Functional coupling, desensitization and internalization of virally expressed mu opioid receptors in cultured dorsal root ganglion neurons from mu opioid receptor knockout mice

Although mu opioid receptors desensitize in various cell lines in vitro, the relationship of this change in signaling efficacy to the development of tolerance in vivo remains uncertain. It is clear that a system is needed in which functional mu opioid receptor expression is obtained in appropriate neurons so that desensitization can be measured, manipulated, and mutated receptors expressed in this environment. We have developed a recombinant system in which expression of a flag-tagged mu opioid receptor is returned to dorsal root ganglia neurons from mu opioid receptor knockout mice in vitro. Flow cytometry analysis showed that adenoviral-mediated expression of the amino-terminal flag-tagged mu opioid receptor in neurons resulted in approximately 1.3x10(6) receptors/cell. Many mu opioid receptor cell lines express a similar density of receptors but this is approximately 7x greater than the number of endogenous receptors expressed by matched wild-type neurons. Inhibition of the high voltage-activated calcium currents in dorsal root ganglia neurons by the mu agonist, D-Ala(2), N-MePhe(4), Gly(5)-ol-enkephalin (DAMGO), was not different between the endogenous and flag-tagged receptor at several concentrations of DAMGO used. Both receptors desensitized equally over the first 6 h of DAMGO pre-incubation, but after 24 h the response of the endogenous receptor to DAMGO had desensitized further than the flag- tagged receptor (71+/-3 vs 29+/-7% respectively; P<0.002), indicating less desensitization in neurons expressing a higher density of receptor. Using flow cytometry to quantify the percentage of receptors remaining on the neuronal cell surface, the flag-tagged receptor internalized by 17+/-1% after 20 min and 55+/-2% after 24 h of DAMGO. These data indicate that this return of function model in neurons recapitulates many of the characteristics of endogenous mu opioid receptor function previously identified in non-neuronal cell lines.     

Mu opioid receptor-effector coupling and trafficking in dorsal root ganglia neurons

Morphine induces profound analgesic tolerance in vivo despite inducing little internalization of the mu opioid receptor (muOR). Previously proposed explanations suggest that this lack of internalization could either lead to prolonged signaling and associated compensatory changes in downstream signaling systems, or that the receptor is unable to recycle and resensitize and so loses efficacy, either mechanism resulting in tolerance. We therefore examined, in cultured neurons, the relationship between muOR internalization and desensitization in response to two agonists, D-Ala2, N-MePhe4, Gly5-ol-enkephalin (DAMGO) and morphine. In addition, we studied the chimeric mu/delta opioid receptor (mu/ partial differentialOR) which could affect internalization and desensitization in neurons. Dorsal root ganglia neurons from muOR knockout mice were transduced with an adenovirus expressing either receptor and their respective internalization, desensitization and trafficking profiles determined. Both receptors desensitized equally, measured by Ca2+ current inhibition, during the first 5 min of agonist exposure to DAMGO or morphine treatment, although the mu/partial differentialOR desensitized more extensively. Such rapid desensitization was unrelated to internalization as DAMGO, but not morphine, internalized both receptors after 20 min. In response to DAMGO the mu/partial differentialOR internalized more rapidly than the muOR and was trafficked through Rab4-positive endosomes and lysosomal-associated membrane protein-1-labeled lysosomes whereas the muOR was trafficked through Rab4 and Rab11-positive endosomes. Chronic desensitization of the Ca2+ current response, after 24 h of morphine or DAMGO incubation, was seen in the DAMGO, but not morphine-treated, muOR-expressing cells. Such persistence of signaling after chronic morphine treatment suggests that compensation of downstream signaling systems, rather than loss of efficacy due to poor receptor recycling, is a more likely mechanism of morphine tolerance in vivo. In contrast to the muOR, the mu/partial differentialOR showed equivalent desensitization whether morphine or DAMGO treated, but internalized further with DAMGO than morphine. Such ligand-independent desensitization could be a result of the observed higher rate of synthesis and degradation of this chimeric receptor.     

