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ANALGESIA

The Effects of Morphine on Human 5-HT_3A Receptors

From the Klink und Poliklinik für Anästhesiologie und Operative Intensivmedizin, Universitätskliniken Bonn, Bonn, Germany.

Address correspondence and reprint requests to Dr. med. Maria Wittmann, Universitätskliniken Bonn, Sigmund-Freud-Straße-Str. 25 D-53105, Bonn, Germany. Address e-mail to maria.wittmann{at}gmx.de.

Abstract

5-HT₃ receptors are ligand-gated ion channels that are involved in the modulation of emesis and pain. In this study, we investigated whether the opioid analgesic, morphine, exerts specific effects on human 5-HT₃ receptors. Whole-cell patches from HEK-293 cells stably transfected with the human 5-HT_3A receptor cDNA were used to determine the effects of morphine on the 5-HT-induced currents using the patch clamp technique. At negative membrane potentials, 5-HT induced inward currents in a concentration-dependent manner. The 5-HT₃ receptor antagonist, ondansetron, (0.3 nM) reversibly inhibited the 5-HT-induced signals. Morphine reversibly suppressed 5-HT-induced peak currents as a function of concentration (IC₅₀ = 1.1 µM, Hill coefficient = 1.2). The block by morphine decreased with increasing 5-HT concentrations, suggesting a competitive effect. In addition, the activation, as well as the inactivation, kinetics of the currents were significantly slowed in the presence of morphine. The morphine antagonist, naloxone, also inhibited 5-HT-induced currents (e.g., at 3 µM by 17%). The effects of morphine and naloxone were not additive. The potency of morphine and the competitivity of the blocking effect points to a specific mechanism at a receptor site rather than an unspecific membrane effect.

Morphine, a naturally occurring alkaloid, is the prototype of opiate analgesics. Although morphine acts through opiate receptors (1), it is unlikely that all clinically observed side effects, e.g., nausea and emesis, are exclusively dependent on this type of signal transduction. Neurophysiological and pharmacological evidence has shown that 5-HT, in addition to opioids, plays an important role in signal transmission at various levels of the pain pathway (2). At the spinal level, intrathecal administration of 5-HT produces an antinociceptive effect via spinal 5-HT₃ receptors (3), which are ligand-gated ion channels, forming a cation-permeable membrane channel. 5-HT₃ receptors are also involved in the mediation of acute chemo-inflammatory pain (4). Intrathecal administration of the 5-HT₃ receptor agonist, 2-methylserotonin, produced dose-related analgesia against formalin-induced chemo-inflammatory pain in male rats (5).

Within the central nervous system, 5-HT₃ receptors are found primarily in the limbic system, brainstem, and spinal cord. In the spinal cord, 5-HT₃ receptors have been localized in the superficial laminae of the dorsal horn (6) and are prominently expressed in a variety of peripheral ganglia, including a subset of neurons in the dorsal root ganglion (7).

The 5-HT₃ receptor is directly and indirectly linked to several physiologic and pathologic processes, including gastrointestinal motility, visceral pain, nausea, and emesis (8).

The 5-HT₃ receptor in the area postrema triggers emesis (9), and 5-HT₃ receptor antagonists such as ondansetron are clinically used to prevent nausea associated with chemotherapy and general anesthesia (4). A variety of drugs affect the 5-HT₃ receptor apart from their primary action at other receptors. For example, cannabinoids (10) also act on 5-HT₃ receptors and it has been suggested that these effects may be linked to in vivo effects such as analgesia and antiemesis.

More recently, morphine has been shown to act as a specific competitive antagonist at rat 5-HT₃ receptors (11). Therefore it was the aim of this study to investigate whether morphine also has a specific effect on human 5-HT₃ receptors, to characterize it, and to elucidate if there are differences between human and rat 5-HT₃ receptors.

METHODS

HEK 293 cells were grown as monolayers on culture plates (NUNC, Wiesbaden, Germany) in DMEM Nutrient Mix F12 (1:1; v v^–1) medium containing 10% heat inactivated fetal calf serum, penicillin (100 I.U./mL), streptomycin (100 µg/mL), geneticine (0.75 µg/mL), and glutamine (292 µg/mL). The cells were cultured at 37°C in a humidified atmosphere (5% CO₂). For electrophysiological experiments cells were transferred to 35 mm Petri dishes (NUNC). They were used 2–6 days after transfer, before the cell layer became confluent.

