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Adenylyl cyclase type II activity is regulated by two different mechanisms: Implications for acute and chronic opioid exposure
Neuropharmacology 50 (2006) 998e1005 www.elsevier.com/locate/neuropharm
Adenylyl cyclase type II activity is regulated by two different mechanisms: Implications for acute and chronic opioid exposure Ester Schallmach, Debora Steiner, Zvi Vogel* Department of Neurobiology, The Weizmann Institute of Science, Hertzel str, Rehovot 76100, Israel Received 7 October 2005; received in revised form 2 January 2006; accepted 25 January 2006
Abstract Acute and chronic activation of opioid receptors differentially regulate the activity of the various adenylyl cyclase (AC) isoforms. In several AC isoforms (I, V, VI and VIII) acute opioid activation (by agonists such as morphine) leads to AC inhibition, while prolonged opioid activation leads to increase in AC activity, a phenomenon known as AC sensitization or superactivation. In several other AC isoforms (II, IV and VII), acute opioid activation leads to AC stimulation, while chronic opioid exposure inhibits AC activity, in a process, which in analogy to the term ‘‘superactivation’’ is referred to as ‘‘superinhibition’’. AC-II is highly regulated by multiple and independent biochemical stimuli, including Gbg, Gas and PKC activation. We investigated the regulation of AC-II by Gas and by PKC under conditions of acute and chronic exposure to opioid agonists in COS-7 transfected cells. We found that acute opioid exposure led to an increase in AC-II activity by either Gas or PKC stimulation. This effect seems to be regulated by Gbg subunits, in both activation pathways, as the increase in AC-II activity was abolished by pertussis toxin treatment and by Gbg scavengers. On the other hand, while chronic opioid exposure led to a decrease in AC-II activity (‘‘superinhibition’’) upon stimulation of the Gas pathway, this superinhibition was not observed when the opioid treated cells were stimulated via PKC activation. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Adenylyl cyclase type II; Opioid receptor; G-proteins; cAMP; PKC; Superinhibition
1. Introduction Mammalian cells possess genes for at least nine different isoforms of adenylyl cyclase (AC), denoted AC types IeIX, which differ in their tissue distribution and biochemical properties as well as in the degrees of sensitivity to different stimulatory or inhibitory agonists (Mons and Cooper, 1995; Simonds, 1999; Sunahara and Taussig, 2002). Little was known about the regulation of specific AC isozymes by agonists of the opioid- and other Gi/o-coupled receptors. In earlier studies, our group and other laboratories found that acute activation of the m-opioid receptor inhibits AC-I, AC-V, AC-VI and AC-VIII and stimulates the activity of AC-II, AC-IV and AC-VII (Tsu et al., 1995; Avidor-Reiss et al., 1997; Nevo et al., 1998). Chronic treatment followed
* Corresponding author. Tel.: þ972 8 934 4539; fax: þ972 8 934 4131. E-mail address: [email protected] (Z. Vogel). 0028-3908/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2006.01.004
by agonist withdrawal leads to an increase in the activity of AC-I, AC-V, AC-VI and AC-VIII, a phenomenon known as AC superactivation, while it leads to reduced activity of the AC-II family of AC isozymes (e.g. AC-II, IV and VII). This was referred to ‘‘superinhibition’’ in analogy to the ‘‘superactivation’’ described above. Similar results were obtained with several other Gi/o-coupled receptors (Nevo et al., 1998; Rhee et al., 1998, 2000). Thus, the effects of acute and chronic exposure to G-protein coupled receptor agonists in a given cell depend on the complement of AC isoforms present in that cell (Avidor-Reiss et al., 1997; Watts and Neve, 2005). Among the AC isozymes, AC-II, which is mainly expressed in the central nervous system (Feinstein et al., 1991), is particularly interesting. Using reconstituted systems it was shown that this isozyme can be stimulated by Gas subunits (Tang and Gilman, 1995), phorbol esters (Jacobowitz and Iyengar, 1994) and Gbg subunits (Taussig et al., 1993). In this regard, Jacobowitz and Iyengar (1994) demonstrated that the basal activity of AC-II was increased following stimulation by the
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PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA). Chakrabarti and Gintzler (2003) showed that in the presence of activated recombinant Gas, recombinant AC-II was dose dependently stimulated by Gbg, the magnitude of this activation was dependent upon its phosphorylation state. In this study, we used AC-II transfected cells to investigate its regulation by opioid agonists following either PKC or Gas activation. This was performed upon either acute or chronic opioid exposure. We found that both PKC and Gas activation pathways increased AC-II activity upon acute opioid exposure, and that this activation was Gbg dependent. Chronic opioid exposure led to superinhibition upon stimulation of AC-II via the Gas pathway, while it did not lead to changes in AC-II activity when the latter was stimulated by PKC.
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otherwise indicated, followed by agonist withdrawal (obtained by one wash and rapid addition of opioid receptor antagonist, i.e. 5 mM naloxone). The antagonist was added together with the AC stimulator (TPA or TSH) and AC activity was assayed for 10 min as described above. We found that the uptake of [3H]adenine into the cells was not affected by the chronic opioid agonist treatment.
2.5. Data analysis Data are expressed as means SD of three independent experiments, each performed in triplicate. The data (in cpm of [3H]cAMP) was transformed into percent of control activity and the percentages of the various experiments were averaged. Results were analyzed using ANOVA and where indicated followed by Dunnett’s post hoc t test comparing control with treated groups.
3. Results 2. Experimental 2.1. Materials [3H]Adenine (24.2 Ci/mmol) was purchased from Perkin Elmer Life Sciences, Inc. (Boston, MA, USA). Opioid ligands were obtained from Research Biochemical International (Natick, MA, USA) and from the National Institute of Drug Abuse, Research Technology Branch (Rockville, MD, USA). The phosphodiesterase inhibitors 1-methyl-3-isobutaylxanthine and RO-20-1724 were from Calbiochem (La Jolla, CA, USA). TPA, bovine serum albumin, thyroid-stimulating hormone (TSH) and cAMP were from Sigma (St. Louis, MO, USA). Pertussis toxin (PTX) was from List Biological Laboratories (Campbell, CA, USA).
2.2. Plasmids Plasmids encoding for AC-II, m-opioid receptor, TSH receptor, and atransducin were previously described (Avidor-Reiss et al., 1997). The Gas wild type was a gift of Dr J.S. Gutkind (NIH, Bethesda, MD, USA). The triple Gas dominant negative mutant, (a3b5/G226A/A366S) was a gift of Prof. C.H. Berlot (Geisinger Clinic, Danville, PA, USA).
2.3. Cell cultures and transfection methods COS-7 cells were cultured and transiently transfected using the DEAEdextran method, as described (Avidor-Reiss et al., 1996, 1997). The method was optimized to reach high transfection efficiency (ranging from 40 to 80%, as determined by cotransfecting a plasmid encoding b-galactosidase and staining the cells for this enzymatic activity). For the assay of AC activity (see below), the cells were transfected in 10-cm plates; 24 h later, the cells were transferred to 24-well plates, and 24 h later, assayed for AC activity.
