Hybrid modes and postmodern uncertainties

Our initial sensitization experiments employed HEK293 cells that were transiently transfected with AC2 and the D2LDR. Consistent with significant AC2 expression and function, HEK293 cells co-expressing AC2 and the D2LDR

displayed a robust increase in cAMP accumulation in response to PMA activation of PKC (2.75±0.48 pmol/well, n = 3) as compared to cells transfected with empty

vector (0.60±0.06 pmol/well, n = 3). To observe D2LDR-mediated sensitization of AC2, cells were pretreated for 2 h with the D2LDR agonist quinpirole, and

subsequently stimulated with PMA to activate AC2. The quinpirole-pretreated cells co-expressing AC2 and the D2LDR displayed PMA-stimulated cAMP accumulation that was 251±34% of the vehicle pretreatment condition, whereas the empty vector transfected cells provided a response that was similar to that of vehicle-treated cells (Figure 3.1). These observations are consistent with our previous studies using cells stably expressing AC2, where persistent D2LDR activation resulted in a sensitized response to AC2 activation via PKC as measured by the enhancement of cAMP accumulation over that of the vehicle pretreatment condition (Chapter 2 and (Cumbay and Watts, 2001)). Studies have suggested an important role for Gi/o protein subunits in the development of AC sensitization (Watts and Neve, 2005). The regulatory properties of AC2 offer a unique system to dissociate the functional roles of Gαi/o and Gβγ subunits in AC sensitization. Specifically, AC2 is thought to be insensitive to functional regulation by Gαi subunits (Tang and Gilman, 1991; Taussig et al., 1994),

thereby allowing the specific observation of Gβγ subunit-modulated AC2 activity.

To directly study the role of Gβγ subunits in AC2 sensitization, the membrane-localized Gβγ subunit-sequestering protein βARKct-CD8 was co-expressed with AC2 and the D2LDR in HEK293 cells. Expression of βARKct-CD8 resulted in a blockade of D2LDR-mediated sensitization of AC2 (124±13% of vehicle response), suggesting a role for Gβγ subunits in sensitization of AC2 (Figure 3.1).

Figure 3.1 Effect of the Gβγ subunit sequestering protein on heterologous sensitization of AC2. HEK293 cells were transiently transfected with AC2, the D2LDR, and either empty vector or βARKct-CD8. Cells were pretreated with quinpirole or vehicle for 2 h and subsequently stimulated with 1 μM PMA. Data are expressed as a percentage of the vehicle condition for each transfection and are the mean ± S.E.M. of three independent experiments. The raw cAMP values for each condition are as follows: AC2+D2L vehicle pretreatment condition = 2.75

± 0.48 pmol/well; AC2 + D2L quinpirole pretreatment condition = 6.97 ± 1.55 pmol/well; AC2 + D2L + βARKct-CD8 vehicle pretreatment condition = 2.73 ± 0.78 pmol/well; AC2 + D2L + βARKct-CD8 quinpirole pretreatment condition = 3.31 ± 0.89 pmol/well.

The observation that βARKct-CD8 prevented heterologous sensitization suggests that the role of Gβγ subunits in AC2 sensitization could be either direct or indirect. In an effort to explore the direct pathway, we tested two small

molecule Gβγ signaling inhibitors for their ability to modulate sensitization of AC2.

HEK293 cells stably expressing AC2 and the D2LDR (HEK-AC2/D2L cells) were pretreated with increasing concentrations of the agonist quinpirole, followed by subsequent AC2 activation by PMA treatment. As expected, quinpirole

pretreatment resulted in a concentration-dependent enhanced responsiveness of AC2 to activation by PMA (Figure 3.2A). Initial studies with the small molecule inhibitors, M119 and gallein, revealed that the quinpirole-induced enhanced response to PMA was not altered by the small molecule Gβγ signaling inhibitors.

The lack of efficacy of M119 and gallein may represent the reported specificity for inhibition of specific Gβγ-effector interfaces (Bonacci et al., 2006). Thus, we examined the ability of M119 and gallein to block conditional acute activation of AC2 by Gβγ subunits (Federman et al., 1992; Taussig et al., 1993b). For these studies HEK-AC2/D2L cells were treated with PMA (to activate AC2) in the presence of quinpirole to activate the D2LDR (allowing for activation of Gαi and release of Gβγ subunits) for Gβγ-dependent potentiation of AC2 activity. The results of these studies revealed that the Gβγ signaling inhibitors, M119 and gallein, had no effect on either PMA-stimulated or Gβγ-dependent potentiation of AC2 (Figure 3.2B).