For stable transfection 20% confluent HEK 293 cells were transfected by the modified calcium phosphate method with the human 5-HT_3A receptor cDNA subcloned into the mammalian expression vector pcDNA3 (Invitrogen, Karlsruhe, Germany) under the control of the human cytomegalovirus promoter. Two days after transfection, cells were selected by the addition of geneticine (800 µg/mL) to the culture medium. The medium was changed every 2 days. After occurrence of single cell colonies these were separated by use of cloning cylinders (Sigma-Aldrich, Munich, Germany) and further subcultured in 24 well plates (Falcon, BD Biosciences, Heidelberg, Germany) until confluence. Approximately 20 to 40 colonies from each transfection experiment were tested for stable expression of the particular cDNA by 5-HT-induced [¹⁴C]-guanidinium influx and binding of the selective 5-HT₃ receptor antagonist [³H]-GR65630. Colonies with the highest receptor expression were used for further experiments. Although most physiological 5-HT_3A receptors are not exclusively composed of this subunit, the homopentameric 5-HT_3A receptor is very suitable for mechanistic studies because of its homogeneous composition.

Currents through the 5-HT₃ receptor were measured with the patch-clamp technique. A patch-clamp amplifier (EPC-7; List Electronic, Darmstadt, Germany) was used with the output filter set between 65 and 500 Hz (sampling rate 125–1000 Hz) and pClamp software (Axon Instruments, Foster City, CA, U.S.A.). 5-HT (3 µM) was applied for 60 s at –30 or –60 mV in a standard voltage-clamp experiment in whole cell configuration. Capacitative transients and series resistance were measured and compensated using the internal compensation circuitry of the amplifier. A series resistance compensation of up to 70% was used.

The baseline control response to 3 µM 5-HT was measured before and after the drug application. To exclude rundown effects, the mean value of these two agonist applications was taken as the average control current. Each current trace was measured three times and averaged to reduce noise effects. A washout time of at least 90 s was allowed for recovery of the receptors from desensitization.

The solutions were applied via a perfusion pipette positioned close to the cell. The solution exchange rate was monitored by "open pipette" experiments. The external solution applied to the patch had the following composition (mM): NaCl 150, KCl 5.6, CaCl₂ 1.8, MgCl₂ 1.0, HEPES 10, pH 7.4. Patch pipettes with resistances of 1.5–3 M were filled with "intracellular" solution containing (mM): KCl 140, EGTA 10, MgCl₂ 5, HEPES 10, adjusted to a pH of 7.4. To minimize loss of drug from interaction with plastic material, drug solutions were stored in glass reservoirs and Teflon tubing was used. The recordings were performed at room temperature (21 ± 1°C).

The currents were digitized with an interface (Digidata 1200; Axon Instruments, Molecular Devices Corporation) and stored on an IBM 586-compatible PC. Data analysis was performed with pClamp^R 6/8 software (Axon Instruments). GraphPad Prism^R 3.03 software (GraphPad, San Diego, CA) was used to create graphics. The concentration-response curves for 5-HT, and analogously for the morphine effect, were fitted by the Hill equation,

i: immediate peak current as fraction of the maximal (control) current,

c: 5-HT concentration,

n: Hill coefficientEC₅₀: 5-HT concentration inducing the half-maximal effect.

To calculate a K_B value for morphine at human 5-HT_3A receptors, the EC₅₀ values of 5-HT in the absence and in the presence of morphine (1 µM) were applied to the Schild Equation:

The time course of agonist-induced activation and desensitization f(t) were fitted either separately using a single exponential function, or simultaneously with a bi-exponential function (according pClamp 6/8, Axon Instruments):

5-hydroxytryptamine (creatinine sulfate) was obtained from Sigma (München, Germany). Morphine (sulfate) was obtained from Mundipharma (Limburg, Germany) and naloxone (hydrochloride) was obtained from Ratiopharm (Ulm, Germany). Drug solutions were prepared daily from aqueous stock solutions (10–50 mM).

RESULTS

At negative membrane potentials the application of 5-HT to single HEK 293 cells induced inward currents (Fig. 1A) in a concentration-dependent manner. Current amplitudes between 500 and 5000 pA were observed in whole-cell patches at –30 mV. The specific human 5-HT_3A receptor antagonist ondansetron (0.3 nM applied before and during the agonist) reversibly inhibited the 5-HT (3 µM)-induced peak currents (Fig. 2A). The 5-HT concentration-response curve was measured and fitted to the Hill equation yielding an EC₅₀ value for 5-HT of 2 µM and a Hill coefficient of 2.2 (Fig. 1B).

