Sumita Chakrabarti

Expression of the μ‐Opioid Receptor in CHO Cells: Ability of μ‐Opioid Ligands to Promote α‐Azidoanilido[32P]GTP Labeling of Multiple G Protein α Subunits

Abstract: The identities of heterotrimeric G proteins that can interact with the μ‐opioid receptor were investigated by α‐azidoanilido[32P]GTP labeling of α subunits in the presence of opioid agonists in Chinese hamster ovary (CHO)‐MORIVA3 cells, a CHO clone that stably expressed μ‐opioid receptor cDNA (MOR‐1). This clone expressed 1.01 × 106μ‐opioid receptors per cell and had higher binding affinity and potency to inhibit adenylyl cyclase for the μ‐opioid‐selective ligands [d‐Ala2,N‐MePhe4,Gly‐ol]‐enkephalin and [N‐MePhe3,d‐Pro4]‐morphiceptin, relative to the δ‐selective opioid agonist [d‐Pen2,d‐Pen5]‐enkephalin or the κ‐selective opioid agonist U‐50,488H. μ‐Opioid ligands induced an increase in α‐azidoanilido[32P]GTP photoaffinity labeling of four Gα subunits in this clone, three of which were identified as Gi3α, Gi2α, and Go2α. The same pattern of simultaneous interaction of the μ‐opioid receptor with multiple Gα subunits was also observed in two other clones, one expressing about three times more and the other 10‐fold fewer receptors as those expressed in CHO‐MORIVA3 cells. The opioid‐induced increase of labeling of these G proteins was agonist specific, concentration dependent, and blocked by naloxone and by pretreatment of these cells with pertussis toxin. A greater agonist‐induced increase of α‐azidoanilido[32P]GTP incorporation into Gi2α (160–280%) and Go2α (110–220%) than for an unknown Gα (G?α) (60%) or Gi3α (40%) was produced by three different μ‐opioid ligands tested. In addition, slight differences were also found between the ability of various μ‐opioid agonists to produce half‐maximal labeling (ED50) of any given Gα subunit, with a rank order of Gi3α > Go2α > Gi2α = G?α. In any case, these results suggest that the activated μ‐opioid receptor couples to four distinct G protein α subunits simultaneously.

Formation of μ-/κ-opioid receptor heterodimer is sex-dependent and mediates female-specific opioid analgesia

Sexually dimorphic nociception and opioid antinociception is very pervasive but poorly understood. We had demonstrated that spinal morphine antinociception in females, but not males, requires the concomitant activation of spinal μ- and κ-opioid receptors (MOR and KOR, respectively). This finding suggests an interrelationship between MOR and KOR in females that is not manifest in males. Here, we show that expression of a MOR/KOR heterodimer is vastly more prevalent in the spinal cord of proestrous vs. diestrous females and vs. males. Cross-linking experiments in combination with in vivo pharmacological analyses indicate that heterodimeric MOR/KOR utilizes spinal dynorphin 1–17 as a substrate and is likely to be the molecular transducer for the female-specific KOR component of spinal morphine antinociception. The activation of KOR within the heterodimeric MOR/KOR provides a mechanism for recruiting spinal KOR-mediated antinociception without activating the concomitant pronociceptive functions that monomeric KOR also subserves. Spinal cord MOR/KOR heterodimers represent a unique pharmacological target for female-specific pain control.

Distribution of neuropeptide receptors: New views of peptidergic neurotransmission made possible by antibodies to opioid receptors

The cloning of receptors for neuropeptides made possible studies that identified the neurons that utilize these receptors. In situ hybridization can detect transcripts that encode receptors and thereby identify the cells responsible for their expression, whereas immunocytochemistry enables one to determine the region of the plasma membrane where the receptor is located. We produced antibodies to portions of the predicted amino acid sequences of delta, mu, and kappa opioid receptors and used them in combination with antibodies to a variety of neurotransmitters in multicolor immunofluorescence studies visualized by confocal microscopy. Several findings are notable: First, the cloned delta opioid receptor appears to be distributed primarily in axons, and therefore most likely functions in a presynaptic manner. Second, the cloned mu and kappa opioid receptors are found associated with neuronal plasma membranes of dendrites and cell bodies and therefore most likely function in a postsynaptic manner. However, in certain, discrete populations of neurons, mu and kappa opioid receptors appear to be distributed in axons. Third, enkephalin-containing terminals are often found in close proximity (although not necessarily synaptically linked) to membranes containing either the delta or mu opioid receptors, whereas dynorphin-containing terminals are often found in proximity to kappa opioid receptors. Finally, a substantial mismatch between opioid receptors and their endogenous ligands was observed in some brain regions. However, this mismatch was characterized by complementary zones of receptor and ligand, suggesting underlying principles of organization that underlie long-distance, nonsynaptic neurotransmission.

