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Effects of sodium and potassium channel blockers on hyperinflation-induced slowly adapting pulmonary stretch receptor stimulation in the rat
Life Sciences 67 (2000) 2167Ð2175
Effects of sodium and potassium channel blockers on hyperinßation-induced slowly adapting pulmonary stretch receptor stimulation in the rat S. Matsumoto*, M. Ikeda, T. Nishikawa Department of Physiology, Nippon Dental University, School of Dentistry at Tokyo, 1-9-20 Fujimi, Chiyoda-ku, Tokyo 102-8159, Japan Received 14 October 1999; accepted 3 February 2000
Abstract The excitatory responses of slowly adapting pulmonary stretch receptor (SAR) activity to hyperinßation (inßation volume 5 3 tidal volumes) for approximately 10 respiratory cycles were examined before and after administration of ßecainide, a Na1 channel blocker, and 4-aminoprydine (4-AP), a K1 channel blocker. The experiments were performed in anesthetized, artiÞcially ventilated rats after unilateral vagotomy. During hyperinßation the SARs increased their activity during inßation and decreased their discharge during deßation. The magnitude of increased SAR activity during inßation became more prominent as compared to that of decreased receptor activity during deßation. Flecainide treatment (6 mg/kg) that was sufÞcient to block veratridine (50 mg/kg)-induced SAR stimulation did not signiÞcantly alter the excitatory response of SAR activity to hyperinßation. Subsequent administration of 3 mg/kg ßecainide (a total dose, 9 mg/kg) resulted in a greater inhibition of hyperinßationinduced SAR stimulation. Although administration of 4-AP (2 mg/kg) usually stimulated SAR activity, particularly in the deßation phase, in the control ventilation, 4-AP treatment had no signiÞcant effect on hyperinßation-induced SAR stimulation. These results suggest that the excitatory effect of hyperinßation on SAR activity may not be involved in the activation of either ßecainide-sensitive Na1 channels or 4-AP-sensitive K1 channels. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Slowly adapting pulmonary stretch receptor; Hyperinßation; Na1 channel; K1 channel; Rat
Introduction As a generalized phenomenon the mechanical deformation through stretch or distension of the airway smooth muscle is thought to stimulate the activity of slowly adapting pulmonary stretch receptors (SARs). The stretch-activated (SA) channels have been identiÞed in a * Corresponding author. Tel.: 81-3-3261-8706; fax: 81-3-3261-8740. 0024-3205/00/$ Ð see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 8 1 3 -4
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number of vertebrate tissues, invertebrates and plants [1,2]. The mechanism mediating the mechanosensitivity of SARs has not yet been determined, but those receptors are believed to have SA channels on the endings. In the mechanoreceptor cells and spider mechanoreceptor neurons, ion channels responsible for the mechanically activated currents are mainly selective for Na1 [3Ð6]. In view of the action potential generation, Na1 conductance plays an important role in depolarization of the nerve cell membranes. However, the relationship between the activation of SA channels on SAR endings and the inßux of Na 1 remains to be determined. Veratridine modiÞcation of Na1 channels in the nerve cell is TTX-sensitive [7, 8], and TTX can block veratridine-induced increase in the intracellular Na1 concentration ([Na1]i) of cultured rat hippocampal neurons [9]. Based on evidence that the Na1 channel blocker ßecainide [10,11] abolished veratridine-induced SAR stimulation [12], it is possible that stimulation of SARs by veratridine is mediated by the fast, TTX-sensitive Na1 channels, but not by the slow, TTX-insensitive Na1 channels. A recent study has demonstrated that a tetrodotoxin (TTX)-resistant channel subtype is expressed in the terminal axonal branches of many of the more slowly conducting dural afferent nerve Þbers such as slow Ad and C Þbers [13]. However, it has not been established whether the function of SA channels on the SAR endings is related to an increase in Na1 inßux via TTX-sensitive Na1 channels. In the present study the activation of SA channels of SARs due to hyperinßation was evaluated by an increase in SAR activity. We therefore investigated whether augmentation of SAR activity seen during hyperinßation is altered by pretreatment with ßecainide in a sufÞcient dose to abolish 50 mg/kg veratridine-induced SAR stimulation. On the other hand, ßecainide in human atrial myocytes is known to block the transient outward K1 current (Ito) [14]. In other series of experiments, responses of SARs to hyperinßation were compared before and after administration of 4-aminoprydine (4-AP) that blocks the Ito related to the action potential repolarization in the myelinated axons of isolated rat sciatic nerve [15]. The experiments were performed in anesthetized, artiÞcially ventilated rats after unilateral vagotomy. In this study, we selected SARs that responded to hyperinßation. Materials and methods Animal preparation Thirteen Wistar rats, weighing 280 to 360 g, were anesthetized with sodium pentobarbital (45Ð50 mg/kg, i.p.). The trachea was exposed through a middle incision in the neck and cannulated below the larynx. In order to obtain a wide space for liquid parafÞn, the trachea and esophagus were dissected free and retracted rostrally. Tracheal pressure (PT) was measured by connecting a polyethylene catheter inserted into the tracheal tube to a pressure transducer. The right carotid artery and jugular vein were cannulated for continuous monitoring of blood pressure (BP) and for administration of drugs or a 0.9% NaCl solution, respectively. During the course of experiments, supplemental doses (9Ð10 mg/kg, i.v.) of sodium pentobarbital were administered to maintain the level of anesthesia, abolishing the corneal reßex and pain reßexes induced by tail pinch. Then the left vagus nerve was exposed and sectioned. The right vagus nerve was left intact. The rectal temperature was maintained at around 378C by
