Agonist-induced Receptor Desensitization Research Articles

Amphetamine pretreatment blunts dopamine-induced D2/D3-receptor occupancy by an arrestin-mediated mechanism: A PET study in internalization compromised mice

While all reversible receptor-targeting radioligands for positron emission tomography (PET) can be displaced by competition with an antagonist at the receptor, many radiotracers show limited occupancies using agonists even at high doses. [11C]Raclopride, a D2/D3 receptor radiotracer with rapid kinetics, can identify the direction of changes in the neurotransmitter dopamine, but quantitative interpretation of the relationship between dopamine levels and radiotracer binding has proven elusive. Agonist-induced receptor desensitization and internalization, a homeostatic mechanism to downregulate neurotransmitter-mediated function, can shift radioligand-receptor binding affinity and confound PET interpretations of receptor occupancy. In this study, we compared occupancies induced by amphetamine (AMP) in drug-naive wild-type (WT) and internalization-compromised β-arrestin-2 knockout (KO) mice using a within-scan drug infusion to modulate the kinetics of [11C]raclopride. We additionally performed studies at 3 h following AMP pretreatment, with the hypothesis that receptor internalization should markedly attenuate occupancy on the second challenge, because dopamine cannot access internalized receptors. Without prior AMP treatment, WT mice exhibited somewhat larger binding potential than KO mice but similar AMP-induced occupancy. At 3 h after AMP treatment, WT mice exhibited binding potentials that were 15 % lower than KO mice. At this time point, occupancy was preserved in KO mice but suppressed by 60 % in WT animals, consistent with a model in which most receptors contributing to binding potential in WT animals were not functional. These results demonstrate that arrestin-mediated receptor desensitization and internalization produce large effects in PET [11C]raclopride occupancy studies using agonist challenges.

Evaluation of salivary alpha-amylase as a potential biomarker of inhaled β2-agonist response

In individuals with asthma, chronic use of short (SABAs) and long-acting beta-2 agonists (LABAs) can lead to loss of bronchoprotection (LOBP) from direct and indirect stimuli.1-3 It is hypothesized that LOBP is the result of beta-2 receptor down-regulation, reduced production or internalization of receptors, and uncoupling from secondary messengers leading to desensitization of beta-2 receptors.4 Assessment of beta-2 receptor activation is a laborious process.5 Therefore, a biomarker that serves as an indirect method of measuring beta-2 receptor activation would facilitate clinical studies on beta-2 agonist-induced LOBP and beta-2 receptor desensitization.

Structural determinants of dual incretin receptor agonism by tirzepatide

SignificanceTirzepatide is a dual agonist of the glucose-dependent insulinotropic polypeptide receptor (GIPR) and the glucagon-like peptide-1 receptor (GLP-1R), which are incretin receptors that regulate carbohydrate metabolism. This investigational agent has proven superior to selective GLP-1R agonists in clinical trials in subjects with type 2 diabetes mellitus. Intriguingly, although tirzepatide closely resembles native GIP in how it activates the GIPR, it differs markedly from GLP-1 in its activation of the GLP-1R, resulting in less agonist-induced receptor desensitization. We report how cryogenic electron microscopy and molecular dynamics simulations inform the structural basis for the unique pharmacology of tirzepatide. These studies reveal the extent to which fatty acid modification, combined with amino acid sequence, determines the mode of action of a multireceptor agonist.

Molecular and Cellular Mechanisms of the Age-Dependency of Opioid Analgesia and Tolerance

The age-dependency of opioid analgesia and tolerance has been noticed in both clinical observation and laboratory studies. Evidence shows that many molecular and cellular events that play essential roles in opioid analgesia and tolerance are actually age-dependent. For example, the expression and functions of endogenous opioid peptides, multiple types of opioid receptors, G protein subunits that couple to opioid receptors, and regulators of G protein signaling (RGS proteins) change with development and age. Other signaling systems that are critical to opioid tolerance development, such as N-methyl-D-aspartic acid (NMDA) receptors, also undergo age-related changes. It is plausible that the age-dependent expression and functions of molecules within and related to the opioid signaling pathways, as well as age-dependent cellular activity such as agonist-induced opioid receptor internalization and desensitization, eventually lead to significant age-dependent changes in opioid analgesia and tolerance development.

