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beta2-Adrenoceptor Density of Human Lymphocytes After Nitroprusside-Induced Hypotension

Heesen, Michael MD; Dietrich, Gerald V. MD; Boldt, Joachim MD; Zeiler, Dirk MD; Alberts, Caspar H.; Hempelmann, Gunter MD

General Article

The present study was undertaken to assess the influence of nitroprusside-induced hypotension on beta2-adrenoceptor density. Twenty-four patients undergoing noseseptum corrections under general anesthesia were allocated randomly to a nitroprusside or control group. beta2-Receptor density on lymphocytes was measured by binding studies using (-)125 I-iodocyanopindolol. Lymphocyte subpopulations B, T, T (helper), Tsuppressor, and natural killer cells were determined simultaneously by flow cytometry. Five of 12 nitroprusside-treated patients developed significant intraoperative increases of epinephrine levels (+ 69% versus preoperatively) which were not seen in the remaining seven patients. In these five patients, beta2-receptor density of unfractionated lymphocytes was 26% lower (P < 0.05) on the first day after surgery compared with preoperative values. Since no changes in proportions of lymphocyte subpopulations were observed, these results are not caused by redistribution phenomena inducing a decrease of subsets with a high number of beta receptors. These findings suggest that beta2-adrenergic responsiveness might be diminished after nitroprusside treatment in some patients.

(Anesth Analg 1995;81:1250-4)

Department of Anesthesiology and Intensive Care Medicine, Justus-Liebig-University Giessen, Giessen, Germany.

Accepted for publication July 18, 1995.

Address correspondence and reprint requests to Michael Heesen, MD, Department of Anesthesiology and Intensive Care Medicine, Klinikstr. 29, Justus-Liebig-University Giessen, D-35385 Giessen, Germany.

Sodium nitroprusside (SNP) is commonly used for induction and maintenance of controlled hypotension [1]. It is a vasodilator with a rapid onset [2]. After contact with hemoglobin, the SNP molecule is immediately degradated [3] and blood pressure rapidly returns to baseline values after discontinuation of SNP infusion [4]. The molecular mechanism of action including increases of cyclic guanosine monophosphate in vascular smooth muscle is well known [5]. Other authors studied cardiovascular and hemodynamic side effects [6]. A decrease of arterial blood pressure was found to activate the sympathoadrenergic system via the baroreceptor reflex with a consequent release of plasma epinephrine [4,7]. Comedication with beta-blocking drugs [8] or clonidine [9] was recommended to blunt the endocrine stress response and to reduce the increase in plasma catecholamines. The responsiveness of the sympathetic nervous system is not reflected by plasma concentrations of adrenergic hormones, since their plasma half-time is only a few minutes. Moreover, norepinephrine is released mainly into the synaptic cleft and its spillover into plasma is not constant [10]. The target of hormone action are adrenoceptors and it became apparent that receptors of the beta subtype, rather than being static entities, are dynamically regulated by a wide variety of physiologic and pathophysiologic conditions [11]. No studies are available dealing with changes of the beta-adrenergic receptor system after SNP-induced hypotension. The present study examined the influence of increases in catecholamine plasma concentrations during SNP-induced hypotension on lymphocyte beta2-receptor density by comparing preoperative values with data obtained on the first postoperative morning.

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After approval by the local ethics committee and written informed consent 24 patients of ASA physical status I or II scheduled for nose-septum correction were allocated randomly to the study group (n = 12, receiving SNP) or the control group (n = 12, without SNP). Only patients scheduled as the first case of the day were included so that pre- and postoperative blood samples could be obtained at approximately the same time (7:00 AM). Before taking the pre- and postoperative sample, patients were in a supine position for at least 30 min during which no manipulations were performed. Patients fasted from midnight before surgery. Patients received 7.5 mg midazolam 45 min before arrival in the operating room. Anesthesia was induced by 0.1 mg fentanyl, 2 mg vecuronium bromide, 0.2 mg/kg etomidate, and 1 mg/kg succinylcholine intravenously and maintained with 1%-1.5% isoflurane in a gas mixture containing 66% nitrous oxide. End-expiratory carbon dioxide concentration was measured by capnometry and adjusted to normocapnia by the attending anesthesiologist. Patients received 6 mL centered dot h-1 centered dot kg-1 body weight of balanced salt solutions during the operation. Routine intraoperative monitoring was used. In addition, a radial artery was cannulated to measure blood pressure continuously in the patients of the study group.