2.4. AC activity AC activity was measured in intact cells prelabeled with [3H]adenine as previously described (Vogel et al., 1993; Avidor-Reiss et al., 1996). In brief, cells cultured in 24-well plates were preincubated for 2 h with 0.25 ml of 5 mCi/ml of [3H]adenine, and then washed with DMEM containing 20 mM HEPES (pH 7.4) and 0.1 mg/ml bovine serum albumin. Fresh medium containing phosphodiesterase inhibitors (0.5 mM RO-20-1724 and 0.5 mM 1methyl-3-isobutylxanthine) was then added, together with TPA (1 mM) or TSH (0.1 mM; in this case, the cells were co-transfected with a plasmid encoding the TSH receptor) to stimulate AC activity, in the presence or absence of opioid ligands. After 10 min of incubation, the medium was removed and the reaction stopped by addition of 2.5% perchloric acid. The supernatant was neutralized and applied to a two-step column separation procedure, as described (Salomon, 1991; Avidor-Reiss et al., 1996). Chronic opioid treatment was achieved by incubating the cells for 18 h with 1 mM morphine, unless
3.1. Acute and chronic m-opioid agonist treatments differentially regulate TPA- and TSH-stimulated AC-II activity As mentioned above, AC-II can be activated by either Gas, Gbg subunits or PKC. COS-7 cells co-transfected with m-opioid receptor, TSH receptor, and where indicated with AC-II, were used to examine TSH- and TPA-induced cAMP accumulation following acute or chronic opioid treatments. In Fig. 1a, it can be seen that exposure to TSH during the assay enhanced AC-II activity (by 14-fold). Activation of AC-II by TPA increased AC-II activity but to a lesser extent (3.5-fold). Simultaneous activation with TSH and TPA increased AC-II activity by 19-fold compared with the background value without stimulation suggesting additive effects of TSH and TPA. Acute exposure to the m-agonist morphine (1 mM, 10 min) led to a similar increase (of ca. 30e45%) in the TSH-, TPA- and TSH þ TPA-stimulated activity. Chronic exposure to morphine followed by withdrawal decreased TSH- and TSH þ TPA-stimulated AC-II activity, leading to superinhibition of AC activity (of 50% and 25% respectively). Interestingly, the TPA-stimulated AC-II activity did not show the superinhibition phenomenon. The TPA stimulated AC-II activity following withdrawal was the same as in non-agonist treated TPA stimulated cells. To insure that the acute and chronic opioid effects were specific for AC-II activation and were not due to COS-7 endogenous adenylyl cyclase(s) the cells were transfected as above (with m-opioid receptor and TSH receptor) but without AC-II and were subsequently stimulated with TSH, TPA, or TSH þ TPA. It can be seen (Fig. 1b), that the endogenous AC activity, while affected by Gas stimulation, is not affected by TPA and by either acute or chronic opioid exposure and thus it does not interfere with the measurement of AC-II activity. Fig. 1c shows the results for AC activity after subtracting the endogenous AC activity. It shows that the stimulation of AC-II by acute opioid exposure is similar in the presence of TSH, TPA, and TSH þ TPA (1.6-, 1.5- and 1.4-fold, respectively). It also shows that the level of superinhibition was equivalent to 66% and to 26% for the TSH and TSH þ TPA stimulated AC-II activity, respectively.
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PKC can also be stimulated by activation of Gaq-coupled receptors (e.g. m3-muscarinic receptor). In the experiment presented in Fig. 2 the cells were transfected with AC-II, m-opioid receptor, and m3-muscarinic receptor cDNAs and the muscarinic receptor was activated (during the AC assay) with 10 mM carbachol. As shown above for TPA (see Fig. 1), carbachol stimulation increased AC-II activation. Acute exposure to morphine in the presence of carbachol increased AC-II activity further, while withdrawal of the chronic opioid agonist did not show the superinhibition phenomenon. This result suggests that, in contrary to stimulation via Gas (by TSH), PKC stimulation, independently of the activation mechanism (by TPA or Gaq), is not able to induce AC-II superinhibition after withdrawal of the chronically applied opioid agonist.
3.2. Time-dependency of AC-II superinhibition upon chronic agonist treatment and its reversal To investigate whether the superinhibition is a time-dependent process cells were treated with opioid agonists for different periods of time and after a quick wash naloxone was added to the cells. Fig. 3a shows that the acquisition of superinhibition is a time dependent biphasic process. The first phase was rapid, requiring w1 h to reach 50% of its maximal effect, while the second phase was much slower and commenced in the 2e6 h time frame. Similar results were observed with the two opioid agonists tested, morphine and DAMGE ([DAla2,N-methyl-Phe4,Gly-ol5]enkephalin). The state of superinhibition can be reversed by prolonged exposure to the opioid antagonist. In the experiment shown in Fig. 3b, the cells were treated chronically (18 h) with either morphine or DAMGE and then for the times indicated with the antagonist naloxone. Similarly to the development of superinhibition, the
Fig. 1. Effects of acute and chronic activation of the m-opioid receptor on the activity of AC-II. COS-7 cells were transfected with the cDNAs of m-opioid receptor (1 mg), TSH receptor (1 mg), and where indicated with AC-II (2 mg). After 48 h, AC activity was stimulated with 0.1 mM TSH or 1 mM TPA or their combination in the presence or absence of opioid ligands. (a) Effect of acute and chronic morphine exposure on Gas (TSH)- and PKC (TPA)stimulated AC-II activity. Basal: AC activity without stimulation or opioid treatment; Stimulation: the effect of TSH and/or TPA on AC-II activity; Acute: 1 mM morphine was present during the 10-min AC assay; Withdrawal: cells were incubated with 1 mM morphine for 18 h, followed by rapid wash and the addition of 5 mM naloxone at the start of the AC assay. (b) Effects of TSH and TPA on the activity of endogenous AC (the same batch of cells were transfected as above but without the cDNA for AC-II). (c) Net AC-II activity after subtraction of the endogenous AC activity. AC activity is expressed in cpm of cAMP accumulation and is presented as mean SD of triplicate determinations of a representative experiment out of three experiments which gave the same results.
Fig. 2. Regulation of Gaq-stimulated AC-II activity. COS-7 cells were transfected with cDNAs of m-opioid receptor, AC-II and m3-muscarinic receptor. Basal: carbachol was not present during the AC assay. Stimulation: carbachol (10 mM)-stimulated AC-II activity (no opioid ligands were present); Acute: 1 mM morphine was present together with carbachol during the 10-min AC assay; Withdrawal: cells were incubated with 1 mM morphine for 18 h, followed by rapid wash and the addition of 5 mM naloxone at the start of the AC assay. cAMP accumulations are expressed in cpm and are mean SD of triplicate determinations of a representative experiment out of three experiments which gave the same results.
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AC-II was not affected by the overexpression of Gas wt, while it was strongly decreased (to 20% of control levels) by the Gas dn mutant (Fig. 4a). The activity of AC-II in the presence of TPA could be further increased by Gas wt, but not by the Gas dn mutant. This suggests additive activation of AC-II by Gas subunits and PKC in agreement with the additive effects of TSH and TPA (see Fig. 1). In the next step, we investigated the effect of Gas wt and Gas dn on the acute and chronic opioid effects. We found (Fig. 4b) that both Gas wt and Gas dn, significantly reduced the acute opiate increase of AC-II activity under both TPA and TSH stimulating conditions. The reduction in the level of acute opioid stimulation of AC-II by the Gas wt and Gas dn mutant could be due to the bg scavenging effect of these overexpressed Gas molecules. The somewhat stronger effect
Fig. 3. Kinetics of AC superinhibition and its reversal. COS-7 cells were transfected and assayed as described in Fig. 1. (a) Kinetics of superinhibition: cells were treated with 1 mM morphine or DAMGE for the indicated periods of time. After a quick wash naloxone (5 mM) was added and AC activity assayed. (b) Kinetics of loss of superinhibition. Cells were chronically treated with morphine or DAMGE (18 h, 1 mM), after a quick wash, naloxone was added (5 mM) for the indicated times and AC activity assayed during the last 10 min of incubation. The data represent the mean SD of three independent experiments. 100% represents AC-II activity under control (TSH-stimulated) conditions.
reversal of the superinhibition was also biphasic. The first 1 h with naloxone led to a quick reversal achieving ca. 65% reversal from the superinhibited state, while the second slower phase, had a very small additional contribution to the reversal of the superinhibition. The total AC activity did not return to the 100% level even after 6 h with naloxone. Again, similar results were observed with cells pretreated with either morphine or DAMGE.
