Department of Pharmacology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523, Japan
Submitted 5 March 2004 ; accepted in final form 13 May 2004
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ABSTRACT |
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G protein-coupled receptor; cystic fibrosis transmembrane conductance regulator gene; cross talk; electrophysiology
It is well known that the -subunits of G protein regulate the activities of several types of effectors, including phospholipase C (PLC), adenylate cyclases (AC), phosphatidylinositol 3-kinases, G protein-coupled receptor kinases, and some ion channels (4, 20, 25). Although G
-subunits are members of all trimeric G proteins, it is generally accepted that high concentrations of free G
-subunits in the cells arise from Gi/o proteins, which are abundantly expressed in the cells compared with other G proteins (20, 25). Thus it is important to elucidate whether the G
-subunits released from Gi/o proteins regulate the activity of various effectors.
The present study was designed to elucidate the involvement of G-subunits released from Gi/o protein in the diverse intracellular signaling pathways. In particular, we focused on the involvement of cAMP signaling and intracellular Ca2+ signaling pathways. To this end, using an electrophysiological assay as well as a cAMP assay with the Xenopus oocyte expression system, we examined whether a set of cloned Gi/o-coupled receptors regulate such signaling pathways.
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MATERIALS AND METHODS |
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Preparation of cRNA.
cDNA from the human CFTR gene was provided by Dr. J. R. Riordan (Mayo Clinic, Scottsdale, AZ). AC type II was obtained from Dr. R. Reed (The Johns Hopkins University, Baltimore, MD), and AC type III was supplied by Dr. K. G. Zinn (California Institute of Technology, Pasadena, CA). Rat PACAP receptor was obtained from Dr. A. Arimura (Tulane University, New Orleans, LA). Rat 5-HT1A receptor (5-HT1AR) and 5-HT2CR, 2-adrenoceptor, and
-opioid receptor were kindly provided by Dr. H. A. Lester (California Institute of Technology). Rat phospholipase C-
1 (PLC-
1) and PLC-
2 were obtained from Dr. S. G. Rhee (National Heart, Lung, and Blood Institute, Bethesda, MD). GABAB(1b) and GABAB2 receptors were supplied by Dr. N. J. Fraser (GlaxoWellcome Research and Development, Stevenage, UK). Each cRNA was synthesized from the respective linearized cDNA using a T7 or SP6 RNA polymerase kit (Ambion, Austin, TX).
Oocyte preparation and cRNA injection.
Immature V and VI stage Xenopus oocytes were enzymatically dissociated as reported previously (28). Isolated oocytes were incubated at 18°C in ND-96 medium (in mM: 96 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, and 5 HEPES, pH 7.4) containing 2.5 mM sodium pyruvate and 50 µg/ml gentamicin. For measurement of CFTR currents induced by stimulation of Gs-coupled receptors, cRNA for CFTR (4 ng) and 2-adrenoceptor (0.1 ng) or PACAP receptor (1 ng) were injected into the oocytes together with or without 5-HT1AR (10 ng),
-opioid receptor (10 ng), GABAB(1b) receptor (10 ng), and GABAB2 receptor (10 ng). In some oocytes, cRNA for 5-HT2CR (1 ng), G transducin
(Gt
; 5 ng), AC type II (5 ng), AC type III (5 ng), PLC-
1 (5 ng), or PLC-
2 (25 ng) was injected. The final injection volume was <50 nl in all cases. Oocytes were incubated in ND-96 medium and used 38 days after injection.
Electrophysiological recordings.
Electrophysiological recordings were performed using the two-electrode voltage-clamp method with a GeneClamp 500 amplifier (Axon Instruments, Foster, CA) at room temperature. Oocytes were clamped at 60 mV and continuously superfused with ND-96 medium in a 0.25-ml chamber at a flow rate of 5 ml/min, and test compounds were added to the superfusion solution. In experiments with niflumic acid, the compound was added 10 min before agonist stimulation and maintained throughout the experiment. Voltage-recording microelectrodes were filled with 3 M KCl and had a tip resistance of 1.02.5 M. Currents were continuously recorded and stored with Mac Lab software (AD Instruments, Castle Hill, Australia) on a Macintosh computer. The ramp protocol was performed as described previously (28).
cAMP assay. The cellular cAMP level in individual oocytes was measured by conventional radioimmunoassay. Briefly, oocytes were washed three times with ND-96 buffer and preincubated with 1 mM IBMX for 30 min followed by another incubation with varying concentrations of ISO or other test compounds containing 1 mM IBMX and 1 mg/ml bovine serum albumin as described previously (31). The reaction was stopped by the addition of 100 mM HCl followed by heating to 95°C for 5 min. The cAMP content of individual oocytes was assayed using a [125I]-labeled cAMP radioimmunoassay kit (Yamasa, Chiba, Japan).
