Introduction

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Opioid receptors are members of the G protein-coupled receptor superfamily (Evans et al., 1992; Raynor et al., 1994) and exert their principal effects by coupling to inhibitory G proteins (G_o/G_i). Opioid-induced dissociation of heterotrimeric G proteins produces diverse effects mediated by both  and subunits. G_i/o inhibits adenylyl cyclase (AC) (McKenzie and Milligan, 1990; Murthy and Makhlouf, 1996), whereas G activates K⁺ channels (Logothetis et al., 1987; Clapham and Neer, 1997) and inhibits voltage-dependent Ca²⁺ channels (Herlitze et al., 1996; Ikeda, 1996). Activation of opioid receptors also appears to stimulate phospholipase C (PLC) (Jin et al., 1992; Miyamae et al., 1993; Jin et al., 1994; Tsu et al., 1995; Smart and Lambert, 1996), although how the activated receptor couples to PLC is not clear.

Activation of -opioid receptors in NG108-15 cells releases Ca²⁺ from intracellular stores via a process that appears to activate PLC, as indicated by inhibition of the response by the PLC inhibitor U73122 (Jin et al., 1994). Furthermore, Smart and Lambert (1996) have shown that -opioids will stimulate the production of 1,4,5-inositol triphosphate (IP₃) in NG108-15 cells. In these studies, both opioid-induced [Ca²⁺]_i increases and the production of IP₃ were prevented by pertussis toxin (PTX), indicating that the response was mediated by an inhibitory G protein. The  subunits of inhibitory G proteins are not thought to couple directly to PLC. However, G liberated by the activation of heterotrimeric G_i can stimulate certain isoforms of PLC (Camps et al., 1992; Katz et al., 1992). G_q may also participate in -opioid-induced [Ca²⁺]_i increases (Okajima et al., 1993), but how this coupling might occur is not clear. In this study, we tested the hypothesis that activation of -opioid receptors in NG108-15 cells mobilizes IP₃-sensitive Ca²⁺ stores via liberation of G.

We used fura-2-based digital imaging to measure opioid-induced [Ca²⁺]_i responses in single NG108-15 cells. Selective inhibitors that bound IP₃ receptors, G, and G_q were microinjected to determine the contribution of these signaling molecules to the [Ca²⁺]_i increase. We determined that activation of -opioid receptors mobilized Ca²⁺ through IP₃ receptors and that G mediated this response.

	Materials and Methods

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Reagents and Peptides. Materials used and companies from which they were purchased are as follows: fura-2 acetoxymethyl ester, Molecular Probes, Eugene, OR; D-Ala²-D-Leu⁵ enkephalin (DADLE), Peninsula Laboratories, Inc., Belmont, CA; CO₂-independent media, hypoxanthine/aminopterin/thymidine supplement, Life Technologies, Inc., Grand Island, NY; all other reagents were purchased from Sigma, St. Louis, MO. QEHA and SKEE peptides were synthesized in the laboratory of Dr. R. P. Elde, using an Applied Biosystems Synergy system (Foster City, CA). The sequences for QEHA peptide and SKEE peptide were QEHAQEPERQYMHIGTMVEFAYALVGK and SKEEKSDKERWQHLADLADFALAMKDT, respectively. QLKK and ADRK peptides were synthesized in the Microchemical Facility at the University of Minnesota, using a Milligen Biosearch BS 9600 system (Novota, CA). The sequences for QLKK peptide and ADRK peptide were QLKKLKEICEKEKKELKKKMDKKRQEKITEAK and ADRKRVETALEACSL, respectively. All peptides were purified by C₁₈ reversed-phase HPLC in 0.1% trifluoroacetic acid and eluted with an H₂O-acetonitrile gradient of 0 to 60% in 30 min. Solvents were removed by lyophilization.

Cell Culture. NG108-15 cells (passage 21-30) were grown in T25 cm² flasks in Dulbecco's modified Eagle's medium with 5% fetal bovine serum, 0.1 mM sodium hypoxanthine, 0.4 µM aminopterine, 16 µM thymidine, 100 mg/l streptomycin, and 100 I.U./ml penicillin in a humidified atmosphere of 90% air and 10% CO₂. Cells from the stock culture were plated onto glass coverslips (25-mm round) at a density of 3 × 10⁴ cells/coverslip and grown until 80% confluent. The cells then were placed in a CO₂-independent medium (phosphate- rather than bicarbonate-buffered) supplemented with 5 µM forskolin for 3 to 6 days at 37°C and atmospheric gas levels.

