Relevance of Mu-Opioid Receptor Splice Variants and Plasticity of Their Signaling Sequelae to Opioid Analgesic Tolerance.

Opioid dose escalation to effectively control pain is often linked to the current prescription opioid abuse epidemic. This creates social as well as medical imperatives to better understand the mechanistic underpinnings of opioid tolerance to develop interventions that minimize it, thereby maximizing the analgesic effectiveness of opioids. Profound opioid analgesic tolerance can be observed in the absence of mu-opioid receptor (MOR) downregulation, aggregate MOR G protein uncoupling, and MOR desensitization, in the absence of impaired G protein coupled receptor kinase phosphorylation, arrestin binding, or endocytosis. Thus, we have explored alternative biochemical sequelae that might better account for opioid analgesic tolerance. Our findings indicate that substantial plasticity among upstream and downstream components of opioid receptor signaling and the emergence of alternative signaling pathways are major contributors to opioid analgesic tolerance. An exemplar of this plasticity is our findings that chronic morphine upregulates the MOR variants MOR-1B2 and MOR-1C1 and phosphorylation of their C-terminal sites not present in MOR-1, events causally associated with the chronic morphine-induced shift in MOR G protein coupling from predominantly Gi/Go inhibitory to Gs-stimulatory adenylyl cyclase signaling. The unique feature(s) of these variants that underlies their susceptibility to adapting to chronic morphine by altering the nature of their G protein coupling reveals the richness and pliability of MOR signaling that is enabled by generating a wide diversity of MOR variants. Furthermore, given differential anatomical expression patterns of MOR variants, MOR splice variant-dependent adaptations to chronic morphine could enable mechanistic underpinnings of tolerance and dependence that are CNS region- and cell-specific.

Phosphorylation of unique C-terminal sites of the mu-opioid receptor variants 1B2 and 1C1 influences their Gs association following chronic morphine.

We recently demonstrated in rat spinal cord that a regimen of escalating doses of systemic morphine, analogous to regimens used clinically for chronic pain management, selectively up-regulates the mu-opioid receptor (MOR) splice variants MOR-1B2 and MOR-1C1 mRNA and functional protein. This study investigated the potential relevance of up-regulating MOR-1B2 and MOR-1C1 to the ability of chronic morphine to shift MOR signaling from predominantly Gi /Go inhibitory to Gs stimulatory. Specifically, we tested the hypotheses that chronic morphine induces phosphorylation of carboxyl terminal sites unique to MOR-1B2 and MOR-1C1, and that this phosphorylation is causally related to augmented association of these variants with Gs α. Hypotheses were validated by (i) abolition of the chronic morphine-induced increment in MOR-1C1 and MOR-1B2 association with Gs α by inhibitors of protein kinase A and Casein kinase 2, respectively; (ii) failure of chronic morphine to augment MOR variant Gs α interactions in Chinese hamster ovary cells transiently transfected with either rat MOR-1C1 or MOR-1B2 in which targeted protein kinase A and Casein kinase 2 serine phosphorylation sites, respectively, were mutated to alanine; (iii) abrogation of chronic morphine-induced augmented MOR Gs α association in spinal cord of male rats following intrathecal administration of dicer substrate small interfering RNAs targeting MOR-1B2/MOR-1C1 mRNA. The ability of chronic morphine to not only up-regulate-specific MOR variants but also their carboxyl terminal phosphorylation and consequent augmented association with Gs α may represent a novel component of opioid tolerance mechanisms, suggesting novel potential targets for tolerance abatement.

Chronic opioid treatment augments caveolin-1 scaffolding: relevance to stimulatory µ-opioid receptor adenylyl cyclase signaling.

