Author + information
- Received August 29, 1999
- Revision received March 1, 2000
- Accepted April 13, 2000
- Published online September 1, 2000.
- Antonino Mazzone, MD∗,* (, )
- Iolanda Mazzucchelli, BSc∗,
- Monia Vezzoli, MD∗,
- Elena Ottini, MD∗,
- Carla Auguadro, MD†,
- Alessandra Serio, MD† and
- Colomba Falcone, MD†
- ↵*Reprint requests and correspondence: Dr. Antonino Mazzone, Adjunct Professor of Immunopharmacology, Department of Internal Medicine and Nephrology, University of Pavia, IRCCS, San Matteo Hospital, P.le Golgi 2, 27100 Pavia, Italy
The purpose of this study was to evaluate benzodiazepine receptor expression on leukocytes from patients with symptomatic or silent myocardial ischemia.
Silent myocardial ischemia is frequently observed in patients with coronary artery disease. Pain can be effectively controlled by various endogenous mechanisms. Benzodiazepines and their receptors play key roles in pain, in interactions with peptide opioids, in inflammation and in the response to stress.
The study group consisted of 57 patients with reproducible exercise-induced myocardial ischemia. The presence of a constant behavior in the anginal pain perception during both exercise-induced ischemia and daily life was the most important inclusion criterion. Venous blood samples were taken from all patients to evaluate the expression of peripheral benzodiazepine receptors by flow cytometry. The study cohort was classified into two groups: 24 patients who had anginal pain both at home and during the exercise stress test and 33 patients who were asymptomatic during both daily life and exercise-induced ischemia.
Flow cytometry analysis showed increased expression of peripheral benzodiazepine receptors on all types of leukocytes in the asymptomatic patients. The difference was statistically significant for lymphocytes (p < 0.005), monocytes (p < 0.001) and granulocytes (p < 0.001).
These data show that expression of peripheral benzodiazepine receptors was higher in patients with silent myocardial ischemia than in symptomatic patients.
Coronary artery disease (CAD) is a progressive atherosclerotic process (1) that results in a spectrum of clinical manifestations ranging from asymptomatic and stable angina to the acute coronary syndromes, unstable angina, acute myocardial infarction (MI) and sudden ischemic death (2). The absence of anginal pain during transitory episodes of myocardial ischemia occurring during both daily life (3,4) and stress tests is frequent (5,6); similarly, acute ischemia during MI and during angioplasty-induced coronary occlusion may not be associated with anginal pain (7,8). The mechanisms responsible for silent ischemia are not well understood, and many theories have been advanced (4). The presence or absence of angina may be partly explained by individual differences in the pain threshold (5,6). A generalized hyposensitivity to different painful stimuli and a higher pain threshold have been reported in patients with silent ischemia compared with symptomatic patients (4–6).
The endogenous opioid system plays an important role in pain modulation (7). An increase in beta-endorphin plasma levels, coinciding with an increase in plasma cortisol, has been documented during exercise as part of the physiologic stress response (3,8). Benzodiazepines could antagonize opioid-induced analgesia by enhancing the action of gamma-aminobutyric acid (GABA) on GABAa receptors in the pain modulation circuits (9). Some evidence connects peripheral benzodiazepine receptors (PBRs) with voltage-dependent calcium channel antagonists in relaxing smooth muscle and inhibiting beta-endorphin release in a cultured pituitary cell line (10). In addition, various investigators have shown that benzodiazepine treatment improves control of angina (11) and that PBRs could play a role in the stress response (12), in the interactions of the opioid system (7) and in steroid biosynthesis (13). Benzodiazepines have been shown to interact with opioid antinociception (9). Furthermore, in the last few years, the roles of inflammation and leukocytes in CAD have been underlined (14,15). In peripheral ischemic and inflamed tissue (16), an interaction between immune cell–derived opioids and other receptors localized on sensory nerve terminals can result in strong, clinically measurable analgesia (17–19). Given that PBRs affect opioid-induced antinociception and modulation of pain in animals, we hypothesized that the expression of PBRs on leukocytes is different in patients with angina than in patients without anginal symptoms during myocardial ischemia. We tested this hypothesis by measuring PBRs expression using flow cytometry on various sets of leukocytes in these two groups of patients.
Our study group consisted of 57 men, 24 of whom reported anginal pain both at home and during two exercise stress tests (group P) and 33 of whom were asymptomatic during both daily life and exercise-induced ischemia (group A). The inclusion criteria were evidence of myocardial ischemia during these two exercise stress tests and reproducibility of the same original pain perception or lack of during both exercise-induced ischemia and daily life (patients always symptomatic or patients always asymptomatic). The patients studied were male because the reproducibility of pain perception in women can be confounded by the effects of neurohormonal changes. Patients with intercurrent inflammatory conditions, as well as patients taking nonsteroidal anti-inflammatory drugs, steroids, benzodiazepine or antiepileptic agents were excluded from the study. All tests were performed in pharmacologic washout; calcium channel blocking agents and nitrates were suspended 48 h before. No patient was receiving beta-blocker therapy at the time of the study. Beta-blockers were gradually reduced and stopped one week before our examination. No patient received digitalis. Informed, written consent for all tests was obtained from all subjects. The study was approved by the Ethical Committee of our hospital.
