Biomedical subjects

Laura M Bohn

Publications and source records attributed to Laura M Bohn.

15 recordsLinked to original sources

PubMed2007

A comparison of noninternalizing (herkinorin) and internalizing (DAMGO) mu-opioid agonists on cellular markers related to opioid tolerance and dependence.

Previous studies established that Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol (DAMGO) and (2S,4aR,6aR,7R,9S,10aS,10bR)-9-(Benzoyloxy)-2-(3-furanyl)dodecahydro-6a,10b-dimethyl-4,10-dioxo-2H-naphtho-[2,1-c]pyran-7-carboxylic acid methyl ester (herkinorin) are fully efficacious mu-agonists. Herkinorin (HERK), unlike DAMGO, does not recruit beta-arrestin and promote mu-receptor internalization, even in cells that over express beta-arrestin. We hypothesized that chronic HERK and DAMGO treatment will differentially affect cellular markers of tolerance and dependence. CHO cells expressing the cloned human mu-receptor were treated for 20 h with 10 microM DAMGO, HERK, morphine, or medium. Both DAMGO and HERK acted as full agonists in the [(35)S]GTP-gamma-S binding assay with E(MAX) values of 230% and EC(50) values of 12.8 and 92.5 nM, respectively. In the cAMP assay, DAMGO and HERK had similar E(MAX) values of approximately 80% and EC(50) values of 3.23 and 48.7 nM, respectively. Chronic exposure to both drugs produced moderate tolerance to both drugs ( approximately 2 to 5 fold) in the [(35)S]GTP-gamma-S binding assay. In the cAMP assay, chronic DAMGO produced tolerance to both drugs ( approximately 3 to 4 fold). Chronic HERK eliminated the ability of either drug to inhibit forskolin-stimulated cAMP accumulation. Chronic DAMGO increased, and chronic HERK decreased, forskolin-stimulated cAMP accumulation. Naloxone, after chronic HERK (but not DAMGO) induced a large increase in forskolin-stimulated cAMP accumulation. Viewed collectively with published data, the current data indicate that both internalizing and noninternalizing mu-agonists produce cellular signs of tolerance and dependence.

Analgesics, Opioid↗

PubMed2006

Opioid receptor signaling: relevance for gastrointestinal therapy.

Opiate drugs, such as morphine, are renowned for their analgesic properties. To date, opioid narcotics represent the largest and most potent class of pain relievers available to treat both acute and chronic pain conditions. The use of opiates, however, is severely limited by several adverse side effects. Upon chronic use, opiates can produce tolerance, physical dependence and addiction. Although these conditions warrant consideration, there are acute effects that present more immediate concerns when choosing opiate narcotics for pain therapy. One of the most prevalent side effects, which continues for as long as the opiate is used for pain control, is constipation. This can impact patient compliance, as it is often one of the top reasons why patients discontinue opiate treatment. The challenge, therefore, is to develop pain therapies that preserve potent analgesia while preventing constipation.

Constipation↗

PubMed2005

Mu opioid receptor regulation and opiate responsiveness.

Opiate drugs such as morphine are well known for their ability to produce potent analgesia as well as such unwanted side effects as tolerance, physical dependence, respiratory suppression and constipation. Opiates act at opioid receptors, which belong to the family of G protein-coupled receptors. The mechanisms governing mu opioid receptor (muOR) regulation are of particular interest since morphine and other clinically important analgesics produce their pharmacological effects through this receptor. Here we review recent advances in understanding how opioid receptor regulation can impart differential agonist efficacy produced in vivo.

Analgesics, Opioid↗

PubMed2005

Morphine side effects in beta-arrestin 2 knockout mice.