View larger version (11K): [in this window] [in a new window]   Figure 1. Electrophysiological response to 5-HT of single HEK 293 cells expressing human 5-HT3A receptors. A, Exemplary whole cell currents recorded from one single cell in response to application of different concentrations of serotonin (–30 mV). B, Concentration–response curve (fitted to the Hill equation) shown for serotonin activation of human 5-HT3A receptors (mean ± sem from 4 to 7 cells) in whole-cell patches.

View larger version (15K): [in this window] [in a new window]   Figure 2. Inhibition of 5-HT (3 µM)-evoked peak-currents (whole cell mode, –30 mV) by morphine. The drug was applied continuously 90 s before and during the 5-HT pulse (equilibrium application). A, Current recordings from one cell show the inhibition of the peak-current by 0.3 nM ondansetron. B, Current recordings from one cell show the inhibition of the peak-current by 1 µM morphine, and a deceleration of the kinetics of activation and inactivation C, Concentration-response curve (Hill equation) for the inhibition of the 5-HT-evoked peak current amplitudes by morphine (mean ± sem, n = 5–9 different cells). Control refers to the current amplitude observed in the absence of morphine.

Morphine inhibited 5-HT (3 µM, EC₇₀)-induced peak currents concentration-dependently and reversibly (Fig. 2B) with an IC₅₀ value of 1.1 µM and a Hill coefficient of 1.2 (Fig. 2C). In these experiments, the opioid was present continuously for 1 min before and also for the duration of the 5-HT stimulus (equilibrium application). In addition to suppressing peak currents, morphine decreased the activation and inactivation time courses of the currents in a concentration–dependent manner: e.g., 1 µM morphine increased the activation time constant (_on) by a factor of 2.3 and the inactivation time constant (_off) by a factor of 2.6 (Fig. 3; paired Student's t-test, P < 0.05). At morphine concentrations larger than 1 µM the inactivation time constant could not be determined because the activation process was not complete even after 10 s of simultaneous 5-HT and morphine application. Morphine (0.1–10 µM) applied alone, to patches that were sensitive to 5-HT, did not evoke any current.

View larger version (10K): [in this window] [in a new window]   Figure 3. Deceleration of the current kinetics by morphine (3 µM 5-HT, whole-cells, –30 mV, biexponential fits, mean ± sem). A, Concentration-dependent (monoexponential fit) deceleration of current-activation (on, n = 2–7). B, Significant deceleration of the current-activation (on) by 1 µM morphine (n = 8, paired Student's t-test, P = 0.006). C, Significant deceleration of the current-inactivation (off) by 1 µM morphine (n = 5–18, unpaired Student's t-test, P = 0.043).

The morphine effect was attenuated by increasing 5-HT concentrations from 1 µM to 100 µM 5-HT and the agonist concentration-response curve was shifted to the right in the presence of 1 µM morphine (Figs. 4 and 5). Applying the EC₅₀ values of 5-HT in the absence and presence of 1 µM morphine (1.97 and 3.2 µM, respectively) to the Schild Equation, a K_B value for morphine of 1.6 µM could be calculated. This value is close to the IC₅₀ value (1.1 µM). In addition, the slowing of current kinetics by morphine is attenuated by increasing 5-HT concentrations.

View larger version (13K): [in this window] [in a new window]   Figure 4. 5-HT concentration-response curves in the absence (filled symbols) and in the presence (open symbols) of morphine (1 µM) (whole cell mode, –30 mV, mean ± sem, n = 3–8; ***P < 0.001). Note that 1 µM morphine induces a shift of the 5-HT concentration-response curve to the right.

View larger version (23K): [in this window] [in a new window]   Figure 5. Effect of naloxone on 5-HT3 receptor-mediated currents A, 3 µM naloxone (but not 1 µM) significantly inhibits currents evoked by 3 µM 5-HT (17% inhibition), (n = 7–19, paired t-test). B, I: Naloxone (3 µM) reduces 5-HT (3 µM)-induced currents less than morphine (II; 1 µM). III: 3 µM naloxone + 1 µM morphine, co-applied reduce currents <1 µM morphine alone (II). IV: calculated, theoretical additive inhibition of 3 µM naloxone + 1 µM morphine.

DISCUSSION

The present investigation addresses the question whether morphine has specific effects on human 5-HT_3A receptors at clinical concentrations. First, however, to validate the whole cell preparation for this purpose, we measured the 5-HT concentration-response curve in whole cells and compared our results with existing data from whole cell patches (12). The EC₅₀ value of 5-HT reported here is in agreement with previous results for whole cell measurements (13). The specific 5-HT₃ antagonist, ondansetron, inhibits the 5-HT-evoked currents at nanomolar concentrations, which proves that the measured currents are exclusively mediated by 5-HT₃ receptors (Fig. 2B).

