Biomedical subjects

Alan R Gintzler

Publications and source records attributed to Alan R Gintzler.

13 recordsLinked to original sources

PubMed2006

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.

Adaptation, Physiological↗

PubMed2006

Phospholipase Cbeta1 modulates pain sensitivity, opioid antinociception and opioid tolerance formation.

Phospholipase C (PLC) activity has been implicated in multiple opioid-induced sequelae. The relevance of PLC-linked pathways to opioid actions is isoform-specific. Chronic morphine augments PLCbeta1 signaling while diminishing that of PLCbeta3. This suggests that PLCbeta1 makes an important contribution to opioid tolerance formation (PNAS 100: 13686-1369, 2003). In the present study, PLCbeta1 knockout animals (-/-) were used to assess the relevance of PLCbeta1 to pain thresholds, morphine antinociception and analgesic tolerance formation. Response latencies to thermal nociceptive stimuli were markedly diminished in -/- animals relative to their wild-type (+/+) and heterozygous (+/-) counterparts; thermal nociceptive thresholds obtained in +/+ and +/- mice did not differ. This suggests that the contribution of PLCbeta1 to thermal pain thresholds requires a critical concentration of PLCbeta1 protein. PLCbeta1 genotype also influenced acute and chronic responsiveness to morphine. Analgesic dose responsiveness and the magnitude of analgesic tolerance formation to morphine were significantly attenuated in -/- vs. +/+ animals. Notably, in contrast to thermal nociceptive thresholds, acute and chronic morphine responsiveness differed significantly only between +/+ and -/- genotypes and not between -/- vs. +/- groups. These data suggest that whereas the contribution of PLCbeta1 to thermal nociceptive response thresholds requires a critical concentration of PLCbeta1 protein, its participation in morphine analgesic and tolerance-producing mechanisms is graded. Importantly, GTPgammaS binding studies revealed that there is no detectable diminution in functional opioid receptors in spinal tissue from -/- animals. This underscores the importance of PLCbeta1 to morphine sequelae that are initiated downstream from the opioid receptor.

Analysis of Variance↗

PubMed2005

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.

Adenylyl Cyclases↗

PubMed2005

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.

Animals↗

PubMed2004

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.

Analgesics, Opioid↗

PubMed2004

Dual effects of DAMGO [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin and CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2) on adenylyl cyclase activity: implications for mu-opioid receptor Gs coupling.

The mu-opioid receptor (MOR) couples to multiple G proteins, of which coupling to Gs has long been debated. As expected, in opioid naive Chinese hamster ovary cells expressing recombinant MOR, the predominant action of [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) is inhibitory. However, inactivation of Gi/Go proteins via pertussis toxin (PTX) unmasks its ability to facilitate forskolin activation of adenylyl cyclase (AC) activity. Tolerance develops to this effect of DAMGO, which can also be attenuated by cholera toxin (CTX). The latter suggests G mediation. D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP), previously considered to be a neutral MOR antagonist, also produces a facilitation of forskolin (FSK) activation of AC that is augmented by chronic morphine. Facilitative effects of CTAP in naive as well as its augmentation in tolerant membranes are both substantially reduced by CTX. This suggests that not only Gs mediation but also G(salpha)-linked signaling is critical to the chronic morphine-induced enhanced facilitative action of CTAP. Interestingly, the (augmented) CTAP facilitation of FSK-stimulated AC activity that is observed in opioid tolerant (but not in naive) membranes is also sensitive to PTX. This can best be explained by postulating the involvement of Gi-derived G(betagamma), which would stimulate type 2 ACs, conditional on the presence of activated G(salpha). The emergence of a G(betagamma) dimension of AC stimulation by CTAP after chronic morphine could explain its ability to augment the stimulatory action of CTAP on AC. These results support putative MOR coupling to Gs and underscore the multifaceted nature and plasticity of MOR G protein coupling.

Adenylyl Cyclases↗

PubMed2004

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.

Adaptation, Physiological↗

PubMed2003

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.

