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Department of Anesthesiology, Penn State University College of Medicine, Hershey, Pennsylvania
Submitted 22 September 2006; accepted in final form 4 December 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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M-6-G (77) endomorphin I (86). On the other hand, the rank order in mutant-expressing neurons was: DAMGO (14) >> morphine (39) >> endomorphin I (74) M-6-G (82), with a twofold leftward shift for both DAMGO and morphine. The DAMGO-mediated Ca2+ current inhibition was abolished by the selective MOR blocker, CTAP, and by pertussis toxin pretreatment of neurons expressing either hMOR subtype. These results suggest that the A118G variant MOR exhibits an altered signal transduction pathway and may help explain the variability of responses to opiates observed with carriers of the mutant allele. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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(Gi/o) protein subunits (Bailey and Connor 2005
). This results in a decrease of both neuronal excitability and neurotransmitter release. However, the long-term use of opioid analgesics can lead to tolerance, drug dependency, and addiction.
The human OPRM1 gene has been reported to undergo at least ten single-nucleotide polymorphisms (SNPs) within the open reading frame and >100 in noncoding regions (for review see Lötsch and Geisslinger 2005). The most common SNP within the OPRM1 coding region occurs at position 118 (A118G) in Exon I and results in an amino acid change from asparagine (N) to aspartate (D) at position 40 of the receptor. Asparagine is one of five putative glycosylation sites located on the extracellular N-terminal domain of the receptor (Mestek et al. 1995). The A118G polymorphism occurs with an allelic frequency ranging from 10 to 40% (Kim et al. 2004; Lötsch and Geisslinger 2005; Szeto et al. 2001; dependent on population studied). Several earlier clinical studies showed that the presence of A118G polymorphism is associated with opiate effectiveness observed in patients (Janicki et al. 2006; Klepstad et al. 2004; Lötsch and Geisslinger 2005; Romberg et al. 2004, 2005; Shi et al. 2002; Skarke et al. 2003) as well as susceptibility to drug addiction (Bond et al. 1998; Szeto et al. 2001). On the other hand, some studies also reported a lack of a correlation between the presence of the A118G SNP and drug addiction (Arias et al. 2006; Franke et al. 2001; Gelernter et al. 1999).
Few in vitro studies have examined the effect of the A118G polymorphism on receptor function and the findings have been conflicting. One report found that the mutant hMOR expressed in AV-12 cells had a threefold higher binding affinity than the wild-type opioid receptor for 
-endorphin (Bond et al. 1998). In addition, the -endorphin–mediated GIRK channel activation was three times more potent in mutant-expressing Xenopus oocytes than those expressing wild-type hMOR. Conversely, two separate studies showed that the mutant hMOR expressed in either COS cells (Befort et al. 2001) or HEK 293 cells (Beyer et al. 2004) did not demonstrate significant changes in binding affinity, potency, or signaling mechanisms compared with wild-type receptors. More recently, it was reported that Chinese hamster ovary (CHO) cells transfected with the mutant hMOR exhibited lower mRNA and protein expression levels (Zhang et al. 2005). The authors also reported that mutant 118G allele mRNA levels were lower than those of the wild-type allele in human brain tissue.
The purpose of the present study was to investigate the role that the A118G polymorphism plays in N-type Ca2+ channel modulation by various MOR agonists in rat sympathetic superior cervical ganglion (SCG) neurons. The majority of Ca2+ current in SCG neurons is carried by N-type Ca2+ channels (Ikeda 1991) and SCG neurons do not natively express µ-opioid receptors. Therefore this model system offers an appropriate null background within a neuronal cellular context. Importantly, N-type Ca2+ channels play a major role in neurotransmitter release and were previously shown to be modulated by MOR in central and sensory neurons (for review see Law et al. 2000). In the present report, wild-type and mutant hMOR were heterologously expressed in SCG neurons and the pharmacological profile of various MOR agonists was determined to ascertain whether the N40D mutation exhibits a differential modulation of N-type Ca2+ channels. In this report, the first-described, or "prototype," hMOR and "A118G variant" gene products are referred to throughout as wild-type and mutant hMOR, respectively.
