Biomedical subjects

Sumita Chakrabarti

Publications and source records attributed to Sumita Chakrabarti.

8 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↗

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

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.

Cost Allocation↗

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↗