Figure 3.2 The effect of Gβγ signaling inhibitors on D2LDR-mediated AC2 signaling. A. The effect of M119 and gallein (10 μM) on quinpirole-induced sensitization was measured in HEK-AC2/D2L cells. Cells were pretreated with increasing concentrations of quinpirole for 2 h in the presence of vehicle, M119, or gallein and subsequently stimulated with 1 μM PMA. Data are representative of two independent experiments. B. The effect of M119 and gallein (10 μM) on acute quinpirole-induced potentiation of PMA-stimulated AC2 activity was measured in HEK-AC2/D2L cells. Data are the mean ± S.E.M. of three independent experiments.

In an effort to rapidly assess the activity of several small molecules for modulation of D2LDR-induced sensitization of AC2, we developed a 96 well sensitization assay (with reduced wash and decant steps) for increased

throughput (Conley et al., in press). The higher throughput sensitization assay provided the ability to efficiently investigate multiple pharmacological modulators simultaneously. The observation that βARKct-CD8 expression inhibits D^2L DR-induced sensitization of AC2 and lack of effect by M119 and gallein suggest the hypothesis that AC2 sensitization is mediated by Gβγ subunits, in an indirect fashion, by a downstream effector of Gβγ subunits. Furthermore, previous sensitization studies with other adenylyl cyclases have provided evidence that a number of kinases may also be involved in the development and expression of sensitization of adenylyl cyclase (Chakrabarti et al., 1998; Johnston et al., 2002).

Thus, several ligands including a peptide Gβγ signaling inhibitor, small molecule inhibitors of Gβγ effectors, and several additional kinase inhibitors were tested for the ability to inhibit quinpirole-induced sensitization of AC2 in HEK-AC2/D2L cells.

Specifically, HEK-AC2/D2L cells were pretreated with inhibitors in the presence of either vehicle or quinpirole, and subsequently stimulated with PMA to promote PKC activation of AC2. The results of these studies are depicted as a

percentage of sensitization (i.e., quinpirole induced sensitization = 100%).

Consistent with the effects of βARKct-CD8, the cell-permeable Gβγ subunit sequestering peptide, QEHA-TAT significantly inhibited sensitization in the HEK-AC2/D2L cells, whereas the small molecule Gβγ subunit inhibitor, gallein, had no effect (negative control) (Table 3.1). The next set of compounds included

reported pharmacological kinase inhibitors that target Gβγ effectors that include c-JNK, Raf-1, PI3K, or MEK. Much to our disappointment, there was no

significant difference observed in the D2LDR-mediated sensitization of AC2 in the presence of the Gβγ-modulated kinase inhibitors (Table 3.1). Additional kinases, including those that have been identified as being involved in sensitization of AC isoforms (i.e., PKC and PKA) were also examined in the same manner

(Chakrabarti et al., 1998; Johnston et al., 2002). As expected, the PKC inhibitor bisindolylmaleimide I (BisI) blocked sensitization of AC2 (3.0±1.7% sensitization).

Inhibitors of PKA (H89) and PI3K/PI4K (phenylarsine oxide, PAO) significantly reduced the level of D2LDR-mediated sensitization of AC2 (29±12% and 29±5%

sensitization, respectively), suggesting roles for these kinases in sensitization of AC2.

Table 3.1 Effects of pharmacological inhibitors on D2LDR-mediated sensitization of AC2. Data are expressed as a percent sensitization with the quinpirole-induced sensitization = 100%. Data are the mean ± S.E.M. of five independent experiments.

Compound Target Mean±S.E.M.

Control 100

10 μM Gallein Gβγ 110±24

30 µM QEHA-TAT Gβγ 62±8.2*

10 μM SP600125 c-JNK 130±13

30 μM GW5074 Raf-1 99±12

300 nM Wortmannin PI3K 95±10

30 μM PD98059 MEK 90±6.2

10 μM H89 PKA 29±12 ***

10 μM PAO PI3K/PI4K 29±5.5 ***

1 μM BisI PKC 3.0±1.7 ***