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Clinical Science  |   September 1998
Acute Depression of Myocardial β-Adrenergic Receptor Signaling during Cardiopulmonary Bypass: Impairment of the Adenylyl Cyclase Moiety
Author Notes
  • for the Duke Heart Center Perioperative Desensitization Group; (Booth) Visiting Associate in Anesthesiology, Department of Anesthesiology.
  • (Landolfo) Assistant Professor of Surgery, Department of Surgery.
  • (Chesnut) Laboratory Research Analyst I, Department of Anesthesiology.
  • (Bennett-Guerrero, Gerhardt, Atwell) Cardiac Anesthesiology Fellow, Department of Anesthesiology.
  • (El-Moalem) Assistant Professor of Biostatistics, Department of Community and Family Medicine.
  • (Smith) Assistant Professor of Anesthesiology, Department of Anesthesiology.
  • (Funk) Anesthesiology Research Nurse, Department of Anesthesiology.
  • (Kuhn) Professor of Pharmacology, Department of Pharmacology.
  • (Kwatra) Assistant Professor of Anesthesiology and Pharmacology/Cancer Biology, Department of Anesthesiology.
  • (Schwinn) Professor of Anesthesiology and Surgery; Associate Professor of Pharmacology/Cancer Biology, Department of Anesthesiology.
  • Received from Duke University Medical Center, Durham, North Carolina. Submitted for publication October 29, 1997. Accepted for publication May 4, 1998. Funded in part by a 1996-1997 Duke Heart Center Award (to Dr. Landolfo), American Heart Association Grant-in-Aid #92009270 (to Dr. Schwinn), and National Institutes of Health grants #HL57447, AG00745, and AG11268 (to Drs. Schwinn and Kwatra). Presented in part at the Society of Cardiovascular Anesthesiologists 19th Annual Meeting, Baltimore, Maryland, May 10 - 14, 1997.
  • John Booth and Kevin Landolfo are equal first authors for their role on this project.
  • Address reprint requests to Dr. Schwinn: Box 3094, Department of Anesthesiology, Duke University Medical Center, Durham, North Carolina 27710. Address electronic mail to: Schwi001@mc.duke.edu
Article Information
Clinical Science
Clinical Science   |   September 1998
Acute Depression of Myocardial β-Adrenergic Receptor Signaling during Cardiopulmonary Bypass: Impairment of the Adenylyl Cyclase Moiety
Anesthesiology 9 1998, Vol.89, 602-611.. doi:
Anesthesiology 9 1998, Vol.89, 602-611.. doi:
Key words: Cardiac surgery; catecholamines; myocardium.
This article is featured in "This Month in Anesthesiology." Please see this issue of Anesthesiology, page 7A.
MYOCARDIAL dysfunction is a significant problem that can occur after cardiac surgery. [1] Possible causes of transient myocardial dysfunction after cardiopulmonary bypass (CPB) include incomplete myocardial protection, myocardial stunning, suboptimal revascularization, ongoing going ischemia, and acute myocardial [Greek small letter beta]-adrenergic receptor ([Greek small letter beta] AR) desensitization. Desensitization is an adaptive mechanism in biological systems that dampens receptor responsiveness to agonist exposure and thereby reduces second messenger formation and biological effect. [2] Myocardial [Greek small letter beta] AR desensitization occurs in chronic diseases such as congestive heart failure, where sympathetic stimulation results in twofold increases in plasma norepinephrine levels. [3] However, CPB represents the only clinical situation in which acute myocardial [Greek small letter beta] AR desensitization occurs. [4] 
Previously we found significant decreases in myocardial [Greek small letter beta] AR coupling to adenylyl cyclase after CPB in transmural ventricular myocardial biopsies obtained during a canine model of CPB. [4] In this model, ventricular [Greek small letter beta] AR density did not change significantly during CPB but declined after CPB, suggesting initial acute uncoupling of the [Greek small letter beta] AR from its signal transduction cascade during CPB followed by [Greek small beta] AR downregulation (receptor internalization and destruction). [4] Despite these data, direct evidence for myocardial [Greek small letter beta] AR dysfunction in adult humans during CPB remains controversial. Indirect evidence for reduced responsiveness of [Greek small letter beta] ARs after heart surgery has been demonstrated using a lymphocyte model, although these studies did not control for the use of perioperative inotropic drugs. [5,6] Furthermore, lymphocytes express only [Greek small letter beta]2ARs, whereas human myocytes express both [Greek small letter beta]1-and[Greek small letter beta]2ARs. [7] Therefore, in this human study, we tested two prospective hypotheses. The first hypothesis is that myocardial [Greek small letter beta] AR function (measured as [Greek small letter beta] AR coupling to adenylyl cyclase in the right atrium) is reduced in adults after undergoing CPB. The second prospective hypothesis is that administration of long-term preoperative [Greek small letter beta] AR antagonist therapy prevents intraoperative decreases in [Greek small letter beta] AR function during CPB.