Mas-related Gene X2 (MrgX2) Is a Novel G Protein-coupled Receptor for the Antimicrobial Peptide LL-37 in Human Mast Cells: RESISTANCE TO RECEPTOR PHOSPHORYLATION, DESENSITIZATION, AND INTERNALIZATION

Human LL-37 is a multifunctional antimicrobial peptide that promotes inflammation, angiogenesis, wound healing, and tumor metastasis. Most effects of LL-37 are mediated via the activation of the cell surface G protein-coupled receptor FPR2 on leukocytes and endothelial cells. Although LL-37 induces chemotaxis, degranulation, and chemokine production in mast cells, the receptor involved and the mechanism of its regulation remain unknown. MrgX2 is a member of Mas-related genes that is primarily expressed in human dorsal root ganglia and mast cells. We found that a human mast cell line LAD2 and CD34(+) cell-derived primary mast cells, which natively express MrgX2, responded to LL-37 for sustained Ca(2+) mobilization and substantial degranulation. However, an immature human mast cell line, HMC-1, that lacks functional MrgX2 did not respond to LL-37. shRNA-mediated knockdown of MrgX2 in LAD2 mast cell line and primary CD34(+) cell-derived mast cells caused a substantial reduction in LL-37-induced degranulation. Furthermore, mast cell lines stably expressing MrgX2 responded to LL-37 for chemotaxis, degranulation, and CCL4 production. Surprisingly, MrgX2 was resistant to LL-37-induced phosphorylation, desensitization, and internalization. In addition, shRNA-mediated knockdown of the G protein-coupled receptor kinases (GRK2 and GRK3) had no effect on LL-37-induced mast cell degranulation. This study identified MrgX2 as a novel G protein-coupled receptor for the antibacterial peptide LL-37 and demonstrated that unlike most G protein-coupled receptors it is resistant to agonist-induced receptor phosphorylation, desensitization, and internalization.

Competitive binding at a nicotinic receptor transmembrane site of two α7-selective positive allosteric modulators with differing effects on agonist-evoked desensitization

Positive allosteric modulators (PAMs) of nicotinic acetylcholine receptors (nAChRs) have attracted considerable interest as a novel area of therapeutic drug discovery. Two types of α7-selective PAMs have been identified (type I and type II). Whilst both potentiate peak agonist-induced responses, they have different effects on the rate of agonist-induced receptor desensitization. Type I PAMs have little or no effect on the rapid rate of desensitization that is characteristic of α7 nAChRs, whereas type II PAMs cause dramatic slowing of receptor desensitization. Previously, we have obtained evidence indicating that PNU-120596, a type II PAM, causes potentiation by interacting with an allosteric transmembrane site. In contrast, other studies have demonstrated the importance of the ‘M2–M3 segment’ in modulating the effects of the type I PAM NS1738 and have led to the proposal that NS1738 may interact with the extracellular N-terminal domain. Here, our aim has been to compare the mechanism of allosteric potentiation of α7 nAChRs by NS1738 and PNU-120596. Functional characterization of a series of mutated α7 nAChRs indicates that mutation of amino acids within a proposed intrasubunit transmembrane cavity have a broadly similar effect on these two PAMs. In addition, we have employed a functional assay designed to examine the ability of ligands to act competitively at either the orthosteric or allosteric binding site of α7 nAChRs. These data, together with computer docking simulations, lead us to conclude that both the type I PAM NS1738 and the type II PAM PNU-120596 bind competitively at a mutually exclusive intrasubunit transmembrane site.

