Orally administered glucose or sucrose is an effective means to decrease the pain response to mildly invasive procedures in newborns. In studies of newborns undergoing heel prick or arm venipuncture for blood collection, oral sucrose has been shown to be an effective analgesic (1–3). The use of sucrose as an analgesic for procedural pain in newborns has been reviewed (4). The mechanism underlying the analgesic effects of sugars may involve the opioid system. For example, in preclinical studies, an intraoral infusion of sucrose nearly doubled the pain threshold in young rats in a heat withdrawal test, and this effect was reversed by naloxone, an opioid receptor antagonist (5). Naloxone was also found to reduce the tail-flick analgesic index, which had been increased in adult rats fed a sucrose solution (6). In other studies, the pain threshold in a hotplate foot withdrawal test was increased in hyperglycemic rats compared with controls, and the administration of naloxone reversed this increase (7). A study of infants born to methadone mothers reported that an orally delivered sucrose solution that could reduce crying behavior in normal infants had no effect on crying in the methadone infants (8).
Involvement of the opioid system in the analgesic actions of sugar solutions could involve a direct effect of glucose on the opioid receptor or it could involve an indirect effect via the release of endogenous opioids. In these studies, we tested the hypothesis that glucose directly activates the mu opioid receptor or modulates the interaction of opioids with the mu receptor. Mu opioid receptors (MOR-1) were expressed in Xenopus oocytes, a well recognized preparation for the expression of receptors and ion channels (9).
The use of Xenopus laevis frogs (Xenopus I, Inc, Dexter, MI) in these experiments was approved by the University of Missouri Animal Care and Use Committee. The synthetic enkephalin, Tyr-D-Ala-Gly-Me-Phe-Gly-ol (DAMGO), and collagenase type IA were from Sigma (St. Louis, MO). Dextrose was from Fisher Scientific (Pittsburgh, PA). Gentamicin was from APP (Los Angeles, CA). All reagents were of the highest reagent grade.
Capped complimentary cRNA transcripts encoding the rat MOR-1, subcloned in the pRc/CMV vector and the mouse G protein-activated inwardly rectifying potassium channel, GIRK2 (Kir3.2), in the pBluescript II KS vector, were prepared by in vitro transcription from the linearized cDNAs using the Message Machine (Ambion, Austin, TX). Commonwealth Biotechnologies, Inc. (Richmond, VA) prepared the MOR-1 and GIRK2 cRNA transcripts. The MOR-1 and GIRK2 cDNA constructs were kindly provided by Dr. R. Adron Harris, University of Texas at Austin. Oocyte removal and cRNA injection followed established procedures (9) and National Institute of Health guidelines. Briefly, frogs were anesthetized in 0.2% tricaine methanesulfonate, with a pH value of 7.4. The oocytes were surgically removed and incubated with 2 mg/mL of collagenase type 1A for 45 min at room temperature with gentle rocking to remove the follicular cell layer. Stage VI oocytes were injected the following day with 50 nL of DEPC water containing 1.4 ng of MOR-1 cRNA and 0.4 ng of GIRK2 cRNA. Oocytes were incubated at 14°C–15°C, with no shaking, in ND96PG containing (in mM): NaCl 96, KCl 2, CaCl2 1.8, MgCl2 1, pyruvate 2.5, 50 μg/mL of gentamicin, and HEPES-Na 5, with a pH value of 7.4, for 3–7 days. ND96PG was changed daily.
For the electrophysiological studies, the oocytes were perfused at a rate of 3 mL/min at room temperature (21°C–23°C) with ND96 containing (in mM): NaCl 96, KCl 2, CaCl2 1.8, MgCl2 1, and HEPES-Na 5, with a pH value of 7.4. The perfusion chamber volume was ∼0.25 mL. To measure inward current through GIRK2 channels, the perfusion solution was changed from ND96 to a high K (HK) solution containing (in mM): KCl 98, CaCl2 1.8, MgCl2 1, and HEPES-K 5, with a pH value of 7.4. Some HK solutions contained 5 mM of NaCl, substituted equimolar with KCl, to increase the stability of the current recording, and this caused no changes in the results. In the glucose experiments, 20 mM of glucose was added to the HK solution. This addition increased the osmolarity of the HK solution from 213 to 230 mOsm but had no effect on resting holding currents. Other studies of Xenopus oocytes have reported no changes in membrane potential or whole cell currents with osmolarity increases or decreases of up to 120 mOsm (10). Current responses were measured using a two-electrode voltage clamp (Model OC 725C; Warner Instruments, Hamden, CT) and monitored with a chart recorder. The oocytes were stable for 1–2 h. Microelectrodes were filled with 3 M KCl and had resistances of 0.5–5.0 MΩ. All experiments were performed at a holding potential of −60 mV.