3.3. Role of Gas in TSH and TPA stimulation pathways A triple Gas mutant, (a3b5/G226A/A366S), which blocks signaling from the luteinizing hormone receptor to Gas by up to 97%, acting as a dominant negative mutant, was described by Berlot (2002). To determine the role of Gas in the TSH and TPA activation pathways, COS-7 cells were transfected with m-opiod receptor, TSH receptor and AC-II cDNAs with or without the cDNA for the Gas wild type (Gas wt) or its dominant negative mutant (Gas dn). The activity of TSH-stimulated
Fig. 4. Effect of dominant negative Gas mutant on TSH- and TPA-stimulated AC-II activity and its opioid regulation. Cells were transfected as described in Fig. 1 with the addition (where indicated) of 2 mg of the cDNA for Gas wt or Gas dn mutant (Gas a3b5/G226A/A366S). (a) Effect of wt and dn Gas on TSHand TPA-stimulated AC-II activity. 100% represents AC stimulated activity in the absence of Gas. (b) Effect of Gas wt and dn and morphine treatments on AC-II activity. 100% represents AC stimulated activity following stimulation by either TSH or TPA as indicated. Data are mean SD of triplicate determinations of three separate experiments. *p < 0.05 versus the appropriate stimulated control; A, #, sp < 0.05 versus the treatment marked with the same symbol (Dunnett’s post-repeated measures ANOVA).
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of the dominant negative molecule is in line with the increased affinity of this mutant (and especially the G226A part of the molecule) to bg (Berlot, 2002; Lee et al., 1992). The superinhibition (upon chronic opioid treatment followed by agonist withdrawal) was significantly decreased (by ca. 50%) by both Gas wt and Gas dn mutant under the conditions of TSH-stimulation. TPA-stimulation did not show the superinhibition phenomenon independently of the presence or absence of the Gas wt or Gas dn mutant. 3.4. Role of Gi/o proteins in AC-II activation PTX is known as an efficient blocker of Gi/o signaling. As shown in Fig. 5, pretreatment with PTX did not induce any changes in the level of stimulation of AC-II by either TSH or TPA. This result demonstrates that there is no involvement of Gi/o proteins under these stimulatory conditions. On the other hand, the increase in AC-II activity obtained by acute opioid exposure was completely eliminated by PTX pretreatment, in both the TSH- and TPA-stimulated pathways. This result is in line with the involvement of Gi/o proteins during opioid receptor activation. Similarly, the superinhibition after withdrawal of the chronic opioid agonist was almost completely eliminated.
Fig. 5. Effect of PTX on TSH- and TPA-stimulated AC-II activity upon opioid exposure. Cells were incubated with PTX (100 ng/ml, 18 h). AC-II activity was determined following acute and chronic (withdrawal) opioid exposure. (a) TSH- and (b) TPA-stimulated cAMP accumulation are expressed in cpm [3H]cAMP formed and are mean SD of triplicate determinations of a representative experiment out of three experiments which gave the same results.
This is easily observed in cells where AC-II was activated using TSH. The TPA activation did not yield superinhibition even under control conditions and this was not changed by the PTX pretreatment. The trend toward a decrease in AC-II activation upon acute exposure to opioids in the presence of PTX is probably due to the reduction, by PTX, of the amount of free Gbg subunits liberated from Gi/o upon opioid exposure. 3.5. The Gbg scavenger a-transducin reduces the activity of AC-II The a subunit of transducin is a known scavenger of Gbg, which can bind to free Gbg, and reduce its concentration (Federman et al., 1992). Fig. 6a shows that the TSH-stimulated activity of AC-II was strongly decreased in cells co-transfected with a-transducin (reaching 30% of control levels), while TPA-stimulated activation was not affected. This result shows that AC-II activation upon TSH stimulation is for the most part
Fig. 6. Effect of a-transducin on AC-II activity upon acute and chronic morphine exposure. COS-7 cells were transfected as in Fig. 1, and where indicated, with the cDNA for a-transducin (2 mg). (a) Effect of a-transducin on AC-II activity. 100% represents AC stimulated activity in the absence of a-transducin. (b) Effect of a-transducin and morphine treatments on AC-II activity. 100% represents AC stimulated activity following stimulation by either TSH or TPA as indicated. The data represent the mean SD of triplicate determinations of three separate experiments. *p < 0.05 versus the appropriate stimulated control; A, #, sp < 0.05 versus the treatment marked with the same symbol (Dunnett’s post-repeated measures ANOVA).
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bg dependent, while TPA activation is not. AC-II activation by acute opioid exposure was strongly decreased by a-transducin when the initial activation was performed with either TSH (by 63%) or TPA (completely eliminated) (Fig. 6b). The level of superinhibition observed following withdrawal from chronic opioid exposure (under TSH activation) was also reduced (from 67% to 42%) but was not completely eliminated. 4. Discussion Among the multiple isoforms of AC cloned to date, the type II family isoforms (AC-II, AC-IV and AC-VII) are unique for their positive responsiveness to G protein bg subunits. The stimulation of AC-II by bg in vitro is conditional, requiring coincidence of a second signal, such as Gas- (Tang and Gilman, 1995; Taussig et al., 1994) or PKC-activation (Lustig et al., 1993). Earlier studies demonstrated that the basal activity of AC-II was increased followed stimulation by TPA or 12,13 phorbol dibutyrate (PdBu) while the inactive isomer of TPA, 4-a-phorbol 12-myristate 13-acetate (4-a-PMA) had no effect (Jacobowitz and Iyengar, 1994; Lustig et al., 1993; Yoshimura and Cooper, 1993). Similar activation by TPA was also reported for AC-VI (Beazely et al., 2005). Moreover, Chakrabarti et al. (2001) showed that the increment in AC-II activity produced by Gbg was increased by approximately 2fold following in vitro phosphorylation by the catalytic subunit of either PKA or PKC. Indeed, we have shown here that AC-II activity was stimulated by the Gas pathway (through the TSH receptor) as well as by PKC activation (e.g. with TPA or by activation of a Gaq-coupled receptor). Simultaneous stimulation of both pathways led to a further increase in AC-II activity. Our results suggest that the activation of AC-II is independent for each of the two pathways, since co-transfection with a Gas dominant negative mutant, led to a decrease in the Gas-activated pathway but did not significantly affect the PKC-activated pathway. Acute opioid exposure increased AC-II activity via both the TPA and Gas activation pathways. This result is in agreement with the finding that HEK 293 cells transiently expressing AC-II and Gi/o-coupled receptors show an efficient receptormediated potentiation of PKC-activated AC-II (Tsu and Wong, 1996). In a similar way, Nasman et al. (2002) reported that the effects of a2A-adrenoreceptor and TPA are additive on AC-II activity. The activation of Gi/o-coupled receptors releases bg subunits, which in turn potentiate the activation of AC-II by Gas or PKC. In this regard, in vitro studies have shown a potentiating effect of bg when AC-II is activated by PKC, albeit with lower potency compared with the effect of bg on the as pathway (Zimmermann and Taussig, 1996). The role of bg subunits in the two pathways is supported by the observation that co-expression of a-transducin, which sequesters the released bg subunits, blocks activation by Gi/o-coupled receptors (Tsu et al., 1995; Tsu and Wong, 1996). These observations are in agreement with our data showing that (i) the acute opioid activation of AC-II was suppressed by PTX pretreatment, and (ii) co-transfection with a-transducin led to a decrease in the opioid agonist AC-II activation in both pathways.