P.4.001 Mechanism and behavioural consequences of agonist-induced upregulation of alpha 7 nicotinic acetylcholine receptor levels in the rat brain

Positive allosteric modulators (PAMs) of nicotinic acetylcholine receptors (nAChRs) have attracted considerable interest as a novel area of therapeutic drug discovery. Two types of α7-selective PAMs have been identified (type I and type II). Whilst both potentiate peak agonist-induced responses, they have different effects on the rate of agonist-induced receptor desensitization. Type I PAMs have little or no effect on the rapid rate of desensitization that is characteristic of α7 nAChRs, whereas type II PAMs cause dramatic slowing of receptor desensitization. Previously, we have obtained evidence indicating that PNU-120596, a type II PAM, causes potentiation by interacting with an allosteric transmembrane site. In contrast, other studies have demonstrated the importance of the ‘M2–M3 segment’ in modulating the effects of the type I PAM NS1738 and have led to the proposal that NS1738 may interact with the extracellular N-terminal domain. Here, our aim has been to compare the mechanism of allosteric potentiation of α7 nAChRs by NS1738 and PNU-120596. Functional characterization of a series of mutated α7 nAChRs indicates that mutation of amino acids within a proposed intrasubunit transmembrane cavity have a broadly similar effect on these two PAMs. In addition, we have employed a functional assay designed to examine the ability of ligands to act competitively at either the orthosteric or allosteric binding site of α7 nAChRs. These data, together with computer docking simulations, lead us to conclude that both the type I PAM NS1738 and the type II PAM PNU-120596 bind competitively at a mutually exclusive intrasubunit transmembrane site.

Purinoceptor Signaling in Arterial Smooth Muscle is Regulated by G-Protein-Coupled Recptor Kinase-2

The regulation of arterial smooth muscle cell (SMC) contraction by adenine and uridine nucleotides, plays a key role in controlling systemic blood pressure. In SMCs, UTP activates P2Y receptors (subtypes 2/4/6), which couple via Gαq/11-proteins to stimulate phospholipase C, increasing IP3/Ca2+ concentrations and leading to SMC contraction. Continuous or repeated receptor stimulation reduces responsiveness to further stimulation, a process termed desensitization. Receptor desensitization is often regulated by G protein-coupled receptor kinases (GRKs), which phosphorylate receptors, enhancing their interaction with β-arrestins and uncoupling them from G-proteins.We investigated the regulation of receptors responding to UTP, which mediates concentration-dependent contraction in rat mesenteric arteries. To characterize adaptations that occur on repeated UTP additions, changes in IP3 and [Ca2+]i were assessed using single-cell imaging. Receptor desensitization was assessed by challenging mesenteric SMCs with an EC50 concentration of UTP (10 μM) for 30 sec before (R1) and after (R2) the addition of a maximal UTP concentration (Rmax, 100 μM, 30 sec) with 5 min washout periods. The change in R2 relative to R1 was used to characterize P2Y receptor desensitization. By extending the washout period after Rmax a time-dependent recovery of IP3/Ca2+ responses were observed. To evaluate the involvement of individual GRKs in this process, cells were transfected with catalytically-inactive, dominant-negative GRK mutants.Using IP3 generation to indicate receptor recovery (R2/R1%), over-expression of D110A,K220RGRK3 (34±4%), K215RGRK5 (38±7%), or K215RGRK6 (29±8%) caused similar reductions in IP3 levels to those in empty-vector-transfected cells (23±5%). In contrast, expression of D110A,K220RGRK2 (58±7%) markedly attenuated receptor desensitization (n=10-19 cells, >3 animals). Furthermore, siRNA-mediated knockdown (>75%) of GRK2 protein also attenuated agonist-induced receptor desensitization compared to control (68±8% versus 29±6%, respectively, n=8-12). In conclusion, this work implicates GRK2 as the pre-eminent GRK isoenzyme regulating UTP signaling in SMCs.

G Protein-coupled Receptor Kinase-mediated Phosphorylation Regulates Post-endocytic Trafficking of the D2 Dopamine Receptor

