). The effects of differentiation on D2L receptor-mediated long-term heterologous sensitization were also examined. The magnitude of quinpirole-mediated sensitization was similar in both control (1.8 ± 0.11–fold, n = 10) and differentiated cells (2.0 ± 0.16–fold, n = 4). As we observed in the acute assays, differentiation robustly enhanced forskolin-stimulated cyclic AMP accumulation (Fig. 2). Basal levels of cyclic AMP accumulation were not altered by quinpirole pretreatment or by differentiation.

image

Figure 2. Effects of differentiation on D2L receptor-induced heterologous sensitization. CAD-D2L cells were grown in presence (control) or absence of serum (differentiated) for 3–4 days. Cells were pretreated for 18 h with vehicle, 10 µm quinpirole (+ Quin), or 10 µm quinpirole and 1 µm spiperone (Quin + Spip). Cells were extensively washed, and forskolin-stimulated cyclic AMP accumulation was determined following a 15-min incubation. Basal levels of cyclic AMP accumulation were 4.0 ± 0.8 pmol/well in control cells and 2.4 ± 0.5 pmol/well in differentiated cells. Basal levels of cyclic AMP were not altered by any of the drug treatment conditions. Data shown are the mean ± SEM of between four and 10 independent experiments, each assayed in duplicate. *p < 0.05 compared with vehicle-treated cells (Dunnett's post hoc repeated measures anova).

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Persistent activation of D2L receptors using quinpirole is predicted to inhibit PKA activity through Gαi/o-mediated inhibition of cyclic AMP accumulation. To examine the role of long-term PKA inhibition in heterologous sensitization, we used two reagents to inhibit directly PKA activity in CAD-D2L cells. Long-term (18 h) treatment with either Rp-8Br-cyclic AMP or the non-nucleoside PKA inhibitor, H89, significantly increased subsequent forskolin-stimulated cyclic AMP accumulation to an extent that was similar to that observed following long-term quinpirole pretreatment (Fig. 3). Basal levels of cyclic AMP were not altered by any agents used to inhibit PKA activity. Additional studies revealed that 18 h pretreatment with a selective inhibitor of PKC, bisindoleaylmide, did not alter forskolin-stimulated cyclic AMP accumulation (results not shown). Thus, long-term treatment with PKA inhibitors (i.e. quinpirole, H89, or Rp-8Br-cAMP) resulted in enhancement of subsequent forskolin-stimulated cyclic AMP accumulation.

image

Figure 3. Effects of protein kinase A (PKA) inhibitors on cyclic AMP accumulation. CAD-D2L cells were treated for 18 h with vehicle, 10 µm quinpirole, 10 µm H89, or 100 µm Rp-8Br-cAMP. Cells were extensively washed, and basal and forskolin-stimulated cyclic AMP accumulations were determined following a 15-min incubation. Data shown are the mean ± SEM of six independent experiments, each assayed in duplicate. **p < 0.01 compared with vehicle-treated cells (Dunnett's post hoc repeated measures anova).

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Effect of PKA activators on heterologous sensitization

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Production and maintenance of cell line
  6. Cyclic AMP accumulation assay
  7. Quantification of cyclic AMP
  8. Saturation binding assays
  9. Immunodetection
  10. Adenylate cyclase isoform identification
  11. Results
  12. Inhibition of adenylate cyclase in CAD-D2L cells
  13. Sensitization of cyclic AMP accumulation in CAD-D2L cells
  14. Effect of PKA activators on heterologous sensitization
  15. Effects of PDE inhibition on cyclic AMP accumulation
  16. Effects of modulators of PKA activity on PKA subunit expression
  17. Analysis of adenylate cyclase isoform expression pattern
  18. Discussion
  19. Acknowledgements
  20. References