Distinct Differences Between Morphine- and [d-Ala2,N-MePhe4,Gly-ol5]-Enkephalin- μ-Opioid Receptor Complexes Demonstrated by Cyclic AMP-Dependent Protein Kinase Phosphorylation

Abstract: The present study demonstrates a conditional, agonist‐dependent phosphorylation of the μ‐opioid receptor (MOR‐1) by cyclic AMP‐dependent protein kinase (PKA) in membrane preparations of MOR‐1‐transfected neuroblastoma Neuro2A cells. Opioid agonist‐dependent phosphorylation occurs in a time‐ and concentration‐dependent manner (EC50∼40 nM) and can be abolished by the receptor antagonist naloxone. Stoichiometric analysis indicates incorporation of a maximum of 6 mol of phosphate/mol of receptor in the presence of 1 µM morphine and 6 nM PKA. Although morphine and related alkaloids as well as some peptide agonists (PLO17 and β‐endorphin) stimulated phosphorylation of MOR‐1 by PKA, the potent μ‐opioid‐selective peptide [d‐Ala2,N‐MePhe4,Gly‐ol5]‐enkephalin (DAMGO) or other enkephalin analogues such as [d‐Ala2]‐Met5‐enkephalinamide (DALA), [d‐Ala2,d‐Leu5]‐enkephalin (DADLE), and Met5‐enkephalin had no effect. The lack of the effect of DAMGO on MOR‐1 phosphorylation state was evident also after chronic pretreatment. These results suggest the existence of different agonist‐dependent conformations of MOR‐1. Furthermore, phosphorylation may be a useful parameter with which to identify different agonist‐receptor conformations.

Chronic morphine induces the concomitant phosphorylation and altered association of multiple signaling proteins: A novel mechanism for modulating cell signaling

Traditional mechanisms thought to underlie opioid tolerance include receptor phosphorylation/down-regulation, G-protein uncoupling, and adenylyl cyclase superactivation. A parallel line of investigation also indicates that opioid tolerance development results from a switch from predominantly opioid receptor Giα inhibitory to Gβγ stimulatory signaling. As described previously, this results, in part, from the increased relative abundance of Gβγ-stimulated adenylyl cyclase isoforms as well as from a profound increase in their phosphorylation [Chakrabarti, S., Rivera, M., Yan, S.-Z., Tang, W.-J. & Gintzler, A. R. (1998) Mol. Pharmacol. 54, 655–662; Chakrabarti, S., Wang, L., Tang, W.-J. & Gintzler, A. R. (1998) Mol. Pharmacol. 54, 949–953]. The present study demonstrates that chronic morphine administration results in the concomitant phosphorylation of three key signaling proteins, G protein receptor kinase (GRK) 2/3, β-arrestin, and Gβ, in the guinea pig longitudinal muscle myenteric plexus tissue. Augmented phosphorylation of all three proteins is evident in immunoprecipitate obtained by using either anti-GRK2/3 or Gβ antibodies, but the phosphorylation increment is greater in immunoprecipitate obtained with Gβ antibodies. Analyses of coimmunoprecipitated proteins indicate that phosphorylation of GRK2/3, β-arrestin, and Gβ has varying consequences on their ability to associate. As a result, increased availability of and signaling via Gβγ could occur without compromising the membrane content (and presumably activity) of GRK2/3. Induction of the concomitant phosphorylation of multiple proteins in a multimolecular complex with attendant modulation of their association represents a novel mechanism for increasing Gβγ signaling and opioid tolerance formation.