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METHODS |
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SCG neurons from adult rats were prepared using the method described previously (Ruiz-Velasco and Ikeda 2000). The experiments carried out were approved by the Institutional Animal Care and Use Committee (IACUC). Male Wistar rats (175–225 g) were anesthetized with CO2 and then decapitated using a laboratory guillotine. The neurons were enzymatically dissociated as described (Ruiz-Velasco and Ikeda 2000). The isolated neurons were resuspended in Minimal Essential Medium (MEM), supplemented with 10% fetal calf serum, 1% glutamine, and 1% penicillin-streptomycin solution (all from Invitrogen, Carlsbad, CA). The dissociated neurons were plated onto 35-mm polystyrene tissue-culture plates coated with poly-L-lysine and stored in a humidified incubator (95% O2-5% CO2) at 37°C.
cDNA microinjection
Microinjection of cDNA plasmids was performed with an Eppendorf 5246 microinjector and 5171 micromanipulator (Brinkmann Instruments, Westbury, NY) 3–5 h after plating the neurons as described previously (Ikeda 2004). Plasmids coding for wild-type (Guthrie cDNA Resource Center, Sayre, PA) and mutant hMOR were subcloned in pcDNA3.1 (Invitrogen) and injected at concentrations of 5, 20, and 200 ng/µl. The mutant hMOR construct was prepared by site-directed mutagenesis (TOP Gene Technologies, Montreal, Canada). Wild-type and mutant plasmid sequences were confirmed by automated oligonucleotide sequencing. The "enhanced" green fluorescent protein (pEGFP-N1; BD Biosciences, Clontech, Palo Alto, CA) cDNA was coinjected at 5 ng/µl to allow for identification of successfully injected neurons.
Electrophysiology and data analysis
Ca2+ currents were recorded at room temperature (21–24°C) using the whole cell patch-clamp technique within 24 h after nuclear microinjection of vectors. The recording pipettes were pulled from borosilicate glass capillaries (Corning 7052; Garner Glass, Claremont, CA) on a Flaming-Brown (P-97) micropipette puller (Sutter Instrument, Novato, CA), coated with Sylgard (Dow Corning, Midland, MI), and fire polished with a microforge. SCG whole cell Ca2+ currents were acquired with a patch-clamp amplifier (Axopatch 200B, Axon Instruments, Foster City, CA), analog filtered at 5–10 kHz (–3 dB; four-pole low-pass Bessel filter), and digitized by use of custom-designed software (S5) on a PowerMacG4 computer (Apple Computer, Cupertino, CA) equipped with an 18-bit A/D converter board (ITC18, Instrutech, Port Washington, NY). The cell's series resistance (80–85%) and membrane capacitance were electronically compensated. Data and statistical analyses were performed with the Igor Pro (WaveMetrics, Lake Oswego, OR) and drc package from the R statistical programming environment (R Development Core Team) software packages, respectively, with P < 0.05 considered statistically significant. Summary graphs and current traces were produced with the Igor Pro and Canvas 8.0 (Deneba Software, Miami, FL) software packages.
The pipette solution contained (in mM): 120 N-methyl-D-glucamine, 20 tetraethylammonium hydroxide (TEA-OH), 11 EGTA, 10 HEPES, 1 CaCl2, 4 Mg-ATP, 0.3 Na2GTP, and 14 Tris creatine phosphate. The pH was adjusted to 7.2 with methanesulfonic acid and the osmolality was 293–302 mosmol/kg. The external solution consisted of (in mM): 145 TEA-OH, 140 methanesulfonic acid, 10 HEPES, 15 glucose, 10 CaCl2, and 0.0003 tetrodotoxin. The pH was adjusted to 7.4 with TEA-OH and the osmolality was 320–325 mosmol/kg.