Caveolin-1 is the predominant structural protein of caveolae, a subset of (lipid) membrane rafts that compartmentalize cell signaling. Caveolin-1 binds most to G protein-coupled receptors and their signaling partners, thereby enhancing interactions among signaling cascade components and the relative activation of specific G protein-coupled pathways. This study reveals that chronic opioid exposure of µ-opioid receptor (MOR) expressing Chinese hamster ovary cells (MOR-CHO) and chronic in vivo morphine exposure of rat spinal cord augmented recruitment of multiple components of MOR-adenylyl cyclase (AC) stimulatory signaling by caveolin-1. Strikingly, in MOR-CHO and spinal cord, blocking the caveolin-1 scaffolding domain substantially attenuated the chronic morphine-induced increased interaction of caveolin-1 with MOR, Gsα, protein phosphatase 2A (PP2A), and AC. Chronic morphine treatment also increased interactions among the above signaling proteins, thus enabling sufentanil to stimulate (rather than inhibit) cAMP production within lipid membrane microdomains. The latter finding underscores the functionality of augmented interactions among MOR, Gs α, PP2A, and AC. In the aggregate, our data strongly suggest that augmented caveolin-1 scaffolding undergirds the ability of chronic opioids to recruit an ancillary signaling pathway by acting as an organizing template for MOR-Gs α-AC signaling and delimiting the membrane compartment(s) in which it occurs. Since caveolin-1 binds to a wide spectrum of signaling molecules, altered caveolin-1 scaffolding following chronic opioid treatment is likely to pertain to most, if not all, MOR signaling partners. The chronic morphine-induced trigger that augments caveolin-1 scaffolding could represent a seminal perturbation that initiates the wide spectrum of adaptations thought to contribute to opioid tolerance and dependence.

Selective up-regulation of functional mu-opioid receptor splice variants by chronic opioids.

We recently reported (Verzillo, et al. J. Neurochem: 130, 790-796, 2014) that chronic systemic morphine selectively up-regulates mRNA encoding two C-terminal µ-opioid receptor (MOR) splice variants, MOR-1C1 and MOR-1B2 (MOR-1B2/-1C1). Given the known disconnects between changes in levels of mRNA and corresponding protein, it is essential to directly demonstrate that chronic opioid treatment elevates functional MOR-1B2/-1C1 protein prior to inferring relevance of these MOR variants to spinal opioid tolerance mechanisms. Accordingly, we investigated the ability of chronic opioid exposure to up-regulate MOR protein in Chinese hamster ovary cells stably transfected with rat MOR variants MOR-1B2, MOR-1C1, or MOR-1 (considered to be the predominant MOR). Findings revealed that chronic treatment with the clinically relevant opioids morphine, oxycodone and hydrocodone substantially up-regulated membrane MOR-1B2/-1C1 protein. This up-regulation was abolished by the protein synthesis inhibitor cycloheximide, eliminating contributions from receptor redistribution. The increment in MOR-1B2/-1C1 protein was paralleled by a significant increment in opioid agonist-stimulated GTPγS-binding (reflective of increased aggregate MOR G protein coupling) indicating that up-regulated MOR-1B2/-1C1 represented functional receptors. Strikingly, these tolerance-associated adaptations of MOR-1B2/-1C1 differed considerably from those of MOR-1. Antithetical regulation of MOR-1B2/-1C1 and MOR-1 by chronic opioids has significant implications for the design of new therapeutic agents to counteract opioid analgesic tolerance and accompanying addiction. Since chronic opioids induce MOR-1B2/-1C1 up-regulation in spinal cord of males, but not females, elucidating cellular compartments and intracellular pathways mediating MOR-1B2/-1C1 up-regulation and defining their unique signaling attributes would enable a precision medicinal approach to pain management and addiction therapy. In the spinal cord of males, but not females, chronic morphine up-regulates mRNA encoding two mu-opioid receptor (MOR) variants, MOR-1B2 and MOR-1C1 (MOR-1B2/-1C1). We now demonstrate that chronic treatment with the clinically relevant opioids morphine, hydrocodone or oxycodone up-regulates MOR-1B2/-1C1 functional protein, which is dependent on de novo protein synthesis. Findings underscore the importance of unique signaling attributes of MOR variants to sexually dimorphic tolerance mechanisms.

Mu-opioid receptor splice variants: sex-dependent regulation by chronic morphine.