The results described above suggest that inhibition of PKA is mechanistically involved in heterologous sensitization of adenylate cyclase activity in CAD-D2L cells. Thus, the ability of several PKA activators to alter drug-induced heterologous sensitization was explored. Reagents acting via three distinct mechanisms to activate PKA activity were utilized: forskolin, a direct activator of adenylate cyclase, isobutylmethylxanthine (IBMX), a cyclic AMP phosphodiesterase (PDE) inhibitor, and dibutyryl cyclic AMP or 8Br-cyclic AMP, cell-permeable analogs of cyclic AMP. Long-term (18 h) treatment of CAD-D2L cells with forskolin, dibutyryl cyclic AMP, 8Br-cyclic AMP, or IBMX attenuated both quinpirole- and H89-induced heterologous sensitization (Fig. 4 and data not shown for 8Br-cyclic AMP). Furthermore, long-term treatment with each of the PKA activators alone resulted in a significant decrease in subsequent forskolin-stimulated cyclic AMP accumulation (Fig. 4). As a negative control, long-term treatment with dideoxyforskolin, an inactive analog of forskolin, did not alter subsequent adenylate cyclase activity or attenuate quinpirole-induced heterologous sensitization. Specifically, the magnitude of quinpirole-induced sensitization in the presence of dideoxyforskolin was 1.7-fold (n = 4) which was similar to the magnitude observed in the absence of dideoxyforskolin (1.7-fold, n = 4 and see Fig. 2). These data indicate that long-term activation of PKA can attenuate drug-induced heterologous sensitization in CAD-D2L cells.

image

Figure 4. Effects of protein kinase A (PKA) activators on heterologous sensitization of adenylate cyclase activity. CAD-D2L cells were treated for 18 h with vehicle (Veh), 10 µm quinpirole (Quin), or 10 µm H89 in the absence or presence of the indicated PKA activator. The PKA activators were used at the following concentrations: (a) 1 µm forskolin (FSK) (b) 300 µm dibutyryl cAMP (dbcAMP), and (c) 500 µm IBMX (IBMX). Following 18 h pretreatment, cells were washed extensively and cyclic AMP was stimulated using 10 µm forskolin for 15 min. Data shown are mean ± SEM of either five or six independent experiments, *< 0.05 compared with vehicle-treated cells (Dunnett's post hoc repeated measures anova).

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Effects of PDE inhibition on cyclic AMP accumulation

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Production and maintenance of cell line
  6. Cyclic AMP accumulation assay
  7. Quantification of cyclic AMP
  8. Saturation binding assays
  9. Immunodetection
  10. Adenylate cyclase isoform identification
  11. Results
  12. Inhibition of adenylate cyclase in CAD-D2L cells
  13. Sensitization of cyclic AMP accumulation in CAD-D2L cells
  14. Effect of PKA activators on heterologous sensitization
  15. Effects of PDE inhibition on cyclic AMP accumulation
  16. Effects of modulators of PKA activity on PKA subunit expression
  17. Analysis of adenylate cyclase isoform expression pattern
  18. Discussion
  19. Acknowledgements
  20. References

Previous reports have indicated that activation of PKA can lead to enhanced cyclic AMP degradation due to an increase in the activity of cyclic AMP phosphodiesterases (Sette and Conti 1996; Oki et al. 2000). Thus, the effects of long-term PKA activation on subsequent PDE activity were examined. Specifically, CAD-D2L cells were pretreated with forskolin for 18 h and subsequent cyclic AMP accumulation was measured in response to several stimulation conditions (Fig. 5). As observed previously (Fig. 4a), forskolin (1 µm) pretreatment (18 h) caused a significant reduction (65 ± 2%) in subsequent forskolin-stimulated cyclic AMP accumulation (Fig. 5). Similarly, forskolin pretreatment also caused a significant reduction (31 ± 5%) of cyclic AMP accumulation in response to the highly selective PDE-4 inhibitor, rolipram. Additional experiments revealed that pretreatment with forskolin also reduced cyclic AMP accumulation when forskolin stimulations were carried out in the presence of rolipram or IBMX (Fig. 5).

image

Figure 5. Effects of forskolin pretreatment (18 h) on subsequent cyclic AMP accumulation in the absence or presence of phosphodiesterase inhibitors. CAD-D2L cells were treated for 18 h with vehicle or 1 µm forskolin. Following pretreatment, cells were washed extensively andcyclic AMP accumulation was measured in the presence of 10 µmforskolin (FSK), 100 µm rolipram (Rol), 10 µm forskolin and 500 µm Isobutylmethylxanthine (FSK + IBMX), or 10 µm forskolin and100 µm rolipram (FSK + Rol) for 15 min. Basal levels of cyclic AMP accumulation were 2.8 ± 0.6 pmol/well in vehicle-treated cells and 3.1 ± 0.8 pmol/well in forskolin-treated cells. Data shown are mean ± SEM of four independent experiments, *< 0.05 compared with vehicle-treated cells under the indicated stimulation condition (Student's paired t-test).