Adenylyl Cyclases↗

PubMed2003

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.

Acclimatization↗

PubMed2003

Facilitative interactions between vasoactive intestinal polypeptide and receptor type-selective opioids: implications for sensory afferent regulation of spinal opioid action.

Afferent tone is known to influence spinal opioid antinociception but the underlying neurochemical events are not well defined. This study investigates the consequence on cAMP formation of the coincident activation of signal transduction sequelae initiated by an afferent transmitter and opioid using dissociated spinal cord tissue. Afferent transmission was simulated via the addition of vasoactive intestinal polypeptide (VIP), a pelvic visceral afferent transmitter. Individually, mu, delta-, or kappa-selective opioids (1 microM each) did not alter basal spinal content of cAMP. However, VIP (1 microM) and the delta-opioid selective agonist, [D-Pen(2,5)] enkephalin (DPDPE; 1 microM), in combination, manifest a striking facilitative interaction to augment spinal levels of cAMP. Facilitative interactions between VIP and kappa- or mu-opioids were of a reduced magnitude or not observed, respectively. Blockade of delta-opioid or VIP receptors using naltrindole or VIP6-28, respectively antagonized the VIP-DPDPE facilitative interaction, as did pertussis toxin treatment. The VIP-DPDPE facilitative interaction was also eliminated by phospholipase Cbeta inhibition and inositol trisphosphate receptor blockade. This suggests that modulation of Ca(2+) trafficking by VIP and delta-opioid agonists is a point of convergence of their respective signal transduction cascades, the concomitant action at which achieves cytosolic Ca(2+) concentrations that are now sufficient for the activation of signaling molecules, e.g. Ca(2+)/calmodulin-stimulated adenylyl cyclase isoforms. These data underscore the plasticity of spinal delta-opioid neurochemical sequelae and their dependence on concomitant afferent transmitter-initiated neurochemical events.

Afferent Pathways↗

PubMed2003

Influence of ovarian sex steroids on spinal methionine-enkephalin release: comparison with dynorphin reveals asymmetrical regulation.

The concomitant activation of spinal kappa- and delta-opioid systems is a prerequisite for the antinociception of gestation and its hormonal simulation [via 17 beta-estradiol and progesterone administration; hormone-simulated pregnancy (HSP)]. However, it is not known whether the release of kappa- and delta-opioids is also concomitantly regulated. This study investigates whether the release of methionine-enkephalin and modulation thereof is altered during HSP, as has been reported for dynorphin. K+-stimulated release of spinal methionine-enkephalin from lumbar spinal tissue obtained from control animals is negatively modulated by nociceptin (orphanin FQ; N/OFQ) in a dose-dependent manner, but not by opioids. Conversely, selective blockade of spinal N/OFQ, but not opioid receptors, augments the K+-induced increase in methionine-enkephalin release, indicating that endogenous N/OFQ also functions as a negative modulator of methionine-enkephalin release. The magnitude of K+-evoked methionine-enkephalin release from spinal tissue obtained from ovarian steroid-treated animals remains unchanged, consistent with the insensitivity of its modulation by N/OFQ to the ovarian sex steroid milieu. These characteristics of methionine-enkephalin release stand in sharp contrast to those previously reported for the evoked release of spinal dynorphin. Dynorphin release is subject to negative modulation by opioid (predominantly delta) as well as N/OFQ, both of which are offset during HSP, resulting in an approximately 2-fold increase in the magnitude of its release. These observations reveal that regulation of spinal dynorphin/kappa- and methionine-enkephalin/delta-spinal opioid antinociceptive systems is independent, divergent, and not symmetrical and support the formulation that spinal methionine-enkephalin/delta-opioid tone acts in a permissive/facilitative capacity to accentuate spinal dynorphin/kappa-activity.

Animals↗

PubMed1996

Estrogen and progesterone activate spinal kappa-opiate receptor analgesic mechanisms.