Methods
Clinical Protocol
Patients undergoing elective aortocoronary surgery were enrolled after we received institutional review board approval and informed patient consent. Patient characteristics were recorded, including demographics, history of chronic disease, and preoperative medications. Preoperative cardiac medications were continued until the time of surgery; of note, patients taking long-term [Greek small letter beta] AR antagonists were given their usual dose on the morning of surgery. Patients received oral premedication consisting of 5 - 10 mg methadone and 5 - 10 mg diazepam 1.5 h before surgery. Hemodynamic monitors were placed immediately before anesthesia and included radial and pulmonary artery cannulation. Anesthesia was induced with fentanyl (5 - 15 [micro sign]g/kg) and midazolam (1 - 5 mg) and maintained with continuous drug infusions (0.05 to 0.1 [micro sign]g [middle dot] kg-1[middle dot] min-1fentanyl and 0.5 to 1 [micro sign]g [middle dot] kg-1[middle dot] min-1midazolam). Pancuronium was used for muscle relaxation and then the trachea was intubated. Ventilation was controlled to maintain normocarbia. A small piece of right atrial appendage (30 - 100 mg wet weight) was obtained immediately before CPB at the time of atrial cannulation. During CPB, patients were cooled to 32 [degree sign]C before administration of cardioplegia (crystalloid cardioplegia: 25 g/l dextrose, 100 mEq/l NaCl, 15 mEq/l KCl, 2 mEq/l CaCl2, mEq/l MgCl2, titrated to pH 7.5 at 4 [degree sign]C with Tris buffer; 362 mOsm/l) and aortic cross-clamping. Just before CPB was terminated (approximately 20 - 30 min after release of the aortic cross-clamp), once the patient's core body temperature had returned to 36 [degree sign]C, a second similar atrial biopsy (10 - 30 mg wet weight) was obtained proximal to the atrial cannulation suture line (from the blood-filled atrium side not excluded by the suture, and therefore nonischemic tissue). No inotropic drugs were administered until after the second biopsy was obtained, and the study was discontinued in any patient who required intraoperative [Greek small letter beta]AR antagonists for acute myocardial ischemia. Blood was obtained at the time of atrial biopsies to determine plasma catecholamine concentrations.
Atrial Tissue Samples
Atrial tissue samples were immediately placed in liquid nitrogen and the stored at -70 [degree sign]C until analysis. Samples were weighed, the homogenized (polytron PT3000, Brinkman Instruments, Westbury, NY at maximum speed for 30 s in 2 ml ice-cold lysis buffer (5 mM Tris, 2 mM EDTA) with protease inhibitors (10 [micro sign]g/ml soybean trypsin inhibitor, 10 [micro sign]g/ml benzamidine, and 5 [micro sign]g/ml leupeptin). The solution was filtered through a 210-[micro sign]m mesh filter (Spectra-mesh, VWR, Boston, MA) to remove any nonhomogenized particulate tissue. Further lysis buffer (10 ml) was added and the homogenate centrifuged at 36,000g for 15 min at 4 [degree sign]C; this step was repeated twice to remove retained [Greek small letter beta] AR antagonist (a fact documented in pilot experiments using extremely high concentrations [10-4M] of several clinically used [Greek small letter beta] AR antagonists). The final pellet was suspended in assay buffer (75 mM Tris, 12.5 mM MgCl2, 2 mM EDTA, pH 7.4 with protease inhibitors at 4 [degree sign]C), 20 [micro sign]l/mg tissue wet weight. The protein concentration was determined using the bicinchoninic acid method (Pierce, Rockford, IL) with bovine serum albumin as the standard.
Adenylyl Cyclase Assays
Atrial membrane adenylyl cyclase activities were assessed using the method of Salomon, [8] as modified and described previously. [4] Briefly, 20 [micro sign]l atrial membranes were incubated in triplicate with either water (basal), 100 [micro sign]M isoproterenol (ISO-MAX), 500 nM isoproterenol (ISO-EC50), [9,10] 100 [micro sign]M zinterol (a selective [Greek small letter beta]2AR agonist), 10 mM sodium fluoride (mixed in a glass tube), or 5 mM manganese for 15 min at 37 [degree sign]C in a 50-[micro sign]l reaction mixture (Figure 1). Manganese (as opposed to forskolin) was chosen as the direct stimulant for adenylyl cyclase in these experiments because it stimulates adenylyl cyclase without being influenced by the presence or activity of G proteins. [11,12] The reaction mixture contained the following final drug concentrations: 30 mM Tris, 5 mM MgCl2, 0.8 mM EDTA, 0.12 mM adenosine triphosphate, 0.06 mM guanosine triphosphate, 2.8 mM phosphoenol-pyruvate, 50 [micro sign]g/ml myokinase, 0.1 mM cyclic adenosine monophosphate (cAMP), 10 [micro sign]g/ml pyruvate kinase, and 1 [micro sign]Ci [Greek small letter alpha] [(32) P]adenosine triphosphate. The reaction was stopped with 1 ml stop buffer (360 [micro sign]m adenosine triphosphate, 285 [micro sign]M cAMP, and 25,000 cpm/ml [(3) H]-cAMP. [(32) P]cAMP was isolated by sequential chromatography over Dowex columns using 1 ml alumina, and individual column recovery was normalized based on the recovery of a known amount of [(3) H]cAMP added to the stop buffer; routine recovery is approximately 75 - 80%. Samples were eluted off alumina columns with 0.1 M imidazole into 15 ml scintillation cocktail and counted with a dual-channel liquid scintillation counter (Wallac Inc., Gaithersburg, MD). This resulted in a linear accumulation of [(32) P]cAMP with respect to time, protein, and temperature. Final results were reported as pmol cAMP [middle dot] mg total protein-1[middle dot] 15 min-1.