GABAB-mediated upregulation of the high-voltage-activated Ca2+ channels in rat dorsal root ganglia

 The mechanism underlying the enhancement of the high-voltage-activated (HVA) Ca²⁺ current (I _Ca) after application of baclofen, a GABA_B agonist, in neurones of the rat dorsal root ganglia was studied by a combined use of the nystatin perforated patch clamp recording and our rapid superfusion system. Baclofen (50 μM) decreased the peak amplitude of HVA I _Ca and slowed the onset of the current, i.e. produced a typical G-protein-mediated inhibition of I _Ca. However, when baclofen was rapidly removed from the medium, the amplitude of the current was rather augmented, exceeding the control value obtained before application of the drug. This enhancement was not due to a shift of the voltage dependence of Ca²⁺ channel activation or a change in ionic permeability to other ions. The enhancement of HVA I _Ca by baclofen was sensitive to pertussis toxin treatment. The enhancement was evident during superfusion of baclofen. Since the inhibitory effect of baclofen on HVA I _Ca was not attenuated, even after a continuous application of baclofen for 10 min, the enhancement was not due to relief from tonic G-protein-mediated inhibition of the current or a desensitization of the GABA_B receptor–effector system. An extremely prolonged time course of the enhancement of HVA I _Ca by baclofen strongly suggests an involvement of some intracellular signal transduction system. Received: 22 May 1996 / Received after revision: 17 October 1996 / Accepted: 7 January 1997     

Epidermal growth factor receptor in adult human dorsal root ganglia

Transforming growth factor- (TGF) enhances neuronal survival and neurite outgrowth in cultured dorsal root ganglia (DRG) sensory neurons. It binds a membrane protein, denominated epidermal growth factor receptor (EGFr). EGFr has been localized in developing and adult human DRG. However, it remains to be elucidated whether all DRG neurons express EGFr or whether differences exist among neuronal subtypes. This study was undertaken to investigate these topics in adult human DRG using immunoblotting, and combined immunohistochemistry and image analysis techniques. A mouse monoclonal antibody (clone F4) mapping within the intracytoplasmic domain of EGFr was used. Immunoblotting revealed two main proteins with estimated molecular masses of - 65 kDa and 170 kDa, and thus consistent with the full-length EGFr. Additional protein bands were also encountered. Light immunohistochemistry revealed specific immunoreactivity (IR) for EGFr-like proteins in most (86%) primary sensory neurons, the intensity of immunostaining being stronger in the small- and intermediate-sized ones. Furthermore, EGFr-like IR was also observed in the satellite glial cells of the ganglia as well as in the intraganglionic and dorsal root Schwann cells. Taken together, our findings demonstrate that EGFr, and other related proteins containing the epitope labeled with the antibody F4, are responsible for the EGFr IR reported in DRG. Furthermore, we demonstrated heterogeneity in the expression of EGFr-like IR in adult human primary sensory neurons, which suggests different responsiveness to their ligands.     

Responses of macrophages in rat dorsal root ganglia following peripheral nerve injury

Summary Immunohistochemical studies with monoclonal antibodies to macrophage antigens were performed on sections of rat lumbar dorsal root ganglia. In confirmation of previous observations, cells with macrophage antigenicity were detected in normal ganglia. Many of these presumptive macrophages were perineuronal in contact with the neuron/satellite cell complex, a few were perivascular, and others were in interstitial position not in apparent contact with either blood vessels or neurons. The number of macrophages in lumbar dorsal root ganglia started to increase 2–4 days after sciatic nerve transection and remained elevated for four weeks. Perineuronal macrophages resembled satellite glial cells in light microscope appearance but were distinguished from glial cells by their lack of S-100 immunoreactivity. Following this sciatic nerve injury, macrophage counts were modestly increased in contralateral lumbar dorsal root ganglia but not in cervical dorsal root ganglia. Thus peripheral nerve injury induces a recruitment and/or proliferation of macrophages in the corresponding dorsal root ganglion. Although the functions of these macrophages are unclear, those in perineuronal position could contribute to the survival or regeneration of axotomized neurons.     

Sprouting of sensory neurons in dorsal root ganglia after transection of peripheral nerves

Morphological reaction of sensory neurons of dorsal root ganglia after peripheral nerve transection was investigated by a nerve tracing method using E. coli lacZ (beta-galactosidase) gene recombinant adenovirus. The sciatic nerve of the rat was transected and inoculated with the gene recombinant adenovirus from the cutting end of nerve fibers. The fixation was accomplished from one to six weeks after inoculation. A whole mount specimen was observed after the reaction in a X-galactocidase substrate. Newly formed sprouting processes of dorsal root ganglion (DRG) cells appeared, all of them sprouting from the primary segment of DRG cells. Developed branches were morphologically categorized in to two types: one was the "linear type" which showed diverged branches running straightly along the major axis of the DRG; the other was the "winding type" which exhibited a random running pattern to the original axons and wound and extended in all directions in dorsal root ganglia with many branches. Many of this type encircled other cell bodies and formed a ring-like structure. There was no difference in the size of cell bodies in either type or between the ring-like structure forming the cells and those cells encircled by them.     