Experimental Procedures. The cells were loaded with 2 µM fura-2 acetoxymethyl ester for digital imaging in HEPES-buffered Hanks' salts solution containing 0.5% BSA for 45 min at 37°C. The HEPES buffer was composed of the following: 20 mM HEPES, 137 mM NaCl, 1.26 mM CaCl₂, 0.4 mM MgSO₄, 0.5 mM MgCl₂, 5.0 mM KCl, 0.4 mM KH₂PO₄, 0.6 mM Na₂HPO₄, 3.0 mM NaHCO₃, and 5.0 mM glucose. The loading was terminated by washing with HEPES-buffered Hanks' solution for 15 min before starting an experiment. The cover glass was then mounted in a flow-through chamber (Thayer et al., 1988), which was superfused at a rate of 2 ml/min with HEPES-buffered Hanks' solution. Solutions were selected with a multiport valve coupled to several reservoirs. The chamber containing the fura-2-labeled cells was mounted on the stage of an inverted microscope (Nikon Diaphot; Nikon, Melville, NY) and alternately excited at 340 or 380 nm by rapidly switching optical filters (10-nm band pass) mounted in a computer-controlled wheel (Sutter Instrument Co., Novato, CA) placed between a 75 W Xe arc lamp and epifluorescence port of the microscope. Excitation light was reflected from a dichroic mirror (400-nm) through a 90× objective (Leitz, numerical aperture 1.15). Fluorescent images (510, 40-nm band pass) were projected (0.5×) onto a cooled charge-coupled device camera (Photometrics, Inc., Tucson, AZ; 384 × 576 binned to 192 × 288 pixels, 12-bit scale) controlled by an IBM-compatible computer. Image pairs were collected every 6 s; exposure to excitation light was always 120 ms/image and the interval between paired images was 385 ms. [Ca²⁺]_i was calculated from the ratio of the two background-subtracted digital images. Cells were delimited by producing a mask that contained pixel values above a threshold applied to the 380-nm image. Background images were collected at the end of each experiment after the cells were removed from the coverslip. Autofluorescence from cells not loaded with the dye was less than 5% and thus, not corrected. Ratio values were converted to free [Ca²⁺]_i by the equation [Ca²⁺]_i = K  (RR_min)/(R_maxR), in which R is the 340/380-nm fluorescence emission ratio and K = 224 nM, the dissociation constant for fura-2 (Grynkiewicz et al., 1985). The maximum ratio (R_max = 3.46), the minimum ratio (R_min= 0.25), and the constant  (the ratio of the fluorescence measured at 380 nm in Ca²⁺-free and saturating solution), 5.17, were determined by treating cells with 10 µM ionomycin in Ca²⁺-free (1 mM EGTA) and saturating (5 mM Ca²⁺) solution.

Microinjection. Ten minutes after an initial control response to agonist, either DADLE or bradykinin, approximately half of the responding cells were microinjected. Pipettes were pulled from borosilicate glass capillary tubes (TW100F-4; WPI, Sarasota, FL). A microinjector (Eppendorf, Hamburg, Germany) was used to apply a holding pressure of 8 hPa and an injection pressure of 11 to 90 hPa for 0.1 to 0.6 s. Cells were allowed to recover for 35 min after injection. Injection solution was composed of dextran-conjugated tetramethylrhodamine (MW 10,000; pipette concentration = 12.5 mg/ml) combined with test agents in distilled water. A single test agent was examined per experiment. None of the agents microinjected in this study affected the resting fura-2 fluorescence intensity values at the end of the 35-min recovery period. The injection volume was quantified by digital imaging of rhodamine fluorescence at an excitation wavelength of 535 nm (50-nm bandpass) and an emission wavelength of 605 nm (55-nm bandpass). Dilution factors for injected substances were estimated by dividing the rhodamine intensity value from the injected cell by the fluorescence from a droplet comparable in size to a single cell containing a known concentration of rhodamine. The mean fluorescence intensity from 10 droplets containing 0.5 mg/ml tetramethylrhodamine dextran injected into immersion oil was 1953 ± 248 arbitrary units (12-bit scale, 10-ms exposure).