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Interestingly, chronic opioid exposure led to a different ACII regulation pattern following either Gas or PKC activation. While chronic exposure to opioid agonist followed by withdrawal decreased TSH- and TSH þ TPA-stimulated AC-II activity, leading to superinhibition, TPA-stimulated AC-II activity did not show the superinhibition phenomenon. Similar results were shown upon activation of PKC via activation of the Gaq coupled m3-muscarinic receptor. This decrease in Gas activated-AC-II activity upon chronic agonist exposure was shown to be also dependent on bg subunits, since PTX pretreatment and a-transducin overexpression led to a large decrease in the level of superinhibition observed. The finding that the superinhibition of AC-II is reduced by this Gbg scavenger suggests that either chronic activation of AC-II per se is needed for AC-II superinhibition, or that Gbg dimers are important for the superinhibition phenomenon, in addition to their role in the acute activation of AC-II. Our laboratory has previously shown that chronic activation of the m-opioid receptor leads to a time-dependent shift in the detergent solubility of both Gai and Gb1 subunits (Bayewitch et al., 2000). It was proposed that these proteins may undergo an agonist-induced time-dependent physical or chemical modification that alters their ability to be solubilized, or that these G protein subunits may interact with other protein partners that prevent their solubilization. It is possible that the superinhibition process, described here, is a consequence of such a shift in the solubility of the bg dimers. The PKC-stimulated AC-II activity in cells chronically treated with the opioid agonist was not affected by the PTX treatment and was not affected by the presence of a-transducin. These results suggest that bg may not be directly involved in this regulatory mechanism. On the other hand, it is also possible that the application of phorbol esters enhances the rate of receptor phosphorylation and down-regulation (Kramer and Simon, 1999), leading to a loss of the chronic receptor effect and hence to a loss of its capacity to regulate AC-II activity (which returns to control level). We found that acquiring the superinhibition of AC-II is a time-dependent biphasic process. The first phase was rapid requiring ca. 1 h to reach 50% of its maximal effect, while the second phase was much slower. Similar results were observed for cells pretreated with two opioid agonists (morphine or DAMGE). Moreover, we have shown that the state of superinhibition is for the most part reversible and can be reversed by prolonged exposure to the opioid antagonist naloxone. This reversal was also biphasic. The first hour with naloxone led to a very rapid reversal of the superinhibition, while the second phase of the reversal was much slower. Interestingly, the biphasic kinetics of acquiring the superinhibition of AC-II is similar to that observed for the superactivation of AC-I and AC-VIII following chronic exposure to the D2-dopaminergic or m-opioid receptor agonists, respectively (Nevo et al., 1998; Steiner et al., 2005). The reasons for the biphasic nature of the chronic agonist-induced AC superactivation or superinhibition are not yet clear but hint to the complexity of the mechanisms involved. This similarity in kinetics could suggest a common mechanism for the superinhibition of AC-II and the
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Fig. 7. Schematic diagram illustrating the effect of acute and chronic agonist exposure on the Gs and PKC activating pathways. Activation via the Gs pathway. (a) AC-II is activated by Gas as well as by the Gbg released by GsPCR stimulation. (b) Upon acute opioid receptor stimulation additional Gbg is released (from Gi/o) leading to further activation of AC-II. (c) Following withdrawal from chronic opioid treatment the activity of AC-II is decreased, due to relocalization or degradation of the bg subunits (those originated from the Gs as well as those from the Gi/o). Activation via the PKC pathway. (d) AC-II is activated by TPA. (e) AC-II is activated by Gbg released by acute opioid receptor stimulation. (f) Withdrawal from chronic opioid receptor activation leads to relocalization or degradation of Gbg subunits (originating from Gi/o) and AC-II activity reaches control levels.
superactivation of AC-VIII, AC-I and probably AC-V. Such a mechanism could involve the translocation of bg subunits following chronic exposure as described above and in Bayewitch et al. (2000) or its degradation following chronic exposure (Mouledous et al., 2005). The existence of such degradation of Gbg could also explain why the reversal of the superinhibition following prolonged antagonist exposure reached a maximal value of 75% and did not reach the 100% value of the AC-II initial activity. The various steps that seem to be involved in the acute and chronic opioid regulation of AC-II when stimulated via either TSH or TPA activation and the possible role of Gbg dimers are presented in a model shown in Fig. 7. In summary, we found that acute opioid exposure led to an increase in AC-II activity via either Gas or PKC stimulation.
This effect seems to be regulated by Gbg subunits in both activation pathways as AC-II activity was decreased by PTX treatment and by a Gbg scavenger. Chronic opioid exposure led to a decrease in AC-II activity (superinhibition) upon stimulation of the Gas pathway. This superinhibition was not observed following PKC activation.
Acknowledgments We thank Professor C.H. Berlot and Dr J.S. Gutkind for providing the plasmids containing the Gas dominant negative mutant and Gas wild type, respectively. This work was supported by the National Institute of Drug Abuse (grant DA-06265) and the Nella and Leon Benoziyo Center for the Neurosciences.
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Z.V. is the incumbent of the Ruth and Leonard Simon Chair for Cancer Research. References Avidor-Reiss, T., Nevo, I., Levy, R., Pfeuffer, T., Vogel, Z., 1996. Chronic opioid treatment induces adenylyl cyclase V superactivation: involvement of Gbg. J. Biol. Chem. 271, 21309e21315. Avidor-Reiss, T., Nevo, I., Saya, D., Bayewitch, M., Vogel, Z., 1997. Opiateinduced adenylyl cyclase superactivation is isozyme specific. J. Biol. Chem. 272, 5040e5047. Bayewitch, M., Nevo, I., Avidor-Reiss, T., Levy, R., Simonds, W.F., Vogel, Z., 2000. Alterations in detergent solubility of heterotrimeric G proteins after chronic activation of G(i/o)-coupled receptors: changes in detergent solubility are in correlation with onset of adenylyl cyclase superactivation. Mol. Pharmacol. 57, 820e825. Beazely, M.A., Alan, J.K., Watts, V.J., 2005. Protein kinase C and epidermal growth factor stimulation of Raf1 potentiates adenylyl cyclase type 6 activation in intact cells. Mol. Pharmacol. 67, 250e259. Berlot, C.H., 2002. A highly effective dominant negative as construct containing mutations that affect distinct functions inhibits multiple Gs-coupled receptor signaling pathways. J. Biol. Chem. 277, 21080e21085. Chakrabarti, S., Oppermann, M., Gintzler, A.R., 2001. Chronic morphine induces the concomitant phosphorylation and altered association of multiple signaling proteins: a novel mechanism for modulating cell signaling. Proc. Natl. Acad. Sci. U.S.A. 98, 4209e4214. Chakrabarti, S., Gintzler, A.R., 2003. Phosphorylation of Gb is augmented by chronic morphine and enhances Gbg stimulation of adenylyl cyclase activity. Brain Res. Mol. Brain Res. 119, 144e151. Federman, A.D., Conklin, B.R., Schrader, K.A., Reed, R.R., Bourne, H.R., 1992. Hormonal stimulation of adenylyl cyclase through Gi-protein bg subunits. Nature 356, 159e161. Feinstein, P.G., Schrader, K.A., Bakalyar, H.A., Tang, W.J., Krupinski, J., Gilman, A.G., Reed, R.R., 1991. Molecular cloning and characterization of a Ca2þ/calmodulin-insensitive adenylyl cyclase from rat brain. Proc. Natl. Acad. Sci. U.S.A. 88, 10173e10177. Jacobowitz, O., Iyengar, R., 1994. Phorbol ester-induced stimulation and phosphorylation of adenylyl cyclase 2. Proc. Natl. Acad. Sci. U.S.A. 91, 10630e10634. Kramer, H.K., Simon, E.J., 1999. Role of protein kinase C (PKC) in agonistinduced mu-opioid receptor down-regulation: I. PKC translocation to the membrane of SH-SY5Y neuroblastoma cells is induced by mu-opioid agonists. J. Neurochem. 72, 585e593. Lee, E., Taussig, R., Gilman, A.G., 1992. The G226A mutant of Gs alpha highlights the requirement for dissociation of G protein subunits. J. Biol. Chem. 267, 1212e1218. Lustig, K.D., Conklin, B.R., Herzmark, P., Taussig, R., Bourne, H.R., 1993. Type II adenylylcyclase integrates coincident signals from Gs, Gi, and Gq. J. Biol. Chem. 268, 13900e13905. Mons, N., Cooper, D., 1995. Adenylate cyclases: critical foci in neuronal signaling. Trends Neurosci. 18, 536e542.