The concentration–response curves were determined by the sequential application of increasing concentrations of the receptor agonist. No more than three different concentrations were used with each cell to avoid desensitization. The results were pooled and each point represents the mean ± SE. The concentration–response curves were fit to the Hill equation: I = IMAX/{1 + (IC50/[ligand])nH}, where I is the percentage inhibition, IMAX is maximum inhibition, IC50 is half-inhibition concentration, [ligand] is agonist concentration, and nH is the Hill coefficient.
Solution and drugs
Stock solutions of norepinephrine (NE)-bitartrate, [D-Ala2,NMe-Phe4,gly-ol5]-enkephalin (DAMGO), morphine-6-glucuronide (M-6-G), morphine, endomorphin I, and D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) (all from Sigma Chemical, St. Louis, MO) were prepared in H2O and diluted in the external solution to their final concentrations before use. Bordetella pertussis toxin (PTX, List Biological Laboratories, Campbell, CA) was added to the culture medium (12–20 h) at a final concentration of 500 ng/ml.
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RESULTS |
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In the present study, we examined the functional effects of the hMOR mutation (N40D) by comparing the modulation of N-type Ca2+ channels by the wild-type and mutant hMOR heterologously expressed in acutely dissociated rat SCG neurons. The initial experiments were carried out to determine the effective concentration of the hMOR cDNA constructs required to obtain coupling with Ca2+ channels without altering other native GPCR signaling pathways (i.e., 2-adrenergic receptors). Ca2+ currents were evoked every 5 s with a double-pulse voltage protocol (shown in Fig. 1A) consisting of two identical test pulses (to +10 mV from a holding potential of –80 mV) separated by a large depolarizing conditioning pulse to +80 mV (Elmslie et al. 1990; Ikeda 1991). The Ca2+ current inhibition was measured isochronally 10 ms after initiation of the prepulse in the absence and presence of the agonist. Figure 1A shows the time course of both pre- and postpulse Ca2+ current amplitude before and after external application of the high-affinity MOR agonists DAMGO and NE. Also shown is the Ca2+ current facilitation, which is the ratio of the postpulse to prepulse currents. Exposure of the cell to DAMGO (10 µM) did not result in Ca2+ channel inhibition (current traces 1 and 3). On the other hand, stimulation of the 2-adrenergic receptor by NE (10 µM) resulted in inhibition of Ca2+ channel currents by 50% (current traces 5 and 7). The NE-induced inhibition was greater during the prepulse (trace 7) than the postpulse (trace 8), indicating a voltage-dependent inhibition of the currents characterized by kinetic slowing of the prepulse current and enhancement of the postpulse current. Thus the post/pre ratio increased from 1.27 to 2.21 in the presence of NE (Fig. 1A).
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) and cannabinoid receptors (Vasquez and Lewis 1999). The NE-mediated Ca2+ current inhibition in neurons microinjected with 5, 20, and 200 ng/µl mutant hMOR cDNA was similar to that observed with wild-type hMOR-expressing cells (data not shown). Figure 1D is a summary graph showing the DAMGO- and NE-mediated Ca2+ current inhibition of uninjected and neurons microinjected with 5, 20, and 200 ng/µl wild-type or mutant hMOR cDNA. Although microinjection of 5 ng/µl did not result in an alteration of coupling components between NE-activated 2-adrenergic receptors and Ca2+ channels, the DAMGO-mediated Ca2+ current inhibition was variable. Also, nuclear microinjection of 200 ng/µl of either hMOR cDNA resulted in a significantly lower (P < 0.05) NE-induced Ca2+ channel inhibition. Thus for all subsequent experiments described, 20 ng/µl hMOR cDNA was chosen as the concentration that would maintain a consistent receptor-channel stoichiometry without altering native signaling pathways.