The concentration–dependent inhibition by morphine of 5-HT-induced currents (IC₅₀ = 1.1 µM) at least partially involves a specific and competitive mechanism. First, the inhibition was attenuated when the agonist concentration was increased, suggesting competition between morphine and 5-HT. This is further supported by the observation that morphine concentration-dependently slows the activation and inactivation kinetics of the 5-HT-induced currents. Second, the potency of morphine observed here is high when compared to its relative low lipophilicity (logP = 0.89), which argues against a pure "unspecific" mechanism. Third, although the specific µ-receptor antagonist naloxone slightly inhibited 5-HT-induced currents, it did not further enlarge but attenuated the inhibition by morphine. This is consistent with the assumption that both structurally related drugs compete for one common binding site at the 5-HT_3A receptor rather than act independently (i.e., additive) from each other. A competitive mechanism with similar potency was also found for rat 5-HT₃ receptors (11). However, the moderate discrepancy between the measured IC₅₀ of 1.1 µM and the calculated K_B value of 1.6 µM suggests an additional inhibitory (noncompetitive) component, which manifests itself as a reduction in efficacy of 5-HT (Fig. 4).

In the present study, morphine reduced the 5-HT-induced early peak current. After this inhibition, the currents were enhanced by a slowing of the inactivation (see traces in Fig. 2B). This long lasting enhancing effect could correspond to an emetic action of morphine. Assuming that the suppression of 5-HT₃ receptors has an antiemetic effect whereas enhancement of 5-HT₃ receptors promotes emesis, these results are initially surprising, but there are reports that clinically morphine can have both an emetic as well as an antiemetic effect (14). For instance, it has been shown, in dogs, that administration of methylnaltrexone blocked the peripheral emetic effect of morphine and unmasked a central antiemetic effect of morphine (15). Therefore we assume that the peripheral emetic effect might be evoked via opioid receptors, but in the central nervous system an inhibition of 5-HT₃ receptors might contribute to an antiemetic effect. The clinically observed emetic action of morphine may be related to the higher potency of morphine at opioid-receptors than at 5-HT₃ receptors.

Concentrations of morphine, which almost completely inhibited 5-HT₃ receptors in the present study (10 µM), are clinically meaningful, in contrast to IV administration observed in the cerebrospinal fluid during epidural or intrathecal administration (4–10 µM) (16). Thus, if one assumes that 5-HT₃ receptors are already completely blocked by morphine after subarachnoid injection, then the administration of additional 5-HT₃ receptor antagonists would not be expected to be a meaningful therapeutic strategy to further reduce emesis. In agreement with this reasoning, a previous study reported that the specific 5-HT₃ receptor antagonist, tropisetron, did not significantly reduce nausea and vomiting after subarachnoid injection of bupivacaine and morphine (17).

Several studies support the idea that, in addition to the opioid system, the serotonergic system is responsible for modulation of nociception (18). The interaction between opioid and serotonergic pathways is further supported by the observation that enkephalin-containing interneurons in the rat spinal trigeminal nucleus and in the rat spinal cord superficial dorsal horn are innervated by 5-HT-containing fibers (19). Furthermore, enkephalinergic inhibitory neurons appear to mediate serotonin-induced spinal analgesia, at least in part, via activation of 5-HT₃ receptors expressed in enkephalinergic neurons (20). Thus the present study adds to the existing evidence of interactions between the opioid and the serotonergic pathways.

Besides direct interactions of morphine with serotonergic receptors, indirect effects could be important: actions of morphine on the reuptake, release or metabolism of 5-HT could also contribute to its emetogenic activity by changes of the free 5-HT concentration and will be the subject of further studies.

The results presented here suggest that the human 5-HT₃ receptor is a sensitive and specific target for morphine and, thus, that opioid actions affecting 5-HT₃ receptors may be relevant for emesis and analgesia.

ACKNOWLEDGMENTS

We thank Dr. M. Brüss for the supply of HEK 293 cells, stably transfected with the cDNA of the human 5-HT_3A receptor.

Footnotes

Accepted for publication May 16, 2006.

Supported, in part, by the DFG (BA 1454).