Statistical analysis. Data are expressed as means ± SE. Differences between two groups were examined for statistical significance using a paired t-test. For comparisons between multiple groups, we performed one-way analysis of variance followed by Scheffés test. P < 0.05 denoted the presence of a statistically significant difference.
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RESULTS |
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DISCUSSION |
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Stimulation of Gi/o-coupled receptors is generally considered to inhibit cAMP accumulation in most cells. On the other hand, stimulation of Gi/o-coupled receptors, including GABAB receptor, dopamine D2 receptor, or 2 adrenoceptor, has been shown to enhance cAMP production induced by Gs-coupled receptors such as
2-adrenoceptor and vasoactive intestinal peptide receptor (9, 12, 23, 30). Some Gi/o-coupled receptors, including opioid receptor,
2-adrenoceptor, muscarinic M4 receptor, and cannabinoid CB1 receptor, are coupled not only to Gi/o protein but also to Gs protein and are known to stimulate cAMP production (5, 7, 8, 11). We have shown in this study that 5-HT-induced enhancement of ISO- and PACAP38-mediated responses was abrogated by pretreatment with PTX in oocytes expressing CFTR,
2-adrenoceptor, or PACAP receptor together with 5-HT1AR. Because PTX interrupts signaling pathways from Gi/o-coupled receptors to Gi/o proteins without affecting Gs protein-mediated responses (9), we conclude that Gi/o proteins, but not Gs proteins, are involved in such enhancement.
Because -subunits of G protein regulate the activities of several types of effectors, including PLC, AC, and some ion channels (4, 20), we investigated whether the
-subunits of Gi/o protein contribute to the enhancement of responses mediated by Gs-coupled receptors. Gt
has been shown to inhibit downstream signaling through G
-subunits by sequestering free
-subunits (9). We previously demonstrated that Gi/o-coupled cannabinoid CB1 and CB2 receptors activated inward rectifying K+ channels through G
-subunits by showing that these effects were inhibited by coexpression of Gt
(13). Our results also showed that coexpression of Gt
prevented 5-HT-induced enhancement of CFTR currents caused by ISO, suggesting that G
-subunits released by Gi/o-coupled receptor stimulation caused the enhancement of Gs-coupled receptor-mediated cAMP production. cAMP is produced by activation of AC, and to date, eight types of AC genes have been cloned, each of which is differentially regulated by Gs
, G
, Ca2+/calmodulin, protein kinase C, and [Ca2+]i (27). The
-subunits are known to activate AC types II and IV, but not AC type III, in the presence of activated Gs
(19, 27). In agreement with these reports, our results showed that enhancement of 5-HT-induced CFTR currents was augmented by coexpression of AC type II, but not AC type III, in oocytes expressing
2-adrenoceptor-CFTR and 5-HT1AR. Thus
-subunits released by activation of Gi/o-coupled receptors contribute to the enhancement of Gs-coupled receptor-mediated responses, presumably by the activation of AC type II or IV present in oocytes. Collectively, these findings demonstrate that activation of Gi/o-coupled receptors enhance the cAMP production induced by Gs-coupled receptors through G
-subunits released from PTX-sensitive Gi/o proteins.
We also have demonstrated that -subunits released by activation of Gi/o-coupled receptors appear to be involved in another signaling pathway, the Ca2+ mobilization signaling pathway. We have clearly shown that 5-HT or DADLE elicited Ca2+-activated Cl currents in oocytes expressing 5-HT1AR or
-opioid receptor together with PLC-
2. It has been shown that G
-subunits activate PLC-
2, but not PLC-
1, in cotransfection assays with COS-7 cells (15). As such, substitution of PLC-
1 for PLC-
2 in the coexpression did not elicit Ca2+-activated Cl currents induced by 5-HT or DADLE in oocytes expressing 5-HT1AR or
-opioid receptor, respectively. In Xenopus oocytes, G
-subunit-sensitive PLC-
-like proteins, namely, PLC-
X, were present (10). Accordingly, G
liberated by activation of Gi/o-coupled receptors may increase [Ca2+]i through activation of endogenously expressed PLC-
X in oocytes. In fact, 5-HT elicited Ca2+-activated Cl currents without additional expression of PLC-
2 or -
3 in oocytes expressing cloned 5-HT1A receptor in the vector, which was designed to overexpress the receptor in oocytes (22). However, we found it necessary to express PLC-
2 to elicit Gi/o-coupled receptor-mediated Ca2+-activated Cl currents, including 5-HT1AR,
-opioid receptor, cannabinoid CB1 and CB2 receptors, and functional GABAB receptors (present study and unpublished observations). The reason for the differences between the results of other investigators and ours is not known at present. It could be the expression levels of cell surface receptors in Xenopus oocytes; extremely overexpressed receptors may liberate G
-subunits sufficiently to activate endogenous PLC-
X.