Exercise stress testing
A multistage bicycle ergometric stress test (EST) was performed in all patients. The initial work load was 25 W, and the subsequent stepwise increments were 25 W every 2 min at a pedalling frequency of 60 rpm. A standard 12-lead electrocardiogram (ECG) and blood pressure were recorded in basal conditions, with the patient lying in a supine position for 10 min and then just before the test, at the end of each stage, at the appearance of ECG signs of ischemia or anginal pain, at peak exercise and every 3 min during recovery. Three ECG leads (D3, V5, V6) were continuously monitored and displayed on an oscilloscope throughout the test. During the test, the patients were continuously asked about the onset of angina or other symptoms. A positive ECG response was defined as the occurrence of at least 1 mm ST segment depression, as compared with the baseline tracing. The ischemic threshold was defined as the appearance of 1 mm flat or downsloping ST segment depression 0.08 s after the J point; the angina threshold was defined as the time of the first report of chest pain during the test. EST was stopped when moderate to severe angina, dyspnea, muscle fatigue, ST segment depression >3 mm or major arrhythmias occurred. The rate–pressure product (RPP: heart rate × systolic blood pressure) was calculated in basal conditions, at the ischemic threshold and at peak exercise.
All patients underwent cardiac ventriculography and Sones selective coronary arteriography within 7 ± 5 days after the exercise stress test. Left ventriculography was performed before coronary arteriography in the 30° right anterior oblique projection; ventricular volumes and left ventricular ejection fraction were calculated by the standard area–length method. Significant CAD was defined as ≥50% narrowing of the lumen of at least one of the three main arteries or their major branches.
Flow cytometry analysis
Expression of PBRs was measured by direct immunofluorescence methods using a fluorescent probe (Bodipy Ro-1986, Molecular Probes, Eugene, Oregon), a fluorescence derived from desdiethylfluorazepam with a high affinity and selectivity for benzodiazepine receptors. Venous blood samples were taken from all patients at 8 am, before the first exercise stress test, in reproducible and standardized conditions. The analysis of PBRs expression on the plasma membrane of the leukocytes was performed as described.
A sample of 100 μl of Na2 EDTA preserved blood was treated with 2 ml of ice-cold erythrocyte-lysing solution (NH4Cl 0.208 g; Na2EDTA 0.1 mg; KHCO3 0.025 g in 250 ml H2O), resuspended in 100 μl of cold phosphate-buffered saline (PBS) (NaCl 8 g/liter; KCl 0.2 g/liter; Na2HPO4, 2.89 g/liter; KH2PO4 0.23 g/liter; BSA 2 g/liter; NaN3 0.2 g/liter) and incubated for 30 min at 0° to 5°C in the dark with 10 μl of Bodipy Ro-1986 at 5 × 10−5 mol/liter.
After incubation, cells were washed three times and then resuspended in 500 μl of cold PBS containing NaN3 and analyzed by flow cytometry (Coulter Epics Profile II). Gating for lymphocytes, monocytes and granulocytes was determined by the dot blot generated by the forward angle scatter versus right angle scatter; 10,000 gated cells per sample were analyzed, and the fluorescence was recorded as a mean channel number over the logarithmic range of 1 to 1,024. The PBRs are expressed on the plasma membrane and on the mitochondrial membrane (20); in our study, we evaluated the plasma membrane sites of PBRs. The expression of membrane receptors was reported as sites per cell as described by Caranyon et al. (21).
Binding specificity assay
To verify the fluorescence specificity, the agonist fluorazepam (without fluorescein) or the agonist Ro5-4684 (Sigma, St. Louis, Missouri) or the antagonist PK-11195 (Sigma) was added to our prelabeled samples. In short, 100 μl of prelabeled cells was incubated for 30 min at 37°C with 10 μl of fluorazepam 10−4 mol/liter or Ro5-4684 3 × 10−4 mol/liter or PK-11195 3 × 10−4 mol/liter. After incubation, cells were washed three times with cold PBS and then resuspended in 250 μl of cold PBS containing NaN3. Samples were analyzed by flow cytometry under the same conditions as samples stained with Bodipy Ro-1986.
Results are expressed as the mean value ± SD. Mann-Whitney U nonparametric analysis was used to investigate differences between the two groups. The Stat View program for Apple Computers was used, and p < 0.05 was considered statistically significant.