Morphine is a potent analgesic, yet, like most opioid narcotics, it exerts unwanted side effects such as constipation and respiratory suppression, thereby limiting its clinical utility. Pharmacological approaches taken to preserve the analgesic properties, while eliminating the unwanted side effects, have met with very limited success. Here, we provide evidence that altering mu opioid receptor regulation may provide a novel approach to discriminate morphine's beneficial and deleterious effects in vivo. We have previously reported that mice lacking the G protein-coupled receptor regulatory protein, beta-arrestin 2, display profoundly altered morphine responses. beta-Arrestin 2 knockout mice have enhanced and prolonged morphine analgesia with very little morphine tolerance. In this report, we examine whether the side effects of morphine treatment are also augmented in this animal model. Surprisingly, the genetic disruption of opioid receptor regulation, while enhancing and prolonging analgesia, dramatically attenuates the respiratory suppression and acute constipation caused by morphine.

Animals↗

PubMed2005

Opioid receptor homo- and heterodimerization in living cells by quantitative bioluminescence resonance energy transfer.

Opioid receptors have been shown to dimerize or oligomerize among themselves and each other, affecting their functional properties. This study used bioluminescence resonance energy transfer (BRET) between the mu, delta, and kappa opioid receptors to study opioid receptor aggregation in transfected human embryonic kidney 293 cells. Titration of receptor levels indicated that all three opioid receptors have a similar affinity to form homo- or hetero-oligomers in combination with any other opioid receptor type. In contrast, none of the opioid receptors formed detectable oligomers with the muscarinic M2 receptor, indicating that interactions among opioid receptors are selective. The formation of opioid receptor dimers, rather than higher order oligomers, is supported by binding kinetics in competition experiments between labeled and unlabeled receptors. Opioid receptor dimerization occurred at physiological temperatures upon receptor biosynthesis, before trafficking to the plasma membrane. Moreover, using BRET, coimmunoprecipitation, receptor binding, and G protein coupling, we demonstrate for the first time functional mu opioid receptor-kappa opioid receptor heterodimerization. These combined results demonstrate that opioid receptors can undergo homo- and heterodimerization, a process with potential implications for opioid physiology and pharmacology.

Animals↗

PubMed2005

Characterization of conditioned place preference to cocaine in congenic dopamine transporter knockout female mice.

RATIONALE: The dopamine transporter (DAT) is thought to play a major role in the rewarding effects of cocaine. Therefore, it is surprising that cocaine reveals conditioned effects in DAT knockout (DAT-KO) mice. OBJECTIVES: To examine these findings further, we obtained complete dose-effect curves for DAT-KO and DAT wild-type (DAT-WT) mice in a cocaine conditioned place preference (CPP) procedure. METHODS: Congenic C57BL6 background female DAT-KO and DAT-WT mice were conditioned in a three-compartment place preference apparatus. Conditioning consisted of three 30-min sessions with cocaine (2.5, 5.0, 10.0, 20.0, or 40.0 mg/kg) and three 30-min sessions with saline. The distribution of time in each choice compartment was determined after each pair of conditioning sessions (one cocaine and one saline session). RESULTS: DAT-WT mice revealed CPP over a wide range of cocaine doses (5.0-40 mg/kg), whereas DAT-KO mice revealed CPP over a more restricted range of doses, with consistent CPP only occurring with 10 mg/kg of cocaine. CONCLUSIONS: CPP for cocaine develops in both DAT-KO and DAT-WT mice; however, the dose range at which CPP develops is much more restricted in DAT-KO mice than in DAT-WT mice. These observations corroborate the significant role of DAT inhibition in cocaine's conditioned effects.

Animals↗

PubMed2004

Desensitization of G protein-coupled receptors and neuronal functions.