The gene encoding the mu-opioid receptor (MOR) generates a remarkable diversity of subtypes, the functional significance of which remains largely unknown. The structure of MOR could be a critical determinant of MOR functionality and its adaptations to chronic morphine exposure. As MOR antinociception has sexually dimorphic dimensions, we determined the influence of sex, stage of estrus cycle, and chronic systemic morphine on levels of MOR splice variant mRNA in rat spinal cord. Chronic systemic morphine influenced the spinal expression of mRNA encoding rMOR-1B2 and rMOR-1C1 in a profoundly sex-dependent fashion. In males, chronic morphine resulted in a twofold increase in expression levels of rMOR-1B2 and rMOR-1C1 mRNA. This effect of chronic morphine was completely absent in females. Increased density of MOR protein in spinal cord of males accompanied the chronic morphine-induced increase in MOR variant mRNA, suggesting that it reflected an increase in corresponding receptor protein. These results suggest that tolerance/dependence results, at least in part, from different adaptational strategies in males and females. The signaling consequences of the unique composition of the C-terminus tip of rMOR-1C1 and rMOR-1B2 could point the way to defining the molecular components of sex-dependent tolerance and withdrawal mechanisms. Chronic systemic morphine increases levels of mRNA encoding two splice variants of mu-opioid receptor (MOR), MOR-1B2 and MOR-1C1, variants differing from rMOR-1 in their C-terminal (and phosphorylation sites therein) and thus possibly signaling sequelae. This adaptation is sex-specific. It occurs in the spinal cord of males, but not females, indicating the importance of sex-specific mechanisms for and treatments of tolerance and addiction.

Pleiotropic opioid regulation of spinal endomorphin 2 release and its adaptations to opioid withdrawal are sexually dimorphic.

We studied adaptations to acute precipitated opioid withdrawal of spinal µ-opioid receptor (MOR)-coupled regulation of the release of endomorphin 2 (EM2). The release of this highly MOR-selective endogenous opioid from opioid-naive spinal tissue of male rats is subjected to MOR-coupled positive as well as negative modulation via cholera toxin-sensitive G(s) and pertussis toxin-sensitive G(i)/G(o), respectively. The net effect of this concomitant bidirectional modulation is inhibitory. MOR-coupled pleiotropic regulation of EM2 release is retained in opioid-withdrawn spinal tissue of male rats, but the balance of MOR-coupled inhibitory and facilitatory regulation shifted such that facilitatory regulation predominates. Augmented coupling of MOR to G(s) is causally associated with this change. Strikingly, pleiotropic characteristics of MOR-coupled regulation of spinal EM2 release and adaptations thereof to opioid withdrawal are male-specific. In females, MOR-coupled regulation of EM2 release from opioid-naive and -withdrawn spinal tissue does not have a significant G(s)-coupled facilitatory component, and MOR-coupled inhibition of EM2 release persists unabated in withdrawn preparations. The male-specific adaptations to chronic morphine that shift the relative predominance of opposing dual G protein-coupled MOR pathways provides a mechanism for mitigating inhibitory MOR signaling without losing MOR-coupled feedback regulation. These adaptations enable using endogenous EM2 as a substitute for morphine that had been precipitously removed. The sexually dimorphic functionality and regulation of spinal EM2/MOR-coupled signaling suggest the clinical utility of using sex-specific treatments for addiction that harness the activity of endogenous opioids.

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-, ß-, 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.

Formation of mu-/kappa-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.

Subcellular localization of mu-opioid receptor G(s) signaling.

In membranes obtained from mu-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 G(s)alpha that was abolished by blocking MOR with naloxone. This unequivocally demonstrates the long-debated functionality of the previously described association of MOR with G(s)alpha. Several complementary observations indicate the relevance of caveolae to MOR-coupled G(s)alpha signaling. 1) In MOR-CHO membranes, sufentanil stimulated the translocation of G(s)alpha into Triton-insoluble membrane compartments. 2) Sufentanil enhanced the coimmunoprecipitation (co-IP) of G(s)alpha 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 G(s) inactivation (via cholera toxin) abolished both the increased trafficking of G(s)alpha into the Triton-insoluble membrane fraction of MOR-CHO and the augmented co-IP from spinal cord membranes of G(s)alpha and AC with caveolin-1. This indicates that these events occurred subsequent to activation of MOR and G(s)alpha. Strikingly, lesser-phosphorylated G(s)alpha, 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 G(s)alpha signaling. MOR-stimulated trafficking of G(s)alpha and AC into caveolae and the likelihood of increased MOR G(s)alpha coupling within caveolae could suggest that they contain the downstream effectors for MOR G(s)alpha AC signaling.