	Results

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Opioids Increase [Ca²⁺]_i by Activating IP₃ Receptors. NG108-15 cells were grown in serum-free, CO₂-independent media supplemented with 5 µM forskolin for 4 to 6 days and [Ca²⁺]_i was recorded with fura-2-based digital imaging as described in Materials and Methods. A brief (90-s) exposure to 100 nM DADLE produced a rapid and transient increase in [Ca²⁺]_i (Fig. 1A). We have shown previously that this response is mediated by an inhibitory G protein and results from Ca²⁺ release from intracellular stores (Jin et al., 1994). In this report we present studies aimed at identification of the signaling pathway that couples inhibitory G proteins to Ca²⁺ mobilization. The general strategy we employed was to elicit an initial control response to 100 nM DADLE (Fig. 1A) and then, after recovery, microinject selective inhibitors into some of the responding cells. Noninjected cells in the same field served as controls. In separate experiments, sham injections or injections of inactive analogs of the inhibitory agents were additional controls. Rhodamine (12.5 mg/ml) was added to the pipette solution and rhodamine fluorescence was quantified by digital imaging to confirm successful microinjection.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Microinjection of heparin inhibits the opioid-induced [Ca2+]i increase. [Ca2+]i was measured in fields of NG108-15 cells, using fura-2-based digital imaging as described in Materials and Methods. Image pairs were collected at 6-s intervals. 100 nM DADLE was applied as indicated by horizontal bars. A, sham injection of rhodamine (pipette concentration = 12.5 mg/ml) into two cells (solid symbols) at the time indicated by the arrow did not influence the amplitude of a second opioid-induced [Ca2+]i increase evoked 35 min after injection. B, microinjection of heparin (pipette concentration = 100 mg/ml) combined with rhodamine blocked the second DADLE-induced [Ca2+]i transient evoked 35 min after microinjection (solid symbols). C, summary of effects of heparin microinjection on DADLE-induced responses. The DADLE-induced response amplitude is presented as a percentage of the initial control responses (peak2/peak1) for control (n = 175), sham-injected (sham, n = 9), and heparin-injected (heparin, n = 21) cells. D, rhodamine fluorescence (arbitrary units) from injected cells. Data are expressed as mean ± S.E. **p < .01, heparin-injected relative to control and sham-injected cells (ANOVA with Bonferroni post-test).

G Couples Opioid Receptors to IP₃-Sensitive Ca²⁺ Stores. We tested the hypothesis that the opioid-induced [Ca²⁺]_i increase was mediated through the subunits of a heterotrimeric G protein. Chen et al. (1995) found that a synthetic peptide (QEHA), which encodes residues 956 to 982 of AC II, binds to G protein subunits and inhibits G-mediated signaling. Synthetic SKEE peptide represents the cognate region of AC III, which does not bind to G. To determine whether the opioid-induced [Ca²⁺]_i increases were mediated through G, we microinjected the QEHA peptide (pipette concentration = 1 mM) into NG108-15 cells and subsequently stimulated the cells with DADLE. As shown in Fig. 2B, microinjection of QEHA peptide into NG108-15 cells inhibited the second opioid-induced [Ca²⁺]_i transient. Injection of 0.1 mM QEHA did not affect the DADLE-evoked response. Pseudocolor images for the experiment plotted in Fig. 2B are displayed in Fig. 2C. The frame numbers correspond to the times indicated along the plot (Fig. 2B) and the two cells injected with 1 mM QEHA are identified by the rhodamine fluorescence shown in the lower left frame (Fig. 2C). The second DADLE-induced [Ca²⁺]_i increase in the QEHA peptide-injected cells was 24 ± 7% of the initial response, significantly less than the 72 ± 4% response recorded for control cells (Fig. 2D). In contrast, microinjection of SKEE peptide (pipette concentration = 1 mM) into NG108-15 cells did not significantly affect the opioid-induced [Ca²⁺]_i increase (Fig. 2A). Rhodamine fluorescence in SKEE peptide- and QEHA peptide-injected cells was not significantly different (Fig. 2E), indicating that the amount of peptide injected was similar. Based on the fluorescence intensities of known concentrations of rhodamine (see Materials and Methods), we estimate that the pipette solution was diluted 19-fold. This dilution corresponds to an approximate peptide concentration of 53 µM, which is well within the 10 to 200 µM concentration range shown by Chen et al. (1995) to selectively inhibit -stimulated type-2 AC activity. These results demonstrate that free G protein subunits are involved in the opioid-induced [Ca²⁺]_i increase in NG108-15 cells.