G protein-coupled receptors (GPCRs) have proven to be the most highly favorable class of drug targets in modern pharmacology. Over 90% of nonsensory GPCRs are expressed in the brain, where they play important roles in numerous neuronal functions. GPCRs can be desensitized following activation by agonists by becoming phosphorylated by members of the family of G protein-coupled receptor kinases (GRKs). Phosphorylated receptors are then bound by arrestins, which prevent further stimulation of G proteins and downstream signaling pathways. Discussed in this review are recent progress in understanding basics of GPCR desensitization, novel functional roles, patterns of brain expression, and receptor specificity of GRKs and beta arrestins in major brain functions. In particular, screening of genetically modified mice lacking individual GRKs or beta arrestins for alterations in behavioral and biochemical responses to cocaine and morphine has revealed a functional specificity in dopamine and mu-opioid receptor regulation of locomotion and analgesia. An important and specific role of GRKs and beta arrestins in regulating physiological responsiveness to psychostimulants and morphine suggests potential involvement of these molecules in certain brain disorders, such as addiction, Parkinson's disease, mood disorders, and schizophrenia. Furthermore, the utility of a pharmacological strategy aimed at targeting this GPCR desensitization machinery to regulate brain functions can be envisaged.

Animals↗

PubMed2004

Drosophila CG8422 encodes a functional diuretic hormone receptor.

Diuretic hormone 44 (DH) is a bioactive neuropeptide that mediates osmotic balance in a wide variety of insects through increases in cAMP. It is structurally similar to mammalian corticotrophin releasing factor (CRF) peptides. In the moth Manduca and the cricket Acheta, functional studies have shown that its cognate receptor (DH-R) is related to the mammalian CRF receptor. The Drosophila genome contains two genes (CG8422 and CG12370) orthologous to Manduca and Acheta DH-Rs. Here, we present multiple lines of evidence to support the hypothesis that the orphan CG8422 G-protein-coupled receptor is a functional DH-R. When expressed in mammalian cells, CG8422 conferred selective sensitivity to DH, as indicated by translocation of a beta-arrestin-2-GFP reporter from the cytoplasm to the cell membrane. Consistent with its in vivo activities in other insects, DH activation of CG8422 elicited increases in a cAMP reporter system (CRE-luciferase), with an EC(50) of 1.7 nmol l(-1). CG8422 activation by DH also led to increases in intracellular calcium but at substantially higher doses (EC(50) approximately 300 nmol l(-1)). By microarray analysis, the CG8422 transcript was detectable in Drosophila head mRNA of different genotypes and under different environmental conditions. The identification of a Drosophila receptor for the DH neuropeptide provides a basis for genetic analysis of this critical factor's roles in maintaining physiological homeostasis.

Animals↗

PubMed2004

G protein-coupled receptor kinase/beta-arrestin systems and drugs of abuse: psychostimulant and opiate studies in knockout mice.

G protein-coupled receptors (GPCRs) currently represent pharmaceutical targets for numerous medicinal compounds that are used to treat conditions ranging from blood pressure dysregulation to depression to pain, demonstrating the wide range of functions mediated by this receptor family. GPCR activation is determined not only by the initiation of signaling cascades but also by regulatory mechanisms that control the extent and duration of their signals. The balance of activation and desensitization dictate the ultimate physiological response to both endogenous and exogenous receptor stimuli. Therefore, these mechanisms may play a particularly relevant role during chronic exposure to agonists such as in conditions when drugs are abused. Two major classes of drugs of abuse, opiates and psychostimulants, both use either direct or indirect GPCR signaling mechanisms to mediate their effects. Therefore, the regulation of GPCRs may have bearing on the neuronal adaptations that underlie the reinforcing properties of drugs of abuse.

Animals↗

PubMed2003

Enhanced rewarding properties of morphine, but not cocaine, in beta(arrestin)-2 knock-out mice.

