David Daunt

Ligand Efficacy and Potency at Recombinant α2 Adrenergic Receptors: Agonist-Mediated [35s]gtpγs Binding

Abstract Alpha-2 adrenergic receptors (α2 AR) mediate incorporation of guanosine 5′-O-(γ-thio)triphosphate ([35S]GTPγS) into isolated membranes via receptor-catalyzed exchange of [35S]GTPγS for GDP. In the current study, we used [35S]GTPγS incorporation to characterize the intrinsic activity and potency of agonists and antagonists at the cloned mouse α2a/d and human α2a, α2b, and α2c ARs. Full agonists increased [35S]GTPγS binding to membranes by 2- to 3-fold. Antagonists did not increase [35S]GTPγS binding but competitively inhibited agonist-stimulated [35S]GTPγS binding. Compounds with intrinsic activities less than that of the full agonists norepinephrine (NE) or epinephrine (EPI) were capable of antagonizing agonist-stimulated [35S]GTPγS binding. The agonistic properties of a number of α2 AR ligands were characterized at each α2 AR subtype. The rank order of agonist potency for selected compounds at the human receptors (with intrinsic activity compared with NE, defined as 1.0) was: α2a: Dexmedetomidine (0.73) > guanabenz (0.38) > UK-14304 (1.02) > clonidine (0.32) > ST-91 (0.63) > NE (1.00). α2b: Dexmedetomidine (1.10) > clonidine (0.18) > guanabenz (0.71) > NE (1.00) > ST-91 (0.44) > UK-14304 (0.59). α2c: Dexmedetomidine (1.03) > NE (1.00) > UK-14304 (0.75) > ST-91 (0.32) ≥ clonidine (0.23) ≫ guanabenz (0). This report provides a functional characterization of adrenergic receptor ligands at human and mouse α2a/d AR. It also illustrates the utility of [35S]GTPγS incorporation as a functional marker of receptor activation.

Endoscopic computer-enhanced beating heart coronary artery bypass grafting

BACKGROUND Telemanipulation systems have enabled coronary revascularization on the arrested heart. The purpose of this study was to develop a technique for computer-enhanced endoscopic coronary artery bypass grafting on the beating heart. METHODS The operation was performed using the daVinci telemanipulation system. Through three ports, the left internal thoracic artery was harvested in 10 mongrel dogs (30 to 35 kg) using single right-lung ventilation and CO2 insufflation. Through a fourth port an articulating stabilizer, manipulated from a second surgical console, was inserted to stabilize the heart. The left anterior descending artery was snared using silicone elastomer slings anchored in the stabilizer cleats and the graft to coronary artery anastomosis was performed. RESULTS In 7 of 10 dogs, total endoscopic beating heart bypass grafting, cardiac stabilization, arteriotomy, and arterial anastomosis were performed using computer-enhanced technology. Endoscopic stabilization and temporary left anterior descending artery occlusion were well tolerated. All grafts were patent although minor strictures were found in 2. In 3 dogs, the procedure could not be completed (1 ventricular arrhythmia, 1 left atrial laceration, and 1 right ventricular outflow tract compression). CONCLUSIONS Endoscopic beating heart coronary artery bypass grafting is possible in a canine model using a computer-enhanced instrumentation system and articulating stabilization.

Alpha-2 adrenergic agonists as analgesics in horses

Administration of alpha-2 agonists to horses produces a variety of behavioral effects (sedation, somnolence, analgesia), and physiological effects. One of the most significant beneficial effects of administering alpha-2 agonists is the degree of analgesia they provide. Alpha-2 agonists have been the mainstay of analgesia for colic pain in horses since their introduction to clinical veterinary medicine. The increased potency of the more recently introduced alpha-2 agonists allows the provision of analgesia for conditions not previously relieved by other drugs. Unfortunately, there are significant side effects associated with alpha-2 agonist administration. Studies are underway to identify the physiologic effects associated with the stimulation of each alpha-2 receptor subtype, in hopes of developing subtype-specific alpha-2 agonists and antagonists.