View larger version (44K): [in this window] [in a new window]   Fig. 2.   Microinjection of QEHA peptide inhibits the opioid-induced [Ca2+]i increase. [Ca2+]i was measured in fields of NG108-15 cells using fura-2-based digital imaging as described in Materials and Methods. Image pairs were collected at 6-s intervals. 100 nM DADLE was applied for 90 s as indicated by horizontal bars. A, microinjection of SKEE peptide (pipette concentration = 1 mM) into two cells at the time indicated by the arrow did not decrease the opioid-induced [Ca2+]i transients (solid symbols). B, microinjection of QEHA peptide (pipette concentration = 1 mM) into NG108-15 cells blocked opioid-induced [Ca2+]i transients (solid symbols). C, pseudocolor representations of [Ca2+]i were derived from fura-2-based digital images for the experiment shown in B. Two of the cells in which DADLE induced an increase in [Ca2+]i were injected with QEHA as indicated by rhodamine fluoresence (lower left frame) and marked with the solid triangles on the bright field image (upper left frame). The images displayed in C were acquired at the times indicated by the frame numbers placed along the plot in B. D, summary of effects of SKEE peptide and QEHA peptide on the DADLE-induced response. The DADLE-induced response amplitude is presented as a percentage of the initial control responses (peak2/peak1) for control (n = 78), SKEE peptide-injected (SKEE, n = 9) and QEHA peptide-injected (QEHA, n = 16) cells. E, rhodamine fluorescence (arbitrary units) from injected cells. There was no significant difference in rhodamine fluorescence between SKEE- and QEHA-injected cells. Data are expressed as mean ± S.E. *p < .05, QEHA-injected relative to control and SKEE-injected cells (ANOVA with Bonferroni post-test).

The G_q-Binding Peptide Inhibits Bradykinin-Induced [Ca²⁺]_i Responses in NG108-15 Cells. Wu et al.(1993) have identified the region of PLC1 that interacts with G_q. A peptide derived from this region (QLKK) inhibited activation of PLC by G_q. We synthesized this peptide and confirmed its activity by testing it against [Ca²⁺]_i responses elicited by 3 nM bradykinin, which are known to be mediated by G_q (Gutowski et al., 1991). Microinjection of QLKK peptide (pipette concentration = 15 mM) inhibited markedly the bradykinin-induced [Ca²⁺]_i transient (Fig. 3B). The bradykinin-induced response in QLKK peptide-injected cells was 20 ± 11% of the initial control response, significantly smaller than the 72 ± 3% response observed in control cells (Fig. 3C). A pipette concentration of 1 mM QLKK was without effect, and 10 mM peptide produced a partial inhibition. A peptide representing residues 180 to 194 of PLC1 (ADRK), which does not interact with G_q, did not affect the bradykinin-induced [Ca²⁺]_i increase when microinjected into NG108-15 cells (Figs. 3, A and C). The response in ADRK peptide-injected cells was 85 ± 7% of the initial control response. Rhodamine fluorescence intensities in the ADRK peptide- and QLKK peptide-injected cells were not significantly different, indicating that a similar amount of peptide was microinjected in each case. Based on the fluorescence intensities of known concentrations of rhodamine, we estimate that the pipette solution was diluted 17-fold. This dilution corresponds to an approximate peptide concentration of 880 µM. Wu et al. (1993) demonstrated sequence specific inhibition of G_q-mediated guanosine 5'-O-(3-thiotriphosphate)- (GTPS-) dependent activation of PLC1 for peptide concentrations up to 1 mM. These results indicate that QLKK peptide acts as an inhibitor of G_q-mediated PLC activation in NG108-15 cells.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Microinjection of QLKK peptide inhibits the bradykinin-induced [Ca2+]i increase. [Ca2+]i was measured in fields of NG108-15 cells using fura-2-based digital imaging as described in Materials and Methods. Image pairs were collected at 6-s intervals. 3 nM bradykinin was applied for 60 s as indicated by horizontal bars. A, microinjection of ADRK peptide (pipette concentration = 15 mM) into a single cell at the time indicated by the arrow did not influence the bradykinin-induced [Ca2+]i transient (solid symbols). B, microinjection of QLKK peptide (pipette concentration = 15 mM) into two cells at the time indicated by the arrow blocked the bradykinin-induced [Ca2+]i transients. C, summary of effects of QLKK peptide on the bradykinin-induced responses. Bradykinin-induced response amplitude is presented as a percentage of initial control responses (peak 2/peak 1) for control (n = 70), ADRK peptide-injected (ADRK, n = 7), and QLKK peptide-injected (QLKK, n = 6) cells. D, rhodamine fluorescence (arbitrary units) for injected cells was not significantly different between ADRK- and QLKK-injected cells. Data are expressed as mean ± S.E. **p < .01 QLKK-injected relative to control and ADRK-injected cells (ANOVA with Bonferroni post-test).