Diverse immunocytochemical expression of opioid receptors in electrophysiologically defined cells of rat dorsal root ganglia

The development of opiate analgesics that do not produce adverse side effects is hampered by the difficulty in developing drugs that are tissue/sensory cell-specific. Previously, our laboratory has demonstrated that small- and medium-diameter dorsal root ganglia (DRG) cells can be subclassified into at least nine distinct cell types based upon their patterns of voltage activated currents [Petruska, J.C., Napaporn, J., Johnson, R.D., Gu, J.G., Cooper, B.Y., 2000. Subclassified acutely dissociated cells of rat DRG: histochemistry and patterns of capsaicin-, proton-, and ATP-activated currents. J. Neurophysiol. 84 (5), 2365–2379; Petruska, J.C., Napaporn, J., Johnson, R.D., Cooper, B.Y., 2002. Chemical responsiveness and histochemical phenotype of electrophysiologically classified cells of the adult rat dorsal root ganglion. Neuroscience 115 (1), 15–30.] Based on their responses to algesic compounds and histochemical phenotype, eight of the nine subtypes are likely nociceptors. In the present study, we examined the immunoreactivity (IR) of delta-, kappa- and mu-opioid receptors (DOR, KOR and MOR, respectively), in 164 electrophysiologically subclassified DRG neurons.

The expression of opioid receptors in the DRG cell types was diverse. Type 1 (25–30 μm cell diameter) and type 9 (35–45 μm) expressed MOR-IR, but were negative for DOR-IR and KOR-IR. Type 2 (25–30 μm) co-expressed DOR-IR and MOR-IR, but did not express KOR-IR. Type 3 (15–20 μm), the non-nociceptive cell type, was not immunoreactive. Type 4 (35–45 μm), type 6 (35–45 μm), and type 7 (15–20 μm) expressed all three opioid receptors. Type 5 (35–45 μm) and type 8 (35–45 μm), co-expressed KOR-IR and MOR-IR, but did not express DOR-IR. The co-expression of opioid receptors in some of the cell types suggests that these sensory afferents might contain heteromeric opioid receptors. Additionally, the diverse expression patterns of opioid receptors between cell types and the consistency of these patterns maintained within each cell type provides further evidence of distinct functional properties of DRG nociceptors.     

Expression pattern of leucine-rich repeat neuronal protein 4 in adult mouse dorsal root ganglia

A member of leucine-rich repeat neuronal protein family, leucine-rich repeat neuronal protein 4 (Lrrn4), is a type I transmembrane protein. Previously, we have reported that Lrrn4 is expressed in various regions of the central nervous system (CNS) and involved in the memory retention. However, little is known about the role of Lrrn4 in the peripheral nervous system (PNS). Northern blot analysis revealed that Lrrn4 mRNA was expressed predominantly in the dorsal root ganglia (DRGs) with low levels in some regions of the CNS. To identify Lrrn4-expressing cells in the DRGs, we performed in situ hybridization histochemistry and LacZ staining in Lrrn4-heterozygous (Lrrn4^+/−) mice generated by the replacement of Lrrn4 gene with β-galactosidase gene. In the adult DRGs, 8% of total DRG neurons contained Lrrn4 mRNA, which was exclusively expressed in the small-sized neurons. LacZ staining combined with immunohistochemistry revealed that approximately 42% and 58% of Lrrn4-positive neurons contained receptor tyrosine kinase A (TrkA)- and Ret-immunoreactivity, respectively. After sciatic nerve axotomy, the expression of Lrrn4 mRNA was reduced in injured side of the DRGs. Thus, Lrrn4 is expressed in a subset of nociceptive neurons and might contribute to the maintenance of nociceptive circuits.     