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Effects of modulators of PKA activity on PKA subunit expression

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Production and maintenance of cell line
  6. Cyclic AMP accumulation assay
  7. Quantification of cyclic AMP
  8. Saturation binding assays
  9. Immunodetection
  10. Adenylate cyclase isoform identification
  11. Results
  12. Inhibition of adenylate cyclase in CAD-D2L cells
  13. Sensitization of cyclic AMP accumulation in CAD-D2L cells
  14. Effect of PKA activators on heterologous sensitization
  15. Effects of PDE inhibition on cyclic AMP accumulation
  16. Effects of modulators of PKA activity on PKA subunit expression
  17. Analysis of adenylate cyclase isoform expression pattern
  18. Discussion
  19. Acknowledgements
  20. References

Long-term activation of PKA has been shown to modulate the expression levels of the catalytic and regulatory subunits of PKA (Boundy et al. 1998), which may ultimately influence the cyclic AMP signaling pathway. Thus, the expression levels of the α and β catalytic subunits as well as the Iβ and IIα regulatory subunits of PKA were examined following long-term activation or inhibition of PKA in CAD-D2L cells. The results of these experiments revealed that the expression levels of both the α and β catalytic subunits of PKA in CAD-D2L cells were not altered following long-term treatment with any of the agents used to activate or inhibit PKA (Table 1). In addition, analysis of both the RIIα and RIβ subunits revealed only one significant drug-induced change in expression (Table 1). Specifically, H89 pretreatment reduced the expression of the RIβ subunit by greater than 25%. Together, these data suggest that the expression levels of the PKA subunits in CAD-D2L cells are not altered markedly following long-term exposure to PKA modulators at the concentrations used here.

Table 1.  Effects of drug exposure on the expression levels of protein kinase A subunits
Drug treatmentProtein kinase A subunit expression level (% of control)
RIIαRIβ
  1. CAD-D2L cells were incubated with vehicle or the indicated drug under normal growth conditions for 18 h at 37°C. The cells were lysed and samples prepared for immunodetection of protein kinase A subunits as described in Materials and methods. Data shown are expressed as the percentage of immunoreactivity measured in control cells prepared on the same day, and represent the mean ±SEM of six to eight independent preparations. *< 0.05 compared with vehicle-treated cells (one-tailed, one-sample t-test).

Quinpirole (10 µm)98 ± 8100 ± 18108 ± 1085 ± 8
H89 (10 µm)96 ± 9 77 ± 10 98 ± 872 ± 7*
Forskolin (1 µm)92 ± 13109 ± 18105 ± 686 ± 7
Isobutylmethylxanthine (500 µm)86 ± 12 96 ± 12 88 ± 1372 ± 8
db cyclic AMP (300 µm)90 ± 8107 ± 8111 ± 1289 ± 6

Analysis of adenylate cyclase isoform expression pattern

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Production and maintenance of cell line
  6. Cyclic AMP accumulation assay
  7. Quantification of cyclic AMP
  8. Saturation binding assays
  9. Immunodetection
  10. Adenylate cyclase isoform identification
  11. Results
  12. Inhibition of adenylate cyclase in CAD-D2L cells
  13. Sensitization of cyclic AMP accumulation in CAD-D2L cells
  14. Effect of PKA activators on heterologous sensitization
  15. Effects of PDE inhibition on cyclic AMP accumulation
  16. Effects of modulators of PKA activity on PKA subunit expression
  17. Analysis of adenylate cyclase isoform expression pattern
  18. Discussion
  19. Acknowledgements
  20. References

The nine membrane-bound isoforms of adenylate cyclase have unique regulatory properties (Taussig and Zimmerman 1998). Thus, the specific expression pattern of the adenylate cyclase isoforms is likely to have significant implications on the interpretation of biochemical data derived from any given individual cell line. We used RT-PCR methodology to identify mRNAs for the individual isoforms of adenylate cyclase expressed in CAD-D2L cells. Degenerate primers were used to amplify a region of approximately 300 bp in the CIIa catalytic domain of the adenylate cyclase cDNA. The PCR products were then subcloned into the pBS SK(–) vector for sequencing. The sequencing of 25 individual colonies confirmed the presence of mRNA for two isoforms of adenylate cyclase in CAD-D2L cells. Specifically, 21 of the clones had DNA sequences that were 95% homologous to the published rat ACVI cDNA sequence (Premont et al. 1992). The remaining four clones had DNA sequences that were 96% homologous to the published human ACIX cDNA sequence (Hacker et al. 1998). Thus, ACVI and ACIX appear to be the predominant isoforms expressed in CAD-D2L cells. As a control, the ability of the degenerate primers to amplify cDNAs for ACI, ACII, ACIV, ACV, and ACVIII was confirmed using plasmid cDNAs encoding for these individual isoforms (results not shown).