The ambiguities of opioid tolerance mechanisms: barriers to pain therapeutics or new pain therapeutic possibilities.

Identification of adaptations to chronic morphine that are causally associated with opioid tolerance formation has long been intensely pursued by the opioid research community. There is an impressive array of components of signaling pathways that are influenced by chronic opioid administration. This underscores the importance to tolerance mechanisms of the complex interplay of cellular adaptations that are downstream from the opioid receptor. A major impetus for this research remains the need to develop opioid agonists that are potent and efficacious activators of analgesic mechanisms without triggering opioid tolerance-producing adaptations. Implicit in most models of opioid tolerance is that their underlying mechanisms are invariant and independent of the system in which they have been observed. Reports that prior acute morphine treatment and pain could influence tolerance mechanisms were not understood on mechanistic levels and, consequently, were not incorporated into commonly used models of opioid tolerance. The recent demonstration that adenylyl cyclase/cAMP-related cellular adaptations to chronic morphine depend on cell state demonstrates that ongoing cell physiology is a critical determinant of tolerance mechanisms. The plasticity and pliability of cellular adaptations that mediate tolerance formation indicate that mechanisms underlying opioid analgesic tolerance could be a moving target. Although this might represent a daunting barrier to developing antitolerance pharmacotherapies, appreciation of this complexity could lead to the development of new pharmacotherapeutic approaches.

Plasticity of adenylyl cyclase-related signaling sequelae after long-term morphine treatment.

Adaptations to long-term morphine treatment resulting in tolerance are protective by counteracting the consequences of sustained opioid receptor activation. Consequently, the manifestation of specific adenylyl cyclase (AC)-related neurochemical sequelae of long-term morphine treatment should depend on the consequences of short-term mu-opioid receptor (MOR) activation. We tested this by comparing complementary chemical sequelae of long-term morphine treatment among cells in which short-term MOR activation inhibited instead of stimulated AC activity. Short-term activation of MOR in Chinese hamster ovary (CHO) cells stably transfected with MOR (MOR-CHO) inhibits AC activity. Long-term morphine treatment of these cells increased AC and Gbeta phosphorylation, membrane protein kinase Cgamma (PKCgamma) translocation, and MOR G(s) association. All converge, shifting the consequences of short-term MOR activation from Galpha(i)/Galpha(o) inhibitory to AC stimulatory signaling. In contrast, overexpression of the Gbetagamma-stimulated AC isoform AC2 (which converted MOR-coupled inhibition to stimulation of AC) eliminated or reversed these adaptations to long-term morphine treatment; it negated the increase in Gbeta phosphorylation and PKCgamma translocation while reversing the increase in AC phosphorylation and MOR G(s) association. These adaptations greatly attenuated MOR-coupled stimulation of AC activity. Altered overexpression of AC protein per se was not a confounding factor because MOR-CHO overexpressing AC1, which is inhibited by short-term MOR activation, manifested adaptations to long-term morphine treatment qualitatively identical with those of MOR-CHO. These results reveal that adaptations elicited by long-term morphine treatment depend on the effects of short-term MOR activation. This dynamic and pliable nature of tolerance mechanisms could represent a new paradigm for pharmacotherapeutics.

Phosphorylation of Galphas influences its association with the micro-opioid receptor and is modulated by long-term morphine exposure.

The recent biochemical demonstration of the association of the mu-opioid receptor (MOR) with Galpha(s) that increases after long-term morphine treatment (Mol Brain Res 135:217-224, 2005) provides a new imperative for studying MOR-Galpha(s) 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-Galpha(s) association. This study demonstrates that 1) Galpha(s) exists as a phosphoprotein, 2) the stoichiometry of Galpha(s) phosphorylation decreases after long-term morphine treatment, and 3) in vitro dephosphorylation of Galpha(s) increases its association with MOR. Furthermore, our data suggest that increased association of Galpha(s) with protein phosphatase 2A is functionally linked to the long-term morphine treatment-induced reduction in Galpha(s) 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 Galpha(s) is a critical determinant of its interaction with MOR. Long-term morphine treatment decreases Galpha(s) phosphorylation, which is a key mechanism underlying the previously demonstrated increased association of MOR and Galpha(s) in opioid tolerant tissue.