Our study group consisted of 57 men (mean age 62.5 ± 8.6 years) classified into two groups: 24 patients (with a history of angina pectoris during daily life (group P: 9 patients with effort angina, 4 patients with angina at rest and 11 patients with mixed angina) and 33 patients with silent ischemia documented during the exercise stress test (group A), including 17 patients who had had nonpainful ischemic episodes during domiciliary Holter recordings. The other clinical characteristics of the two groups of patients are reported in Table 1. There were no statistically significant differences in age, ECG-documented severity of angina, hypertension, hyperlipidemia, diabetes or smoking. All patients in group P reported anginal pain during exercise; moderate to severe dyspnea was experienced during EST in group A patients. The entity of ST segment depression at peak exercise was similar between the two groups.
Patients with silent ischemia had greater expression of PBRs on leukocytes than did patients reporting angina pectoris. The binding specificity of PBRs was evaluated using a high concentration of the agonist Ro5-4684 (Fig. 1).
On lymphocytes, the PBRs expression in group A was 2.0 ± 0.4 × 105 sites/cell, and in group P it was 1.2 ± 0.4 × 105 sites/cell (p < 0.005). On monocytes, the PBRs expression in Group A was 13.4 ± 0.9 × 105 sites/cell, and in group P it was 8.0 ± 0.5 × 105 sites/cell (p < 0.001). On granulocytes, the PBRs expression in group A was 7.22 ± 0.8 × 105 sites/cell, and in group P it was 4.25 ± 0.9 × 105 sites/cell (p < 0.001) (Fig. 2).
Our data show that the expression of PBRs on leukocytes from patients with documented ischemia is significantly greater in patients with silent ischemia than in patients with symptomatic ischemia.
The PBRs are expressed on all human peripheral blood leukocyte subsets, but mainly on monocytes and granulocytes, whereas natural killer cells and B- and T-lymphocytes display lower levels of PBRs (20). The PBRs have been found to modulate immunocyte functions such as chemotaxis (22), respiratory burst (21) and cytokine secretion (23). Modulation of chemotaxis limits the spread of chemotactic agents and preserves cells from free radical damage (21).
Various endogenous substances, such as diazepam-binding inhibitor (DBI), are able to bind to PBRs; these substances have been found in human blood and urine and are involved in pain control (7,9,12,13). The interaction between PBRs and morphine-induced antinociception is abolished by benzodiazepine antagonists such as flumazenil (9,13), indicating, again, that PBRs are involved in pain control. Opioid receptors on peripheral sensory nerves are upregulated during inflammation. Opioid receptors are expressed on immune cells and can modulate the behavior and release of cytokines, inducing local analgesia (7). Moreover, neuroactive substances and related receptors could inhibit nociception of the compromised heart by directly influencing and modulating the microenvironment (4,7,19).
The absence of pain in certain patients with myocardial ischemia could be linked to an increased expression of PBRs. The possible functional significance of the increased number of benzodiazepine-binding sites on the plasma membrane could be related to DBI, which enhances the production of interleukin-1 and tumor necrosis factor (24); these cytokines are able to increase the expression of selectins, which have a key role in inflammation-induced pain (17). A balanced control of pain with PBRs and opioids may take place through cytokine release.
It is interesting that in epileptic patients, increased DBI and decreased PBRs are related to increased excitability and resistance to specific therapy (25), whereas increased PBRs expression on all leukocyte subsets is related to a decrease of DBI, which raises the pain threshold (4,6,12,13). Thus, the concentrations of DBI, opioids and benzodiazepines are fundamental in the control of pain responses and threshold of pain. The PBRs may be a critical element in the interrelation between brain, behavior, immunity and modulation of pain circuits.
Our study has some limitations. Only one determination at a single time point was made, but the samples were analyzed in duplicate. Different endogenous substances capable of binding to PBRs have been found in extracts of various rat organs and in human blood and urine, and a benzodiazepine receptor–mediated model of pain has also been advanced. We did not test DBI, benzodiazepines or endorphins in our patients. In this study, we did not measure the algogenic stimulus in myocardial ischemia, but we have studied this in a previous report (5).
Increased or decreased PBRs density may reflect endogenenous ligand activity or regulatory processes such as DBI or endogenous benzodiazepines. Modifications of the concentration of DBI or endogenous benzodiazepines in silent myocardial ischemia could explain the observed PBRs upregulation. The higher density of PBRs on leukocytes of patients with asymptomatic ischemia is related to qualitative differences in central and/or peripheral pain mechanism regulation. Further studies are needed to explain whether the origin of these different expressions of PBRs are genetically determined or secondary to the stressful events and neurohormonal response.
☆ This study was supported by Grant 96/03, current reserach at IRCCS, San Matteo Hospital, Pavia, Italy.
- coronary artery disease
- diazepam-binding inhibitor
- electrocardiogram or electrocardiographic
- ergometric stress test
- γ-aminobutyric acid
- myocardial infarction
- peripheral benzodiazepine receptors
- phosphate-buffered saline
- Received August 29, 1999.
- Revision received March 1, 2000.
- Accepted April 13, 2000.
- American College of Cardiology
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