Figure 1. A schematic of the [Greek small letter beta]-adrenergic receptor ([Greek small letter beta] AR) signal transduction cascade highlighting the site of action for each drug used in adenylyl cyclase assays. ISO, isoproterenol (nonselective [Greek small letter beta] AR agonist); zinterol (selective [Greek small letter beta]2AR agonist); Gs [Greek small letter alpha], [Greek small letter alpha] subunit of stimulatory G protein; [Greek small letter beta][Greek small letter gamma], stimulatory G protein [Greek small letter beta][Greek small letter gamma] subunit; NaF, sodium fluoride (stimulates Gs); AC, adenylyl cyclase; Mn, manganese (stimulates adenylyl cyclase moiety directly); ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate.
Figure 1. A schematic of the [Greek small letter beta]-adrenergic receptor ([Greek small letter beta] AR) signal transduction cascade highlighting the site of action for each drug used in adenylyl cyclase assays. ISO, isoproterenol (nonselective [Greek small letter beta] AR agonist); zinterol (selective [Greek small letter beta]2AR agonist); Gs [Greek small letter alpha], [Greek small letter alpha] subunit of stimulatory G protein; [Greek small letter beta][Greek small letter gamma], stimulatory G protein [Greek small letter beta][Greek small letter gamma] subunit; NaF, sodium fluoride (stimulates Gs); AC, adenylyl cyclase; Mn, manganese (stimulates adenylyl cyclase moiety directly); ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate.
Figure 1. A schematic of the [Greek small letter beta]-adrenergic receptor ([Greek small letter beta] AR) signal transduction cascade highlighting the site of action for each drug used in adenylyl cyclase assays. ISO, isoproterenol (nonselective [Greek small letter beta] AR agonist); zinterol (selective [Greek small letter beta]2AR agonist); Gs [Greek small letter alpha], [Greek small letter alpha] subunit of stimulatory G protein; [Greek small letter beta][Greek small letter gamma], stimulatory G protein [Greek small letter beta][Greek small letter gamma] subunit; NaF, sodium fluoride (stimulates Gs); AC, adenylyl cyclase; Mn, manganese (stimulates adenylyl cyclase moiety directly); ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate.
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Ligand Binding Assays
The [Greek small letter beta] AR density was determined using standard ligand-binding techniques. Briefly, ligand binding was performed in triplicate (20 - 30 [micro sign]g of membrane protein per tube) in a final volume of 500 [micro sign]l assay buffer (75 mM Tris, 12.5 mM MgCl2mM EDTA, pH 7.4 with protease inhibitors [10 [micro sign]g/ml soybean trypsin inhibitor, 10 [micro sign]g/ml benzamidine, and 5 [micro sign]g/ml leupeptin] at 4 [degree sign]C) using a saturating concentration (275 pM) of [(125) I]-cyanopindolol (Dupont, Boston, MA); Propranolol (1 [micro sign]M; Sigma Chemical Co., St. Louis, MO) was used to determine nonspecific binding. The reaction was incubated and agitated for 2 h at room temperature (25 [degree sign]C). Bound [(125) I]-cyanopindolol was seperated from free by rapid vacuum filtration onto glass fiber (GF/C) filters (Whatmann International, Maidstone, UK). Filters were rinsed rapidly three times with 3 or 4 ml ice-cold 50 mM Tris, pH 7.4, using a Brandel cell harvester (Brandel, Gaithersburg, MD) and counted in a gamma counter (Packard, Downers Grove, IL).
Plasma Catecholamines
Blood samples obtained during surgery were collected on ice and immediately centrifuged (834g at 4 [degree sign]C); plasma was placed, using a pipette, into storage vials, and immediately placed in liquid nitrogen; plasma was stored at -70 [degree sign]C until analysis. Catecholamines were assayed by high-pressure liquid chromatography using on-line trace enrichment onto a cation exchange resin, followed by elution onto a C18 column and thin-layer electrochemical detection as previously described by Kilts. [13] Lower limits of assay detection are 10 pg for both norepinephrine and epinephrine; intraassay variability is +/-3-5%.
Statistical Analysis
Changes in serum catecholamine concentrations before CPB and at the end of CPB were tested using paired t tests; P < 0.05 was considered significant. [Greek small letter beta] AR dysfunction was defined a priori as decreased (>or= to 15% based on our original canine study [4]) ISO-MAX or ISO-EC50-stimulated adenylyl cyclase activity.
Mechanisms underlying impaired [Greek small letter beta] AR dysfunction were examined using [Greek small letter beta] AR density as well as isoproterenol-, zinterol-, sodium fluoride-, and manganese-stimulated adenylyl cyclase activity. The mean percentage change was used for the analysis. Power calculations for each hypothesis were performed using a two-tailed paired t test to test the null hypothesis of no mean percentage change using Solo Power Analysis [14] and Muller's software. [15] Because data in some of the adenylyl cyclase subgroups was not normal, logarithmic transformation was performed to ensure normality, followed by analysis using paired t tests and a general linear multivariate model. To determine whether receptor-mediated or G protein-mediated changes in adenylyl cyclase activity differed in magnitude from direct manganese stimulation of the adenylyl cyclase moiety, equality of mean percentage change of variates was tested using a generalized estimating Equation modelRF 16,17* with identity link and exchangeable correlation. To evaluate the effect of long-term use of preoperative [Greek small letter beta] AR antagonists on myocardial [Greek small letter beta] AR dysfunction after CPB, the data were divided into two subsets and characteristics of the two groups were inspected to assess similarity. Unless otherwise stated, data are presented to two significant figures as means +/- SD; because some subgroups of adenylyl cyclase data are not normally distributed, these data are presented as medians [interquartile range].