DADLE-Induced [Ca²⁺]_i Responses Are Not Significantly Inhibited by a G_q-Binding Peptide. It has been suggested that the G_q subunit is also involved in the -opioid-induced [Ca²⁺]_i increase (Okajima et al., 1993). Thus, we determined whether the G_q subunit was involved in the opioid-induced [Ca²⁺]_i increase in NG108-15 cells. Microinjection of the QLKK peptide (pipette concentration = 15 mM), which interacts with the G_q subunit and inhibits G_q-mediated signaling, did not significantly inhibit the opioid-induced [Ca²⁺]_i transient (Fig. 4B). The DADLE-induced response in QLKK peptide-injected cells was 58 ± 10% (n = 12) of the initial control response, which was not significantly different than the 86 ± 6% (n = 58) and 71 ± 16% (n = 8) observed in control cells and ADRK peptide-injected cells, respectively (Fig. 4, A and C). Rhodamine fluorescence in the ADRK peptide- and QLKK peptide-injected cells was not significantly different, indicating that a similar amount of peptide was microinjected. Based on the fluorescence intensities of known concentrations of rhodamine, we estimate that the pipette solution was diluted 16-fold. This dilution corresponds to an approximate peptide concentration of 940 µM, which is 3-fold greater than the concentration Wu et al. (1993) found to completely inhibit G_q-mediated GTPS- dependent activation of PLC1. Thus, even a supramax imal concentration of QLKK failed to significantly inhibit DADLE-induced Ca²⁺ mobilization. These results suggest that activation of G_q is not required for the -opioid-induced [Ca²⁺]_i increase in NG108-15 cells.

View larger version (30K): [in this window] [in a new window]   Fig. 4.   Microinjection of QLKK did not inhibit the opioid-induced [Ca2+]i increase. [Ca2+]i was measured in fields of NG108-15 cells using fura-2-based digital imaging as described in Materials and Methods. Image pairs were collected at 6-s intervals. 100 nM DADLE was applied for 90-s as indicated by horizontal bars. ADRK peptide (pipette concentration = 15 mM) or QLKK peptide (pipette concentration = 15 mM) was injected into the cells indicated by the solid symbols at the times marked by arrows. Microinjection of ADRK peptide (A) or QLKK peptide (B) did not inhibit DADLE-induced [Ca2+]i transients. C, summary of effects of QLKK peptide on the DADLE-induced responses. The DADLE-induced response amplitude is presented as a percentage of the initial control response (peak 2/peak 1) for control (n = 58), ADRK-injected (ADRK, n = 8), and QLKK-injected (QLKK, n = 12) cells. D, rhodamine fluorescence (arbitrary units) for injected cells was not significantly different between ADRK- and QLKK-injected cells. Data are expressed as mean ± S.E.

	Discussion

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In this report, we demonstrate through the use of selective antagonists that opioid-induced increases in [Ca²⁺]_i result from the mobilization of IP₃-sensitive Ca²⁺ stores via the liberation of the G subunits. Thus, we suggest the signaling pathway outlined in Fig. 5. The essential elements of this model include mobilization of the IP₃-sensitive Ca²⁺ store and opioid-induced liberation of G.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Hypothetical mechanism of opioid-induced Ca2+ release from intracellular stores in differentiated NG108-15 cells. Upon binding of DADLE to the -opioid receptor (-OR), a PTX-sensitive heterotrimeric G protein dissociates into i and subunits. i binds to and inhibits AC, and binds to and activates PLC. Activation of PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate into IP3 and diacylglycerol. IP3 binds to IP3 receptors located on intracellular stores releasing Ca2+ into the cytosol. Heparin is a competitive antagonist for IP3 receptors. QEHA binds free G and QLKK binds free Gq.