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Production and maintenance of cell line
  6. Cyclic AMP accumulation assay
  7. Quantification of cyclic AMP
  8. Saturation binding assays
  9. Immunodetection
  10. Adenylate cyclase isoform identification
  11. Results
  12. Inhibition of adenylate cyclase in CAD-D2L cells
  13. Sensitization of cyclic AMP accumulation in CAD-D2L cells
  14. Effect of PKA activators on heterologous sensitization
  15. Effects of PDE inhibition on cyclic AMP accumulation
  16. Effects of modulators of PKA activity on PKA subunit expression
  17. Analysis of adenylate cyclase isoform expression pattern
  18. Discussion
  19. Acknowledgements
  20. References

Heterologous sensitization of adenylate cyclase activity has been proposed to be a neuroadaptive response to persistent inhibitory neurotransmission (Thomas and Hoffman 1987; Nestler and Aghajanian 1997). Although the exact mechanism of this phenomenon is unknown, a number of studies have provided evidence for the role of various G protein subunits in the development of heterologous sensitization (Watts and Neve 1996; Watts et al. 1998; Bayewitch et al. 2000; Rhee et al. 2000). The present studies were designed to examine the role of long-term Gαi/o activation and the PKA signaling pathway in D2L receptor-induced heterologous sensitization in a novel neuronal environment. Agonist-induced sensitization in CAD-D2L cells appears to differ from sensitization in a number of other cell types. Specifically, sensitization appeared to be dependent on the inhibition of PKA and was attenuated in presence of PKA activators. Subsequent analysis of the adenylate cyclase isoforms provided evidence that the PKA-dependent mechanism may be linked to the expression of ACVI.

Initial experiments compared D2L receptor function in control and differentiated CAD-D2L cells using an acute inhibition assay and a long-term sensitization assay. These experiments revealed that morphological differentiation of CAD-D2L cells did not alter D2L receptor signaling in either assays examining acute or long-term receptor activation. In contrast, differentiation of CAD-D2L cells markedly enhanced forskolin-stimulated cyclic AMP accumulation (Figs 1 and 2). This increased responsiveness to forskolin may be important to mechanisms of differentiation in CAD cells because prolonged (> 72 h) exposure to cell permeable analogs of cyclic AMP will also induce differentiation in CAD cells (results not shown). On the other hand, the mechanism of enhanced forskolin responsiveness following differentiation may be related to changes in the expression of adenylate cyclase isoforms (see Hanoune and Defer 2001). The effects of differentiation on cyclic AMP signaling in CAD-D2L cells are currently under investigation. However, since both acute and long-term D2L receptor-mediated responses did not appear to be altered following differentiation, we chose to conduct all subsequent investigations of heterologous sensitization in undifferentiated cells.

The results of the present study examining sensitization in CAD-D2L cells differed from a number of our earlier studies. Specifically, short-term (2 h) agonist treatment of CAD-D2L cells did not lead to an enhanced forskolin response, which contrasts previous studies in C6-D2L, HEK-D2L, and NS20Y-D2L cells (Watts and Neve 1996; Watts et al. 1998). We also found that the magnitude of long-term (18 h) sensitization in CAD-D2L cells is much less than observed in HEK-D4 (Watts et al. 1999) and NS20Y-D2L cells (Johnston et al. 2001). These observations may suggest that the mechanism for long-term heterologous sensitization in CAD-D2L cells differs from that observed in other cell types. One potential mechanism concerns the role of PKA in heterologous sensitization of adenylate cyclase. The potential role of PKA is based on a previous study where PKA activators blocked short-term sensitization following adenosine receptor activation (Port et al. 1992) and the hypothesis that persistent activation of Gαi/o-coupled receptors induces sensitization through prolonged inhibition of PKA. However, several studies have provided evidence that sensitization actually occurs independent of PKA in C6-D2L, HEK-D2L, HEK-D4, and NS20Y-D2L cells (Watts and Neve 1996; Watts et al. 1999; Johnston et al. 2001). On the other hand, one early study of D2 receptor-mediated sensitization found that PKA activators actually enhanced agonist-induced sensitization in Ltk fibroblast cells (Bates et al. 1991). Thus, a strong role for PKA in heterologous sensitization had not been established. Data suggesting differences among cell types and the unique properties of CAD-D2L cells prompted us to explore the role of PKA in the current system.