Next, we wanted to determine whether the signaling proteins that couple N-type Ca2+ channels and the N40D mutant receptor were different from the wild-type hMOR. As mentioned earlier, MOR are coupled with members of the Gi/Go subfamily that are pertussis toxin (PTX) sensitive. Figure 2, Ai and Bi shows current traces of neurons expressing wild-type and mutant hMOR, respectively. Bath application of DAMGO (10 µM) resulted in inhibition of Ca2+ currents by 75 and 78%, respectively. On the other hand, Fig. 2, Aii and Bii shows that overnight PTX pretreatment of the neurons decreased the DAMGO-mediated Ca2+ current inhibition. The mean Ca2+ current inhibition (±SE) in PTX-treated cells was significantly (P < 0.01) reduced in both wild-type (56 ± 6 vs. 9 ± 3%) and mutant (61 ± 9 vs. 9 ± 2%) expressing neurons (Fig. 1C). These results suggest that both receptor subtypes modulate N-type Ca2+ channels by Gi/o G-protein subunits.
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In the next set of experiments, the concentration-dependent Ca2+ current inhibition by DAMGO was determined in neurons heterologously expressing wild-type or mutant hMOR. Ca2+ currents were evoked using the voltage protocol described in Fig. 1A. Figure 3A shows the time course of Ca2+ current inhibition by 0.003, 0.03, and 3 µM DAMGO in neurons expressing the wild-type hMOR. The time course shown in Fig. 3B is that of a neuron expressing mutant hMOR receptors exposed to 0.03, 0.3, and 3 µM DAMGO. Again, both plots show that the DAMGO-mediated Ca2+ current inhibition is voltage dependent. The DAMGO concentration–response curves for the wild-type (closed circles) and mutant (open circles) hMOR are plotted in Fig. 3C. The data were fit to the Hill equation. The EC50, maximum inhibition, and Hill coefficient (±SE) obtained were 30.8 ± 8.5 and 14.2 ± 3.7 nM, 64.4 ± 3.4 and 61.6 ± 2.5%, and 0.8 and 1.2 for wild-type (n = 4–11) and mutant (n = 4–16) hMOR-expressing cells, respectively. Thus the data plotted in Fig. 3C show that DAMGO displayed a significantly higher potency (P = 0.002) for mutant hMOR-expressing neurons but similar efficacy for both receptor subtypes.
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). Therefore in the next set of experiments, the concentration-dependent Ca2+ current inhibition by morphine was determined in SCG neurons expressing each receptor subtype. The time course of morphine-mediated Ca2+ current of wild-type and mutant hMOR-expressing neurons is shown in Fig. 5, A and B, respectively. Ca2+ currents were evoked as described above and increasing concentrations of morphine were applied to the external bath solution. Figure 5C illustrates the morphine concentration–response relationship for both hMOR-expressing group of cells. The Hill equation was again used for data analysis. The EC50, maximum inhibition (± SE), and Hill coefficient values for wild-type hMOR-expressing neurons were 75.5 ± 26.0 nM, 50.2 ± 3.4%, and 1.3, respectively (n = 3–11). On the other hand, the EC50, maximum inhibition (±SE), and Hill coefficient values of neurons microinjected with mutant hMOR cDNA were 39.5 ± 18.9 nM, 61.1 ± 5.2%, and 0.8, respectively (n = 3–8). Overall, these results demonstrate that morphine exhibits a higher potency (P = 0.13) with mutant hMOR-expressing cells than those expressing wild-type hMOR, as well as a slightly higher efficacy (Fig. 5C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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; Mayer and Höllt 2006). Although pain perception and bioavailability contribute to the variability observed clinically, genetics is now considered an important factor that can affect patient response to analgesics. For example, the gene coding for the hMOR was previously found to undergo several SNPs. The most frequently occurring SNP occurs at nucleotide 118 (A
G) and leads to a change of the amino acid asparagine to aspartate (N40D) and elimination of a putative glycosylation site at the N-terminus (Bond et al. 1998; Lötsch and Geisslinger 2005). It is believed that the A118G SNP may play a significant role in the variability of the clinical effectiveness of opiates, susceptibility for drug addiction, and sensitivity of pain patients to develop long-term pain symptoms (Janicki et al. 2006; Lötsch and Geisslinger 2005).
