Anesth Analg 1999;88:1232
© 1999 International Anesthesia Research Society
CARDIOVASCULAR ANESTHESIA
The Direct Effects of Heparin and Protamine on Canine Tracheal Smooth Muscle Tone
Michiaki Yamakage, MD, PhD,
Takashi Matsuzaki, MD, PhD,
Naoki Tsujiguchi, MD,
Tomohisa Mori, MD, and
Akiyoshi Namiki, MD, PhD
Department of Anesthesiology, Sapporo Medical University School of Medicine, Sapporo, Japan
Address correspondence and reprint requests to Michiaki Yamakage, MD, PhD, Department of Anesthesiology, Sapporo Medical University School of Medicine, South 1, West 16, Chuo-ku, Sapporo, Hokkaido, 060-8543, Japan. Address e-mail to yamakage @sapmed.ac.jp.
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Abstract
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Heparin and protamine are used for cardiopulmonary bypass in cardiac surgery; however, the direct effects and mechanisms of these drugs on airway smooth muscle tone are still not fully known. We investigated the in vitro effects of these drugs on canine tracheal smooth muscle by measuring the muscle tension and intracellular Ca2+ concentration ([Ca2+]i) and by measuring inward Ca2+ currents (ICa) through voltage-dependent Ca2+ channels. [Ca2+]i was monitored by the 500-nm light emission ratio of preloaded Ca2+ indicator fura-2. Isometric tension was measured simultaneously. Whole-cell patch clamp recording techniques were used to investigate the effects of the drugs on ICa in freshly dispersed smooth muscle cells. Heparin (0.12–120 U/mL), protamine (0.15–150 U/mL), or heparin-protamine complex (4:5 U/U) was introduced into a bath solution. Protamine and heparin- protamine complex dose-dependently inhibited both carbachol-induced contraction of the muscle and increase in [Ca2+]i. These drugs also decreased the ICa of the muscle cells and shifted the inactivation curve to a more negative potential. Heparin itself had a slight enhancing effect on carbachol-induced muscle contraction without changing [Ca2+]i. Protamine and heparin-protamine complex can decrease the agonist-induced increase in [Ca2+]i by the inhibition of voltage-dependent Ca2+ channels both in the activated and inactivated states.
Implications: Protamine and heparin-protamine complex inhibited carbachol-induced canine tracheal smooth muscle contraction by inhibiting the increase in intracellular concentration of free Ca2+. These drugs can decrease the agonist-induced increase in intracellular Ca2+ by the inhibition of voltage-dependent Ca2+ channels in both the activated and inactivated states.
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Introduction
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Heparin is used as an anticoagulant during cardiopulmonary bypass and thrombolytic therapy. Inhaled heparin prevents methacholine-induced asthma (1), but the direct effect of this drug on airway smooth muscle contractility is still controversial (1,2). Protamine is used to reverse heparin anticoagulation. During this reversal, protamine may cause a number of adverse responses, including bronchoconstriction (3), which is achieved by the generation of anaphylatoxic complements induced by the heparin-protamine complex (3,4). Although the direct vasodilatory effect of protamine has been clarified in the rabbit artery (5), the direct effect of protamine and/or heparin on airway smooth muscle is still unclear.
Activation of membrane-associated receptors and channels of airway smooth muscle alters the intracellular concentration of free Ca2+ ([Ca2+]i), which itself controls the contractile state of the muscle (6). Because sustained contraction of airway smooth muscle requires the continued entry of extracellular Ca2+ and the blockade of voltage-dependent Ca2+ channels (VDCCs) suppressed the sustained increase in [Ca2+]i (7), VDCCs are important in regulating [Ca2+]i (7,8). We therefore undertook the present study to clarify the direct effects of heparin, protamine, and heparin-protamine complex on canine tracheal smooth muscle using the following methods: measuring [Ca2+]i simultaneously with muscle tension in a smooth muscle strip and measuring inward Ca2+ currents through VDCCs in a freshly dispersed single smooth muscle cell.