Serotonin receptor mRNA expression in rat dorsal root ganglion neurons

In the present study, we have used in situ hybridization to examine the distribution of serotonin (5-HT) receptors in rat dorsal root ganglion (DRG) neurons. Within DRG neurons, mRNAs for 5-HT1B, 5-HT1D, 5-HT2A, 5-HT2B, 5-HT3B and 5-HT4 receptors were readily detected in small (<25 microm), medium (25-45 microm) and large (>45 microm) diameter neurons. In contrast mRNAs for 5-HT1A, 5-HT1E, 5-HT2C, 5-HT5A, 5-HT5B, 5-HT6 and 5-HT7 receptors were undetectable in these neurons. The present study provides an insight into the molecular profile of 5-HT receptor subtypes in neurons responsible for modulating sensory information.     

Influence of peripheral inflammation on growth-associated phosphoprotein (GAP-43) expression in dorsal root ganglia and on nerve recovery after crush injury

An experimental study was performed to investigate the influence of the inflammation in peripheral target tissue on growth-associated phosphoprotein (GAP-43) expression in dorsal root ganglia (DRG) and on recovery of crushed nerve. Fifty-four male Wistar rats were used for this study. The sciatic nerve was operatively crushed unilaterally with an aneurysm clip. Inflammation in peripheral target tissue was induced by injection of complete Freund's adjuvant (CFA) at 1 week before crush. In crushed or crushed with arthritis rats DRGs were examined in immunohistochemistry for GAP-43 and the sciatic nerves were observed in Epon embedded sections with toluidine blue stain. In addition, electrophysiological studies of the nerves were performed to evaluate the recovery of function. Immunohistochemical studies showed the ratio of GAP-43 immunopositive cells in crushed with arthritis rats was significantly lower than that in crushed rats at 1 week after crush (P<0.01). Electrophysiological studies at 4 weeks after crush showed functional nerve recovery in crushed with arthritis rats was significantly suppressed compared with that in crushed rats (P<0.01). Histological studies showed the mean diameter of the axons in crushed with arthritis rats was significantly smaller than that in crushed rats (P<0.01). All these findings indicate that inflammation in peripheral target tissue suppresses GAP-43 expression in DRG and eventually suppresses functional and morphological recovery of the crushed nerve.     

Down-regulation of mu opioid receptor expression within distinct subpopulations of dorsal root ganglion neurons in a murine model of bone cancer pain

Although micro opioid receptor (MOR) agonists are used for treatment of most types of pain, a recent study has suggested that the sensitivity of bone cancer pain to systemic morphine was lower than that of inflammatory pain. However, the reasons for this have remained unclear. In this study, MOR expression and the analgesic effects of morphine in a bone cancer model were compared with those in an inflammatory pain model. A bone cancer pain model and an inflammatory pain model were made by implantation of sarcoma cells into the intramedullary space of the femur and hind-paw injection of complete Freund's adjuvant (CFA), respectively. In a behavioral study, sarcoma-implanted mice showed flinching behavior of magnitude comparable to that induced by CFA injection. The flinching behavior of sarcoma-implanted mice was less sensitive to intrathecal morphine than that of CFA-injected mice. Western blot analysis showed that MOR expression in the dorsal root ganglion (DRG) ipsilateral to sarcoma implantation was significantly reduced, while that in the DRG ipsilateral to CFA injection was increased. In sarcoma-implanted mice, the percentage of MOR-positive DRG neuronal profiles was lower than that in control mice (30.3% vs. 45.2%). In particular, MOR expression was reduced among calcitonin gene-related peptide- and transient receptor potential vanilloid subfamily 1-positive DRG neuronal profiles, which are considered to be involved in the generation of bone cancer pain (from 61.5% to 41.5% and from 72.1% to 48.4%, respectively). These results suggest that down-regulation of MOR in the distinct populations of DRG neurons contributes to the fact that higher doses of morphine are needed to produce analgesia in bone cancer as compared with those used in non-malignant inflammatory situations.     

