Lee E. Limbird

Central Hypotensive Effects of the α2a-Adrenergic Receptor Subtype

α2-Adrenergic receptors (α2ARs) present in the brainstem decrease blood pressure and are targets for clinically effective antihypertensive drugs. The existence of three α2AR subtypes, the lack of subtype-specific ligands, and the cross-reactivity of α2AR agonists with imidazoline receptors has precluded an understanding of the role of individual α2AR subtypes in the hypotensive response. Gene targeting was used to introduce a point mutation into the α2aAR subtype in the mouse genome. The hypotensive response to α2AR agonists was lost in the mutant mice, demonstrating that the α2aAR subtype plays a principal role in this response.

The α2a Adrenergic Receptor Subtype Mediates Spinal Analgesia Evoked by α2 Agonists and Is Necessary for Spinal Adrenergic–Opioid Synergy

Agonists acting at α2 adrenergic and opioid receptors have analgesic properties and act synergistically when co-administered in the spinal cord; this synergy may also contribute to the potency and efficacy of spinally administered morphine. The lack of subtype-selective pharmacological agents has previously impeded the definition of the adrenergic receptor subtype(s) mediating these effects. We therefore exploited a genetically modified mouse line expressing a point mutation (D79N) in the α2a adrenergic receptor (α2aAR) to investigate the role of the α2aAR in α2 agonist-evoked analgesia and adrenergic–opioid synergy. In the tail-flick test, intrathecal administration of UK 14,304, a nonsubtype-selective α2AR agonist, had no analgesic effect in D79N mice, whereas the analgesic potency of morphine (intrathecal) in this assay was not affected by the mutation. The mutation also decreased α2-agonist-mediated spinal analgesia and blocked the synergy seen in wild-type mice with both the δ-opioid agonist deltorphin II and the μ-opioid agonist [d-ALA2,N-Me-Phe4,Gly-ol5]-Enkephalin (DAMGO) in the substance P behavioral test. In addition, the potency of spinally administered morphine was decreased in this test, suggesting that activation of descending noradrenergic systems impinging on the α2aAR contributes to morphine-induced spinal inhibition in this model. These results demonstrate that the α2aAR subtype is the primary mediator of α2 adrenergic spinal analgesia and is necessary for analgesic synergy with opioids. Thus, combination therapies targeting the α2aAR and opioid receptors may prove useful in maximizing the analgesic efficacy of opioids while decreasing total dose requirements.

G protein-coupled receptor interacting proteins: Emerging roles in localization and signal transduction

The mechanism by which G protein-coupled receptors (GPCRs) translate extracellular signals into cellular changes initially was envisioned as a simple linear model: activation of the receptor by agonist binding leads to dissociation of the heterotrimeric GTP-binding G protein into its alpha and betagamma subunits, both of which can activate or inhibit various downstream effector molecules. The plethora of recently described multidomain scaffolding proteins and accessory/chaperone molecules that interact with GPCR, including GPCR themselves as homo- or heterodimers, provides for diverse molecular mechanisms for ligand recognition, signalling specificity, and receptor trafficking. This review will summarize the recently described GPCR-interacting proteins and their individual functional roles, as understood. Implicit in the search for the functional relevance of these interactions is the expectation that enhancement or disruption of target cell-specific events could serve as highly selective therapeutic opportunities.

The Importance of Identification of the Myocardial-Specific Isoenzyme of Creatine Phosphokinase (MB Form) in the Diagnosis of Acute Myocardial Infarction

Serial plasma determinations of the isoenzymes of CPK were performed in all patients (376) admitted to a coronary care unit during a 12-month period with diagnosis of possible acute myocardial infarction. Results were compared with data from other enzyme studies and from the electrocardiogram. An attempt was made to determine the incidence of falsely positive CPK-MB (myocardial-specific form). “No acute infarction” was diagnosed in all patients in whom neither total CPK nor the isoenzymes of LDH indicated myocardial necrosis, and in whom there were no QRS changes on ECG. Incidence of falsely negative CPK isoenzyme data was also determined. All patients, in whom total CPK was transiently elevated, and LDH1 exceeded LDH2, and new QRS changes occurred, were termed “definite” acute infarction. CPK-MB form was present in all 55 of these (0% false negative). Therefore, determination of the isoenzymes of CPK by this method provides both a sensitive and specific indication of acute myocardial infarction.