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Mouledous, L., Neasta, J., Uttenweiler-Joseph, S., Stella, A., Matondo, M., Corbani, M., Monsarrat, B., Meunier, J.C., 2005. Chronic morphine treatment enhances proteasome-dependent degradation of Gb in human neuroblastoma SH-SY5Y cells: correlation with onset of adenylate cyclase sensitization. Mol. Pharmacol. 68, 467e476. Nasman, J., Kukkonen, J.P., Holmqvist, T., Akerman, K.E., 2002. Different roles for Gi and Go proteins in modulation of adenylyl cyclase type-2 activity. J. Neurochem. 83, 1252e1261. Nevo, I., Avidor-Reiss, T., Levy, R., Bayewitch, M., Heldman, E., Vogel, Z., 1998. Regulation of adenylyl cyclase isozymes upon acute and chronic activation of inhibitory receptors. Mol. Pharmacol. 54, 419e426. Rhee, M.-H., Bayewitch, M.L., Avidor-Reiss, T., Levy, R., Vogel, Z., 1998. Cannabinoid receptor activation differentially regulates the various adenylyl cyclase isozymes. J. Neurochem. 71, 1525e1534. Rhee, M.H., Nevo, I., Avidor-Reiss, T., Levy, R., Vogel, Z., 2000. Differential superactivation of adenylyl cyclase isozymes after chronic activation of the CB1 cannabinoid receptor. Mol. Pharmacol. 57, 746e752. Salomon, Y., 1991. Cellular responsiveness to hormones and neurotransmitters: conversion of [3H]adenine to [3H]cAMP in cell monolayers, cell suspensions, and tissue slices. Methods Enzymol 195, 22e28. Simonds, W.F., 1999. G protein regulation of adenylate cyclase. Trends Pharmacol. Sci. 20, 66e73. Steiner, D., Avidor-Reiss, T., Schallmach, E., Butovsky, E., Lev, N., Vogel, Z., 2005. Regulation of adenylyl cyclase type VIII splice variants by acute and chronic Gi/o-coupled receptor activation. Biochem. J. 386, 341e348. Sunahara, R.K., Taussig, R., 2002. Isoforms of mammalian adenylyl cyclase: multiplicities of signaling. Mol. Interv. 2, 168e184. Tang, W.J., Gilman, A.G., 1995. Construction of a soluble adenylyl cyclase activated by Gsa and forskolin. Science 268, 1769e1772. Taussig, R., Iniguez-Lluhi, J., Gilman, A.G., 1993. Inhibition of adenylyl cyclase by Gia. Science 261, 218e221. Taussig, R., Tang, W.J., Hepler, J.R., Gilman, A.G., 1994. Distinct patterns of bidirectional regulation of mammalian adenylyl cyclases. J. Biol. Chem. 269, 6093e6100. Tsu, R.C., Chan, J.S.C., Wong, Y.H., 1995. Regulation of multiple effectors by the cloned d-opioid receptor: stimulation of phospholipase C and type II adenylyl cyclase. J. Neurochem. 64, 2700e2707. Tsu, R.C., Wong, Y.H., 1996. Gi-mediated stimulation of type II adenylyl cyclase is augmented by Gq-coupled receptor activation and phorbol ester treatment. J. Neurosci. 16, 1317e1323. Vogel, Z., Barg, J., Levy, R., Saya, D., Heldman, E., Mechoulam, R., 1993. Anandamide, a brain endogenous compound, interacts specifically with cannabinoid receptors and inhibits adenylate cyclase. J. Neurochem. 61, 352e355. Yoshimura, M., Cooper, D.M., 1993. Type-specific stimulation of adenylylcyclase by protein kinase C. J. Biol. Chem. 268, 4604e4607. Watts, V.J., Neve, K.A., 2005. Sensitization of adenylate cyclase by Gai/ o-coupled receptors. Pharmacol. Ther. 106, 405e421. Zimmermann, G., Taussig, R., 1996. Protein kinase C alters the responsiveness of adenylyl cyclases to G protein a and bg subunits. J. Biol. Chem. 271, 27161e27166.
Adenylyl cyclase type II activity is regulated by two different mechanisms: Implications for acute and chronic opioid exposure Ester Schallmach, Debora Steiner, Zvi Vogel* Department of Neurobiology, The Weizmann Institute of Science, Hertzel str, Rehovot 76100, Israel Received 7 October 2005; received in revised form 2 January 2006; accepted 25 January 2006
Abstract Acute and chronic activation of opioid receptors differentially regulate the activity of the various adenylyl cyclase (AC) isoforms. In several AC isoforms (I, V, VI and VIII) acute opioid activation (by agonists such as morphine) leads to AC inhibition, while prolonged opioid activation leads to increase in AC activity, a phenomenon known as AC sensitization or superactivation. In several other AC isoforms (II, IV and VII), acute opioid activation leads to AC stimulation, while chronic opioid exposure inhibits AC activity, in a process, which in analogy to the term ‘‘superactivation’’ is referred to as ‘‘superinhibition’’. AC-II is highly regulated by multiple and independent biochemical stimuli, including Gbg, Gas and PKC activation. We investigated the regulation of AC-II by Gas and by PKC under conditions of acute and chronic exposure to opioid agonists in COS-7 transfected cells. We found that acute opioid exposure led to an increase in AC-II activity by either Gas or PKC stimulation. This effect seems to be regulated by Gbg subunits, in both activation pathways, as the increase in AC-II activity was abolished by pertussis toxin treatment and by Gbg scavengers. On the other hand, while chronic opioid exposure led to a decrease in AC-II activity (‘‘superinhibition’’) upon stimulation of the Gas pathway, this superinhibition was not observed when the opioid treated cells were stimulated via PKC activation. Ó 2006 Elsevier Ltd. All rights reserved. Keywords: Adenylyl cyclase type II; Opioid receptor; G-proteins; cAMP; PKC; Superinhibition
1. Introduction Mammalian cells possess genes for at least nine different isoforms of adenylyl cyclase (AC), denoted AC types IeIX, which differ in their tissue distribution and biochemical properties as well as in the degrees of sensitivity to different stimulatory or inhibitory agonists (Mons and Cooper, 1995; Simonds, 1999; Sunahara and Taussig, 2002). Little was known about the regulation of specific AC isozymes by agonists of the opioid- and other Gi/o-coupled receptors. In earlier studies, our group and other laboratories found that acute activation of the m-opioid receptor inhibits AC-I, AC-V, AC-VI and AC-VIII and stimulates the activity of AC-II, AC-IV and AC-VII (Tsu et al., 1995; Avidor-Reiss et al., 1997; Nevo et al., 1998). Chronic treatment followed
* Corresponding author. Tel.: þ972 8 934 4539; fax: þ972 8 934 4131. E-mail address: [email protected] (Z. Vogel). 0028-3908/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2006.01.004
by agonist withdrawal leads to an increase in the activity of AC-I, AC-V, AC-VI and AC-VIII, a phenomenon known as AC superactivation, while it leads to reduced activity of the AC-II family of AC isozymes (e.g. AC-II, IV and VII). This was referred to ‘‘superinhibition’’ in analogy to the ‘‘superactivation’’ described above. Similar results were obtained with several other Gi/o-coupled receptors (Nevo et al., 1998; Rhee et al., 1998, 2000). Thus, the effects of acute and chronic exposure to G-protein coupled receptor agonists in a given cell depend on the complement of AC isoforms present in that cell (Avidor-Reiss et al., 1997; Watts and Neve, 2005). Among the AC isozymes, AC-II, which is mainly expressed in the central nervous system (Feinstein et al., 1991), is particularly interesting. Using reconstituted systems it was shown that this isozyme can be stimulated by Gas subunits (Tang and Gilman, 1995), phorbol esters (Jacobowitz and Iyengar, 1994) and Gbg subunits (Taussig et al., 1993). In this regard, Jacobowitz and Iyengar (1994) demonstrated that the basal activity of AC-II was increased following stimulation by the
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PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA). Chakrabarti and Gintzler (2003) showed that in the presence of activated recombinant Gas, recombinant AC-II was dose dependently stimulated by Gbg, the magnitude of this activation was dependent upon its phosphorylation state. In this study, we used AC-II transfected cells to investigate its regulation by opioid agonists following either PKC or Gas activation. This was performed upon either acute or chronic opioid exposure. We found that both PKC and Gas activation pathways increased AC-II activity upon acute opioid exposure, and that this activation was Gbg dependent. Chronic opioid exposure led to superinhibition upon stimulation of AC-II via the Gas pathway, while it did not lead to changes in AC-II activity when the latter was stimulated by PKC.