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means of a heating lamp. The animals were paralyzed with an intravenous administration of gallamine (5Ð10 mg/kg) for 30Ð60 min and additional doses (3Ð5 mg/kg) of gallamine were administered to eliminate spontaneous respiratory movements as needed. The stroke volume of the respirator was set at 10 ml/kg and its frequency ranged from 50 to 60 cycles/min. Measurement of slowly adapting pulmonary stretch receptors The peripheral end of the cut left vagus nerve was desheathed. To record the single unit activity of SARs, thin strands containing afferent nerve Þbers were separated, placed on a unipolar silver electrode and submerged in a pool with warm liquid parafÞn (37Ð388C). The SARs were identiÞed, on the basis of their Þring behavior during inßation, as follows: 1) The SARs (low-threshold) increased their activity during inßation and decreased their discharge during deßation. 2) The increase in SAR activity was proportional to that in the inßation volume of the respirator. 3) The discharge of SARs continued as long as the tracheal tube was occluded in a hyperinßated condition. The SAR activity was ampliÞed and selected by means of a window discriminator for counting the number of impulses. Experimental design The experiments were designed to test the roles of generalized Na1 and K1 channels in the responses of SARs to hyperinßation. 1) In 8 SAR Þbers in 8 rats, changes in SAR activity and PT in response to hyperinßation (inßation volume 5 3 VT) for about 10 respiratory cycles were determined. Ten minutes after intravenous administration of ßecainide (6 mg/kg), a Na1 channel blocker, sufÞcient for blocking the excitatory response of SAR activity to the Na1 channel opener veratridine (50 mg/kg, i.v.), the same sets of experiments were repeated. After subsequent administration of ßecainide (3 mg/kg, a total dose 5 9 mg/kg), the experiments were performed under the same conditions. 2) In 5 SAR Þbers in 5 rats, responses of SAR activity and PT to hyperinßation for about 10 respiratory cycles were examined before and after intravenous administration of 4-AP (2 mg/kg). Drugs The drugs used in this study were veratridine (Sigma Chemical, St Louis, MO), ßecainide (Eizai Pharmaceutical Co. Ltd. Tokyo, Japan) and 4-AP (Sigma). Veratridine (10 mg) was dissolved in a small amount of weak HCl and diluted with a 0.9% NaCl solution. Flecainide (10 mg) was dissolved in a 5% glucose solution. 4-AP (20 mg) was dissolved in a 0.9% NaCl solution. Statistical analysis During control condition, the Þring rate of SARs during one whole respiratory cycle was measured over several respiratory cycles and expressed as imp/s. The SAR responses to hyperinßation (inßation volume 53 VT) for approximately ten respiratory cycles and to veratridine administration were obtained by counting the Þring rates of receptors during hyperinßation and between onset of the increased receptor activity and recovery to the control level, respectively, and the average activities of SARs during one whole respiratory cycle were expressed as imp/s. Similarly, control values for PT were averaged over several respiratory cy-
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cles and expressed as cmH2O. The responses of PT to hyperinßation and veratridine administration were obtained by measuring the respiratory parameter, as described above, and the average values of PT were expressed as cmH2O. The statistical signiÞcance of the effects of ßecainide (a total dose, 6 and 9 mg/kg) and 4-AP (2 mg/kg) on the responses of SAR activity and PT to hyperinßation was calculated by a two-way ANOVA. All values were expressed as mean 6 S.E. A value of P,0.05 was considered statistically signiÞcant. Results Administration of veratridine (50 mg/kg) caused excitation of the SAR activity during both inßation and deßation, and this excitation lasted for about 150 s. Under these conditions, the value of PT did not change signiÞcantly (Fig. 1A). Flecainide administration (6 mg/ kg) usually inhibited SAR activity during deßation, and 10 min later the receptor discharge showed a decrease in the SAR activity during both inßation and deßation but the response was not associated with any signiÞcant change in PT. The veratridine-induced SAR stimulation was blocked by pretreatment with ßecainide (Fig. 1B). In a ßecainide (6 mg/kg)-treated animal, as shown in Fig. 2A and 2B, this Na1 channel blocker caused a small inhibition on the responses of SAR activity to normal inßation and hyperinßation. The effects of ßecainide (6 mg/kg) on the responses of SAR activity to hyperinßation in 8 rats are summarized in Fig.