Neurotrophin-3 administration alters neurotrophin, neurotrophin receptor and nestin mRNA expression in rat dorsal root ganglia following axotomy

In the months following transection of adult rat peripheral nerve some sensory neurons undergo apoptosis. Two weeks after sciatic nerve transection some neurons in the L4 and L5 dorsal root ganglia begin to show immunoreactivity for nestin, a filament protein expressed by neuronal precursors and immature neurons, which is stimulated by neurotrophin-3 (NT-3) administration. The aim of this study was to examine whether NT-3 administration could be compensating for decreased production of neurotrophins or their receptors after axotomy, and to determine the effect on nestin synthesis. The levels of mRNA in the ipsilateral and contralateral L4 and L5 dorsal root ganglia were analyzed using real-time polymerase chain reaction, 1 day, 1, 2 and 4 weeks after unilateral sciatic nerve transection and NT-3 or vehicle administration via s.c. micro-osmotic pumps. In situ hybridization was used to identify which cells and neurons expressed mRNAs of interest, and the expression of full-length trkC and p75NTR protein was investigated using immunohistochemistry. Systemic NT-3 treatment increased the expression of brain-derived neurotrophic factor, nestin, trkA, trkB and trkC mRNA in ipsilateral ganglia compared with vehicle-treated animals. Some satellite cells surrounding neurons expressed trkA and trkC mRNA and trkC immunoreactivity. NT-3 administration did not affect neurotrophin mRNA levels in the contralateral ganglia, but decreased the expression of trkA mRNA and increased the expression of trkB mRNA and p75NTR mRNA and protein. These data suggest that systemically administered NT-3 may counteract the decrease, or even increase, neurotrophin responsiveness in both ipsi- and contralateral ganglia after nerve injury.     

Nicotinic receptor mediated stimulation of NO-generation in neurons of rat thoracic dorsal root ganglia

The contribution of nicotinic acetylcholine receptors (nAChRs) to stimulation of NO-production was investigated in isolated rat dorsal root ganglion (DRG) neurons utilizing an NO-sensitive fluorescent indicator 4,5-diaminofluorescein-diacetate (DAF-2DA) and appropriate channel blockers. RT-PCR and immunohistochemical analysis of NOS isoforms in cultured neurons revealed the expression of eNOS in the vast majority of neurons, nNOS in about 5-10%, and iNOS only exceptionally. Application of nicotine resulted in an abrupt increase in DAF-2T fluorescence in 65% of neuronal cell bodies that was fully sensitive to the general nAChR antagonist mecamylamine. Methyllycaconitine reduced the number of nicotine-sensitive neurons and the extent of NO-generation. Thus, alpha7- and/or alpha9/10-nAChRs are required for nicotine-induced NO-production in a subpopulation of DRG neurons, and appear to be partially involved in the remaining, larger subpopulation.     

Macrophage and lymphocyte invasion of dorsal root ganglia after peripheral nerve lesions in the rat

The distribution of major histocompatibility complex class II (MHC II)-positive non-neuronal cells and T-lymphocytes was examined immunohistochemically in dorsal root ganglia (DRGs) up to 12 weeks following transection of one sciatic or lumbar spinal nerve in adult rats. Unlike within the brain, MHC II immunopositive (+) and T-cells are normally present within DRGs. After nerve transection, MHC II+ cell density increased (by about four times after each lesion) in DRGs projecting into lesioned nerves. Subsequently the number declined after sciatic but not spinal nerve transection. MHC II+ cells did not contain glial markers, even when these were up-regulated after the lesions. Initially, MHC II+ cells lay outside the satellite glia but, by 11 weeks, they had moved through them to lie against the somata. T-cells invaded the lesioned DRGs earlier than the MHC II+ cells. They achieved greater numbers after spinal (30 x control) than after sciatic (12 x control) nerve transection. They also increased in undamaged ganglia adjacent to the spinal nerve injury. T-cell density progressively declined after spinal but not sciatic nerve transection. Both cell types appeared to invade the DRGs initially through blood vessels and the meninges, particularly near the subarachnoid angle. At later stages, occasional neurones had dense aggregations of T-cell receptor+ and MHC II+ cells associated with them.We conclude that the magnitude and time course of changes in MHC II expression and T-cell numbers in lesioned DRGs differ from the responses within motor nuclei after axotomy. The influx of inflammatory cells may contribute to neurone survival in the short term. Their long-term presence has implications for patients. These cells have the potential to release excitatory cytokines that may generate ectopic impulse activity in sensory neurones after nerve injury and so may play a role in the generation of chronic neuropathic pain.     