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otherwise indicated, followed by agonist withdrawal (obtained by one wash and rapid addition of opioid receptor antagonist, i.e. 5 mM naloxone). The antagonist was added together with the AC stimulator (TPA or TSH) and AC activity was assayed for 10 min as described above. We found that the uptake of [3H]adenine into the cells was not affected by the chronic opioid agonist treatment.
2.5. Data analysis Data are expressed as means SD of three independent experiments, each performed in triplicate. The data (in cpm of [3H]cAMP) was transformed into percent of control activity and the percentages of the various experiments were averaged. Results were analyzed using ANOVA and where indicated followed by Dunnett’s post hoc t test comparing control with treated groups.
3. Results 2. Experimental 2.1. Materials [3H]Adenine (24.2 Ci/mmol) was purchased from Perkin Elmer Life Sciences, Inc. (Boston, MA, USA). Opioid ligands were obtained from Research Biochemical International (Natick, MA, USA) and from the National Institute of Drug Abuse, Research Technology Branch (Rockville, MD, USA). The phosphodiesterase inhibitors 1-methyl-3-isobutaylxanthine and RO-20-1724 were from Calbiochem (La Jolla, CA, USA). TPA, bovine serum albumin, thyroid-stimulating hormone (TSH) and cAMP were from Sigma (St. Louis, MO, USA). Pertussis toxin (PTX) was from List Biological Laboratories (Campbell, CA, USA).
2.2. Plasmids Plasmids encoding for AC-II, m-opioid receptor, TSH receptor, and atransducin were previously described (Avidor-Reiss et al., 1997). The Gas wild type was a gift of Dr J.S. Gutkind (NIH, Bethesda, MD, USA). The triple Gas dominant negative mutant, (a3b5/G226A/A366S) was a gift of Prof. C.H. Berlot (Geisinger Clinic, Danville, PA, USA).
2.3. Cell cultures and transfection methods COS-7 cells were cultured and transiently transfected using the DEAEdextran method, as described (Avidor-Reiss et al., 1996, 1997). The method was optimized to reach high transfection efficiency (ranging from 40 to 80%, as determined by cotransfecting a plasmid encoding b-galactosidase and staining the cells for this enzymatic activity). For the assay of AC activity (see below), the cells were transfected in 10-cm plates; 24 h later, the cells were transferred to 24-well plates, and 24 h later, assayed for AC activity.
2.4. AC activity AC activity was measured in intact cells prelabeled with [3H]adenine as previously described (Vogel et al., 1993; Avidor-Reiss et al., 1996). In brief, cells cultured in 24-well plates were preincubated for 2 h with 0.25 ml of 5 mCi/ml of [3H]adenine, and then washed with DMEM containing 20 mM HEPES (pH 7.4) and 0.1 mg/ml bovine serum albumin. Fresh medium containing phosphodiesterase inhibitors (0.5 mM RO-20-1724 and 0.5 mM 1methyl-3-isobutylxanthine) was then added, together with TPA (1 mM) or TSH (0.1 mM; in this case, the cells were co-transfected with a plasmid encoding the TSH receptor) to stimulate AC activity, in the presence or absence of opioid ligands. After 10 min of incubation, the medium was removed and the reaction stopped by addition of 2.5% perchloric acid. The supernatant was neutralized and applied to a two-step column separation procedure, as described (Salomon, 1991; Avidor-Reiss et al., 1996). Chronic opioid treatment was achieved by incubating the cells for 18 h with 1 mM morphine, unless
3.1. Acute and chronic m-opioid agonist treatments differentially regulate TPA- and TSH-stimulated AC-II activity As mentioned above, AC-II can be activated by either Gas, Gbg subunits or PKC. COS-7 cells co-transfected with m-opioid receptor, TSH receptor, and where indicated with AC-II, were used to examine TSH- and TPA-induced cAMP accumulation following acute or chronic opioid treatments. In Fig. 1a, it can be seen that exposure to TSH during the assay enhanced AC-II activity (by 14-fold). Activation of AC-II by TPA increased AC-II activity but to a lesser extent (3.5-fold). Simultaneous activation with TSH and TPA increased AC-II activity by 19-fold compared with the background value without stimulation suggesting additive effects of TSH and TPA. Acute exposure to the m-agonist morphine (1 mM, 10 min) led to a similar increase (of ca. 30e45%) in the TSH-, TPA- and TSH þ TPA-stimulated activity. Chronic exposure to morphine followed by withdrawal decreased TSH- and TSH þ TPA-stimulated AC-II activity, leading to superinhibition of AC activity (of 50% and 25% respectively). Interestingly, the TPA-stimulated AC-II activity did not show the superinhibition phenomenon. The TPA stimulated AC-II activity following withdrawal was the same as in non-agonist treated TPA stimulated cells. To insure that the acute and chronic opioid effects were specific for AC-II activation and were not due to COS-7 endogenous adenylyl cyclase(s) the cells were transfected as above (with m-opioid receptor and TSH receptor) but without AC-II and were subsequently stimulated with TSH, TPA, or TSH þ TPA. It can be seen (Fig. 1b), that the endogenous AC activity, while affected by Gas stimulation, is not affected by TPA and by either acute or chronic opioid exposure and thus it does not interfere with the measurement of AC-II activity. Fig. 1c shows the results for AC activity after subtracting the endogenous AC activity. It shows that the stimulation of AC-II by acute opioid exposure is similar in the presence of TSH, TPA, and TSH þ TPA (1.6-, 1.5- and 1.4-fold, respectively). It also shows that the level of superinhibition was equivalent to 66% and to 26% for the TSH and TSH þ TPA stimulated AC-II activity, respectively.
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PKC can also be stimulated by activation of Gaq-coupled receptors (e.g. m3-muscarinic receptor). In the experiment presented in Fig. 2 the cells were transfected with AC-II, m-opioid receptor, and m3-muscarinic receptor cDNAs and the muscarinic receptor was activated (during the AC assay) with 10 mM carbachol. As shown above for TPA (see Fig. 1), carbachol stimulation increased AC-II activation. Acute exposure to morphine in the presence of carbachol increased AC-II activity further, while withdrawal of the chronic opioid agonist did not show the superinhibition phenomenon. This result suggests that, in contrary to stimulation via Gas (by TSH), PKC stimulation, independently of the activation mechanism (by TPA or Gaq), is not able to induce AC-II superinhibition after withdrawal of the chronically applied opioid agonist.
3.2. Time-dependency of AC-II superinhibition upon chronic agonist treatment and its reversal To investigate whether the superinhibition is a time-dependent process cells were treated with opioid agonists for different periods of time and after a quick wash naloxone was added to the cells. Fig. 3a shows that the acquisition of superinhibition is a time dependent biphasic process. The first phase was rapid, requiring w1 h to reach 50% of its maximal effect, while the second phase was much slower and commenced in the 2e6 h time frame. Similar results were observed with the two opioid agonists tested, morphine and DAMGE ([DAla2,N-methyl-Phe4,Gly-ol5]enkephalin). The state of superinhibition can be reversed by prolonged exposure to the opioid antagonist. In the experiment shown in Fig. 3b, the cells were treated chronically (18 h) with either morphine or DAMGE and then for the times indicated with the antagonist naloxone. Similarly to the development of superinhibition, the
Fig. 1. Effects of acute and chronic activation of the m-opioid receptor on the activity of AC-II. COS-7 cells were transfected with the cDNAs of m-opioid receptor (1 mg), TSH receptor (1 mg), and where indicated with AC-II (2 mg). After 48 h, AC activity was stimulated with 0.1 mM TSH or 1 mM TPA or their combination in the presence or absence of opioid ligands. (a) Effect of acute and chronic morphine exposure on Gas (TSH)- and PKC (TPA)stimulated AC-II activity. Basal: AC activity without stimulation or opioid treatment; Stimulation: the effect of TSH and/or TPA on AC-II activity; Acute: 1 mM morphine was present during the 10-min AC assay; Withdrawal: cells were incubated with 1 mM morphine for 18 h, followed by rapid wash and the addition of 5 mM naloxone at the start of the AC assay. (b) Effects of TSH and TPA on the activity of endogenous AC (the same batch of cells were transfected as above but without the cDNA for AC-II). (c) Net AC-II activity after subtraction of the endogenous AC activity. AC activity is expressed in cpm of cAMP accumulation and is presented as mean SD of triplicate determinations of a representative experiment out of three experiments which gave the same results.