Results
Patient Characteristics
Fifty-two patients were enrolled in the study. Patient characteristics (demographics, history of chronic disease, and preoperative medications) are shown in Table 1for all 52 patients and for subgroups of patients receiving (n = 31) or not receiving (n = 21) long-term preoperative [Greek small letter beta] AR therapy. The CPB time was 90 +/- 26 min (91 +/- 28 [Greek small letter beta] AR antagonist group, 88 +/- 21 non-[Greek small letter beta] AR antagonist group). The aortic cross-clamp time was 45 +/- 11 min (43 +/- 10 [Greek small letter beta] AR anatagonist group, 46 +/- 14 non-[Greek small letter beta] AR antagonist group), reflecting use of the first 20 - 30 min of CPB to dissect distal coronary artery anastomosis sites and size coronary grafts before the aortic cross-clamp was applied, thus minimizing myocardial ischemic time. No difference in patient characteristics existed between groups, except for the use of preoperative [Greek small letter beta] AR antagonists.
Table 1. Preoperative Patient Characteristics
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Table 1. Preoperative Patient Characteristics
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Plasma Catecholamines and Acute Myocardial [Greek small letter beta] AR Desensitization during Cardiopulmonary Bypass
Plasma catecholamine levels increased significantly (P < 0.005) during CPB (Figure 2). Isoproterenol-stimulated adenylyl cyclase activity decreased 20% during CPB (19% decrease ISO-CE50, P = 0.0032; 21% decrease ISO-MAX, P = 0.0062; Table 2, Figure 3). Zinterol-stimulated adenylyl cyclase activity ([Greek small letter beta](2) AR-selective agonist) was similar to that mediated by an ISO-EC50 concentration, decreasing 24% during CPB (P = 0.0020, Table 2). The CPB also decreased basal (unstimulated) adenylyl cyclase activity (P < 0.0001, Table 2).
Figure 2. Plasma catecholamine concentrations during CPB. Both plasma epinephrine and norepinephrine levels increased significantly during CPB. ***P < 0.005 compared with before cadiopulmonary bypass. Mean +/- SD.
Figure 2. Plasma catecholamine concentrations during CPB. Both plasma epinephrine and norepinephrine levels increased significantly during CPB. ***P < 0.005 compared with before cadiopulmonary bypass. Mean +/- SD.
Figure 2. Plasma catecholamine concentrations during CPB. Both plasma epinephrine and norepinephrine levels increased significantly during CPB. ***P < 0.005 compared with before cadiopulmonary bypass. Mean +/- SD.
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Table 2. Adenylyl Cyclase Activity (pmol cAMP/mg Total Protein/15 min) and [Greek small letter beta] AR Density (fmol/mg Protein) before and after CPB
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Table 2. Adenylyl Cyclase Activity (pmol cAMP/mg Total Protein/15 min) and [Greek small letter beta] AR Density (fmol/mg Protein) before and after CPB
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Figure 3. Myocardial ([Greek small letter beta] AR) function before cardiopulmonary bypass (CPB) and at the end of CPB. (A) Maximal isoproterenol-stimulated [Greek small letter beta] AR responsiveness (100 [micro sign]M, measured by adenylyl cyclase activity) is depressed at the end of CPB (end-CPB) compared with baseline (left axis). In comparison, [Greek small letter beta] AR density remains constant during CPB (right axis). (B) Although patients receiving long-term preoperative [Greek small letter beta] AR antagonist therapy have higher baseline isoproterenol-stimulated adenylyl cyclase activity median effective concentration (EC50), both groups undergo significant myocardial [Greek small letter beta] AR desensitization during CPB (see the text for details); ISO-EC50 data is presented for this comparison. *P < 0.05, **P < 0.01 compared with before CPB. Mean +/- SEM is used for clarity of presentation.
Figure 3. Myocardial ([Greek small letter beta] AR) function before cardiopulmonary bypass (CPB) and at the end of CPB. (A) Maximal isoproterenol-stimulated [Greek small letter beta] AR responsiveness (100 [micro sign]M, measured by adenylyl cyclase activity) is depressed at the end of CPB (end-CPB) compared with baseline (left axis). In comparison, [Greek small letter beta] AR density remains constant during CPB (right axis). (B) Although patients receiving long-term preoperative [Greek small letter beta] AR antagonist therapy have higher baseline isoproterenol-stimulated adenylyl cyclase activity median effective concentration (EC50), both groups undergo significant myocardial [Greek small letter beta] AR desensitization during CPB (see the text for details); ISO-EC50 data is presented for this comparison. *P < 0.05, **P < 0.01 compared with before CPB. Mean +/- SEM is used for clarity of presentation.
Figure 3. Myocardial ([Greek small letter beta] AR) function before cardiopulmonary bypass (CPB) and at the end of CPB. (A) Maximal isoproterenol-stimulated [Greek small letter beta] AR responsiveness (100 [micro sign]M, measured by adenylyl cyclase activity) is depressed at the end of CPB (end-CPB) compared with baseline (left axis). In comparison, [Greek small letter beta] AR density remains constant during CPB (right axis). (B) Although patients receiving long-term preoperative [Greek small letter beta] AR antagonist therapy have higher baseline isoproterenol-stimulated adenylyl cyclase activity median effective concentration (EC50), both groups undergo significant myocardial [Greek small letter beta] AR desensitization during CPB (see the text for details); ISO-EC50 data is presented for this comparison. *P < 0.05, **P < 0.01 compared with before CPB. Mean +/- SEM is used for clarity of presentation.