Data were analyzed with Prism software (GraphPad, San Diego, CA) and are presented as mean ± sem. Results were normalized to the same oocyte because of the inherent variability in expression of injected cRNA in Xenopus oocytes. For the glucose inhibition studies, the currents activated by the glucose and DAMGO solution were bracketed by responses with DAMGO alone applied before and after the glucose solution. These 2 DAMGO responses were averaged and represented a 100% response. For the desensitization studies, the data were fitted to a single exponential function. The number of oocytes used in the experiments is indicated by N in the figures. Each set of experiments used oocytes from three or more animals because of the inherent variability in the preparation. Differences between experimental and control means were analyzed with the Student’s t-test and assumed to be statistically different if the P value was 0.05 or less.
MOR-1 were expressed in Xenopus oocytes to test the effects of glucose on opioid receptor activation in a functional cell assay. Because the MOR-1 is a G-protein coupled receptor that activates G-proteins upon agonist binding, we co-expressed GIRK2, a G-protein activated, inward rectifying potassium channel. Thus, we could monitor agonist-induced changes in G-protein concentrations by measuring changes in potassium channel currents as a functional assay of opioid receptor activation.
Figure 1A shows a representative voltage-clamp current recording of an oocyte co-injected with the cRNAs for the MOR-1 and GIRK2. To test our hypothesis that glucose directly activates the MOR-1, we added 20 mM glucose to the HK solution perfusing the oocyte. Plasma glucose ranges from approximately 6 mM during fasting to more than 20 mM in low insulin states. As seen in Figure 1A, glucose had no effect on the oocyte holding current. A subsequent addition of 10 nM DAMGO reversibly activated an inward current through the GIRK2 potassium channels as a result of their activation by the MOR-1 via G-proteins. In Fig. 1B, the results from six experiments show that glucose did not activate the receptor directly.
Although glucose did not activate the MOR-1 directly, it is possible that it could modulate the interaction between a MOR-1 agonist and the opioid receptor. Therefore, we tested in HK solution the effect of 20 mM glucose on the activation of the opioid receptor by 10 nM DAMGO. At this concentration, DAMGO activates the receptor to approximately 60% of the maximal response, which is a region of the activation curve that is sensitive to modulators of agonist-receptor interactions (11; data not shown). Figure 2A shows a representative current recording where 10 nM DAMGO reversibly and reproducibly activated opioid receptors. The addition of 20 mM glucose by itself had no effect on the current, as also observed in the experiments of Figure 1, and slightly decreased the current activated by 10 nM DAMGO. After glucose was washed out with HK, the DAMGO activation of current persisted. Figure 2B shows that glucose decreased the activation of oocyte current by DAMGO to 0.89 ± 0.05 (n = 6) of the control values, although the difference was not statistically significant.
Receptor desensitization in the continued presence of an agonist is a common characteristic of G-protein coupled receptors, including opioid receptors (12,13). The analgesic effects of glucose might be explained if glucose were to decrease the rate of agonist-induced desensitization of the G protein-coupled MOR-1. We therefore measured the acute desensitization of the expressed opioid receptors by DAMGO with or without glucose. Figures 3, A and B, show representative current recordings of cRNA injected oocytes when 10 nM DAMGO or 10 nM DAMGO plus 20 mM glucose, respectively, were added to the HK perfusion solutions. DAMGO rapidly activated inward currents, which then desensitized, with exponential time courses, toward steady-state levels in the continued presence of DAMGO. After washout of the agonist with HK, the currents deactivated and returned to baseline levels. The desensitizing, agonist-activated currents were fitted to single exponential functions from which the rates of desensitization and the steady-state levels of desensitization were determined over a period of 6.4 min. Figure 3C shows that the mean rate of desensitization for the DAMGO-activated inward current was 30 ± 3% per minute (n = 12), and the DAMGO-plus-glucose-activated currents was 31% ± 5% per minute (n = 8). These means were not significantly different by t-test. Figure 3D shows that the DAMGO-activated current desensitized to a steady-state level that was 15 ± 5% (n = 12) of the peak current, whereas the current activated by DAMGO plus glucose reached a steady-state that was 28 ± 6% (n = 8) of its peak current over this same period. Although there was less desensitization to DAMGO in the presence of glucose, the difference in steady-state levels of these desensitizations was not statistically significant.