β-Adrenergic receptors: Evidence for negative cooperativity

Abstract The specific binding of [ 3 H] (−)alprenolol to sites in frog erythrocyte membranes provides a tool for directly assessing ligand binding to adenylatecyclase coupled β-adrenergic receptors. Hill Plots of such binding data yield slopes (n H =“Hill Coefficients”) less than 1.0, suggesting that negatively cooperative interactions among the β-adrenergic receptors may occur. The existence of such negative cooperativity was confirmed by a direct kinetic method. The dissociation of receptor bound [ 3 H] (−)alprenolol was studied under two conditions: 1) with dilution of the ligand-receptor complex sufficient to prevent rebinding of the dissociated tracer and 2) with this same dilution in the presence of excess unlabeled (−)alprenolol. If the sites are independent, the dissociation rates must be the same in both cases. However, the presence of (−)alprenolol increases the rate of [ 3 H] (−)alprenolol dissociation, indicating that negatively cooperative interactions among the β-adrenergic receptor binding sites do occur.

Localization and trafficking of α2-adrenergic receptor subtypes in cells and tissues

The three alpha2-adrenergic receptor (alpha2AR) subtypes, all of which couple to multiple effectors via Gi/Go proteins, perform various functions, including the mediation of decreases in adenylyl cyclase activity, activation of receptor-mediated K+ channels, and inhibition of voltage-gated Ca2+ channels. The alpha2ARs are polarized in many target cells, such as neurons in the peripheral and central nervous system and in intestinal and renal epithelia. Precise targeting and polarization of molecules are crucial for many physiological processes, and may confer a degree of specificity that, in the case of the adrenergic receptors, could represent a reasonable strategy by which catecholamines coordinate cellular function in a highly specific way. Receptors also redistribute in response to agonist occupancy by means of sequestration, endocytosis, recycling, or, alternatively, down-regulation (degradation). The focus of this review is to compare the similarities and differences among the three alpha2AR subtypes in terms of specificity, signaling, and trafficking. It is anticipated that a molecular understanding of receptor trafficking will lead to novel therapeutic strategies for diseases linked to aberrant adrenergic receptor function or localization.

Stimulation of Mitogen-activated Protein Kinase by G Protein-coupled α2-Adrenergic Receptors Does Not Require Agonist-elicited Endocytosis

Agonist-elicited receptor sequestration is strikingly different for the α2A-versus α2B-adrenergic receptor (α2-AR) subtypes; the α2B-AR undergoes rapid and extensive disappearance from the HEK 293 cell surface, whereas the α2A-AR does not (Daunt, D. A., Hurt, C., Hein, L., Kallio, J., Feng, F., and Kobilka, B. K. (1997)Mol. Pharmacol. 51, 711–720; Eason, M. G., and Liggett, S. B. (1992) J. Biol. Chem. 267, 25473–25479). Since recent reports suggest that endocytosis is required for some G protein-coupled receptors to stimulate the mitogen-activated protein (MAP) kinase cascade (Daaka, Y., Luttrell, L. M., Ahn, S., Della Rocca, G. J., Ferguson, S. S., Caron, M. G., and Lefkowitz, R. J. (1998) J. Biol. Chem. 273, 685–688; Luttrell, L. M., Daaka, Y., Della Rocca, G. J., and Lefkowitz, R. J. (1997) J. Biol. Chem. 272, 31648–31656; Ignatova, E. G., Belcheva, M. M., Bohn, L. M., Neuman, M. C., and Coscia, C. J. (1999)J. Neurosci. 19, 56–63), we evaluated the differential ability of these two subtypes to activate MAP kinase. We observed no correlation between subtype-dependent agonist-elicited receptor redistribution and receptor activation of the MAP kinase cascade. Furthermore, incubation of cells with K+-depleted medium eliminated α2B-AR internalization but did not eliminate MAP kinase activation, suggesting that receptor internalization is not a general prerequisite for activation of the MAP kinase cascade via Gi-coupled receptors. We also noted that neither dominant negative dynamin (K44A) nor concanavalin A treatment dramatically altered MAP kinase activation or receptor redistribution, indicating that these experimental tools do not universally block G protein-coupled receptor internalization.