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Methods
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This protocol was approved by the our committee on animal research. The tracheas were excised from adult mongrel dogs (weighing 9–12 kg), and the epithelium, cartilage, and connective tissue were stripped from the smooth muscle. For the measurements of the muscle tension and [Ca2+]i, the tissue was cut into small strips (1 mm wide and 8 mm long). The muscle strips were pretreated with 5 µM acetoxymethyl ester of fura-2, an indicator of Ca2+, in a physiological salt solution containing 0.02% (v/v) cremophor EL for 6–7 h at room temperature (22–24°C). The fura-2–loaded muscle strip was held in a temperature-controlled (37°C) organ bath, and one end of the muscle strip was connected to a strain-gauge transducer. Experiments were performed with a fluorescence spectrometer (CAF-100; Japan Spectroscopic, Tokyo, Japan). Excitation light was passed through a rotating filter wheel (48 Hz) that contained 340- and 380-nm filters. The light emitted from the muscle strip at 500 nm was measured with a photomultiplier. The ratio of the fluorescence due to excitation at 340 nm to that at 380 nm (R340/380) was calculated and used as an indicator of [Ca2+]i (7,8). Contractions were induced by carbachol (1 µM), a potent muscarinic receptor agonist. Heparin (0.12–120 U/mL), protamine (0.15–150 U/mL), or heparin-protamine complex was introduced accumulatively into the tissue bath in the presence of carbachol. For the experiment using the heparin-protamine complex, a moderate excess of protamine (4:5 U/U) was used in the neutralization of heparin (9).
For the measurements of whole-cell inward Ca2+ currents (ICa), canine tracheal smooth muscle tissue was minced and digested for 20 min at 37°C in Ca2+-free Tyrode's solution to which 0.08% (wt/v) collagenase, 0.05% trypsin inhibitor, and 0.03% protease had been added (10). Cells were then dispersed by trituration, filtered through nylon mesh, and centrifuged. The pellet was resuspended in a modified Kraftbrühe solution (11) and stored at 4°C for up to 5 h before use. The modified Kraftbrühe solution contained (in mM): KCl 85, K2HPO4 30, MgSO4 5.0, Na2ATP 5.0, pyruvic acid 5.0, creatine 5.0, taurine 20, ß-hydroxybutyrate 5.0, and 0.1% (wt/v) fatty acid-free bovine serum albumin; pH was adjusted to 7.25 with 0.5 M tris [hydroxymethyl]aminomethane (Tris).
The experiments were performed at room temperature (22–24°C). Micropipettes were pulled from hematocrit tubing and had resistances of 3–5 M . The pipette solution contained (in mM): CsCl 130, MgCl2 4.0, EGTA 10, Na2ATP 5.0, and HEPES 10, with pH adjusted to 7.2 with Tris. The bath solution contained (in mM): tetraethylammonium chloride 130, MgCl2 1.0, CaCl2 10, glucose 10, and HEPES 10, with pH adjusted to 7.4 with Tris. An aliquot (approximately 0.5 mL) of the cell suspension was placed in a chamber on the stage of an inverted microscope. A micromanipulator was used to position the patch pipette against the membrane of a tracheal smooth muscle cell. After obtaining a high-resistance seal (> 5 G), the patch membrane was disrupted by strong negative pressure. Membrane currents were monitored using a patch clamp amplifier (CEZ-2400; Nihon Kohden, Tokyo, Japan), and the amplifier output was low-pass filtered at 2000 Hz.
ICa were elicited by 100-ms depolarizing pulses (-50 to 40 mV) from a holding potential of -70 mV. Leak currents were subtracted, and membrane capacitance and series resistance were compensated. Inactivation curves were determined by using a double-pulse protocol that consisted of a 3-s prepulse to a potential in the range of -70 to 20 mV, followed by a 100-ms depolarization to 20 mV. The peak change in the current was expressed as a fraction of that obtained with the -70 mV prepulse, and this quantity was least-squares fitted to a Boltzman expression (12) to estimate the potential of half-maximal inactivation (V1/2) and the slope factor (k). After obtaining a stable baseline of peak ICa, heparin, protamine, or heparin-protamine complex was introduced accumulatively into the tissue bath. After a 5-min exposure, the perfusate was reswitched to the control solution.
