Anesth Analg 2005;100:33-37
© 2005 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000139357.87358.94
CARDIOVASCULAR ANESTHESIA
Protamine After Heparin Produces Hypotension Resulting from Decreased Sympathetic Outflow Secondary to Increased Nitric Oxide in the Central Nervous System
Yoshikazu Hamada, MD*,
Yoshiyuki Kameyama, MD*,
Hideyuki Narita, MD*,
Kirk T. Benson, MD
, and
Hiroshi Goto, MD
*Department of Anesthesiology, Tokyo Medical University, Shinjuku, Tokyo, Japan;
Department of Anesthesiology, University of Kansas Medical Center, Kansas City, Kansas.
Address correspondence and reprint requests to Yoshikazu Hamada, MD, Department of Anesthesiology, Tokyo Medical University, 6–7–1 Nishi-shinjuku, Shinjuku, Tokyo, 160–0023 Japan. Address e-mail to yhamada{at}mbg.ocn.ne.jp
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Abstract
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To elucidate whether there are linkages among protamine-induced hypotension, nitric oxide (NO), and sympathetic nerve activity, we administered 3 mg/kg protamine sulfate after 300 U/kg heparin after 20 mg/kg of NG-nitro-D-arginine methyl ester (D-NAME) or NG-nitro-L-arginine methyl ester (L-NAME) as a pretreatment to baroreceptor-denervated rabbits and compared changes in hemodynamic variables and renal sympathetic nerve activity (RSNA). In the D-NAME group, heart rate (HR), mean arterial blood pressure (MAP), and RSNA significantly decreased to 93.7% ± 0.7%, 75.0% ± 5.1% and 65.2% ± 4.6% (mean ± SE), respectively. In the L-NAME group, the pretreatment of L-NAME significantly inhibited the depressant effects of protamine on these variables. Because the animals were totally baroreceptor-denervated, decreased RSNA was attributable to the central depressant effect of protamine, and decreased sympathetic outflow could have contributed to the reduction of HR and MAP. The depressant effect of protamine on sympathetic outflow was inhibited by the pretreatment with L-NAME, a NO synthase inhibitor, suggesting that decreased sympathetic outflow secondary to a protamine-induced increase in NO concentration in the central nervous system may contribute to protamine-induced cardiovascular depression.
IMPLICATIONS: Using baroreceptor-denervated rabbits it was shown that decreased sympathetic outflow resulting from increased nitric oxide is one mechanism of protamine-induced hypotension.
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Introduction
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Protamine (P) sulfate often causes systemic hypotension when used to reverse the anticoagulant effect of heparin (H). The mechanisms of P-induced hypotension are multifactorial, including direct vasodilation and negative inotropic action (1–2). P is rich in the basic amino acid arginine, which is the precursor of synthesis of nitric oxide (NO). NO is a direct vasodilator, and data suggest that NO decreases central sympathetic tone, leading to systemic hypotension (3–4). The purpose of the study was to elucidate whether there are linkages among P-induced hypotension, NO, and sympathetic nerve activity. For that purpose, P was administered IV after H in baroreceptor-denervated rabbits with and without pretreatment of an active NO synthesis inhibitor, and mean arterial blood pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA) were measured and recorded.
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Methods
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This study was approved by the Kansas University Institutional Animal Care and Use Committee. Appropriate guidelines for the use of animals were observed during all aspects of this study.
Adult male New Zealand White rabbits (2.8–3.3kg) were anesthetized with urethane (1 g/kg) administered via an ear vein. Anesthesia was maintained with supplemental administration of IV urethane (100 mg · kg–1 · h–1) throughout the experiment. The rabbits had a tracheotomy and were ventilated with an infant ventilator (Model LS 104 150; Bourns Life Systems, Riverside, CA) using oxygen in nitrogen (FIO2, 0.4) at a tidal volume of 10–15 mL/kg and a frequency of 25–30 cycles/min. Arterial blood gas analyses was performed periodically and the values were maintained within normal ranges (PaCO2, 35–45 mm Hg; pH, 7.35–7.45). Core body temperature was maintained between 36.5°C and 37.5°C with an external warming apparatus. The animals were paralyzed with vecuronium (0.1 mg/kg) to avoid artifacts in the measurement of sympathetic nerve activity secondary to muscular movement. Polyethylene catheters were inserted into a femoral artery and vein for measurement of MAP and drug administration, respectively.