Nitrous Oxide Produces Antinociceptive Response via α2Band/or α2CAdrenoceptor Subtypes in Mice

BACKGROUND Opiate receptors in the periaqueductal gray region and alpha2 adrenoceptors in the spinal cord of the rat mediate the antinociceptive properties of nitrous oxide (N2O). The availability of genetically altered mice facilitates the detection of the precise protein species involved in the transduction pathway. In this study, the authors establish the similarity between rats and mice in the antinociceptive action of N2O and investigate which alpha2 adrenoceptor subtypes mediate this response. METHODS After obtaining institutional approval, antinociceptive dose-response and time-course to N2O was measured in wild-type and transgenic mice (D79N), with a nonfunctional alpha2A adrenoceptor using tail-flick latency. The antinociceptive effect of N2O was tested after pretreatment systemically with yohimbine (nonselective alpha2 antagonist), naloxone (opiate antagonist), L659,066 (peripheral alpha2-antagonist) and prazosin (alpha2B- and alpha2C-selective antagonist). The tail-flick latency to dexmedetomidine (D-med), a nonselective alpha2 agonist, was tested in wild-type and transgenic mice. RESULTS N2O produced antinociception in both D79N transgenic and wild-type litter mates, although the response was less pronounced in the transgenic mice. Antinociception from N2O decreased over time with continuing exposure, and the decrement was more pronounced in the transgenic mice. The antinociceptive response could be dose dependently antagonized by opiate receptor and selective alpha2B-/alpha2C-receptor antagonists but not by a central nervous system-impermeant alpha2 antagonist (L659,066). Whereas dexmedetomidine exhibited no antinociceptive response in the D79N mice, the robust antinociceptive response in the wild-type litter mates could not be blocked by a selective alpha2B-/alpha2C-receptor antagonist. CONCLUSION These data confirm that the antinociceptive response to an exogenous alpha2-agonist is mediated by an alpha2A adrenoceptor and that there appears to be a role for the alpha2B- or alpha2C-adrenoceptor subtypes, or both, in the analgesic response to N2O.

The zeta isoform of 14-3-3 proteins interacts with the third intracellular loop of different alpha2-adrenergic receptor subtypes.

The α2-adrenergic receptors (α2ARs) are localized to and function on the basolateral surface in polarized renal epithelial cells via a mechanism involving the third cytoplasmic loop. To identify proteins that may contribute to this retention, [35S]Met-labeled Gen10 fusion proteins with the 3i loops of the α2AAR (Val217–Ala377), α2BAR (Lys210–Trp354), and α2CAR (Arg248–Val363) were used as ligands in gel overlay assays. A protein doublet of ∼30 kDa in Madin-Darby canine kidney cells or pig brain cytosol (α2B ≥ α2C≫ α2A) was identified. The interacting protein was purified by sequential DEAE and size exclusion chromatography, and subsequent microsequencing revealed that they are the ζ isoform of 14-3-3 proteins. [35S]Met-14-3-3ζ binds to all three native α2AR subtypes, assessed using a solid phase binding assay (α2A≥α2B> α2C), and this binding depends on the presence of the 3i loops. Attenuation of the α2AR-14-3-3 interactions in the presence of a phosphorylated Raf-1 peptide corresponding to its 14-3-3 interacting domain (residues 251–266), but not by its non-phosphorylated counterpart, provides evidence for the functional specificity of these interactions and suggests one potential interface for the α2AR and 14-3-3 interactions. These studies represent the first evidence for G protein-coupled receptor interactions with 14-3-3 proteins and may provide a mechanism for receptor localization and/or coordination of signal transduction.

Localization of G-protein-coupled receptors in health and disease

G-protein-coupled receptors (GPCRs) represent a superfamily of proteins, characterized by seven transmembrane alpha-helices, that signal through interactions with a family of heterotrimeric GTP-binding proteins, referred to as G proteins. The broad range of physiological functions associated with GPCRs indicates that a better understanding of these receptors and their regulation can provide a solid foundation for novel pharmacological interventions in a variety of disease states.