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To determine whether myocardial [Greek small letter beta] AR dysfunction during CPB occurs at the receptor or more distal in the [Greek small letter beta] AR signal transduction cascade, [Greek small letter beta] AR density, sodium fluoride, and manganese were used. [Greek small letter beta] AR density did not change during CPB (Table 2, Figure 3). Sodium fluoride-stimulated adenylyl cyclase (representing G protein activity) decreased 14% (P = 0.0044, Table 2) and manganese-stimulated adenylyl cyclase activity (representing direct stimulation of the adenylyl cyclase moiety itself) decreased 21% (P = 0.0001, Table 2). Taking into account individual variability in various adenylyl cyclase determinations, the power to detect the observed difference (percentage change) with the final patient number is as follows: ISO-MAX, 81.5% ISO-EC50, 86.4%; zinterol, 90.4%; sodium fluoride, 84.9%; manganese, 99.9%. The observation that neither receptor-mediated nor G protein-mediated decreases in adenylyl cyclase activity were greater than the decrease seen with direct manganese stimulation suggests that the defect is localized to the adenylyl cyclase moiety itself.
Effect of Long-term Preoperative [Greek small letter beta] AR Antagonist Therapy on Acute Myocardial [Greek small letter beta] AR Dysfunction during Cardiopulmonary Bypass
Patients receiving long-term preoperative [Greek small letter beta] AR antagonists had a small but significant increase in atrial [Greek small letter beta] AR density at baseline (before CPB, P = 0.012, Table 2). Consistent with this increased [Greek small letter beta] AR density, ISO-stimulated adenylyl cyclase activity was higher before CPB in patients receiving long-term preoperative [Greek small letter beta] AR antagonists (using assay conditions in which antagonist is no longer present; Figure 3B). However, myocardial [Greek small letter beta] AR response to CPB was similar to both groups, with significantly decreased adenylyl cyclase activity without change in [Greek small letter beta] AR density regardless of long-term preoperative [Greek small letter beta]AR antagonist therapy (Table 2).
Discussion
This study shows that atrial myocardial [Greek small letter beta] AR signal transduction is reduced during CPB in adult humans. providing support for our first hypothesis. Dampened receptor responsiveness (also called desensitization) can occur as a result of changes in the receptor, in proteins involved in the signal transduction pathway, or both. Components of the [Greek small letter beta] AR signal transduction pathway include the cell-surface [Greek small letter beta] AR, intermediary G protein, and the effector (adenylyl cyclase moiety; Figure 1). Three mechanisms are involved in [Greek small letter beta] AR desensitization at the receptor level - uncoupling (disruption of receptor/G protein complex), sequestration (movement of receptor from the cell surface to intracellular compartments), and downregulation (decrease in receptor number resulting from a complex interplay between depressed receptor synthesis and destruction of sequestered receptors). [2,18] These processes are thought to result from receptor phosphorylation by various kinases, such as second messenger stimulated kinases protein kinase A and protein kinase C, as well as G protein-coupled receptor kinases. [18] Receptor desensitization is considered homologous when only the stimulated receptor is desensitized, or heterologous when multiple receptors systems are desensitized indirectly as a result of second messenger activation. Although heterologous desensitization historically has been defined at the receptor level, more recently the definition of heterologous desensitization has been expanded to include impairment of nonreceptor components of the signal transduction cascade [19]; in the [Greek small letter beta] AR signal transduction system, this reflects changes in G protein subtypes and adenylyl cyclase isoforms. [19,20] 
Work in the past two decades examining mechanisms underlying chronic [Greek small letter beta] AR desensitization in heart failure suggests that sympathetic activation leads to increased transmyocardial concentrations of norepinephrine [21] and dampened [Greek small letter beta] AR signal transduction. [2,22] Because these changes occur at both the receptor (decreased number and function) and signal transduction pathway (increased levels of Gi and decreased levels of adenylyl cyclase V and VI), both homologous and heterologous desensitization occurs with chronic heart failure. [22-25] Despite strong evidence for chronic [Greek small letter beta] AR desensitization, a paucity of data exists indicating that acute myocardial [Greek small letter beta] AR desensitization occurs clinically. Because plasma catecholamines increase dramatically during CPB (from 2 to 20 times), [4,26] and application of the aortic cross-clamp results in myocardial ischemia (with resultant release of norepinephrine from myocardial sympathetic nerve fibers) despite protective maneuvers such as hypothermic cardioplegic arrest, CPB provides a potentially good model to examine acute myocardial [Greek small letter beta] AR desensitization. Previously we found acute myocardial [Greek small letter beta] AR desensitization in a canine model of CPB. [4] However, because of species differences in myocardial [Greek small letter beta] AR subtype expression, functional activity, and regulation, [10,27,28] documentation that similar process are present in humans is important. One human study has reported the occurrence of acute myocardial [Greek small letter beta] AR dysfunction in children during heart surgery using atrial biopsies, [29] although another study using lymphocytes contradicts this finding. [30] Furthermore, the relevance of these studies for adults is unclear, because the distribution of myocardial [Greek small letter beta] AR subtypes and coupling to adenylyl cyclase is altered in children with congenital heart disease. [31,32] In the current study, although the CPB period was relatively short, doubling of both plasma norepinephrine and epinephrine occurred. Forty-five minutes of aortic cross-clamping potentially exposed myocardial [Greek small letter beta] ARs to ischemia and elevated myocardial catecholamines, followed by release of the cross-clamp and exposure to blood containing the highest plasma catecholamine concentrations present during CPB. Myocardial ischemia has been shown to variability affect [Greek small letter beta] AR activity, with most studies demonstrating initial acute increases in [Greek small letter beta] AR signaling resulting from sympathetic stimulation, externalization of sequestered receptors, and acute inhibition of Gi activity [33]; however more prolonged ischemia (10-60 min) results in dampened [Greek small letter beta] AR function. [33] When [Greek small letter beta] AR function was examined during CPB in the current study, decreased isoproterenol-stimulated adenylyl cyclase activity occurred, providing evidence of acute impairment of the myocardial [Greek small letter beta] AR signal transduction pathway during CPB in adults undergoing heart surgery.