The following drugs and chemicals were used: trypsin inhibitor, ß-hydroxybutyrate, cremophor EL, nifedipine, heparin (sodium salt), protamine, Bay K 8644 (Sigma Chemical, St. Louis, MO), acetoxymethlester of fura-2 (Dojindo Laboratories, Kumamoto, Japan), type-I collagenase (Gibco Laboratories, Grand Island, NY), and protease (Calbiochem, La Jolla, CA).
Data are expressed as mean ± SD. For the measurement of [Ca2+]i and muscle tension, carbachol-induced sustained changes in [Ca2+]i (indicated by R340/380) and muscle tension were used as references (100%). Changes in measured variables with exposure to each drug were compared at each point (concentrations or applied potential) by using the paired two-tailed t-test. One-way analysis of variance for repeated measurements and Fisher's test were used to determine the concentration-dependent effects. In all comparisons, P < 0.05 was considered significant.
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Results
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R340/380, an indicator of [Ca2+]i, was rapidly increased by carbachol with a concomitant contraction (Fig. 1). Heparin per se enhanced carbachol-induced muscle contraction by 8% without changing [Ca2+]i at the highest concentration of 120 U/mL. The effect of this drug on muscle tension was dose-dependent, but high concentrations (>36 U/mL) were needed to produce significant effects (Fig. 2A). Protamine per se significantly suppressed carbachol-induced contraction of the muscle and increase in [Ca2+]i by 32% and 38%, respectively, at the highest concentration of 150 U/mL. These effects on muscle tension and [Ca2+]i were dose-dependent, but high concentrations (>15 U/mL) were needed to produce significant effects (Fig. 2B). The heparin-protamine complex significantly suppressed carbachol-induced contraction of the muscle and increase in [Ca2+]i by 54% and 58%, respectively, at the highest concentration tested. Both of these effects were dose-dependent and were significant at heparin concentrations of >1.2 U/mL (Fig. 2C). We confirmed that heparin, protamine, and heparin-protamine complex all had no effect on the intensity of fura-2 fluorescence itself within the concentration range tested.
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Figure 1. Representative effects of heparin (A), protamine (B), and heparin-protamine complex (C) in the presence of carbachol (1 µM) on the tension and ratio of the fluorescence due to excitation at 340 nm to that at 380 nm (R340/380) of canine tracheal smooth muscle strips. R340/380 is an indicator of intracellular concentration of free Ca2+ ([Ca2+]i). Both protamine and heparin-protamine complex altered [Ca2+]i and attenuated muscle contraction.
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Figure 2. Relationships between the percent response of the tension ( ) or ratio of the fluorescence due to excitation at 340 nm to that at 380 nm (R340/380; •) of canine tracheal smooth muscle strips and the bath concentrations of the drugs tested. Symbols represent mean ± SD (n = 7). *P < 0.05). The effects of protamine and heparin-protamine complex on the muscle tension and [Ca2+]i were dose-dependent at the concentrations of the drugs studied.
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ICa seen in canine tracheal smooth muscle cells peaked at approximately 10 ms and slowly became inactivated (Fig. 3A, control). In 147 cells, the maximal peak ICa at 20 mV was -305 ± 25 pA (range -198 to -589 pA). The addition of 10-6 M nifedipine substantially attenuated ICa, and 10-6 M Bay K 8644, an L-type Ca2+ channel agonist, enhanced the magnitude of ICa of canine tracheal smooth muscle cells (n = 3 in each case, data not shown).
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Figure 3. Effects of protamine on depolarization-induced inward Ca2+ currents (ICa). A, Typical recordings of ICa induced by pulses to 20 mV in the absence and presence of 150 U/mL protamine. Dashed line denotes zero current. Protamine inhibited the magnitude of ICa but did not alter the time course of the current. B, Representative time course of peak ICa at 20 mV before and after exposure to 150 U/mL protamine. The onset of the inhibition was rapid, and the effect was reversible.
