guanylyl-imidodiphosphate and Burns

The myocardial response to catecholamines is significantly diminished in many types of shock or heart failure. The guinea pig heart is an ideal model for the study of shock, as it is relatively inexpensive, and the cardiovascular system of the guinea pig most closely resembles that of the human. Using this model, we have developed techniques to characterize and quantitate changes in beta-adrenergic receptors (beta AR) in the guinea pig heart after burn injury. Preliminary experiments were performed to determine the optimum binding conditions, e.g., incubation time and conditions, protein concentrations, rinsing, etc. Additional experiments were conducted using agonists and antagonists to characterize the rank order of potency and stereospecificity of the beta AR. Crude membrane preparations (50 micrograms/250 microliters) from sham-burned and burned hearts were incubated with 8-10 concentrations of 125I-cyanopindolol (10-450 pM) at 37 degrees C for 1 hr. Under these conditions, binding assays were linear with respect to protein concentration and time. Alprenolol (10 microM) was used to determine nonspecific binding. The membrane preparations used in this study bound both agonists and antagonists with a rank order of potency and stereospecificity characteristic of a beta-adrenergic receptor. Finally, agonist competition curves were performed with isoproterenol in the presence and absence of Gpp(NH)p to determine receptor regulation by the Gs protein. Analysis using computer-assisted techniques suggests that the fraction of high-affinity beta-receptors is significantly reduced after burn injury (41.2 +/- 4.7%) compared to sham-burned controls (54 +/- 2%, P < or = 0.023).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Alprenolol; Animals; Binding, Competitive; Burns; Cell Membrane; Filtration; Guanylyl Imidodiphosphate; Guinea Pigs; Iodine Radioisotopes; Iodocyanopindolol; Isoproterenol; Male; Myocardium; Pindolol; Receptors, Adrenergic, beta; Shock

Previous studies in our laboratory have demonstrated that burn injury (45% total body surface area, 3rd-degree scald burn) diminishes contractile and relaxation function in the isolated perfused guinea pig heart. The mechanisms responsible for the burn-mediated dysfunction are not well understood. Therefore the purpose of this study was to examine the inotropic response to isoproterenol, a beta-adrenergic agonist, and burn-induced alterations in beta-adrenergic receptors (beta-AR) in adult guinea pig hearts. Isoproterenol dose-response curves were generated in isolated perfused hearts from sham-burned and burned guinea pigs. In addition, binding studies were performed using [125I]iodocyanopindolol on hearts from sham-burned and burned guinea pigs. Both the functional response and sensitivity to isoproterenol were significantly diminished 24 h after burn injury. beta-AR density (binding capacity, Bmax) and affinity were determined by Scatchard analysis. Agonist competition curves were performed in the presence or absence of 0.1 mM 5'-guanylyl imidodiphosphate. There was no difference in Bmax in membranes from sham-burned and burned hearts; however, the affinity of beta-AR was significantly decreased after burn injury compared with sham burn [dissociation constant = 32.5 +/- 1.9 (mean +/- SE), n = 10, vs. 26.7 +/- 1.7 pM, n = 10, P = 0.039]. Furthermore, the fraction of receptors in a high-affinity state (those functionally coupled to Gs protein) was significantly decreased after burn injury compared with sham burn (41.2 +/- 4.7%, n = 9, vs. 54 +/- 2%, n = 9, P = 0.023).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adrenergic beta-Agonists; Animals; Binding, Competitive; Burns; Catecholamines; Dose-Response Relationship, Drug; Guanylyl Imidodiphosphate; Guinea Pigs; Isoproterenol; Male; Myocardium; Receptors, Adrenergic, beta