Post-opioid receptor adaptations to chronic morphine; altered functionality and associations of signaling molecules.

Opioid desensitization/tolerance mechanisms have largely focused on adaptations that occur on the level of the mu-opioid receptor (MOR) itself. These include opioid receptor phosphorylation and ensuing trafficking events. Recent research, however, has revealed additional adaptations that occur downstream from the opioid receptor, which involve covalent modification of signaling molecules and altered associations among them. These include augmented isoform-specific synthesis of adenylyl cyclase (AC) and their phosphorylation as well as augmented phosphorylation of the G(beta) subunit of G(beta gamma). The aggregate effect of these changes is to shift mu-opioid receptor-coupled signaling from predominantly G(i alpha) inhibitory to (G(i)-derived) G(beta gamma) stimulatory AC signaling. Most recently, chronic morphine has been shown to enhance the association (interaction) between MOR and G(s), which should provide an additional avenue for offsetting inhibitory MOR signaling sequelae. The unfolding complexity of chronic morphine-induced sequelae demands an evolving broader and more encompassing perspective on opioid tolerance-producing mechanisms. This should facilitate understanding tolerance within the context of physiological plasticity that is activated by chronic exposure to drugs of abuse. Additional research is required to integrate the various tolerance-producing adaptations that have been elucidated to date. Specifically, the relative contribution to opioid tolerance of identified adaptations is still unknown as is the extent to which they vary among different regions of the central nervous system.

Chronic morphine acts via a protein kinase Cgamma-G(beta)-adenylyl cyclase complex to augment phosphorylation of G(beta) and G(betagamma) stimulatory adenylyl cyclase signaling.

Chronic morphine augments protein kinase C (PKC) phosphorylation of G(beta), which enhances the potency of G(betagamma) to stimulate adenylyl cyclase II (ACII) activity. The present study demonstrates an in vivo association between phosphorylated G(beta) and a specific PKC isoform, PKCgamma. We investigated the association of G(beta) and PKCgamma by assessing the ability of anti-PKCgamma antibodies to co-immunoprecipitate G(beta) from (32)P-radiolabeled Chinese Hamster Ovary cells stably transfected with a mu-opioid receptor (MOR-CHO). PKCgamma immunoprecipitate (IP) obtained from MOR-CHO membranes contained radiolabeled signals of approximately equals 33 and 36--38 kDa that were subsequently identified as G(beta)(s). Chronic morphine significantly increased ( approximately equals 75%) the magnitude of (32)P incorporated into G(beta) present in PKCgamma IP. This suggests that G(beta) is an in vivo substrate for PKCgamma, which mediates the chronic morphine-induced increment in G(beta) phosphorylation. In order to evaluate AC as a putative effector for phosphorylated G(betagamma), its presence in IP obtained using anti-AC antibodies was evaluated. Autoradiographic analyses of AC IP also revealed the presence of phosphorylated G(beta)(s), the magnitude of which was significantly enhanced ( approximately equals 60%) following chronic morphine treatment. This indicates that phosphorylated G(betagamma) associates and presumably interacts in vivo with AC, indicating that it is a target for the enhanced phosphorylated G(betagamma) that is generated following chronic morphine treatment. This would contribute to the previously observed shift from predominantly G(ialpha) inhibitory to G(betagamma) stimulatory AC signaling following chronic morphine. The PKCgamma-G(beta)-AC complex identified in this study provides an organizational framework for understanding the well-documented participation of PKCgamma in opioid tolerance-producing mechanisms.

Biochemical demonstration of mu-opioid receptor association with Gsalpha: enhancement following morphine exposure.