Measurement and recording of RSNA have been described elsewhere (5). Briefly, the left kidney was exposed, and the renal sympathetic nerves were isolated and placed on a bipolar silver electrode. Electrical impulses recorded from the renal sympathetic nerves were amplified, rectified, and integrated (time constant, 2.0 s) and recorded. To quantify RSNA, the resting spontaneous nerve discharge before P administration was defined as 100% control value. All variables were measured continuously and recorded on a DAT tape PCM recorder (RD-100T; TEAC, Montebello, CA), and played back on a multichannel chart recorder (Omnicorder 8M14; San-ei, Japan).
All animals received a combined denervation of carotid sinus, aortic, and vagal nerves to eliminate both arterial and cardiopulmonary baroreflexes. Complete denervation was verified by the lack of reflex changes in RSNA and HR in response to IV injections of phenylephrine (4 µg/kg) and nitroglycerin (75 µg/kg). After hemodynamic stabilization was achieved, baseline HR, MAP, and RSNA were recorded. We performed two preliminary studies. First, H and then (3 min later) P were administered IV in 3 consecutively increasing doses in 6 animals (H 100 U/kg and P 1 mg/kg initial doses; 20 min later H 300 U/kg and P 3 mg/kg; and again 20 min later H 900 U/kg and P 9 mg/kg final doses).
Second, 18 rabbits were divided into three groups (n = 6 each). They were given 2, 6, or 20 mg/kg of NG-nitro-D-arginine methyl ester (D-NAME), an inactive isomer of NG-nitro-L-arginine methyl ester (L-NAME) and 15 min later, each group received the same dose of L-NAME, a competitive inhibitor of NO synthase (NOS). Maximum percentage changes of variables were recorded after each administration of drugs in these preliminary studies. In the main study, 16 rabbits were divided into 2 groups (D-NAME and L-NAME, n = 8). After stabilization of hemodynamics, the rabbits received 20 mg/kg of D-NAME or 20 mg/kg of L-NAME as a pretreatment. Fifteen minutes later, all rabbits were heparinized (300 U/kg) and then 3 min later P (3 mg/kg) was administered over 30 s. HR, MAP, and RSNA were recorded continuously for at least 5 min after P administration.
All data were expressed as mean ± SE. The preinjection and postinjection values in the first and second studies were compared and analyzed by repeated-measures analysis of variance with post hoc Bonferroni corrections. Time course changes of variables and between-group differences (L-NAME group versus D-NAME group in the main study) were also compared using the same statistical analysis. Statistical significance was set at P < 0.05.
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Results
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In the first preliminary study, HR, MAP, and RSNA did not change after H administration. However, all variables decreased dose-dependently after P (Fig. 1). RSNA decreased with P (1, 3, and 9 mg/kg) to 77.7% ± 3.5%, 51.3% ± 2.2%, and 39.2% ± 1.9%, respectively. In the second preliminary study, D-NAME did not change HR, MAP, or RSNA. L-NAME (2, 6, and 20 mg), however, increased MAP to 112.8% ± 1.4%, 122.7% ± 2.0% and 132.7% ± 3.1%, respectively and increased RSNA to 116.2% ± 1.4%, 124.7% ± 2.0% and 132.7% ± 3.1%, respectively (Fig. 2). A significant increase in MAP and RSNA persisted for at least 15 min after IV L-NAME.
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Figure 1. Maximum percent changes in heart rate (HR), mean arterial blood pressure (MAP), and renal sympathetic nerve activity (RSNA) induced by IV heparin (100 to 900 U/kg) or protamine (1 to 9 mg/kg). *P < 0.05 versus values immediately before each injection.
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Figure 2. Maximum percent changes in heart rate (HR), mean arterial blood pressure (MAP), and renal sympathetic nerve activity (RSNA) induced by IV NG-nitro-D-arginine methyl ester (D-NAME: 2 to 20 mg/kg) or NG-nitro-L-arginine methyl ester (L-NAME: 2 to 20 mg/kg). *P < 0.05 versus values immediately before each injection.
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Time course changes of HR, MAP, and RSNA in the main study are shown in Figure 3. In the D-NAME group, HR, MAP, and RSNA decreased to 93.7% ± 0.7%, 75.0% ± 5.1%, and 65.2% ± 4.6%, respectively. The pretreatment of L-NAME significantly inhibited the depressant effects of P on these variables.
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Figure 3. Time course percent changes in heart rate (HR), mean arterial blood pressure (MAP), and renal sympathetic nerve activity (RSNA) after 3 mg/kg of IV protamine in both NG-nitro-D-arginine methyl ester (D-NAME) and NG-nitro-L-arginine methyl ester (L-NAME) groups. *P < 0.05 versus baseline value; P < 0.05 versus NG-nitro-D-arginine methyl ester (D-NAME) group.
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Discussion
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In this study, we found that P decreased sympathetic outflow, which could be one of the contributing factors responsible for P-induced hypotension and bradycardia. The decreased sympathetic outflow is likely the result of increased NO concentration in the central nervous system (CNS) because the P-induced reduction of RSNA was abolished by pretreatment of L-NAME, a NOs inhibitor.