The reinforcing and psychomotor effects of morphine involve opiate stimulation of the dopaminergic system via activation of mu-opioid receptors (muOR). Both mu-opioid and dopamine receptors are members of the G-protein-coupled receptor (GPCR) family of proteins. GPCRs are known to undergo desensitization involving phosphorylation of the receptor and the subsequent binding of beta(arrestins), which prevents further receptor-G-protein coupling. Mice lacking beta(arrestin)-2 (beta(arr2)) display enhanced sensitivity to morphine in tests of pain perception attributable to impaired desensitization of muOR. However, whether abrogating muOR desensitization affects the reinforcing and psychomotor properties of morphine has remained unexplored. In the present study, we examined this question by assessing the effects of morphine and cocaine on locomotor activity, behavioral sensitization, conditioned place preference, and striatal dopamine release in beta(arr2) knock-out (beta(arr2)-KO) mice and their wild-type (WT) controls. Cocaine treatment resulted in very similar neurochemical and behavioral responses between the genotypes. However, in the beta(arr2)-KO mice, morphine induced more pronounced increases in striatal extracellular dopamine than in WT mice. Moreover, the rewarding properties of morphine in the conditioned place preference test were greater in the beta(arr2)-KO mice when compared with the WT mice. Thus, beta(arr2) appears to play a more important role in the dopaminergic effects mediated by morphine than those induced by cocaine.

Animals↗

PubMed2003

Identification of Drosophila neuropeptide receptors by G protein-coupled receptors-beta-arrestin2 interactions.

Activation of G protein-coupled receptors (GPCR) leads to the recruitment of beta-arrestins. By tagging the beta-arrestin molecule with a green fluorescent protein, we can visualize the activation of GPCRs in living cells. We have used this approach to de-orphan and study 11 GPCRs for neuropeptide receptors in Drosophila melanogaster. Here we verify the identities of ligands for several recently de-orphaned receptors, including the receptors for the Drosophila neuropeptides proctolin (CG6986), neuropeptide F (CG1147), corazonin (CG10698), dFMRF-amide (CG2114), and allatostatin C (CG7285 and CG13702). We also de-orphan CG6515 and CG7887 by showing these two suspected tachykinin receptor family members respond specifically to a Drosophila tachykinin neuropeptide. Additionally, the translocation assay was used to de-orphan three Drosophila receptors. We show that CG14484, encoding a receptor related to vertebrate bombesin receptors, responds specifically to allatostatin B. Furthermore, the pair of paralogous receptors CG8985 and CG13803 responds specifically to the FMRF-amide-related peptide dromyosuppressin. To corroborate the findings on orphan receptors obtained by the translocation assay, we show that dromyosuppressin also stimulated GTPgammaS binding and inhibited cAMP by CG8985 and CG13803. Together these observations demonstrate the beta-arrestin-green fluorescent protein translocation assay is an important tool in the repertoire of strategies for ligand identification of novel G protein-coupled receptors.

Animals↗

PubMed2003

Sustained elevation of extracellular dopamine causes motor dysfunction and selective degeneration of striatal GABAergic neurons.

Dopamine is believed to contribute to the degeneration of dopamine-containing neurons in the brain. However, whether dopamine affects the survival of other neuronal populations has remained unclear. Here we document that mice with persistently elevated extracellular dopamine, resulting from inactivation of the dopamine transporter gene, sporadically develop severe symptoms of dyskinesia concomitant with apoptotic death of striatal dopamine-responsive gamma-aminobutyric acidergic neurons. Chronic inhibition of dopamine synthesis prevents the appearance of motor dysfunction. The neuronal death is associated with overactivation of dopaminergic signaling as evidenced by the robust up-regulation of striatal DeltaFosB, cyclin-dependent kinase 5, and p35. Moreover, hyperphosphorylation of the tau protein, a phenomenon associated with the activation of cyclin-dependent kinase 5 in several neurodegenerative disorders, is observed in symptomatic mice. These findings provide in vivo evidence that, in addition to its proposed role in the degeneration of dopamine neurons, dopamine can also contribute to the selective death of its target neurons via a previously unappreciated mechanism.

Animals↗

PubMed2003

Dopaminergic supersensitivity in G protein-coupled receptor kinase 6-deficient mice.