In an effort to address the role of PKA inhibition in sensitization, we used drugs that act via three distinct mechanisms of action to inhibit PKA. Inhibition of PKA following quinpirole activation of the D2L receptor is indirect, whereas H89 and Rp-8Br-cyclic AMP act directly on the catalytic and regulatory subunits, respectively (Schwede et al. 2000). Each PKA inhibitor produced a similar magnitude of enhancement of forskolin-stimulated cyclic AMP accumulation following 18 h treatment. The similarity in magnitude may suggest that each agent inhibits constitutive PKA activity in CAD-D2L cells to a similar extent. The results seen with direct PKA inhibitors added support for the hypothesis that inhibition of PKA induces sensitization and prompted us to ask whether activators of PKA block drug-induced sensitization. This question was addressed using three agents that activate PKA at distinct steps in the activation pathway. Forskolin, a direct activator of adenylate cyclase was used to stimulate cyclic AMP accumulation. The PDE inhibitor, IBMX, was used to prevent the degradation of cyclic AMP. Lastly, dibutyryl cyclic AMP was used to directly activate PKA (Schwede et al. 2000). Each agent reduced the sensitization induced by either quinpirole or H89 treatment. In addition, pretreatment with each PKA activator alone inhibited subsequent forskolin-stimulated cyclic AMP accumulation. Together, these observations highlight a key role of PKA in drug-induced sensitization in CAD-D2L cells, where inhibition of PKA leads to an enhanced forskolin-stimulated cyclic AMP accumulation that is attenuated by activation of PKA.

The present studies suggest an important balance between PKA activity and adenylate cyclase activity in CAD-D2L cells. Currently, there are nine known membrane-bound isoforms of adenylate cyclase. Each isoform exhibits distinct regulatory properties and appears to display differential sensitization (Thomas and Hoffman 1996; Watts and Neve 1996; Avidor-Reiss et al. 1997; Nevo et al. 1998; Rhee et al. 2000). There is also evidence to support that this differential sensitization is due to the differences in the regulatory properties of the individual isoforms (Cumbay and Watts 2001; Watts et al. 2001). This hypothesis, along with the present data, would suggest that the isoform(s) of adenylate cyclase in CAD-D2L cells are sensitive to regulation by PKA, therefore the observed heterologous sensitization may in part be explained by a PKA-dependent mechanism. Of the nine known adenylate cyclase isoforms, only ACV and ACVI have been shown to be negatively regulated (via phosphorylation) by PKA in vitro (Iwami et al. 1995; Chen et al. 1997). In addition, preliminary studies from our laboratory have shown that long-term treatment with forskolin reduces subsequent forskolin-stimulated cyclic AMP accumulation in HEK cells expressing ACVI (Beazely M. A., Johnston C. A. and Watts V. J., unpublished observations). Such observations are consistent with the ability of PKA activators to negatively regulate forskolin-stimulated cyclic AMP accumulation in CAD-D2L cells. Additional support for this hypothesis was revealed when we examined the adenylate cyclase isoform expression patterns in CAD-D2L cells. Using degenerate primers in an RT-PCR protocol (Premont 1994; Varga et al. 1998) to amplify a 300-bp region of the CIIa domain of adenylate cyclase, we confirmed the presence of mRNA for ACVI and ACIX in CAD-D2L cells. In addition, immunoblotting experiments have confirmed the expression of ACV/ACVI-like immunoreactivity as well as ACIX immunoreactivity in CAD-D2L cells (results not shown). Because ACIX has not been shown to be regulated by PKA, these observations support the idea that heterologous sensitization in CAD-D2L cells may be dependent on the ability of PKA to modulate ACVI.