Rats and humans manifest elevated response thresholds to aversive stimuli during gestation and parturition. This pregnancy-associated antinociception is mediated, in part, by a spinal cord dynorphin/kappa antinociceptive system. Simulating the maternal pregnancy blood concentration profile (in non-pregnant animals) of 17-beta-estradiol (E2) and progesterone (P) produces an opioid antinociception which closely approximates that of actual pregnancy. The current study was initiated in order to determine whether sex steroid-induced antinociception involves a spinal cord kappa-opiate receptor-coupled system (as does the antinociception of actual gestation). Additionally, sex steroid modulation of the intrathecal (i.t.) antinociceptive effectiveness of a kappa agonist was investigated. The opioid antinociception associated with simulating the pregnancy blood concentration profile of E2 and P (hormone-stimulated pregnancy, HSP) is significantly antagonized by i.t. administration of nor-binaltorphimine, an antagonist highly specific for the kappa-opiate receptor. This indicates that exposure (of non-pregnant animals) to the pregnancy blood profile of E2 and P activates a spinal cord kappa-opiate receptor analgesic system, as occurs during actual gestation. Furthermore, during HSP, antinociceptive responsiveness to i.t. U50,488H (kappa-selective) is significantly enhanced (approximately 40%). This effect is abolished in animals treated concomitantly with steroid hormones and systemic naltrexone or i.t. nor-binaltorphimine. In contrast to the effects of steroid treatment on antinociceptive responsiveness to i.t. U50,488H, no alteration in antinociceptive responsiveness to i.t. sufentanil was observed on day 19 of HSP over all doses tested (0.1-1 nmol). Thus, during HSP (and actual gestation), a less robust constituent of intrinsic opioid pain-attenuating systems in the spinal cord is recruited. pF to mediate, at least in part, the maternal antinociception of gestation. pF, positive modulation of the spinal cord kappa analgesic system occurs post-synaptically. This laboratory previously reported that simulating the pregnancy blood concentration profile of E2 and P also positively modulates spinal dynorphin content and the processing of its precursor, suggesting a presynaptic loci of action. Thus, female rats possess a spinal dynorphin/kappa analgesic system that can be positively modulated, pre-synaptically as well as post-synaptically, by circulating sex steroids.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗

PubMed1996

Estrogen and progesterone activate spinal kappa-opiate receptor analgesic mechanisms.

Rats and humans manifest elevated response thresholds to aversive stimuli during gestation and parturition. This pregnancy-associated antinociception is mediated, in part, by a spinal cord dynorphin/kappa antinociceptive system. Simulating the maternal pregnancy blood concentration profile (in non-pregnant animals) of 17-beta-estradiol (E2) and progesterone (P) produces an opioid antinociception which closely approximates that of actual pregnancy. The current study was initiated in order to determine whether sex steroid-induced antinociception involves a spinal cord kappa-opiate receptor-coupled system (as does the antinociception of actual gestation). Additionally, sex steroid modulation of the intrathecal (i.t.) antinociceptive effectiveness of a kappa agonist was investigated. The opioid antinociception associated with simulating the pregnancy blood concentration profile of E2 and P (hormone-simulated pregnancy, HSP) is significantly antagonized by i.t. administration of nor-binaltorphimine, an antagonist highly specific for the kappa-opiate receptor. This indicates that exposure (of non-pregnant animals) to the pregnancy blood profile of E2 and P activates a spinal cord kappa-opiate receptor analgesic system, as occurs during actual gestation. Furthermore, during HSP, antinociceptive responsiveness to i.t. U50,488H (kappa-selective) is significantly enhanced (approximately 40%). This effect is abolished in animals treated concomitantly with steroid hormones and systemic naltrexone or i.t. nor-binaltorphimine. In contrast to the effects of steroid treatment on antinociceptive responsiveness to i.t. U50,488H, no alteration in antinociceptive responsiveness to i.t. sufentanil was observed on day 19 of HSP over all doses tested (0.1-1 nmol). Thus, during HSP (and actual gestation), a less robust constituent of intrinsic opioid pain-attenuating systems in the spinal cord is recruited.

Animals↗