We investigated the role of G protein-coupled receptor kinase (GRK)-mediated phosphorylation in agonist-induced desensitization, arrestin association, endocytosis, and intracellular trafficking of the D(2) dopamine receptor (DAR). Agonist activation of D(2) DARs results in rapid and sustained receptor phosphorylation that is solely mediated by GRKs. A survey of GRKs revealed that only GRK2 or GRK3 promotes D(2) DAR phosphorylation. Mutational analyses resulted in the identification of eight serine/threonine residues within the third cytoplasmic loop of the receptor that are phosphorylated by GRK2/3. Simultaneous mutation of these eight residues results in a receptor construct, GRK(-), that is completely devoid of agonist-promoted GRK-mediated receptor phosphorylation. We found that both wild-type (WT) and GRK(-) receptors underwent a similar degree of agonist-induced desensitization as assessed using [(35)S]GTPgammaS binding assays. Similarly, both receptor constructs internalized to the same extent in response to agonist treatment. Furthermore, using bioluminescence resonance energy transfer assays to directly assess receptor association with arrestin3, we found no differences between the WT and GRK(-) receptors. Thus, phosphorylation is not required for arrestin-receptor association or agonist-induced desensitization or internalization. In contrast, when we examined recycling of the D(2) DARs to the cell surface, subsequent to agonist-induced endocytosis, the GRK(-) construct exhibited less recycling in comparison with the WT receptor. This impairment appears to be due to a greater propensity of the GRK(-) receptors to down-regulate once internalized. In contrast, if the receptor is highly phosphorylated, then receptor recycling is promoted. These results reveal a novel role for GRK-mediated phosphorylation in regulating the post-endocytic trafficking of a G protein-coupled receptor.

Cholinergic Agonists as Novel Treatments for Schizophrenia: The Promise of Rational Drug Development for Psychiatry

Cholinergic Agonists as Novel Treatments for Schizophrenia: The Promise of Rational Drug Development for Psychiatry

The role of the N-terminal and mid-region residues of substance P in regulating functional selectivity at the tachykinin NK 1 receptor

Previous studies have shown that tachykinin peptide ligands of the tachykinin NK 1 receptor exhibit functional selectivity with respect to signal activation and desensitization. The differences are most dramatic between the naturally occurring peptides substance P (RPKPQQFFGLM-NH 2) and ranatachykinin C (HNPASFIGLM-NH 2). To understand the structural features of the peptides that underlie these differences, four peptide analogs have been designed and tested. The analogs were designed to assess the major structural differences between substance P and ranatachykinin C, including the role of the N-terminal Arg and the substitution of the mid-region Glns with Ala and Ser (Q5 replaced with A and/or Q6 replaced with S). Receptor binding, receptor activation of intracellular calcium fluxes, and receptor desensitization of the rat tachykinin NK 1 receptor were quantified for each ligand. All of the peptides bound to the rat tachykinin NK 1 receptor with high affinity, produced robust calcium signal activation, and led to agonist-induced receptor desensitization. It was found that deletion of the N-terminal Arg of substance P or replacement of either or both Q5 and Q6 altered the functional selectivity of substance P based on the relationship of receptor binding to receptor activation and activation to desensitization. When considered in light of our previously published nuclear magnetic resonance structure data, the data presented herein suggest that the one, five and six positions of the substance P backbone are key structural residues that govern the relative degree of tachykinin peptide-mediated receptor signaling and desensitization.

Role of receptor internalization in the agonist‐induced desensitization of cannabinoid type 1 receptors

Agonist-induced internalization of G protein-coupled receptors (GPCRs) is an important mechanism for regulating signaling transduction of functional receptors at the plasma membrane. We demonstrate here that both caveolae/lipid-rafts- and clathrin-coated-pits-mediated pathways were involved in agonist-induced endocytosis of the cannabinoid type 1 receptor (CB1R) in stably transfected human embryonic kidney (HEK) 293 cells and that the internalized receptors were predominantly sorted into recycling pathway for reactivation. The treatment of CB1 receptors with the low endocytotic agonist Delta9-THC induced a faster receptor desensitization and slower resensitization than the high endocytotic agonist WIN 55,212-2. In addition, the blockade of receptor endocytosis or recycling pathway markedly enhanced agonist-induced CB1 receptor desensitization. Furthermore, co-expression of phospholipase D2, an enhancer of receptor endocytosis, reduced CB1 receptor desensitization, whereas co-expression of a phospholipase D2 negative mutant significantly increased the desensitization after WIN 55,212-2 treatment. These findings provide evidences for the importance of receptor endocytosis in counteracting CB1 receptor desensitization by facilitating receptor reactivation. Moreover, in primary cultured neurons, the low endocytotic agonist Delta9-THC or anandamide exhibited a greater desensitization of endogenous CB1 receptors than the high endocytotic agonist WIN 55,212-2, CP 55940 or 2-arachidonoyl glycerol, indicating that cannabinoids with high endocytotic efficacy might cause reduced development of cannabinoid tolerance to some kind cannabinoid-mediated effects.