Fig. 2. Regulation of Gaq-stimulated AC-II activity. COS-7 cells were transfected with cDNAs of m-opioid receptor, AC-II and m3-muscarinic receptor. Basal: carbachol was not present during the AC assay. Stimulation: carbachol (10 mM)-stimulated AC-II activity (no opioid ligands were present); Acute: 1 mM morphine was present together with carbachol during the 10-min AC assay; Withdrawal: cells were incubated with 1 mM morphine for 18 h, followed by rapid wash and the addition of 5 mM naloxone at the start of the AC assay. cAMP accumulations are expressed in cpm and are mean SD of triplicate determinations of a representative experiment out of three experiments which gave the same results.
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AC-II was not affected by the overexpression of Gas wt, while it was strongly decreased (to 20% of control levels) by the Gas dn mutant (Fig. 4a). The activity of AC-II in the presence of TPA could be further increased by Gas wt, but not by the Gas dn mutant. This suggests additive activation of AC-II by Gas subunits and PKC in agreement with the additive effects of TSH and TPA (see Fig. 1). In the next step, we investigated the effect of Gas wt and Gas dn on the acute and chronic opioid effects. We found (Fig. 4b) that both Gas wt and Gas dn, significantly reduced the acute opiate increase of AC-II activity under both TPA and TSH stimulating conditions. The reduction in the level of acute opioid stimulation of AC-II by the Gas wt and Gas dn mutant could be due to the bg scavenging effect of these overexpressed Gas molecules. The somewhat stronger effect
Fig. 3. Kinetics of AC superinhibition and its reversal. COS-7 cells were transfected and assayed as described in Fig. 1. (a) Kinetics of superinhibition: cells were treated with 1 mM morphine or DAMGE for the indicated periods of time. After a quick wash naloxone (5 mM) was added and AC activity assayed. (b) Kinetics of loss of superinhibition. Cells were chronically treated with morphine or DAMGE (18 h, 1 mM), after a quick wash, naloxone was added (5 mM) for the indicated times and AC activity assayed during the last 10 min of incubation. The data represent the mean SD of three independent experiments. 100% represents AC-II activity under control (TSH-stimulated) conditions.
reversal of the superinhibition was also biphasic. The first 1 h with naloxone led to a quick reversal achieving ca. 65% reversal from the superinhibited state, while the second slower phase, had a very small additional contribution to the reversal of the superinhibition. The total AC activity did not return to the 100% level even after 6 h with naloxone. Again, similar results were observed with cells pretreated with either morphine or DAMGE.
3.3. Role of Gas in TSH and TPA stimulation pathways A triple Gas mutant, (a3b5/G226A/A366S), which blocks signaling from the luteinizing hormone receptor to Gas by up to 97%, acting as a dominant negative mutant, was described by Berlot (2002). To determine the role of Gas in the TSH and TPA activation pathways, COS-7 cells were transfected with m-opiod receptor, TSH receptor and AC-II cDNAs with or without the cDNA for the Gas wild type (Gas wt) or its dominant negative mutant (Gas dn). The activity of TSH-stimulated
Fig. 4. Effect of dominant negative Gas mutant on TSH- and TPA-stimulated AC-II activity and its opioid regulation. Cells were transfected as described in Fig. 1 with the addition (where indicated) of 2 mg of the cDNA for Gas wt or Gas dn mutant (Gas a3b5/G226A/A366S). (a) Effect of wt and dn Gas on TSHand TPA-stimulated AC-II activity. 100% represents AC stimulated activity in the absence of Gas. (b) Effect of Gas wt and dn and morphine treatments on AC-II activity. 100% represents AC stimulated activity following stimulation by either TSH or TPA as indicated. Data are mean SD of triplicate determinations of three separate experiments. *p < 0.05 versus the appropriate stimulated control; A, #, sp < 0.05 versus the treatment marked with the same symbol (Dunnett’s post-repeated measures ANOVA).
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of the dominant negative molecule is in line with the increased affinity of this mutant (and especially the G226A part of the molecule) to bg (Berlot, 2002; Lee et al., 1992). The superinhibition (upon chronic opioid treatment followed by agonist withdrawal) was significantly decreased (by ca. 50%) by both Gas wt and Gas dn mutant under the conditions of TSH-stimulation. TPA-stimulation did not show the superinhibition phenomenon independently of the presence or absence of the Gas wt or Gas dn mutant. 3.4. Role of Gi/o proteins in AC-II activation PTX is known as an efficient blocker of Gi/o signaling. As shown in Fig. 5, pretreatment with PTX did not induce any changes in the level of stimulation of AC-II by either TSH or TPA. This result demonstrates that there is no involvement of Gi/o proteins under these stimulatory conditions. On the other hand, the increase in AC-II activity obtained by acute opioid exposure was completely eliminated by PTX pretreatment, in both the TSH- and TPA-stimulated pathways. This result is in line with the involvement of Gi/o proteins during opioid receptor activation. Similarly, the superinhibition after withdrawal of the chronic opioid agonist was almost completely eliminated.
Fig. 5. Effect of PTX on TSH- and TPA-stimulated AC-II activity upon opioid exposure. Cells were incubated with PTX (100 ng/ml, 18 h). AC-II activity was determined following acute and chronic (withdrawal) opioid exposure. (a) TSH- and (b) TPA-stimulated cAMP accumulation are expressed in cpm [3H]cAMP formed and are mean SD of triplicate determinations of a representative experiment out of three experiments which gave the same results.
This is easily observed in cells where AC-II was activated using TSH. The TPA activation did not yield superinhibition even under control conditions and this was not changed by the PTX pretreatment. The trend toward a decrease in AC-II activation upon acute exposure to opioids in the presence of PTX is probably due to the reduction, by PTX, of the amount of free Gbg subunits liberated from Gi/o upon opioid exposure. 3.5. The Gbg scavenger a-transducin reduces the activity of AC-II The a subunit of transducin is a known scavenger of Gbg, which can bind to free Gbg, and reduce its concentration (Federman et al., 1992). Fig. 6a shows that the TSH-stimulated activity of AC-II was strongly decreased in cells co-transfected with a-transducin (reaching 30% of control levels), while TPA-stimulated activation was not affected. This result shows that AC-II activation upon TSH stimulation is for the most part
Fig. 6. Effect of a-transducin on AC-II activity upon acute and chronic morphine exposure. COS-7 cells were transfected as in Fig. 1, and where indicated, with the cDNA for a-transducin (2 mg). (a) Effect of a-transducin on AC-II activity. 100% represents AC stimulated activity in the absence of a-transducin. (b) Effect of a-transducin and morphine treatments on AC-II activity. 100% represents AC stimulated activity following stimulation by either TSH or TPA as indicated. The data represent the mean SD of triplicate determinations of three separate experiments. *p < 0.05 versus the appropriate stimulated control; A, #, sp < 0.05 versus the treatment marked with the same symbol (Dunnett’s post-repeated measures ANOVA).