To elucidate the underlying mechanism(s) for myocardial [Greek small letter beta] AR dysfunction during CPB, we first examined [Greek small letter beta] AR density. No change in receptor density occurred with CPB. This result is not surprising because crude membrane preparations isolate [Greek small letter beta] ARs from both the cell membrane and sequestered vesicles. Many studies have shown that 3 h of agonist exposure are required before significant [Greek small letter beta] AR downregulation is apparent using [(125) I]-cyanopindolol in crude membrane preparations. Previously we detected downregulation in a canine model of CPB only at the final time point 30 min after CPB; this time point represented exactly 3 h of agonist exposure. [4] However, in the current study CPB only lasted 90 min, so constant [Greek small letter beta] AR density was expected (demonstrating lack of downregulation). Two methods can be used to distinguish membrane receptors from sequestered receptors. In whole-cell experiments, comparison of ligand binding with hydrophilic radioligands (which cannot cross the lipid membrane and therefore only bind receptors at the cell surface) and hydrophobic radioligands (which do cross the lipid membrane and therefore bind surface and sequestered receptors) identifies the fraction of sequestered receptors. In harvested tissues, differential centrifugation can also be used to separate membrane fractions from light vesicle fractions; unfortunately there was insufficient tissue available in the current study to perform these experiments. Furthermore, identification of sequestered receptors during CPB does not identify a potentially important subpopulation of receptors present at the cell surface that are acutely uncoupled from G proteins. Thus we chose to examine these two possibilities (uncoupling and sequestration) indirectly using functional assays; dampened [Greek small letter beta] AR function above and beyond that seen for other proteins involved further downstream in the [Greek small letter beta] AR signal transduction cascade (G proteins and adenylyl cyclase moiety) would provide indirect evidence of either uncoupling or sequestration.
To explore [Greek small letter beta] AR function before- and at the end of CPB, we examined receptor, intermediary G protein, and manganese-stimulated adenylyl cyclase and found an [almost equal to] 20% decrease (P < 0.007) in function at all levels after exposure to CPB. Because isoproterenol-stimulated adenylyl cyclase activity (receptor level) is dampened no further than that seen with sodium fluoride (G protein level) or manganese (adenylyl cyclase level), and >80% power existed to detect such a difference at all levels, the defect appears to be localized to the adenylyl cyclase moiety itself. Consistent with this notion, during CPB we also found a decrease in basal adenylyl cyclase activity. Thus heterologous desensitization of the [Greek small letter beta] AR signal transduction pathway occurs during CPB. However, these results to not eliminate the possibility of [Greek small letter beta] AR desensitization at the receptor level. Although human heart contains relatively few spare receptors compared with other animal models, [34] even a few spare receptors might prevent detection of a small population of desensitized receptors. Longer agonist exposure, as might be present during the entire postoperative period, might unmask such [Greek small letter beta] AR desensitization with time. However, our data show that the primary defect associated with CPB appears to reside at the level of the adenylyl cyclase moiety.
A decrease in adenylyl cyclase activity could occur via several mechanisms; these include generalized decreases in catalytic function, decreased total protein concentration, or alterations in specific adenylyl cyclase isoforms. For example, decreased concentrations of adenylyl cyclase types V and VI (predominant isoforms in the heart [24]) occur in chronic pacing-induced heart failure, [25-35] whereas changes in activity of these isoforms occur with activation of PKC, [36] PKA, [19] aging, [37] and in pressure-overloaded failing right ventricles. [38] In our study, preservation of adenylyl cyclase catalytic efficiency (evidenced by increases in manganese-stimulated activity before CPB and at the end of CPB of similar magnitudes) suggests that decreased catalytic function is not the cause of depressed adenylyl cyclase activity after CPB. Rather, changes in isoforms (density or activity) or overall decreased adenylyl cyclase concentrations may be involved. In fact, in a study published while this article was being reviewed suggests that intermittent warm blood cardioplegia preserves myocardial [Greek small letter beta] AR function compared with cold crystalloid cardioplegia [39]; because cold potassium cardioplegia results in increases in intracellular calcium, a condition that inhibits adenylyl cyclase isoform VI present in heart, [24] this provides another possible mechanism for our results. Each of these mechanisms are being investigated in our laboratory.