Clinical trials have demonstrated the potential analgesic effects of the oral feeding of sugar solutions. Oral sucrose solutions can alleviate the pain associated with immunizations, heel stick, and circumcision (2,14,15). A systematic review of studies using sucrose analgesia in newborns has demonstrated the clinical efficacy of doses of 0.05–2 mL of 12%–50% sucrose solutions (16). Although the efficacy of this technique has been demonstrated, the mechanisms of these effects have not been fully elucidated. Although nonnutritive sucking of a pacifier or nonlactating nipple have been shown to impart some analgesic effect, the effect is less than that seen with oral sucrose (1,17). In crossover evaluations, two studies demonstrated either decreased crying time or a less marked increase in heart rate with oral sucrose versus oral sterile water in response to heel-stick procedures in neonates, further demonstrating that much of the analgesic effect is related to the sucrose itself (18,19). Two studies have evaluated the efficacy of sucrose (0.5 g), glucose (0.1 and 0.3 g), and breast milk administered before a painful procedure (20,21). Both studies demonstrate the superiority of either glucose or sucrose over breast milk, suggesting that the active component may be either glucose or sucrose.
There are several potential mechanisms that could account for the analgesic effects of oral glucose or sucrose solutions in neonates. These include a direct activation of opioid receptors by the sugar, an enhancement of the effects of endogenous opioids on their receptor systems, or an indirect effect promoting the release of endogenous opioids in the central nervous system. Although it is unlikely that a non-opioid such as glucose would activate the MOR-1, it has been reported in radioligand-binding studies and tissue bioassays that non-opioids, such as ketamine and phencyclidine, can directly bind to and activate this receptor (22,23). Reports in the literature on the effect of 20 mM glucose on radiolabeled agonist binding to mouse membranes containing MOR-1 are inconsistent, with one showing a small but significant decrease in receptor affinity and the other showing no effect (24,25). The analgesic effects of sugar solutions could also be related to an increase in plasma insulin, because insulin has been shown to have analgesic effects and decrease isoflurane minimum alveolar anesthetic concentration (26). Insulin has also been shown to enhance the effect of DAMGO on MOR-1 expressed in Xenopus oocytes (27). In addition, there are indications that sucrose and morphine have similar effects on dopamine receptor expression in the rat forebrain (28) and that dopaminergic mechanisms may play a role in pain modulation (29).
The present study is the first to investigate potential mechanisms of sugar analgesia in an opioid receptor functional assay. Our studies used the expressed rat MOR-1; there may be species differences between the rat and human MOR-1. Nevertheless, in our studies we found that increased glucose concentrations by themselves do not directly activate MOR-1. Additionally, we found that there was no enhancement of the effect of the opioid peptide mu agonist, DAMGO, in the presence of a glucose solution, thus making this mechanism of sugar analgesia unlikely as well. Finally, we tested the possibility that glucose could decrease the rate of desensitization of the MOR-1 in the presence of a specific mu receptor peptide agonist, thus enhancing the effect of the opioid. Opioid receptor desensitization can be affected by receptor phosphorylation, internalization, and association with regulators of G-proteins or other membrane proteins (13). We found that glucose had no significant effect on either the rate of mu receptor desensitization or the steady-state level of desensitization. Based on our findings, we suggest that the sugar solutions must act indirectly, perhaps through the release of endogenous opioids.
Evidence that the mechanism of sugar solution analgesia involves the release of endogenous opioids is supported by animal studies (6,7). However, there is little evidence supporting this mechanism in neonates. One study reporting indirect evidence for an endogenous opioid release mechanism for sugar solution analgesia in neonates is based on observations of the infants of methadone or opioid-addicted mothers who show no response to oral sugar solution analgesia (8). Another study indirectly supporting an endogenous opioid mechanism found an approximate two-minute delay before the maximum analgesic effect of oral sucrose was realized, and this delay coincides with the time required for endogenous opioid release (1,4). A study looking for direct evidence for an increase in serum β endorphin concentrations in response to sugar-solution administration in preterm infants failed to see any increase (30). However, the possibility remains that cerebrospinal fluid endorphin levels increase during sugar-solution analgesia or that other endogenous opioid levels increase. Thus, further studies are required on the mechanism of sugar-solution analgesia in neonates and will likely yield useful information about this useful method for preventing procedural pain in neonates.
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