Cloning and expression of the mouse homolog of the human α2-C2 adrenergic receptor

Summary Three subtypes of α2 adrenergic receptors have been identified in the human and rat. The subtype located on human chromosome 2 (α2-C2) is unique in that it is expressed mainly in the peripheral tissues and lacks sites for N-linked glycosylation. We isolated the gene encoding the mouse homolog of the human α2-C2 adrenergic receptor (Mα2-2H). The deduced amino acid sequence of the Mα2-2H shows 82% and 96% identity to the human α2-C2 and the rat RNGα2 adrenergic receptors, respectively. Southern blot analysis demonstrated that the Mα2-2H was encoded by a single copy gene and was distinct from the mouse homologs of the α2-C4 and α2-C10 adrenergic receptors. When expressed in COS-7 cells, the Mα2-2H exhibited a pharmacological profile similar to the human α2-C2 and rat RNGα2 receptors.

Regulation of G-Protein Coupled Receptor Traffic by an Evolutionary Conserved Hydrophobic Signal

Plasma membrane (PM) expression of G‐protein coupled receptors (GPCRs) is required for activation by extracellular ligands; however, mechanisms that regulate PM expression of GPCRs are poorly understood. For some GPCRs, such as alpha2c‐adrenergic receptors (α2c‐ARs), heterologous expression in non‐native cells results in limited PM expression and extensive endoplasmic reticulum (ER) retention. Recently, ER export/retentions signals have been proposed to regulate cellular trafficking of several GPCRs. By utilizing a chimeric α2a/α2c‐AR strategy, we identified an evolutionary conserved hydrophobic sequence (ALAAALAAAAA) in the extracellular amino terminal region that is responsible in part for α2c‐AR subtype‐specific trafficking. To our knowledge, this is the first luminal ER retention signal reported for a GPCR. Removal or disruption of the ER retention signal dramatically increased PM expression and decreased ER retention. Conversely, transplantation of this hydrophobic sequence into α2a‐ARs reduced their PM expression and increased ER retention. This evolutionary conserved hydrophobic trafficking signal within α2c‐ARs serves as a regulator of GPCR trafficking.

Supportive Therapy in the Anesthetized Horse

In conclusion, vigilant supportive care is necessary to prevent morbidity and death in the anesthetized horse. Because some of the equipment and drugs are specialized and the consequences of some postanesthetic complications are severe, availability of those items must be confirmed prior to anesthesia. Proper positioning and padding will help to reduce the incidence of postanesthetic myopathy-neuropathy syndrome in these large patients. Adequate tissue perfusion is important and can be achieved by controlling anesthetic depth, increasing intravascular volume with fluid administration, and by administering sympathomimetic agents.

Validation of in vivo MR measurement of oxygen saturation after resuscitation with a hemoglobin- based oxygen carrier in a rabbit model

RATIONALE AND OBJECTIVES The authors tested whether noninvasive magnetic resonance (MR) oximetry is accurate in the in vivo measurement of oxygen saturation in a stroma-free, hemoglobin-based oxygen carrier (HBOC). MATERIALS AND METHODS A central venous catheter was placed in the inferior vena cava (IVC) of 10 New Zealand white rabbits (weight range, 2.5-3.2 kg). Each rabbit underwent removal of 20% of blood volume followed by resuscitation with 10 mL/kg of bovine HBOC-200. Oxygen saturation of the blood mixture was measured in vivo at the IVC with MR oximetry, with separate in vitro calibration for each animal. Blood drawn from the IVC was measured with ex vivo oximetry, which was used as the standard of reference. The in vivo and ex vivo measurements were compared. RESULTS There was no significant difference (P > .1) between measurements obtained with MR oximetry and ex vivo oximetry. The results with in vivo MR oximetry demonstrated excellent correlation with those from ex vivo oximetry (r = 0.99) over a wide range of physiologic oxygen saturation values (16.7%-74.9%) in venous blood. CONCLUSION Noninvasive in vivo MR measurement of oxygen saturation is valid for whole blood mixed with stroma-free hemoglobin. Therefore, MR oximetry may be clinically useful for assessing the oxygenation status in patients resuscitated with a HBOC.