Spinal Synthesis of Estrogen and Concomitant Signaling by Membrane Estrogen Receptors Regulate Spinal κ- and μ-Opioid Receptor Heterodimerization and Female-Specific Spinal Morphine Antinociception

We previously demonstrated that the spinal cord κ-opioid receptor (KOR) and μ-opioid receptor (MOR) form heterodimers (KOR/MOR). KOR/MOR formation and the associated KOR dependency of spinal morphine antinociception are most robust during proestrus. Using Sprague Dawley rats, we now demonstrate that (1) spinal synthesis of estrogen is critical to these processes, and (2) blockade of either estrogen receptor (ER) α-, β-, or G-protein-coupled ER1 or progesterone receptor (PR) substantially reduces KOR/MOR and eliminates mediation by KOR of spinal morphine antinociception. Effects of blocking ERs were manifest within 15 min, whereas those of PR blockade were manifest after 18 h, indicating the requirement for rapid signaling by estrogen and transcriptional effects of progesterone. Individual or combined blockade of ERs produced the same magnitude of effect, suggesting that they work in tandem as part of a macromolecular complex to regulate KOR/MOR formation. Consistent with this inference, we found that KOR and MOR were coexpressed with ERα and G-protein-coupled ER1 in the spinal dorsal horn. Reduction of KOR/MOR by ER or PR blockade or spinal aromatase inhibition shifts spinal morphine antinociception from KOR dependent to KOR independent. This indicates a sex steroid-dependent plasticity of spinal KOR functionality, which could explain the greater analgesic potency of KOR agonists in women versus men. We suggest that KOR/MOR is a molecular switch that shifts the function of KOR and thereby endogenous dynorphin from pronociceptive to antinociceptive. KOR/MOR could thus serve as a novel molecular target for pain management in women.

Mobilization of Ca+ from Intracellular Stores in transfected Neuro2a cells by activation of multiple opioid receptor subtypes

In neuronal cell lines, activation of opioid receptors has been shown to mobilize intracellular Ca2+ stores. In this report, we describe the excitatory actions of opioid agonists on murine neuroblastoma neuro2a cells stably expressing either delta, mu, or kappa opioid receptors. Fura-2-based digital imaging was used to record opioid-induced increases in intracellular Ca2+ concentration ([Ca2+]i). Repeated challenges of delta, mu, or kappa opioid receptor expressing cells with 100 nM [D-Ala2,D-Leu5]-enkephalin (DADLE), [D-Ala2,N-Me-Phe4,Gly-ol]-enkephalin (DAMGO), or trans-(+/-)-3,4-dichloro N-methyl-N-(2-[1-pyrollidinyl] cyclohexyl) benzene acetamide (U-50488H), respectively, elicited reproducible Ca2+ responses. Non-transfected neuro2a cells did not respond to opioid agonists. Removal of extracellular Ca2+ from the bath prior to and during agonist challenge did not affect significantly the agonist-evoked increase in [Ca2+]i, indicating that the response resulted from the release of Ca2+ from intracellular stores. Naloxone reversibly inhibited responses in all three cell lines, confirming that they were mediated by opioid receptors. Expression of cloned opioid receptors in neuro2a cells, coupled with digital [Ca2+]i imaging, provides a model system for the study of opioid receptors and opioid-activated signaling processes. The fact that all three receptors coupled to the same intracellular signaling mechanism suggests that the primary functional difference between opioid responses in vivo results from their selective localization.

Phosphorylation of Gαs Influences Its Association with the μ-Opioid Receptor and Is Modulated by Long-Term Morphine Exposure

The recent biochemical demonstration of the association of the μ-opioid receptor (MOR) with Gαs that increases after long-term morphine treatment (Mol Brain Res 135:217–224, 2005) provides a new imperative for studying MOR-Gαs interactions and the mechanisms that modulate it. A persisting challenge is to elucidate those neurochemical parameters modulated by long-term morphine treatment that facilitate MOR-Gαs association. This study demonstrates that 1) Gαs exists as a phosphoprotein, 2) the stoichiometry of Gαs phosphorylation decreases after long-term morphine treatment, and 3) in vitro dephosphorylation of Gαs increases its association with MOR. Furthermore, our data suggest that increased association of Gαs with protein phosphatase 2A is functionally linked to the long-term morphine treatment-induced reduction in Gαs phosphorylation. These findings are observed in MOR-Chinese hamster ovary and F11 cells as well as spinal cord, indicating that they are not idiosyncratic to the particular cell line used or a “culture” phenomenon and generalize to complex neural tissue. Taken together, these results indicate that the phosphorylation state of Gαs is a critical determinant of its interaction with MOR. Long-term morphine treatment decreases Gαs phosphorylation, which is a key mechanism underlying the previously demonstrated increased association of MOR and Gαs in opioid tolerant tissue.