Administration of anesthetic agents can also alter adenylyl cyclase activity. For example, fentanyl (and other opioids) couple via Gi to inhibit adenylyl cyclase, and so do other compounds generated with ischemia, such as adenosine. With regard to anesthetic agents, these drugs were administered as a continuous infusion during the study, so similar amounts should have been present before and at the end of CPB. However, drugs are less well metabolized during hypothermia; in contrast fentanyl binds to the plastic tubing in the CPB circuit, which tends to result in lower than expected plasma levels during CPB. If fentanyl levels were lower at the end of CPB and significant Gi effects occur with fentanyl anesthesia, then this would have biased our results to show no desensitization. Instead we found very reproducible impaired [Greek small letter beta] AR signalling in every patient. Furthermore, anesthetics and metabolic products are washed out during myocardial membrane preparation in the laboratory. Therefore in this study we do not believe that the anesthetic background influenced the results significantly.
Our second prospective hypothesis was that administration of long-term preoperative [Greek small letter beta] AR antagonists would prevent the reduction in [Greek small letter beta] AR signalling associated with CPB. [Greek small letter beta] AR antagonist therapy has been shown to improve myocardial function in a dose-dependent manner in congestive heart failure. [40-42] Although the exact mechanism(s) for improved myocardial function have not been elucidated, regression of chronic [Greek small letter beta] AR desensitization has been postulated. In support of this mechanism, metoprolol ([Greek small letter beta]1-selectiveantagonist) upregulates [Greek small letter beta] AR receptor density and improves myocardial response to dobutamine. [41,42] However, [Greek small letter beta] AR upregulation is not an absolute requirement for the beneficial effects of [Greek small letter beta] AR antagonists in congestive heart failure because carvedilol improves myocardial function without changing [Greek small letter beta] AR density. [43] In the current study, [approximately] 70% of patients in the [Greek small letter beta] AR antagonist group received long-term metoprolol before surgery. Thus our finding of increased myocardial [Greek small letter beta] AR density and concomitant higher baseline isoproterenol-stimulated adenylyl cyclase activity in patients receiving long-term [Greek small letter beta] AR antagonist therapy is expected (the higher [Greek small letter beta] AR density naturally results in higher adenylyl cyclase activity in an assay in which residual [Greek small letter beta] AR antagonist is eliminated). However, because patients in both groups show statistically significant decreases in isoproterenol-stimulated adenylyl cyclase activity with CPB, the second hypothesis is disproved. An explanation for this finding might be that clinically effective doses of preoperative [Greek small letter beta] AR antagonists may not be present in sufficient concentration during CPB to prevent binding of extremely high concentrations of myocardial catecholamines generated during aortic cross-clamp, [44,45] especially because cold inactivates monoamine oxidase and catechol-O-methyltransferase, [46] further increasing myocardial catecholamine levels. Interestingly, a recent canine study performed in our laboratory shows that acute intraoperative administration of esmolol during CPB prevents acute myocardial [Greek small letter beta] AR desensitization and results in improved myocardial function after CPB. [47] 
In conclusion, we provide evidence that transmembrane myocardial [Greek small letter beta] AR dysfunction occurs in adults during aortocoronary surgery requiring hypothermic CPB, regardless of the administration of long-term preoperative [Greek small letter beta] AR antagonists. The defect appears to be localized to the adenylyl cyclase moiety.
We gratefully acknowledge the secretarial assistance of Beth Barbee and Jessica Harris, technical assistance of Robert D. Symanski, and tireless statistical counseling by Elizabeth DeLong, Ph.D. Members of the Duke Heart Center Perioperative Desensitization Group include Terry L. Ainsworth, R.N., Robert W. Anderson, M.D., Mark P. Anstadt, M.D., Joseph E. Arrowsmith, M.B., Ch.B., Beatrice I. Baldwin, C.R.N.A., Hartmuth B. Bittner, M.D., Fiona M. Clements, M.D., Narda D. Croughwell, C.R.N.A., Duane Davis, M.D., Norbert P. DeBruijn, M.D., Elizabeth R. DeLong, Ph.D., J. Michael DiMaio, M.D., Francis Duhaylongsod, M.D., Joseph M. Forbess, M.D., Donald D. Glower, M.D., Katherine P. Grichnik, M.D., Hilary P. Grocott, M.D., F.R.C.P.C., Steven C. Hendrickson, M.D., James Jaggers, M.D., Robert H. Jones, M.D., Bruce J. Leone, M.D., James E. Lowe, M.D., James R. Mault, M.D., Cary H. Meyers, M.D., Carmelo A. Milano, M.D., Michael G. Mythen, M.D., M.B.B.S., F.R.C.A., Mark F. Newman, M.D., Clarence H. Owen, M.D., David S. Peterseim, M.D., Joseph G. Reves, M.D., Corey T. Sawchuk, M.D., Lynne K. Skaryak, M.D., Robert N. Sladen, M.B., Ch.B., Peter K. Smith, M.D., Barbara E. Tardiff, M.D., Mark Tedder, M.D., Christopher M. Watke, M.D., Blake E. Wendelburg, M.D., and Walter G. Wolfe, M.D.
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Figure 1. A schematic of the [Greek small letter beta]-adrenergic receptor ([Greek small letter beta] AR) signal transduction cascade highlighting the site of action for each drug used in adenylyl cyclase assays. ISO, isoproterenol (nonselective [Greek small letter beta] AR agonist); zinterol (selective [Greek small letter beta]2AR agonist); Gs [Greek small letter alpha], [Greek small letter alpha] subunit of stimulatory G protein; [Greek small letter beta][Greek small letter gamma], stimulatory G protein [Greek small letter beta][Greek small letter gamma] subunit; NaF, sodium fluoride (stimulates Gs); AC, adenylyl cyclase; Mn, manganese (stimulates adenylyl cyclase moiety directly); ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate.