Physiological changes in GRK2 regulate CCL2‐induced signaling to ERK1/2 and Akt but not to MEK1/2 and calcium

G protein-coupled receptor (GPCR) kinase 2 (GRK2) regulates G protein-coupled receptor signaling via agonist-induced receptor phosphorylation and desensitization. GRK2 can also modulate cellular activation by interacting with downstream signaling molecules. The intracellular GRK2 level changes during inflammatory conditions. We investigated how IL-1beta-induced changes in endogenous GRK2 expression influence chemokine receptor signaling in primary astrocytes. Culturing astrocytes with IL-1beta for 24 h induced a 2-3-fold increase in GRK2 and decreased C-C chemokine ligand 2 (CCL2)-induced ERK1/2 activation. Conversely, the 45% decrease in GRK2 expression in astrocytes from GRK2+/- animals resulted in a more pronounced CCL2-induced ERK1/2 phosphorylation. Increased GRK2 inhibited CCL2-induced Akt phosphorylation at Thr308 and Ser473 as well as pPDK-1 translocation. In contrast, altered GRK2 levels did not change the CCL2-induced increase in intracellular calcium or MEK1/2 phosphorylation. These data suggest that altered GRK2 expression modulates chemokine signaling downstream of the receptor. We found that GRK2 kinase activity was not required to decrease chemokine-induced ERK1/2 phosphorylation, whereas regulation of CCL2-induced Akt phosphorylation did require an active GRK2 kinase domain. Collectively, these data suggest that changes in endogenous GRK2 expression in primary astrocytes regulate chemokine receptor signaling to ERK1/2 and to PDK-1-Akt downstream of receptor coupling via kinase-dependent and kinase-independent mechanisms, respectively.

N-Glycosylation of the Human κ Opioid Receptor Enhances Its Stability but Slows Its Trafficking along the Biosynthesis Pathway

We examined glycosylation of FLAG-hKOR expressed in CHO cells and determined its functional significance. FLAG-hKOR was resolved as a broad and diffuse 55-kDa band and a less diffuse 45-kDa band by immunoblotting, indicating that the receptor is glycosylated. Endoglycosidase H cleaved the 45-kDa band to approximately 38 kDa but did not change the 55-kDa band, demonstrating that the 45-kDa band is N-glycosylated with high-mannose or hybrid-type glycan. Peptide-N-glycosidase F digestion of solubilized hKOR or incubation of cells with tunicamycin resulted in two species of 43 and 38 kDa, suggesting that the 43-kDa band is O-glycosylated. FLAG-hKOR was reduced to lower Mr bands by neuraminidase and O-glycosidase, indicating that the hKOR contains O-linked glycan. Mutation of Asn25 or Asn39 to Gln in the N-terminal domain reduced the Mr by approximately 5 kDa, indicating that both residues were glycosylated. The double mutant hKOR-N25/39Q was resolved as a 43-kDa (mature form) and a 38-kDa (intermediate form) band. When transiently expressed, hKOR-N25/39Q had a lower expression level than the wild type. In CHO cells stably expressing the hKOR-N25/39Q, pulse-chase experiments revealed that the turnover rate constants (ke) of the intermediate and mature forms were approximately 3 times those of the wild type. In addition, the maturation rate constant (ka) of the 43-kDa form of hKOR-N25/39Q was 6 times that of the mature form of the wild type. The hKOR-N25/39Q mutant showed increased agonist-induced receptor phosphorylation, desensitization, internalization, and downregulation, without changing ligand binding affinity or receptor-G protein coupling. Thus, N-glycosylation of the hKOR plays important roles in stability and trafficking along the biosynthesis pathway of the receptor protein as well as agonist-induced receptor regulation.