Reciprocal modulation of phospholipase Cβ isoforms: Adaptation to chronic morphine

Phosphoinositide turnover and calcium mobilization are fundamental determinants of acute and chronic opioid effects. Phosphoinositide-specific phospholipase C (PLC) are key signaling enzymes that play a pivotal role in mediating opioid modulation of inositol trisphosphate production and cytosolic calcium distribution, substrates for many acute and chronic opioid effects. Notably, phosphorylation of the β isoforms of PLC, by kinases that are up-regulated after chronic morphine, is a potent modality for their regulation. Direct assessment of PLCβ1 and PLCβ3 phosphorylation in the guinea pig longitudinal muscle myenteric plexus tissue revealed substantial alterations after the induction of opioid tolerance. Notably, the direction of this modulation is isoform-specific. Phosphorylation of PLCβ1 is significantly reduced, whereas that of PLCβ3 is substantially augmented, changes not accompanied by altered content of PLCβ1 or PLCβ3 protein. In contrast to chronic morphine, acute morphine treatment of opioid naïve longitudinal muscle myenteric plexus tissue attenuates PLCβ3 phosphorylation, an effect also manifested by endogenous opioids that is reflected by the ability of acute naloxone to substantially augment PLCβ3 phosphorylation. This indicates that PLCβ phosphorylation is dynamically regulated. PLCβ1 and PLCβ3 activities are negatively modulated by phosphorylation. Thus, their concomitant reciprocal phosphorylation would alter the relative contribution of these isoforms to PLC/Ca2+ signaling, a significant shift in light of their differential regulatory characteristics. Reciprocal modulation of the phosphorylation (activity) of two isoforms within the same subclass of signaling enzyme, proteins that have a high degree of structural similarity and subserve the same biological function, represents an adaptation modality to chronic morphine that has heretofore not been recognized.

Sub-Cellular localization of mu-opioid receptor Gs signaling

In membranes obtained from μ-opioid receptor (MOR) expressing Chinese hamster ovary (CHO) cells (MOR-CHO), the MOR-selective agonist sufentanil produced a concentration-dependent stimulation of guanosine 5′-O-(3-[35S]thio)triphosphate binding to Gsα that was abolished by blocking MOR with naloxone. This unequivocally demonstrates the long-debated functionality of the previously described association of MOR with Gsα. Several complementary observations indicate the relevance of caveolae to MOR-coupled Gsα signaling. 1) In MOR-CHO membranes, sufentanil stimulated the translocation of Gsα into Triton-insoluble membrane compartments. 2) Sufentanil enhanced the coimmunoprecipitation (co-IP) of Gsα and adenylyl cyclase (AC) with caveolin-1 (a marker for caveolae) from the Triton-insoluble membrane fraction of spinal cord and MOR-CHO. 3) MOR blockade (via naloxone) or Gs inactivation (via cholera toxin) abolished both the increased trafficking of Gsα into the Triton-insoluble membrane fraction of MOR-CHO and the augmented co-IP from spinal cord membranes of Gsα and AC with caveolin-1. This indicates that these events occurred subsequent to activation of MOR and Gsα. Strikingly, lesser-phosphorylated Gsα, which preferentially couple to MOR (Mol Brain Res 135:217–224, 2005; Mol Pharmacol 72:753–760, 2007; Mol Pharmacol 73:868–879, 2008), are concentrated in caveolae, underscoring their relevance to MOR Gsα signaling. MOR-stimulated trafficking of Gsα and AC into caveolae and the likelihood of increased MOR Gsα coupling within caveolae could suggest that they contain the downstream effectors for MOR Gsα AC signaling.