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Protamine (150 U/mL) inhibited the magnitude of ICa but did not alter the time course of the current (Fig. 3A). Peak ICa obtained with repeated steps to 20 mV decreased rapidly by 30% during exposure to protamine and recovered completely with washout (Fig. 3B). Although heparin had no effect on the peak ICa range at concentrations up to 120 U/mL, protamine (150 U/mL) and heparin-protamine complex (120 U/mL heparin) both significantly inhibited ICa at step potentials in the range of -10 or -20 to 40 mV and reduced peak ICa at 20 mV by 30% and 50%, respectively (Fig. 4). There was no apparent shift in the voltage-dependence of induced ICa with either protamine or heparin-protamine complex. Both protamine and heparin-protamine complex significantly inhibited peak ICa in a dose-dependent manner (Fig. 5). Protamine per se required somewhat higher concentrations to achieve the same inhibitory effect as that of heparin-protamine complex.
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Figure 4. Relationships between peak inward Ca2+ currents (ICa) and applied potential before (•) and after () exposure to heparin (A), protamine (B), and heparin-protamine complex (C). Symbols represent mean ± SD (n = 7). *P < 0.05. Although heparin had no effect on the peak ICa range, protamine and heparin-protamine complex both significantly inhibited ICa at step potentials in the range of –10 or –20 to 40 mV and reduced peak ICa at 20 mV by 30% and 50%, respectively.
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The inactivation variables obtained in 21 cells under control conditions were V1/2 = -19.5 ± 1.5 mV and k = 7.1 ± 0.7 mV. Protamine and heparin-protamine complex both shifted the inactivation curve to a more negative potential, and the changes in V1/2 induced by these drugs were statistically significant in each case (-19.8 to -26.8 mV and -19.5 to -29.8 mV, respectively) (Fig. 6). The slope factor k was not changed by exposure to either drug. In the heparin experiment, there was no change in either V1/2 or k.
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Figure 6. Effects of heparin (A), protamine (B), and heparin-protamine complex (C) on voltage-dependent steady-state inactivation of inward Ca2+ currents. Inactivation curves were generated under control conditions (•), then repeated in the presence of one of or a complex of the drugs tested (). Symbols represent mean ± SD (n = 7). Both protamine and heparin-protamine complex significantly shifted the inactivation curves to more negative potentials without changing the sigmoid shapes of the curve.
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Discussion
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One of the major findings of our study is that both protamine and heparin-protamine complex altered [Ca2+]i and attenuated the muscle contraction induced by muscarinic receptor stimulation in canine tracheal smooth muscle in vitro. Because intracellular free Ca2+ mainly controls the contractile state of airway smooth muscle (6–8), this indicates that protamine and heparin-protamine complex both inhibited the carbachol-induced tracheal smooth muscle contraction, in part, by decreasing [Ca2+]i. An increase in [Ca2+]i can be triggered by two mechanisms: the release of Ca2+ from the sarcoplasmic reticulum and the entry of extracellular Ca2+ through VDCCs or receptor-operated Ca2+ channels (6,8). It is well known that airway smooth muscle contraction is maintained by a Ca2+ influx through the membrane-associated Ca2+ channels (6) and that L-type VDCC antagonists decrease [Ca2+]i almost to the resting level in the presence of carbachol (7,8). This implies that the increase in [Ca2+]i through VDCCs may play an important, perhaps a controlling, role in maintaining airway smooth muscle contraction. Therefore, protamine and heparin-protamine complex may decrease [Ca2+]i by regulating VDCCs.
Heparin per se enhanced muscle contraction without changing [Ca2+]i. Heparin has a competitive effect on the inositol 1,4,5-triphosphate (IP3) receptor of the sarcoplasmic reticulum, which releases Ca2+ by agonist stimulation (13,14). Because heparin cannot pass through the cell membrane because of its negative charge, the competitive effect of this drug on the IP3 receptor cannot be the cause of an increase in muscle tension by this drug. It is assumed that heparin enhances agonist-induced muscle contraction by some Ca2+-independent mechanism.
The ICa measured in this study are presumed to reflect the activity of L-type VDCCs based on their time- and voltage-dependencies, their sensitivity to a nifedipine blockade, and their enhancement by the Ca2+ channel against Bay K 8644 (15,16). Protamine and heparin-protamine complex each inhibited ICa through VDCCs of canine tracheal smooth muscle cells without an apparent change in the kinetics of activation or inactivation. Neither protamine nor heparin-protamine complex altered the voltage-dependence of ICa, which suggests that these drugs have no effect on the membrane surface charge or on the voltage sensor for activation of the channel. These data are consistent with the above-described results showing that these drugs inhibited canine tracheal smooth muscle contraction with a decrease in [Ca2+]i.