Knock-out Mice Reveal the Contributions of P2Y and P2X Receptors to Nucleotide-induced Ca2+ Signaling in Macrophages

Immune cell function is modulated by changes in extracellular nucleotide levels. Here we used reverse transcription-PCR analyses, single cell Ca2+ imaging, and knock-out mice to define the receptors mediating nucleotide-induced Ca2+ signaling in resident peritoneal macrophages. In Ca2+-free buffer, the potent (K0.5<1 microm) stimulatory effect of UTP (or ATP) on endoplasmic reticulum (ER) Ca2+ release was abolished in cells isolated from P2Y2/P2Y4 double knock-out mice. Moreover, P2Y4(0/-), but not P2Y2-/-, macrophages responded to UTP. In P2Y2-/- macrophages, we could elicit Ca2+ responses to "pure" P2X receptor activation by applying ATP in buffer containing Ca2+. Purified UDP and ADP were ineffective agonists, although modest UDP-induced Ca2+ responses could be elicited in macrophages after "activation" with lipopolysaccharide and interferon-gamma. Notably, in Ca2+-free buffer, UTP-induced Ca2+ transients decayed within 1 min, and there was no response to repeated agonist challenge. Measurements of ER [Ca2+] with mag-fluo-4 showed that ER Ca2+ stores were depleted under these conditions. When extracellular Ca2+ was available, ER Ca2+ stores refilled, but Ca2+ increased to only approximately 40% of the initial value upon repeated UTP challenge. This apparent receptor desensitization persisted in GRK2+/- and GRK6-/- macrophages and after inhibition of candidate kinases protein kinase C and calmodulin-dependent kinase II. Initial challenge with UTP also reduced Ca2+ mobilization by complement component C5a (and vice versa). In conclusion, homologous receptor desensitization is not the major mechanism that rapidly dampens Ca2+ signaling mediated by P2Y2, the sole Gq-coupled receptor for UTP or ATP in macrophages. UDP responsiveness (P2Y6 receptor expression) increases following macrophage activation.

Interaction of the μ-Opioid Receptor with Synaptophysin Influences Receptor Trafficking and Signaling

There is increasing evidence that the signal transduction of opioid receptors is modulated by receptor-associated proteins. In the search for proteins regulating mu-opioid receptor (MOPr) endocytosis, synaptophysin was found to bind to the rat micro-opioid receptor in yeast two-hybrid assay. Coimmunoprecipitation experiments and bioluminescence resonance energy transfer assays confirmed that the micro-opioid receptor constitutively interacts with synaptophysin in human embryonic kidney 293 cells overexpressing MOPr and synaptophysin. In this study, we show that overexpression of synaptophysin enhances the micro-opioid receptor endocytosis. One explanation for the observed effects is that synaptophysin recruits dynamin to the plasma membrane, facilitating fission of clathrin-coated vesicles. This suggestion is supported by our finding that overexpression of a synaptophysin truncation mutant, which breaks the interaction between synaptophysin and dynamin, prevents agonist-mediated micro-opioid receptor endocytosis. In addition, the synaptophysin-augmented micro-opioid receptor trafficking leads to attenuated agonist-induced receptor desensitization and faster receptor resensitization. Taken together, our findings strongly suggest that synaptophysin plays an important role in the regulation of micro-opioid receptor trafficking and signaling.

Role of cellular receptor density in the differential agonist-induced receptor desensitization in specific glucagon receptor-transfected HEK-293 clones: Tadashi Ikegami, Bernard Bouscarel, The George Washington University, Washington, DC

Role of cellular receptor density in the differential agonist-induced receptor desensitization in specific glucagon receptor-transfected HEK-293 clones: Tadashi Ikegami, Bernard Bouscarel, The George Washington University, Washington, DC

Endogenous regulator of g protein signaling proteins reduce {mu}-opioid receptor desensitization and down-regulation and adenylyl cyclase tolerance in C6 cells.