The purpose of the present study was to examine the role that the mutant (N40D) hMOR plays in N-type Ca2+ channel modulation in sympathetic neurons. Because opioid alkaloids, such as morphine, and opioid peptides mediate pain inhibition throughout the nervous system partly by activating GIRK channels and inhibiting high-voltage–gated Ca2+ channels, we took advantage of our expression system (i.e., SCG neurons) that would allow us to study the coupling mechanisms within a neuronal context. The pharmacological profile of the high affinity agonist DAMGO showed that mutant hMOR-expressing cells exhibited a twofold higher potency in Ca2+ channel inhibition than neurons expressing the wild-type receptor, whereas both group of neurons displayed similar efficacies. In addition, the DAMGO-mediated Ca2+ current inhibition was blocked by the MOR blocker CTAP and by pretreatment with PTX. Our results are also consistent with those observed with coupling of mutant hMOR and another G-protein effector, GIRK channels (Bond et al. 1998). In that study, evidence was also provided to show that the binding of the endogenous opioid, -endorphin, had a threefold higher binding affinity for the mutant hMOR than for the wild-type hMOR.
Three in vitro studies previously reported that DAMGO binding parameters in cell membrane preparations were not different between wild-type and mutant hMOR in AV-12 (Bond et al. 1998), COS (Befort et al. 2001), and HEK293 cells (Beyer et al. 2004; in this study mutant hMOR expression levels were lower, discussed in the following text). Thus the N40D mutation does not appear to affect the binding of agonists to the mutant receptor, but rather alters the signal transduction events or receptor dimerization. For instance, the study by Befort et al. (2001) also reported that the DAMGO binding characteristics to another hMOR SNP, (T802C), were not different from wild-type hMOR-expressing COS cells. However, they found that [35S]GTP-
S binding (a measure of G-protein signaling) was reduced in the mutant-hMOR–expressing cells. Our results are consistent with an apparent change in signaling mechanism(s) that couple N40D hMOR and Ca2+ channels. It should be noted that under our experimental conditions we are not able to determine protein levels, and thus a decrease in surface expression (i.e., less receptor reserve) of mutant hMOR relative to wild-type receptors cannot be ruled out.
Alternatively, it may be that substitution of the putative glycosylation site at the N-terminus alters the ability of mutant MOR to form dimers. Homo- and heterodimerization is a phenomenon found to occur with several GPCRs, including MOR (Rios et al. 2001). For instance, 1-adrenergic receptors contain one glycosylation site on the N-terminus (N15) and it was previously shown that the N15A mutant receptor exhibited a decreased ability to form homodimers when compared with wild-type receptors as well as a reduction in cell surface expression (He et al. 2002). In a subsequent study, it was demonstrated that dimer formation between the N15A mutant 1-adrenergic receptor and 2-adrenergic receptor (containing a double mutation to block glycosylation) was significantly enhanced when compared with dimerization of both wild-type receptors (Xu et al. 2003). Whether dimerization of mutant hMOR is altered or inhibited by the loss of this sugar moiety requires further investigation.
Because morphine is the most commonly used opiate analgesic, the coupling of Ca2+ channels to morphine-activated wild-type and mutant hMOR was also examined in this study. Neurons expressing mutant receptors exhibited a greater than twofold increase in potency when compared with wild-type hMOR-expressing cells, whereas the efficacy was similar in both groups. A similar observation was reported to occur with oocytes heterologously expressing GIRK channels and mutant hMOR, although -endorphin was the agonist used (Bond et al. 1998). The EC50 value for -endorphin–mediated GIRK channel activation was three times lower in mutant hMOR-expressing oocytes. Reports from clinical studies are conflicting with respect to the presence of the 118G allele and morphine's analgesic effect (for review see Lötsch and Geisslinger 2005). In a subgroup of chronic pain patients homozygous for the wild-type allele, we observed that the morphine requirement for pain relief was significantly higher than that for patients carrying the mutant allele (Janicki et al. 2006). On the other hand, in a group of healthy volunteers, it was shown that the amount of morphine required to achieve pupil-constricting effects was not different between carriers of either allele (Lötsch et al. 2002a). In another report, however, a 2.1 and 3.6 rightward shift of morphine potency was observed in heterozygous and homozygous carriers of the mutant allele, respectively (Skarke et al. 2003). A study of cancer pain patients reported that a higher dose of morphine was necessary for pain relief of those homozygous for the 118G allele (Klepstad et al. 2004). The mechanism for these differences remains unclear.