The ability of PKA to regulate ACVI implicates a potential role for drug-induced changes in PKA expression in regulating cyclic AMP accumulation. For example, Boundy et al. (1998) found that long-term forskolin treatment resulted in a down regulation of both the catalytic and regulatory subunits of PKA in Cath.a and SH-SY5Y cells (Boundy et al. 1998). They also demonstrated that inhibition of PKA by Rp-8Br-cyclic AMP caused a marked increase in the expression of the regulatory subunit in both cell types (Boundy et al. 1998). Although such changes would likely influence ACVI activity, the present study failed to observe consistent changes in the expression of catalytic or regulatory PKA subunits in CAD-D2L cells following treatment with either activators or inhibitors of PKA. Our data suggest that changes in PKA subunit expression are not involved in heterologous sensitization in CAD-D2L cells.

In addition to modulating directly select isoforms adenylate cyclase, PKA is a potent regulator of PDE activity. Specifically, PKA activation has been shown to phosphorylate and subsequently activate several PDE isoforms which leads to enhanced degradation of intracellular cyclic AMP (Sette and Conti 1996; Oki et al. 2000). According to this model, PKA activators would cause an apparent decrease in cyclic AMP accumulation in response to forskolin stimulation. In contrast, PKA inhibitors would decrease PDE activity and potentiate forskolin-stimulated cyclic AMP accumulation. The initial observations with each series of PKA modulators appeared to support this model (see Figs 3 and 4). To further investigate the role of PDE activation in cyclic AMP signaling following long-term forskolin pretreatment, we examined drug-stimulated cyclic AMP accumulation in the presence of rolipram or IBMX. Although the presence of rolipram and IBMX seemed to diminish the effects of forskolin pretreatment on subsequent forskolin-stimulated cyclic AMP accumulation, the reduced responsiveness was not completely attenuated (see Fig. 5). These observations indicate a potential role for PDEs in regulating subsequent cyclic AMP accumulation, however, the effects of PKA activators on adenylate cyclase activity appear to dominate. For example, pretreatment with IBMX, a direct inhibitor of PDE, caused a decrease in subsequent forskolin-stimulated cyclic AMP accumulation presumably through the activation of PKA (Fig. 4c). This net decrease in subsequent adenylate cyclase activity suggests that the effect of PKA activation (presumably via the actions of IBMX elevating cyclic AMP) is a primary determinant in subsequent drug-stimulated cyclic AMP accumulation. Together, these observations suggest that forskolin-induced desensitization of adenylate cyclase is PKA-mediated and that one component may involve a PKA-induced increase in PDE activity.

An up-regulation of adenylate cyclase and PKA have been linked to chronic administration of a number of drugs of abuse (Nestler and Aghajanian 1997). For example, chronic morphine increases the expression of PKA as well as ACI and ACVIII in the locus coeruleus (Lane-Ladd et al. 1997). The fact that PKA does not negatively regulate ACI or ACVIII would provide for a marked up-regulation of cyclic AMP-PKA signaling pathway upon the activation of a Gαs-coupled receptor. On the other hand, the present study would suggest that increased expression of PKA could dampen Gαs-stimulated cyclic AMP signaling in brain regions expressing ACVI such as the ventral tegmental area, substantia nigra, striatum, or nucleus accumbens (Liu et al. 1998). The observations described above support the hypothesis that increased adenylate cyclase activity will increase PKA activity, however, the effect of increased PKA activity on adenylate cyclase will be determined by the isoform expression patterns.

In summary, long-term (18 h) sensitization studies suggested that the mechanisms for heterologous sensitization may be unique in CAD-D2L cells and revealed that treatment with PKA inhibitors induces heterologous sensitization. Molecular analysis of the isoforms of adenylate cyclase indicated that CAD-D2L cells express ACVI, which has been shown to be negatively regulated by PKA. These results suggest that one potential mechanism for heterologous sensitization in CAD-D2L cells may involve PKA-dependent modulation of ACVI.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Materials
  5. Production and maintenance of cell line
  6. Cyclic AMP accumulation assay
  7. Quantification of cyclic AMP
  8. Saturation binding assays
  9. Immunodetection
  10. Adenylate cyclase isoform identification
  11. Results
  12. Inhibition of adenylate cyclase in CAD-D2L cells
  13. Sensitization of cyclic AMP accumulation in CAD-D2L cells
  14. Effect of PKA activators on heterologous sensitization
  15. Effects of PDE inhibition on cyclic AMP accumulation
  16. Effects of modulators of PKA activity on PKA subunit expression
  17. Analysis of adenylate cyclase isoform expression pattern
  18. Discussion
  19. Acknowledgements
  20. References
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