RSNA has been measured in animal experiment models because the renal sympathetic nerve is easily isolated anatomically. Decreased RSNA reflects an overall decrease in the central sympathetic nerve activity and sympathetic outflow. We used urethane as an anesthetic because it does not affect the arterial baroreflex response (6) and it produces long-lasting and satisfactory anesthesia. Among nondepolarizing muscle relaxants, vecuronium was used for muscle relaxation because of its minimal sympathetic ganglionic blocking effect (7). Tsuchida et al. (8) reported that vecuronium did not affect the baroreflex response in humans. As the aim of our present study was to evaluate the effects of IV P after H on the sympathetic nervous system and to elucidate the mechanism of P-induced hypotension, we used one dose of P and compared the hemodynamic variables and RSNA using baroreceptor-denervated rabbits. In the first preliminary study (Fig. 1), P 1 mg/kg caused a slight but significant decrease of RSNA. P 9 mg/kg caused marked and prolonged arterial hypotension. We chose, therefore, the 3 mg/kg dose of P after 300 U/kg H for the main study. Because the animals were totally baroreceptor-denervated, decreased RSNA was most likely attributable to the direct central depressant effect of P. We did not measure preganglionic nerve activity. Thus, a possibility of decreased sympathetic ganglionic transmission with P could not be evaluated in our experiment. It has been shown that H might induce arterial hypotension (9). However, none of the variables changed after H administration in our study.
In the second preliminary study, L-NAME, a competitive inhibitor of NOS increased RSNA and MAP dose-dependently (Fig. 2). It can be speculated that NO formation in the cerebral vascular endothelium and/or in the CNS was inhibited by L-NAME. NO plays a role in the central regulation of sympathetic tone in a manner that decreased NO concentration in the CNS increases sympathetic nerve activity and vice versa (4,10–14). Our results are similar to some findings that have been reported. Zanzinger et al. (11) and Hirai et al. (12) administered L-NAME IV in baroreceptor-denervated animals and observed that sympathetic outflow and MAP increased significantly. In their studies as in ours, HR did not change after L-NAME administration. It is possible that HR was maximally increased due to total barodenervation, including bilateral vagotomy and that HR could not increase further despite the increased sympathetic outflow. We chose the dose 20 mg/kg of these agents for the main study.
The main finding of the study was that pretreatment with L-NAME, an active NOS inhibitor, completely counteracted P-induced reductions of MAP, HR, and RSNA (Fig. 3). This suggests that P can increase NO concentration in the CNS by enhancing NOS activity, leading to decreased sympathetic nerve activity. Decreased MAP and HR after protamine could be attributable, at least in part, to decreased sympathetic outflow from the CNS.
It has been reported that sympathetic nerve activity and MAP were decreased by L-arginine administered either IV (4,13) or injected into the CNS (10,14). This occurs because L-arginine is a precursor of NO formation and NO concentration will increase in the CNS. However, the mechanism for P-related increases in NO concentration in the CNS is unknown. Pearson et al. (15) and Evora et al. (16) demonstrated that P releases NO from the systemic arterial endothelium. Increased NO concentration produces vascular skeletal muscle relaxation, leading to arterial hypotension. Thus, it may be possible that P increases NO production not only in the peripheral arterial endothelium but also in the cerebral vascular endothelium, presumably by increasing NOS activity. It can be speculated that increased NO molecules in the cerebral vasculature diffuse into the CNS, resulting in the reduction of sympathetic nerve activity.
There may be another link between P and NO formation. P sulfate is a polycationic peptide rich in the amino acid L-arginine, a precursor for NO formation. Thus, if P sulfate liberates free L-arginine, NO concentration in the CNS will increase, and central sympathetic nerve activity will be suppressed.
It is unknown which region of the CNS is affected by NO to suppress sympathetic nerve activity. Tseng et al. (10) and Shapoval et al. (14) showed that microinjection of L-arginine into the nucleus tractus solitarius or rostral ventrolateral medulla produced hypotension and inhibition of sympathetic nerve activity.
A limitation of an experimental model is that the central and peripheral effects of P after H could not be differentiated. Our data only suggest a possible central effect of P mechanism to decrease MAP and HR.
In summary, it is speculated in our study that P-induced arterial hypotension could be in part attributable to decreased sympathetic outflow secondary to increased NO concentration in the CNS. Further study is necessary to investigate the degree of the contribution of the decreased sympathetic outflow to overall P-induced hemodynamic depression.
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References
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Accepted for publication June 24, 2004.
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Abstract
Introduction