Brain dopaminergic transmission is a critical component in numerous vital functions, and its dysfunction is involved in several disorders, including addiction and Parkinson's disease. Responses to dopamine are mediated via G protein-coupled dopamine receptors (D1-D5). Desensitization of G protein-coupled receptors is mediated via phosphorylation by members of the family of G protein-coupled receptor kinases (GRK1-GRK7). Here we show that GRK6-deficient mice are supersensitive to the locomotor-stimulating effect of psychostimulants, including cocaine and amphetamine. In addition, these mice demonstrate an enhanced coupling of striatal D2-like dopamine receptors to G proteins and augmented locomotor response to direct dopamine agonists both in intact and in dopamine-depleted animals. The present study indicates that postsynaptic D2-like dopamine receptors are physiological targets for GRK6 and suggests that this regulatory mechanism contributes to central dopaminergic supersensitivity.

Amphetamine↗

PubMed2002

Differential mechanisms of morphine antinociceptive tolerance revealed in (beta)arrestin-2 knock-out mice.

Morphine induces antinociception by activating mu opioid receptors (muORs) in spinal and supraspinal regions of the CNS. (Beta)arrestin-2 (beta)arr2), a G-protein-coupled receptor-regulating protein, regulates the muOR in vivo. We have shown previously that mice lacking (beta)arr2 experience enhanced morphine-induced analgesia and do not become tolerant to morphine as determined in the hot-plate test, a paradigm that primarily assesses supraspinal pain responsiveness. To determine the general applicability of the (beta)arr2-muOR interaction in other neuronal systems, we have, in the present study, tested (beta)arr2 knock-out ((beta)arr2-KO) mice using the warm water tail-immersion paradigm, which primarily assesses spinal reflexes to painful thermal stimuli. In this test, the (beta)arr2-KO mice have greater basal nociceptive thresholds and markedly enhanced sensitivity to morphine. Interestingly, however, after a delayed onset, they do ultimately develop morphine tolerance, although to a lesser degree than the wild-type (WT) controls. In the (beta)arr2-KO but not WT mice, morphine tolerance can be completely reversed with a low dose of the classical protein kinase C (PKC) inhibitor chelerythrine. These findings provide in vivo evidence that the muOR is differentially regulated in diverse regions of the CNS. Furthermore, although (beta)arr2 appears to be the most prominent and proximal determinant of muOR desensitization and morphine tolerance, in the absence of this mechanism, the contributions of a PKC-dependent regulatory system become readily apparent.

Alkaloids↗

PubMed2002

Apparent loss-of-function mutant GPCRs revealed as constitutively desensitized receptors.

The DRY motif is a triplet amino acid sequence (aspartic acid, arginine, and tyrosine) that is highly conserved in G protein-coupled receptors (GPCRs). Recently, we have shown that a molecular determinant for nephrogenic diabetes insipidus, the vasopressin receptor with a substitution at the DRY motif arginine (V2R R137H), is a constitutively desensitized receptor that is unable to couple to G proteins due to its constitutive association with beta-arrestin [Barak, L. S. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 93-98]. Additionally, the mutant receptors are localized in endocytic vesicles, identical to wild-type receptors stimulated with agonist. In this study, we asked whether the constitutively desensitized phenotype observed in the V2R R137H represents a general paradigm that may be extended to other GPCRs. We show that arginine substitutions in the DRY motifs of the alpha(1B) adrenergic receptor (alpha(1B)-AR) and angiotensin II type 1A receptor (AT(1A)R) result in receptors that are uncoupled from G proteins, associated with beta-arrestins, and found localized in endocytic vesicles rather than at the plasma membrane in the absence of agonists. The localization of the alpha(1B)-ARs and AT(1A)Rs with arginine substitutions can be restored to the plasma membrane by either using selective antagonists or preventing the endocytosis of the beta-arrestin-receptor complexes. These results indicate that the arginine residue of the DRY motif is essential for preserving the localization of the inactive receptor complex. Furthermore, constitutive desensitization may underlie some loss-of-function receptor phenotypes and represent an unappreciated mechanism of hormonal resistance.

Amino Acid Motifs↗