The modulation of Ca2+ currents by the active morphine metabolite M-6-G or the opioid peptide endomorphin I was not significantly different in neurons expressing either hMOR subtype. These results are consistent with the observations reported to occur in HEK293 (Beyer et al. 2004) and AV-12 cells (Bond et al. 1998). Nevertheless, a volunteer-based study found that carriers of the mutant allele showed a decreased potency with respect to M-6-G–induced pupil constriction when compared with homozygous wild-type carriers (Lötsch et al. 2002a). Nonetheless, another study found that 118G-carrying healthy volunteers reported less nausea and vomited less frequently when M-6-G was used as the opioid agonist (Skarke et al. 2003). Moreover, in a study of two patients with renal failure, it was observed that the patient carrying the G118 allele was able to better tolerate increased plasma levels of M-6-G than the homozygous wild-type patient (Lötsch et al. 2002b). These studies suggest that some of the side effects associated with M-6-G may offer some protection to 118G carriers. The mechanism is presently unknown.
A recent in vitro study reported that CHO cells transfected with the mutant hMOR had significantly lower MOR mRNA levels and protein expression than those of wild-type–transfected cells (Zhang et al. 2005). The authors suggested that their findings were indicative of a loss of function by the N40D hMOR. Lower mutant hMOR expression levels were also observed to occur in HEK293 cells (Beyer et al. 2004). The results of the present study and those previously reported (Befort et al. 2001; Bond et al. 1998) are not consistent with a loss of MOR function. The discrepancies in the studies indicate that characterization of the mutant hMOR is dependent on the cell system used. In fact, Befort and colleagues (2001) found that the expression levels of wild-type and mutant hMOR in COS cell membranes were not different, nor was there a significant change in the DAMGO-induced downregulation of both receptor subtypes. These differences, however, highlight the advantage of using SCG neurons as an expression system to study the functional coupling of the N40D hMOR to natively expressed ion channels and G-protein subunits. The temporal resolution (i.e., seconds) in our study allows for the measurement of the G-mediated, membrane-delimited, and voltage-dependent modulation of Ca2+ channels. On the other hand, biochemical assays are normally determined over longer periods. Finally, the dissimilarities may be explained by the fact that some cell lines do not express proteins that are involved in mRNA and/or protein processing, folding, or trafficking normally occurring in neurons (for review see Bailey and Connor 2005). For instance, in rat and bovine tissue it was previously reported that tissue expression of the atrial natriuretic peptide receptor, guanylyl cyclase-A, is influenced by the degree of N-glycosylation and is not uniform across tissues, such as brain, kidney, and lung (Müller et al. 2002).
In summary, our results indicate that wild-type and mutant hMOR can be successfully expressed in rat SCG neurons, an expression system that can be used to further examine the signal transduction elements that couple N40D hMOR and N-type Ca2+ channels. The potencies of the DAMGO- and morphine-mediated Ca2+ channel inhibition were shifted leftward in mutant hMOR-expressing neurons when compared with cells expressing the wild-type receptor. Coupling of both receptor subtypes to Ca2+ channels was PTX sensitive and blocked by pretreatment of CTAP. Finally, no significant differences were observed in Ca2+ channel modulation by M-6-G and endomorphin I in neurons expressing either hMOR subtype.
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FOOTNOTES |
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Address for reprint requests and other correspondence: V. Ruiz-Velasco, Department of Anesthesiology, H187, 500 University Drive, Penn State University College of Medicine, Hershey, PA 17033-0850 (E-mail: vruizvelasco{at}psu.edu)
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) acquired from the sequential application of DAMGO (0.003–3 µM). Currents were evoked every 5 s with the "double-pulse" voltage protocol (shown in 