Figure 1. A schematic of the [Greek small letter beta]-adrenergic receptor ([Greek small letter beta] AR) signal transduction cascade highlighting the site of action for each drug used in adenylyl cyclase assays. ISO, isoproterenol (nonselective [Greek small letter beta] AR agonist); zinterol (selective [Greek small letter beta]2AR agonist); Gs [Greek small letter alpha], [Greek small letter alpha] subunit of stimulatory G protein; [Greek small letter beta][Greek small letter gamma], stimulatory G protein [Greek small letter beta][Greek small letter gamma] subunit; NaF, sodium fluoride (stimulates Gs); AC, adenylyl cyclase; Mn, manganese (stimulates adenylyl cyclase moiety directly); ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate.
Figure 1. A schematic of the [Greek small letter beta]-adrenergic receptor ([Greek small letter beta] AR) signal transduction cascade highlighting the site of action for each drug used in adenylyl cyclase assays. ISO, isoproterenol (nonselective [Greek small letter beta] AR agonist); zinterol (selective [Greek small letter beta]2AR agonist); Gs [Greek small letter alpha], [Greek small letter alpha] subunit of stimulatory G protein; [Greek small letter beta][Greek small letter gamma], stimulatory G protein [Greek small letter beta][Greek small letter gamma] subunit; NaF, sodium fluoride (stimulates Gs); AC, adenylyl cyclase; Mn, manganese (stimulates adenylyl cyclase moiety directly); ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate.
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Figure 2. Plasma catecholamine concentrations during CPB. Both plasma epinephrine and norepinephrine levels increased significantly during CPB. ***P < 0.005 compared with before cadiopulmonary bypass. Mean +/- SD.
Figure 2. Plasma catecholamine concentrations during CPB. Both plasma epinephrine and norepinephrine levels increased significantly during CPB. ***P < 0.005 compared with before cadiopulmonary bypass. Mean +/- SD.
Figure 2. Plasma catecholamine concentrations during CPB. Both plasma epinephrine and norepinephrine levels increased significantly during CPB. ***P < 0.005 compared with before cadiopulmonary bypass. Mean +/- SD.
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Figure 3. Myocardial ([Greek small letter beta] AR) function before cardiopulmonary bypass (CPB) and at the end of CPB. (A) Maximal isoproterenol-stimulated [Greek small letter beta] AR responsiveness (100 [micro sign]M, measured by adenylyl cyclase activity) is depressed at the end of CPB (end-CPB) compared with baseline (left axis). In comparison, [Greek small letter beta] AR density remains constant during CPB (right axis). (B) Although patients receiving long-term preoperative [Greek small letter beta] AR antagonist therapy have higher baseline isoproterenol-stimulated adenylyl cyclase activity median effective concentration (EC50), both groups undergo significant myocardial [Greek small letter beta] AR desensitization during CPB (see the text for details); ISO-EC50 data is presented for this comparison. *P < 0.05, **P < 0.01 compared with before CPB. Mean +/- SEM is used for clarity of presentation.
Figure 3. Myocardial ([Greek small letter beta] AR) function before cardiopulmonary bypass (CPB) and at the end of CPB. (A) Maximal isoproterenol-stimulated [Greek small letter beta] AR responsiveness (100 [micro sign]M, measured by adenylyl cyclase activity) is depressed at the end of CPB (end-CPB) compared with baseline (left axis). In comparison, [Greek small letter beta] AR density remains constant during CPB (right axis). (B) Although patients receiving long-term preoperative [Greek small letter beta] AR antagonist therapy have higher baseline isoproterenol-stimulated adenylyl cyclase activity median effective concentration (EC50), both groups undergo significant myocardial [Greek small letter beta] AR desensitization during CPB (see the text for details); ISO-EC50 data is presented for this comparison. *P < 0.05, **P < 0.01 compared with before CPB. Mean +/- SEM is used for clarity of presentation.
Figure 3. Myocardial ([Greek small letter beta] AR) function before cardiopulmonary bypass (CPB) and at the end of CPB. (A) Maximal isoproterenol-stimulated [Greek small letter beta] AR responsiveness (100 [micro sign]M, measured by adenylyl cyclase activity) is depressed at the end of CPB (end-CPB) compared with baseline (left axis). In comparison, [Greek small letter beta] AR density remains constant during CPB (right axis). (B) Although patients receiving long-term preoperative [Greek small letter beta] AR antagonist therapy have higher baseline isoproterenol-stimulated adenylyl cyclase activity median effective concentration (EC50), both groups undergo significant myocardial [Greek small letter beta] AR desensitization during CPB (see the text for details); ISO-EC50 data is presented for this comparison. *P < 0.05, **P < 0.01 compared with before CPB. Mean +/- SEM is used for clarity of presentation.
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Table 1. Preoperative Patient Characteristics
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Table 1. Preoperative Patient Characteristics
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Table 2. Adenylyl Cyclase Activity (pmol cAMP/mg Total Protein/15 min) and [Greek small letter beta] AR Density (fmol/mg Protein) before and after CPB
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Table 2. Adenylyl Cyclase Activity (pmol cAMP/mg Total Protein/15 min) and [Greek small letter beta] AR Density (fmol/mg Protein) before and after CPB
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