Chronic exposure of cells to mu-opioid agonists leads to tolerance which can be measured by a reduced ability to activate signaling pathways in the cell. Cell signaling through inhibitory G proteins is negatively regulated by RGS (regulator of G protein signaling) proteins. Here we examine the hypothesis that the GTPase accelerating activity of RGS proteins, by altering the lifetime of Galpha and Gbetagamma, plays a role in the development of cellular tolerance to mu-opioids. C6 glioma cells were stably transfected with mu-opioid receptor and pertussis toxin (PTX)-insensitive Galpha(o) that was either sensitive or insensitive to endogenous RGS proteins. Cells were treated with PTX to uncouple endogenous Galpha proteins followed by exposure to the mu-opioid agonists [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) or morphine. Receptor desensitization as measured by agonist-stimulated [(35)S]GTPgammaS binding and receptor down-regulation as measured by [(3)H]diprenorphine binding were increased in cells expressing RGS-insensitive Galpha(o). Exposure to high concentrations of morphine or the peptidic mu-opioid agonist DAMGO led to a tolerance to inhibit adenylyl cyclase activity in both cell types with a rapid (30 min) and a slower component. Using a submaximal concentration of DAMGO to induce a reduced level of tolerance, a shift in the concentration-effect curve for DAMGO to inhibit adenylyl cyclase activity was seen in the cells expressing RGS-insensitive Galpha(o), but not in the cells expressing RGS-sensitive Galpha(o), which can be partly explained by an increased supersensitization of the adenylyl cyclase response. The results show that RGS proteins endogenously expressed in C6 cells reduce agonist-induced mu-opioid receptor desensitization, down-regulation, and sensitivity to tolerance to inhibit adenylyl cyclase activity.

GABAergic systems modulate nicotinic receptor-mediated seizures in mice.

The pharmacology of nicotinic receptor-mediated seizures was investigated in C3H mice. Eleven nicotinic agonists and six antagonists were administered centrally (i.c.v.). Epibatidine and epiboxidine were the most potent agonists tested, whereas acetylcholine and the alpha7*-selective compounds 3-(2,4-dimethoxybenzylidene)-anabaseine (GTS-21) and anabasine, were the least potent. Nicotine-induced seizures were blocked by cotreatment with either the nonselective antagonist mecamylamine or the alpha7*-selective antagonist methyllycaconitine. The alpha4beta2*-selective antagonist dihydro-beta-erythroidine was ineffective at blocking seizures. However, high doses of all six antagonists tested were fully efficacious in producing seizures, with d-tubocurarine being the most potent and mecamylamine the least potent. Potential relationships between nicotinic receptor-mediated seizures and drug effects on GABA function were also investigated. No correlation was seen between potencies of the agonists in producing seizures and stimulating [3H]GABA release or between potencies of the antagonists in producing seizures and antagonist inhibition of nicotine-stimulated [3H]GABA release. However, a robust correlation was detected between potencies of the agonists in producing seizures and the IC50 values for inhibition of nicotine-stimulated [3H]GABA release produced by agonist-induced receptor desensitization. We also compared inbred mouse strain sensitivity to nicotine, picrotoxin, bicuculline, and kainate-induced seizures. Robust positive correlations were revealed for nicotine-induced seizures and seizures induced by either picrotoxin or bicuculline, both GABAA receptor antagonists. No correlation was found between nicotine-induced seizures and those induced by the excitatory amino acid receptor agonist kainate. Based on these findings, we present a model for nicotinic receptor-mediated seizures mediated through GABAergic systems.

Intramuscular hypoperfusion, adrenergic receptors, and chronic muscle pain

Abstract: Despite a high prevalence of chronic muscle pain disorders such as fibromyalgia and regional myofascial pain, there is still limited knowledge about the factors that initiate and perpetuate these pain states. Although there are also likely to be downstream neuropathic changes in the central nervous system and spinal cord that sustain and exacerbate the pain states known as fibromyalgia, the focus of this critical review is on studies that examined the connection between both fibromyalgia and regional myofascial pain and sympathetic function. Specifically, we looked at studies that described Raynaud-like symptoms, cardiovascular dysfunction and altered intramuscular perfusion in chronic muscle pain. Our analysis showed that although the first 2 phenomena were intermittently present, a prominent and consistent feature for regional myofascial pain and to a lesser degree for fibromyalgia was intramuscular hypoperfusion. Several hypotheses can be offered why this hypoperfusion exists, and additional studies comparing and contrasting these theories are needed. This review focuses on one of these theories, namely, agonist-induced β-adrenergic receptor desensitization as an explanatory model for hypoperfusion. What cannot be done at this time and is needed in the future is to compare and contrast to what degree the regional muscle pain disorder (myofascial) is similar or different from the more generalized disorder (fibromyalgia). © 2002 by the American Pain Society