That activation of -opioid receptors will mobilize IP₃-sensitive Ca²⁺ stores is consistent with a number of studies including our own (Jin et al., 1994; Smart and Lambert, 1996). We showed previously that DADLE evoked increases in [Ca²⁺]_i in NG108-15 cells that did not require extracellular Ca²⁺ and were blocked by thapsigargin, an inhibitor of the ATP-dependent Ca²⁺ pump that is responsible for loading Ca²⁺ into intracellular stores. The PLC inhibitor U73122 also inhibited the response. Thus, the inhibition produced by heparin lends further support to the idea that -opioid receptors activate PLC to generate IP₃ (Berridge, 1993). Indeed, several laboratories have measured opioid-induced increases in IP₃ directly (Tsu et al., 1995; Smart and Lambert, 1996; Lee et al., 1998).

All previously described -opioid receptor-mediated effects in NG108-15 cells, including opioid-induced increases in [Ca²⁺]_i and IP₃ (Jin et al., 1994; Tsu et al., 1995; Smart and Lambert, 1996), were inhibited by PTX and are thus mediated by inhibitory G proteins. However, there is no precedent for G_i/o-type subunits coupling to PLC. Thus, we explored the possibility that the subunits liberated from the dissociation of the G_i/o heterotrimer were mediating the opioid-induced increase in [Ca²⁺]_i. It has been shown previously that G will activate certain  isoforms of PLC (Camps et al., 1992; Katz et al., 1992). The selective inhibition of the DADLE-induced [Ca²⁺]_i increase by QEHA peptide is consistent with this scenario. There are multiple forms of each of the subunits that make up heterotrimeric G proteins, and in some signaling pathways specific isoforms selectively couple receptor to effector (Kleuss et al., 1991, 1992, 1993). The approach applied in this study could not distinguish the relative contributions of specific G subunits to opioid-induced Ca²⁺ mobilization, although several G isoforms have been identified in NG108-15 cells (Ueda et al., 1998). G₂, one of the predominant isoforms expressed in NG108-15 cells, has been shown to activate PLC2 (Zhang et al., 1996).

In some studies, opioid-induced increases in [Ca²⁺]_i required costimulation with G_q-generating agonists such as bradykinin or ATP (Okajima et al., 1993). However, the opioid-induced [Ca²⁺]_i increase described here did not result from opioid-induced liberation of G_q, because microinjection of the QLKK peptide did not significantly inhibit the response, in contrast to the response elicited by bradykinin. This observation is consistent with a similar experiment in smooth muscle cells in which -opioid receptor-induced activation of PLC was not blocked by an antibody to G_q (Murthy and Makhlouf, 1996). However, we hesitate to completely rule out a role for G_q in this response because the QLKK peptide appeared to attenuate the response, although the effect did not reach statistical significance. It was not experimentally feasible to increase the statistical power sufficient to determine whether the small and variable effects of QLKK on the DADLE-evoked response indicated a small contribution from G_q. It is possible that the DADLE-induced [Ca²⁺]_i increase in NG108-15 cells is mediated by an isoform of PLC that is stimulated by the combined action of G_q and G and that in our paradigm G_q was prebound to the enzyme and, thus, activation of PLC was not readily reversed by the inhibitory peptide. Strassheim et al. (1998) have shown that the 3 isoform of PLC, an isoform activated by both G_q and G (Rhee and Choi, 1992; Singer et al., 1997), is present in NG108-15 cells. The idea that PLC3 might need to be primed with G_q to observe an opioid-induced [Ca²⁺]_i increase would reconcile the results from several laboratories in which the specific culture conditions or the presence of a subthreshold concentration of a G_q-generating agonist was required.

In summary, we have shown that activation of -opioid receptors in NG108-15 cells mobilize IP₃-sensitive Ca²⁺ stores by a G-dependent mechanism. These results are consistent with an opioid signaling pathway that couples to PLC to produce IP₃.