REFERENCES

1. Gomes I, Gupta A, Filipovska J, et al. A role for heterodimerization of mu and delta opiate receptors in enhancing morphine analgesia. Proc Natl Acad Sci U S A 2004;101:5135–9.[Abstract/Free Full Text]
2. Kesim M, Duman EN, Kadioglu M, et al. The different roles of 5-HT₍₂₎ and 5-HT₍₃₎ receptors on antinociceptive effect of paroxetine in chemical stimuli in mice. J Pharmacol Sci 2005;97:61–6.[Web of Science][Medline]
3. Hamon M, Gallissot MC, Menard F, et al. 5-HT₃ receptor binding sites are on capsaicin-sensitive fibres in the rat spinal cord. Eur J Pharmacol 1989;164:315–22.[Web of Science][Medline]
4. Greenshaw AJ. Differential effects of ondansetron, haloperidol and clozapine on electrical self-stimulation of the ventral tegmental area. Behav Pharmacol 1993;4:479–85.[Web of Science][Medline]
5. Giordano J. Analgesic profile of centrally administered 2-methylserotonin against acute pain in rats. Eur J Pharmacol 1991;199:233–6.[Web of Science][Medline]
6. Glaum SR, Proudfit HK, Anderson EG. Reversal of the antinociceptive effects of intrathecally administered serotonin in the rat by a selective 5-HT₃ receptor antagonist. Neurosci Lett 1988;95:313–7.[Web of Science][Medline]
7. Tecott LH, Maricq AV, Julius D. Nervous system distribution of the serotonin 5-HT₃ receptor mRNA. Proc Natl Acad Sci U S A 1993;90:1430–4.[Abstract/Free Full Text]
8. Gyermek L. 5-HT₃ receptors: pharmacologic and therapeutic aspects. J Clin Pharmacol 1995;35:845–55.[Abstract]
9. Karim F, Roerig SC, Saphier D. Role of 5-hydroxytryptamine₃ (5-HT₃) antagonists in the prevention of emesis caused by anticancer therapy. Biochem Pharmacol 1996;52:685–92.[Web of Science][Medline]
10. Barann M, Molderings G, Bruss M, et al. Direct inhibition by cannabinoids of human 5-HT_3A receptors: probable involvement of an allosteric modulatory site. Br J Pharmacol 2002;137:589–96.[Web of Science][Medline]
11. Fan P. Nonopioid mechanism of morphine modulation of the activation of 5-hydroxytryptamine type 3 receptors. Mol Pharmacol 1995;47:491–5.[Abstract]
12. Brown AM, Hope AG, Lambert JJ, Peters JA. Ion permeation and conduction in a human recombinant 5-HT₃ receptor subunit (h5-HT_3A). J Physiol 1998;507:653–65.[Abstract/Free Full Text]
13. Sessoms-Sikes JS, Hamilton ME, Liu LX, et al. A mutation in transmembrane domain II of the 5-hydroxytryptamine_(3A) receptor stabilizes channel opening and alters alcohol modulatory actions. J Pharmacol Exp Ther 2003;306:595–604.[Abstract/Free Full Text]
14. Krueger H, Eddy N, Sumwalt M. The pharmacology of the opium alkaloids. Public Health Rep 1940;704:165.
15. Foss JF, Yuan CS, Roizen MF, Goldberg LI. Prevention of apomorphine- or cisplatin-induced emesis in the dog by a combination of methylnaltrexone and morphine. Cancer Chemother Pharmacol 1998;42:287–91.[Web of Science][Medline]
16. Nordberg G, Hedner T, Mellstrand T, Dahlstrom B. Pharmacokinetic aspects of intrathecal morphine analgesia. Anesthesiology 1984;60:448–54.[Web of Science][Medline]
17. Pitkanen MT, Niemi L, Tuominen MK, Rosenberg PH. Effect of tropisetron, a 5-HT₃ receptor antagonist, on analgesia and nausea after intrathecal morphine. Br J Anaesth 1993;71:681–4.[Abstract/Free Full Text]
18. Gupta S, Vaswani R, Verma D. The 5HT₃ receptors and the descending nociceptive pathway: a review. Middle East J Anesthesiol 1999;15:247–58.[Medline]
19. Nikfar S, Abdollahi M, Kebriaeezadeh A, Chitsaz M. Effect of granisetron on morphine-induced analgesia in mice by formalin test. Gen Pharmacol 1998;31:55–8.[Web of Science][Medline]
20. Tsuchiya M, Yamazaki H, Hori Y. Enkephalinergic neurons express 5-HT₃ receptors in the spinal cord dorsal horn: single cell RT-PCR analysis. Neuroreport 1999;10:2749–53.[Web of Science][Medline]

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Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999