We also studied the effects of these drugs on steady-state, voltage-dependent inactivation of ICa. During prolonged prepulse depolarization, a fraction of the VDCCs enters an unavailable or inactivated state. Protamine and heparin-protamine complex each significantly shifted the inactivation curves to more negative potentials without changing the sigmoid shapes of the curve, whereas heparin had no effect on the curve. A qualitatively similar shift induced by a dihydropyridine-sensitive Ca2+ channel antagonist, such as nifedipine, in porcine tracheal smooth muscle cells (17) has been interpreted as evidence of drug-induced stabilization of the inactivated state (18). We therefore speculate that protamine and heparin-protamine complex both have a dihydropyridine-sensitive Ca2+ channel antagonist-like inhibitory effect on VDCCs in canine tracheal smooth muscle cells, reflecting the stabilization of the VDCCs in both their activated and inactivated states (18). The strong cationic property of protamine is neutralized or substantially diminished by heparin. The negative or positive charge of these drugs therefore could not have contributed to the inhibition of VDCCs seen in the heparin-protamine complex study.
Protamine and heparin-protamine complex showed dose-dependent inhibition of both carbachol-induced contraction of tracheal smooth muscle and increase in [Ca2+]i. These drugs also inhibited ICa of tracheal smooth muscle cells in a dose-dependent manner. The potency order of these drugs in terms of the variables measured is heparin-protamine complex > protamine. Extrapolation of our data to the clinical situation must be viewed with caution because of possible species differences, in vivo/in vitro differences, and the techniques we used in this study. First, the plasma concentrations of these drugs in cardiac surgery are approximately 3.6–6.0 U/mL (4,5). In the clinical range of plasma concentrations, heparin and protamine thus had no direct effect either on muscle tension or on [Ca2+]i in vitro. Ceyhan and Celikel (1) showed that heparin inhaled by human asthmatic volunteers prevented methacholine-induced bronchoconstriction. Abraham et al. (19) demonstrated that heparin blocked immunologically induced tracheal smooth muscle contraction in sheep without affecting the contractile response to the airway smooth muscle agonist acetylcholine. These results suggest that heparin inhibits airway smooth muscle contraction by blocking the neural pathway (1) and/or inhibiting mast cell-mediator release (19). The heparin-protamine complex suppressed carbachol-induced contraction of smooth muscle and increase in [Ca2+]i at significantly lower concentrations than did protamine. Morel et al. (3) and Horiguchi et al. (20) demonstrated that the heparin-protamine complex induced bronchoconstriction and pulmonary hypertension in vivo, respectively. These constrictive phenomena are thought to be induced by thromboxane A2 generation after protamine reversal of heparin (3,20). Thus, the direct inhibitory effect of heparin-protamine complex on the carbachol-induced contraction of airway smooth muscle seen in this study is not incompatible with these data.
In conclusion, protamine and heparin-protamine complex inhibited contractions of carbachol-induced canine tracheal smooth muscle and increase in [Ca2+]i. These drugs also decreased the ICa of dispersed canine tracheal smooth muscle cells, indicating inhibition of VDCCs. These responses can account for the ability of these drugs to relax airway smooth muscle in vitro. A shift of the inactivation curve by these drugs to more negative potentials can be interpreted as evidence for drug-induced stabilization of the inactivated state. Heparin itself had a slight enhancing effect on carbachol-induced muscle contraction without changing [Ca2+]i, which indicates that heparin has an activating effect on the Ca2+-independent mechanism.
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Acknowledgments
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Supported in part by Grant 10770762 for research from the Ministry of Education, Science and Culture of Japan.
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Accepted for publication February 12, 1999.
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Abstract
Introduction
), protamine (
), and heparin-protamine complex (•). Symbols represent mean ± SD (n = 7). * P < 0.05, percentage comparison of control of peak ICa without the drugs. Both protamine and heparin-protamine complex significantly inhibited peak ICa in a dose-dependent manner.