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bg dependent, while TPA activation is not. AC-II activation by acute opioid exposure was strongly decreased by a-transducin when the initial activation was performed with either TSH (by 63%) or TPA (completely eliminated) (Fig. 6b). The level of superinhibition observed following withdrawal from chronic opioid exposure (under TSH activation) was also reduced (from 67% to 42%) but was not completely eliminated. 4. Discussion Among the multiple isoforms of AC cloned to date, the type II family isoforms (AC-II, AC-IV and AC-VII) are unique for their positive responsiveness to G protein bg subunits. The stimulation of AC-II by bg in vitro is conditional, requiring coincidence of a second signal, such as Gas- (Tang and Gilman, 1995; Taussig et al., 1994) or PKC-activation (Lustig et al., 1993). Earlier studies demonstrated that the basal activity of AC-II was increased followed stimulation by TPA or 12,13 phorbol dibutyrate (PdBu) while the inactive isomer of TPA, 4-a-phorbol 12-myristate 13-acetate (4-a-PMA) had no effect (Jacobowitz and Iyengar, 1994; Lustig et al., 1993; Yoshimura and Cooper, 1993). Similar activation by TPA was also reported for AC-VI (Beazely et al., 2005). Moreover, Chakrabarti et al. (2001) showed that the increment in AC-II activity produced by Gbg was increased by approximately 2fold following in vitro phosphorylation by the catalytic subunit of either PKA or PKC. Indeed, we have shown here that AC-II activity was stimulated by the Gas pathway (through the TSH receptor) as well as by PKC activation (e.g. with TPA or by activation of a Gaq-coupled receptor). Simultaneous stimulation of both pathways led to a further increase in AC-II activity. Our results suggest that the activation of AC-II is independent for each of the two pathways, since co-transfection with a Gas dominant negative mutant, led to a decrease in the Gas-activated pathway but did not significantly affect the PKC-activated pathway. Acute opioid exposure increased AC-II activity via both the TPA and Gas activation pathways. This result is in agreement with the finding that HEK 293 cells transiently expressing AC-II and Gi/o-coupled receptors show an efficient receptormediated potentiation of PKC-activated AC-II (Tsu and Wong, 1996). In a similar way, Nasman et al. (2002) reported that the effects of a2A-adrenoreceptor and TPA are additive on AC-II activity. The activation of Gi/o-coupled receptors releases bg subunits, which in turn potentiate the activation of AC-II by Gas or PKC. In this regard, in vitro studies have shown a potentiating effect of bg when AC-II is activated by PKC, albeit with lower potency compared with the effect of bg on the as pathway (Zimmermann and Taussig, 1996). The role of bg subunits in the two pathways is supported by the observation that co-expression of a-transducin, which sequesters the released bg subunits, blocks activation by Gi/o-coupled receptors (Tsu et al., 1995; Tsu and Wong, 1996). These observations are in agreement with our data showing that (i) the acute opioid activation of AC-II was suppressed by PTX pretreatment, and (ii) co-transfection with a-transducin led to a decrease in the opioid agonist AC-II activation in both pathways.
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Interestingly, chronic opioid exposure led to a different ACII regulation pattern following either Gas or PKC activation. While chronic exposure to opioid agonist followed by withdrawal decreased TSH- and TSH þ TPA-stimulated AC-II activity, leading to superinhibition, TPA-stimulated AC-II activity did not show the superinhibition phenomenon. Similar results were shown upon activation of PKC via activation of the Gaq coupled m3-muscarinic receptor. This decrease in Gas activated-AC-II activity upon chronic agonist exposure was shown to be also dependent on bg subunits, since PTX pretreatment and a-transducin overexpression led to a large decrease in the level of superinhibition observed. The finding that the superinhibition of AC-II is reduced by this Gbg scavenger suggests that either chronic activation of AC-II per se is needed for AC-II superinhibition, or that Gbg dimers are important for the superinhibition phenomenon, in addition to their role in the acute activation of AC-II. Our laboratory has previously shown that chronic activation of the m-opioid receptor leads to a time-dependent shift in the detergent solubility of both Gai and Gb1 subunits (Bayewitch et al., 2000). It was proposed that these proteins may undergo an agonist-induced time-dependent physical or chemical modification that alters their ability to be solubilized, or that these G protein subunits may interact with other protein partners that prevent their solubilization. It is possible that the superinhibition process, described here, is a consequence of such a shift in the solubility of the bg dimers. The PKC-stimulated AC-II activity in cells chronically treated with the opioid agonist was not affected by the PTX treatment and was not affected by the presence of a-transducin. These results suggest that bg may not be directly involved in this regulatory mechanism. On the other hand, it is also possible that the application of phorbol esters enhances the rate of receptor phosphorylation and down-regulation (Kramer and Simon, 1999), leading to a loss of the chronic receptor effect and hence to a loss of its capacity to regulate AC-II activity (which returns to control level). We found that acquiring the superinhibition of AC-II is a time-dependent biphasic process. The first phase was rapid requiring ca. 1 h to reach 50% of its maximal effect, while the second phase was much slower. Similar results were observed for cells pretreated with two opioid agonists (morphine or DAMGE). Moreover, we have shown that the state of superinhibition is for the most part reversible and can be reversed by prolonged exposure to the opioid antagonist naloxone. This reversal was also biphasic. The first hour with naloxone led to a very rapid reversal of the superinhibition, while the second phase of the reversal was much slower. Interestingly, the biphasic kinetics of acquiring the superinhibition of AC-II is similar to that observed for the superactivation of AC-I and AC-VIII following chronic exposure to the D2-dopaminergic or m-opioid receptor agonists, respectively (Nevo et al., 1998; Steiner et al., 2005). The reasons for the biphasic nature of the chronic agonist-induced AC superactivation or superinhibition are not yet clear but hint to the complexity of the mechanisms involved. This similarity in kinetics could suggest a common mechanism for the superinhibition of AC-II and the
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Fig. 7. Schematic diagram illustrating the effect of acute and chronic agonist exposure on the Gs and PKC activating pathways. Activation via the Gs pathway. (a) AC-II is activated by Gas as well as by the Gbg released by GsPCR stimulation. (b) Upon acute opioid receptor stimulation additional Gbg is released (from Gi/o) leading to further activation of AC-II. (c) Following withdrawal from chronic opioid treatment the activity of AC-II is decreased, due to relocalization or degradation of the bg subunits (those originated from the Gs as well as those from the Gi/o). Activation via the PKC pathway. (d) AC-II is activated by TPA. (e) AC-II is activated by Gbg released by acute opioid receptor stimulation. (f) Withdrawal from chronic opioid receptor activation leads to relocalization or degradation of Gbg subunits (originating from Gi/o) and AC-II activity reaches control levels.
superactivation of AC-VIII, AC-I and probably AC-V. Such a mechanism could involve the translocation of bg subunits following chronic exposure as described above and in Bayewitch et al. (2000) or its degradation following chronic exposure (Mouledous et al., 2005). The existence of such degradation of Gbg could also explain why the reversal of the superinhibition following prolonged antagonist exposure reached a maximal value of 75% and did not reach the 100% value of the AC-II initial activity. The various steps that seem to be involved in the acute and chronic opioid regulation of AC-II when stimulated via either TSH or TPA activation and the possible role of Gbg dimers are presented in a model shown in Fig. 7. In summary, we found that acute opioid exposure led to an increase in AC-II activity via either Gas or PKC stimulation.
This effect seems to be regulated by Gbg subunits in both activation pathways as AC-II activity was decreased by PTX treatment and by a Gbg scavenger. Chronic opioid exposure led to a decrease in AC-II activity (superinhibition) upon stimulation of the Gas pathway. This superinhibition was not observed following PKC activation.
Acknowledgments We thank Professor C.H. Berlot and Dr J.S. Gutkind for providing the plasmids containing the Gas dominant negative mutant and Gas wild type, respectively. This work was supported by the National Institute of Drug Abuse (grant DA-06265) and the Nella and Leon Benoziyo Center for the Neurosciences.
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