Biochemical data indicate mu-opioid receptor (MOR) coupling predominantly to the G(i) and G(o) family. Additionally, MOR coupling to G(s) is suggested by pharmacological assessments that have revealed excitatory MOR effects, which are resistant to pertussis toxin and sensitive to cholera toxin. However, biochemical evidence for such interactions remains elusive; G(salpha) has not been shown to be present in immunoprecipitate obtained using anti-MOR antibodies. In the current study, the presence of MOR in immunoprecipitate obtained with anti-G(salpha ) antibodies was investigated using Chinese hamster ovary cells stably transfected with MOR (MOR-CHO). MOR Western analyses of opioid naive MOR-CHO membranes immunoprecipitated using anti-G(salpha) antibodies reveal the presence of an approximately 75-80 kDa MOR species. Interestingly, acute and chronic morphine treatment markedly enhances the magnitude of MOR that co-immunoprecipitates with G(salpha), despite the concomitant down-regulation of membrane MOR protein. Enhanced co-precipitation of MOR with G(salpha) occurs without a concomitant increase in the immunoprecipitated G(salpha) protein indicating their increased association. In contrast, chronic morphine diminishes the co-immunoprecipitation of MOR with G(ialpha). Moreover, although only a single MOR species co-immunoprecipitated with G(salpha), MOR Western analysis of MOR-CHO membranes as well as immunoprecipitate obtained with either anti-MOR or anti-G(ialpha) antibodies reveals the presence of multiple molecular mass species of MOR. These data reveal the existence of a subset of MORs whose association with G(salpha) can be enhanced by morphine exposure. Notably, the regulation by chronic morphine of MOR association with G(salpha) and G(ialpha) is reciprocal. The relevance of MOR-Gs(alpha) coupling to opioid tolerance formation is discussed.

Chronic morphine-induced plasticity among signalling molecules.

Most formulations of the consequences of the persistent activation of opioid receptors have centred on the diminution or loss of opioid receptor-coupled signalling mechanisms. Activation of opposing compensatory circuits remains another of the adaptations proposed to underlie the extreme loss of the antinociceptive potency of narcotics following their chronic administration. Recent research has revealed that adaptations to chronic morphine involve not only the impairment of opioid receptor functionality but also the altered consequences of its G protein coupling. Pre-eminent among the biochemical perturbations that underlie the chronic morphine-induced emergence of new signalling strategies are enhanced phosphorylation and altered expression of key signalling molecules. These molecular changes include the up-regulation and augmented phosphorylation of adenylyl cyclase type II isoforms, which underlies the ability of morphine to shift opioid receptor G protein signalling from predominantly Gialpha inhibitory to Gbetagamma stimulatory. Persistent morphine exposure also enhances the concomitant phosphorylation of G protein receptor kinase, beta arrestin and the G protein Gbeta subunit, one consequence of which is to further enhance G protein receptor signalling via the Gbetagamma subunit. This review will focus on our increasing understanding of the importance of qualitative changes among components of opioid receptor-coupled signalling pathways, as opposed to the interruption of such signalling, as the predominant mode of adapting to the presence of opioids.

Applying a new conceptual framework to evaluate tuberculosis surveillance and action performance and measure the costs, Hillsborough County, Florida, 2002.

PURPOSE: Tuberculosis (TB) elimination is an important US public health goal and improving the performance of TB surveillance and action and reducing the costs will help achieve it. But, there exists the need to better evaluate the performance and measure the costs. METHODS: We pilot tested an evaluation strategy in Hillsborough County, Florida using a conceptual framework of TB surveillance and action with eight core and four support activities. To evaluate performance, we developed indicators and validated their accuracy, usefulness, and measurability. To measure the costs, we obtained financial information. RESULTS: In 2001, Hillsborough County reported 78 (7%) of the 1145 Florida TB cases. Nineteen (24%) were previously arrested. While 13 (68%) of the 19 were incarcerated during the 2 years prior to being reported, only 1 (5%) of 19 was reported from the jail. From 111 TB suspects, 219 (25%) of 894 sputum specimens were inadequately collected. Of the $1.08 million annual budget, 22% went for surveillance, 29% for support, and 49% for action. CONCLUSIONS: This conceptual framework allowed measurement of TB surveillance and action performance and cost. The evaluation performed using it revealed missed opportunities for detection of TB cases and wasted resources. This conceptual framework could serve as a model for evaluation of TB surveillance and action.

Chronic morphine-induced loss of the facilitative interaction between vasoactive intestinal polypeptide and delta-opioid: involvement of protein kinase C and phospholipase Cbetas.