Mechanical and thermal hyperalgesia and ectopic neuronal discharge after chronic compression of dorsal root ganglia

Chronic compression of the dorsal root ganglion (CCD) was produced in adult rats by implanting a stainless steel rod unilaterally into the intervertebral foramen, one rod at L(4) and another at L(5). Two additional groups of rats received either a sham surgery or an acute injury consisting of a transient compression of the ganglion. Withdrawal of the hindpaw was used as evidence of a nocifensive response to mechanical and thermal stimulation of the plantar surface. In addition, extracellular electrophysiological recordings of spontaneous discharges were obtained from dorsal root fibers of formerly compressed ganglia using an in vitro nerve-DRG-dorsal root preparation. The mean threshold force of punctate indentation and the mean threshold temperature of heating required to elicit a 50% incidence of foot withdrawal ipsilateral to the CCD were significantly lower than preoperative values throughout the 35 days of postoperative testing. The number of foot withdrawals ipsilateral to the CCD during a 20-min contact with a temperature-controlled floor was significantly increased over preoperative values throughout postoperative testing when the floor was 4 degrees C (hyperalgesia) and, to a lesser extent, when it was 30 degrees C (spontaneous pain). Stroking the foot with a cotton wisp never elicited a reflex withdrawal before surgery but did so in most rats tested ipsilateral to the CCD during the first 2 postoperative weeks. In contrast, the CCD produced no changes in responses to mechanical or thermal stimuli on the contralateral foot. The sham operation and acute injury produced no change in behavior other than slight, mechanical hyperalgesia for approximately 1 day, ipsilateral to the acute injury. Ectopic spontaneous discharges generated within the chronically compressed ganglion and, occurring in the absence of blood-borne chemicals and without an intact sympathetic nervous system, were recorded from neurons with intact, conducting, myelinated or unmyelinated peripheral nerve fibers. The incidence of spontaneously active myelinated fibers was 8.61% for CCD rats versus 0.96% for previously nonsurgical rats. We hypothesize that a chronic compression of the dorsal root ganglion after certain injuries or diseases of the spine may produce, in neurons with intact axons, abnormal ectopic discharges that originate from the ganglion and potentially contribute to low back pain, sciatica, hyperalgesia, and tactile allodynia.     

Noradrenergic receptor mRNA expression in adult rat superficial dorsal horn and dorsal root ganglion neurons

Noradrenaline (NAdr) has well documented analgesic actions at the level of the spinal cord. Released from bulbospinal projections onto superficial dorsal horn (SDH) neurons, NAdr modulates the excitability of these neurons through the activation of ₁, ₂ or β adrenoceptors. This study utilised in situ hybridisation to determine the specific expression of adrenoceptors within adult rat lumbar SDH and dorsal root ganglion (DRG) neurons, and reports the presence of _1A, _1B, _2B, β₁ and β₂ adrenoceptor mRNA within SDH neurons, and the presence of _1A, _1B and _2C adrenoceptor mRNA within DRG neurons. The present study provides an insight into the modulation of sensory processing at the level of the spinal cord following adrenoceptor activation.     

Stimulatory effect of stevioside on peripheral mu opioid receptors in animals

Stevioside is a dietary supplement widely used as a sweetener to prevent hyperglycemic disorders. However, the action mechanisms of this substance for glucose homeostasis remain obscure. In the present study, a dose-related plasma glucose reduction was observed in Wistar rats receiving intraperitoneally injections of stevioside. Similar to the regulation of glucose metabolism by the activation of mu opioid receptors, this action of stevioside was reversed by naloxonazine under the blockade of mu opioid receptors. We also found that stevioside increased glycogen synthesis in isolated hepatocytes, which was concentration-dependently blocked by naloxonazine. Stevioside did not modify the plasma beta-endorphin levels in Wistar rats but it directly increased the phosphorylation of mu opioid receptors in Chinese hamster ovary cells transfected with mu opioid receptors. Unlike morphine, chronic administration of stevioside did not induce the withdrawal signs in mice. Furthermore, stevioside by intraperitoneal injections did not influence the feeding behaviors of rats. By contrast, intracerebroventricular injections of stevioside increased the rats’ food intake, which was also inhibited by pretreatment with naloxonazine. These results showed that it is difficult for stevioside to enter the brain. Stevioside has the ability to activate peripheral mu opioid receptors for lowering plasma glucose and to increase glycogen synthesis in liver. Thus, the stimulation of peripheral mu opioid receptors is responsible for the action of stevioside in the regulation of glucose homeostasis.