SNP was infused through a central venous catheter. The SNP doses applied were within the permitted range from 0.5 to 10.0 micro gram centered dot kg-1 centered dot min-1 and were adjusted to reduce mean arterial blood pressure to 50 mm Hg. SNP infusion was started at the beginning of the operation and was discontinued after 60 min.

Blood samples were taken from a central venous catheter before induction of anesthesia (PRE) and on the morning of the first postoperative day (POST) for measurement of lymphocyte subpopulations and beta2-receptor density. Catecholamine plasma concentrations were determined at these two time-points and additionally four times per hour during surgery by means of a high-performance liquid chromatographic method with electrochemical detection [12]. Normal values are 30-85 pg/mL for epinephrine and 180-285 pg/mL for norepinephrine plasma concentrations. Patients were considered to experience adrenergic activation when more than one of the intraoperative catecholamine plasma concentrations exceeded normal values. For determination of lymphocyte subpopulations [13] whole blood was incubated with murine antihuman monoclonal antibodies (Becton Dickinson, Heidelberg, Germany) to identify T and B lymphocytes, Thelper and Tsuppressor cells and natural killer cells. After lysing erythrocytes, stained lymphocytes were analyzed in a flow cytometer (FACScan; Becton Dickinson). For analysis of beta receptors, lymphocytes were isolated by centrifugation through a Ficoll-Hypaque gradient (500g for 30 min) according to a modification of the method of Boyum [14]. After three washings with phosphate-buffered saline, a population of 90% lymphocytes was obtained, with >95% viability as assessed by trypan blue exclusion. To prepare plasma membranes the lymphocytes were resuspended in ice-cold water and broken by ultrasonication. Aliquots were centrifuged at 40,000g for 20 min at 4 degrees C. Samples were stored at -80 degrees C until used for radioligand binding studies which were performed within 14 days after samples were taken. Lymphocyte beta2-adrenoceptor density and affinity were measured by radioligand binding studies with (-)125 I-iodocyanopindolol (ICYP; specific activity 2200 Ci/mmol; New England Nuclear, Dreieich, Germany) at 6 ICYP concentrations ranging from 10 to 150 pmol. Unspecific binding was defined as radioactivity bound to lymphocytes that was not displaced by 1 micro Meter of (+/-)CGP 12177 (Biotrend, Cologne, Germany) [15]. The equilibrium dissociation constant (Kd) and the maximum number of binding sites (Bmax) for ICYP were calculated from Scatchard plots [16]. Lymphocyte membranes were used and protein concentrations were measured by the method from Bradford [17] using bovine serum albumin as standard. Details of the methods have been described previously [18].

Data are given as mean +/- SEM unless otherwise designated. Statistical evaluation was performed with two-way analysis of variance for repeated measurements. P values <0.05 were considered significant.

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Demographic data (age, weight, sex ratio) and duration of surgery were not different between the groups. In 5 of 12 patients receiving SNP intraoperative epinephrine concentrations increased significantly (+ 69%) compared with values obtained before induction of anesthesia (SNP 1, Table 1). These patients needed significantly (P < 0.05) higher doses of SNP than the other seven patients (SNP 2) to achieve a reduction in mean arterial pressure to 50 mm Hg (8.5 +/- 1.3 micro gram centered dot kg-1 centered dot min-1 compared with 5.3 +/- 0.9 micro gram centered dot kg-1 centered dot min-1). In SNP 1, beta2-receptor density was 26% lower (P < 0.05) on the first postoperative day compared with preoperative values Table 2. In SNP 2 and control groups, no alteration in plasma levels of catecholamines or beta-receptor density was observed throughout the study period. Percentages of lymphocytes subpopulations Table 3 and Kd (data not shown) were not significantly changed in any of the three groups.

Table 1

Table 1

Table 2

Table 2

Table 3

Table 3

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Other authors have suggested that lymphocytes bearing beta2-receptors may be used as a model reflecting beta-receptor changes in the human heart [19] since biochemical and pharmacologic properties are quite comparable. However, criticism against this model [20] arose because radioligand binding studies are performed in unfractionated lymphocytes, while receptor density was shown to vary with different lymphocyte subpopulations [21]. Epinephrine was demonstrated to induce alterations in the relative proportions of lymphocyte subsets [22]. Apparent up- or down-regulation thus could be caused by changes in the distribution of lymphocyte subsets without any alteration in receptor sites per cell.