We have further demonstrated that 5-HT1AR- and -opioid receptor-mediated Ca2+-activated Cl currents were suppressed by pretreatment with PTX and by coexpression of Gt
. These findings suggest that
-subunits released by activation of Gi/o-coupled receptors could activate PLC-
2 and then stimulate intracellular Ca2+ mobilization. A recent study (34) in Xenopus oocytes showed that G
-subunits caused direct activation of inositol 1,4,5-trisphosphate (IP3) receptors in addition to activation of PLC-
, and both pathways consequently increased [Ca2+]i. However, it may be less likely that G
-subunits liberated by 5-HT1AR or
-opioid receptor stimulation directly activated IP3 receptors to increase in [Ca2+]i in our experimental condition, because exogenously expressed PLC-
2 was required to elicit Ca2+-activated Cl currents. Nonetheless, further detailed studies are necessary to clarify the involvement of this novel signaling pathway in Gi/o-coupled receptor-mediated Ca2+ mobilization in the Xenopus oocyte and also in the mammalian cell expression systems.
The present study demonstrates that the -subunits released from Gi/o proteins participate in diverse signal pathways such as enhancement of the Gs-coupled receptor-mediated cAMP system and mobilization of [Ca2+]i, provided that a set of particular effectors, such as AC type II or PLC-
2, is present in the cells. The number of G protein subunits identified to date includes 27 subunits of G
, 5 of G
, and 14 of G
(1). As expected, various combinations of G
and G
are possible, and they appear to lead to a diversity of cellular functions. Using the G
and G
subunit reconstitution assay, investigators in several studies have examined cellular functions mediated by particular combinations of G
-subunits. For instance, the G
1
2 subunit activates, but the G
5
2 subunit inhibits, AC type II (2); the G
5
2 subunit activates PLC-
1 and PLC-
2 (18); and all combinations of G
15
2 except G
5
2 activate AC type II and inhibit AC type I (19, 20). The present study demonstrates that G
-subunits released by activation of Gi/o-coupled receptors activate both AC type II and PLC-
2. Because our experiments were performed with the Xenopus oocyte expression system without expression of any G protein subunits, the expressed Gi/o-coupled receptors in oocytes used endogenously expressed G proteins. Thus the combination of G
and G
subunits responsible for such signaling pathways remains to be elucidated. Further studies are necessary to determine the combinations of G
and G
that are responsible for cellular signaling pathways activated by Gi/o-coupled receptors.
Previous studies reported that some Gi/o-coupled receptor-mediated effects were diverse and closely correlated to biological and biomedical effects. Analgesic µ-opioid receptors couple to Gi/o proteins to inhibit AC activity, but they stimulate AC activity under certain circumstances. In a study measuring paw withdrawal thresholds to mechanical stimuli in Sprague-Dawley rats, Khasar et al. (17) reported that preadministration of the µ-opioid receptor agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) attenuated hyperalgesia induced by prostaglandin E2 (PGE2), an agonist of Gs-coupled PGE2 receptors. On the other hand, postadministration of DAMGO just after PGE2 administration enhanced PGE2-induced hyperalgesia. Khasar et al. concluded that the DAMGO-induced enhancement was mediated by G from Gi/o-coupled µ-opioid receptors and activated PGE2 receptors. Yao et al. (32) showed that ethanol consumption in the rat could be mediated by activation of cAMP-dependent protein kinases by synergistic stimulation of G
-subunits released from Gi/o-coupled dopamine type 2 receptors and activation of Gs-coupled adenosine type 2 receptors, both of which were induced by having the rats drink ethanol. In another study at the same laboratory, Yao et al. (33) reported that some addictive effects caused by activation of
-opioid or cannabinoid CB1 receptors were possibly due to enhanced activation of cAMP-dependent kinases by a synergy of such Gi/o-coupled
-opioid or CB1 receptors with adenosine type 2 receptors. Yao and coworkers proposed that drugs that target G
-subunits or the synergy of several Gi/o-coupled receptors with adenosine type 2 receptors might prevent or attenuate excessive drinking of ethanol and the addictive effects of ethanol and other drugs (32, 33). The present results and further experimental studies should clarify the roles of G
-subunits in such physiological phenomena.
In conclusion, using a set of cloned Gi/o-coupled receptors, we have demonstrated that G-subunits liberated by activation of Gi/o-coupled receptors are involved in diverse signaling pathways such as enhancement of cAMP pathways and intracellular Ca2+ mobilization.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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