This laboratory recently demonstrated a multiplicative interaction between the pelvic visceral afferent transmitter vasoactive intestinal polypeptide (VIP) and the delta-opioid receptor (DOR)-selective agonist [D-Pen2,5] enkephalin (DPDPE) to regulate cAMP levels in spinal cord [Brain Res. 959 (2003) 103]. Although DOR activation is required for the manifestation of the VIP-DPDPE facilitative interaction, its relevance to opioid antinociception remains unclear. The current study investigates whether or not the VIP-DPDPE facilitation of cAMP formation is subject to tolerance formation, a hallmark characteristic of opioid antinociception. Chronic morphine exposure abolishes the VIP-DPDPE facilitative interaction, consistent with its relevance to DOR antinociception. However, acute in vitro inhibition of protein kinase C (PKC) reinstates the VIP-DPDPE multiplicative interaction characteristic of opioid naïve spinal tissue. This suggests that its chronic morphine-induced loss requires a PKC phosphorylation. PKC phosphorylation negatively modulates phospholipase C (PLC)beta, enzymes intimately associated with phosphoinositide turnover and calcium trafficking. These are essential determinants of acute and chronic opioid effects. Accordingly, the effect of chronic morphine on their state of phosphorylation was also investigated. Central nervous system opioid tolerance is associated with the reciprocal phosphorylation (regulation) of two PLCbeta isoforms, PLCbeta1 and PLCbeta3. However, although chelerythrine reinstates the chronic morphine-induced loss of the multiplicative VIP-DPDPE interaction, it does not alter the associated changes in PLCbeta phosphorylation, possibly indicating different time courses of restitution of function and/or involvement of different kinases for different components of tolerance. These results could provide a mechanistic rubric for understanding positive modulation of opioid antinociception by afferent transmission.

Phosphorylation of Gbeta is augmented by chronic morphine and enhances Gbetagamma stimulation of adenylyl cyclase activity.

We previously demonstrated (Chakrabarti, et al., 2001) that in vivo phosphorylation of the Gbeta subunit of G proteins, via protein kinase A (PKA) and protein kinase C (PKC), is dramatically increased following chronic morphine. The present study investigates the PKC isoform selectivity of Gbeta phosphorylation and the consequences thereof on the ability of Gbetagamma to stimulate adenylyl cyclase II (ACII). The catalytic subunit of PKC and PKA, as well as the conventional PKC isoform PKCgamma, was effective in phosphorylating Gbeta. In contrast, Gbeta was only minimally phosphorylated by another conventional isoform, PKCalpha or the atypical isoform PKCzeta. In the presence of activated recombinant Gsalpha, ACII activity was dose dependently stimulated by G(betagamma), the magnitude of which was dependent upon its phosphorylation state. The increment in ACII activity produced by Gbetagamma was increased approximately 2-fold following in vitro phosphorylation by the catalytic subunit of either PKA or PKC. In contrast, the concomitant or sequential phosphorylation of Gbetagamma by PKA and PKC catalytic subunits did not result in an additive enhancement of its ability to stimulate ACII and, in fact, negated the observed enhancing effect of each kinase, individually. Threonine phosphorylated G(beta) occurs naturally in the spinal cord, the levels of which are augmented (approximately 60%) by chronic morphine. The natural occurrence of phosphorylated Gbeta in spinal cord, its up-regulation following chronic morphine and the augmented ability of phosphorylated Gbetagamma to stimulate ACII activity, in the aggregate, indicate that phosphorylation of Gbeta could be a regulatory mechanism causally associated with altered cellular signaling.

Reciprocal modulation of phospholipase Cbeta 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 beta isoforms of PLC, by kinases that are up-regulated after chronic morphine, is a potent modality for their regulation. Direct assessment of PLCbeta1 and PLCbeta3 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 PLCbeta1 is significantly reduced, whereas that of PLCbeta3 is substantially augmented, changes not accompanied by altered content of PLCbeta1 or PLCbeta3 protein. In contrast to chronic morphine, acute morphine treatment of opioid naïve longitudinal muscle myenteric plexus tissue attenuates PLCbeta3 phosphorylation, an effect also manifested by endogenous opioids that is reflected by the ability of acute naloxone to substantially augment PLCbeta3 phosphorylation. This indicates that PLCbeta phosphorylation is dynamically regulated. PLCbeta1 and PLCbeta3 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.