In the present study we simultaneously determined lymphocyte subpopulations by means of flow cytometry. No alterations in distribution of subsets could be found. Thereby one prerequisite might be fulfilled to extrapolate changes of lymphocyte beta receptors to those in the human heart. It should also be taken into account that cardiac beta receptors are divided into beta1 and beta2 subtypes while in lymphocytes beta receptors are exclusively from the beta2 subtype. Since a significantly better correlation between lymphocyte beta2 and cardiac beta (2) receptors was found than with beta1-receptor density [23] lymphocytes should be considered as a suitable model to reflect subtype-selective receptor changes in the human heart.

In the present study, beta receptors were down-regulated on the first postoperative morning in 5 of 12 patients who had significantly higher intraoperative epinephrine concentrations. The exact mechanism leading to receptor down-regulation could not be assessed by the methods used in the current study. The fact that receptor density changes were seen only in patients with intraoperative increases in epinephrine plasma concentrations whereas receptor density was unaffected in individuals without adrenergic activation points tentatively to a homologous desensitization, which is caused by exposure of the receptors to agonists. In the first step of desensitization receptors are uncoupled from their functional response [20]; this step can be followed by a decrease in the number of receptors as observed in our study on the first postoperative morning. Since surgical procedure and demographic data were similar in all groups, whereas alterations in receptor sites were observed in only one group, these factors are unlikely to explain this result. However, we cannot state with certainty that the magnitude of adrenergic activation, i.e., a 69% increase in epinephrine plasma concentrations, would be sufficient to cause the decrease in receptor density.

Our results showed a sympathoadrenergic hyporesponsiveness on the day after surgery in some individuals. These patients required higher concentrations of SNP to reduce mean arterial pressure. The most probable reason is that these patients reacted to the induction of hypotension with release of high concentrations of renin [24] deliberating angiotensin II, which counteracted the reduction in arterial pressure. Consequently, the attending anesthesiologist increased the SNP dose which promoted catecholamine release and thus down-regulated beta-receptor density.

However, a direct effect of SNP molecules or its metabolites on beta (2) receptors should be taken into account. Marty et al. [25] described a 10% decrease of Bmax after in vitro incubation of lymphocyte membranes with halothane. Exposure of dogs to lipopolysaccharide led to reduction in hepatic beta receptor number and affinity due to an activation of phospholipase A [26]. Since SNP is an important source of nitric oxide, which is identical with endothelium-derived relaxing factor, the effect of SNP on second messenger systems was studied extensively. No in vitro influence was found on intracellular cyclic adenosine monophosphate (cAMP) levels of intact human polymorphonuclear cells [27]. cAMP is a second messenger molecule formed by adenylate cyclase, an enzyme which is linked via stimulatory G proteins with beta2 receptors on the membrane of polymorphonuclear cells. A direct molecular effect of SNP on lymphocyte beta receptors thus appears very unlikely.

In previous studies on the effect of cardiac surgery on beta2-receptor function of lymphocytes, a decrease in receptor affinity for isoproterenol was found at the end of cardiopulmonary bypass [28]. Smiley and Vulliemoz [29] described a decrease in cAMP synthesis of lymphocytes after isoproterenol stimulation on the day after cardiac surgery. General surgery can also produce alterations when a pronounced activation of the adrenergic system occurs [30]. In contrast to the other two studies, this group found an increase in receptor density immediately after determination of surgery. However, in the studies cited above no determination of lymphocyte subsets was performed, and it may be argued that a redistribution of lymphocyte subsets could be the reason for discrepancy in the results mentioned.

Since, in the present study, changes in lymphocyte subpopulations were excluded, perhaps cardiac beta2-receptor density was also diminished in those patients with reduced lymphocyte beta-receptor density. This speculation could have clinical implications. Under physiologic conditions, the force of contraction is regulated by beta1 receptors. In the failing human heart only beta1-receptor density is reduced, while the number of beta2 receptors in right atrium and the left and right ventricles is unaffected [31]. Therefore, beta2 receptors compensate, at least partly, the loss of beta1 receptors and thereby may provide myocardial contractility [32]. As our results show, SNP administration can reduce the beta2-receptor density, and thus could be problematic in patients with cardiac insufficiency whose contractility depends especially on the beta2 system. However, this hypothesis must be considered with caution, since it is unclear whether a 26% reduction in beta2-receptor density leads to a substantial and clinically relevant decrease in the production of the second messenger cAMP.

We thank Professor O. E. Brodde, University of Halle, Germany and Dr. M. C. Michel, University of Essen, for assistance in establishing radioligand binding studies and fruitful cooperation.

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