Fig. 1. Responses in PT and SAR activity to veratridine (. 50 mg/kg) before (A) and after (B) administration of ßecainide (6 mg/kg). 100 s indicates the elapsed time between recordings.
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Fig. 2. Responses in PT and SAR activity to control inßation (inßation volume 5 1 VT) and hyperinßation (inßation volume 5 3 VT) before (A) and after administration of ßecainide at a total dose of 6 (B) and 9 mg/kg (C). D: Changes in SAR activity and PT in response to hyperinßation (inßation volume 5 3 VT) before (h) and after pretreatment with ßecainide at a total dose of 6 (h) and 9 mg/kg (j). Vertical bars show mean 6 standard error (n58). * Statistically signiÞcant different from control values (P,0.05). # Statistically signiÞcant different from the values before pretreatment of ßecainide (P,0.05).
2D. The discharges of SARs during control and after ßecainide treatment were 49.2 6 4.6 and 46.2 6 5.1 imp/s, respectively, and, during hyperinßation in the absence and presence of ßecainide, were 83.4 6 6.2 and 79.4 6 4.9 imp/s, respectively. However, the Na1 channel blocker ßecainide (6 mg/kg) did not signiÞcantly alter the excitatory effect of hyperinßation on SAR activity (percent excitation: absence, 77.4 6 6.9, n58; in the presence of ßecainide, 6 mg/kg, 78.1 6 7.2, n58, P.0.05). Subsequent administration of ßecainide (3 mg/kg) caused a greater inhibition on the SAR responses to both normal ventilation and hyperinßation (Fig. 2A and 2C). Fig. 2D summarizes the effect of ßecainide at a total dose of 9 mg/kg on the SAR response to hyperinßation. The discharges of SARs during control and after ßecainide administration (a total dose, 9 mg/kg) were 49.2 6 4.6 and 38.2 6 3.5 imp/s, respectively, and, during hyperinßation in the absence and presence of ßecainide were 83.4 6 6.2 and 42.2 6 3.4 imp/s, respectively. Flecainide at a total dose of 9 mg/kg greatly inhibited hyperinßation-induced SAR stimulation (percent excitation: absence, 77.4 6 6.9, n58; in the presence of ßecainide, a total dose of 9 mg/kg, 10.4 6 2.8, n58, P,0.05). The changes in PT in responses to normal ventilation and hyperinßation were not signiÞcantly altered by administration of ßecainide at any dose (Fig. 2D).
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Fig. 3. Responses in PT and SAR activity to control inßation (inßation volume 5 1 VT) and hyperinßation (inßation volume 5 3 VT) before (A) and after (B) the administration of 4-AP (2 mg/kg). C: Changes in SAR activity and PT in response to hyperinßation (inßation volume 5 3 VT) before (h) and after (j) pretreatment with 4-AP. Vertical bars show mean 6 standard error (n55). * Statistically signiÞcant difference from control values (P,0.05). # Statistically signiÞcant difference from the values before pretreatment with 4-AP.
Figs. 3A and 3B show the responses of SAR activity and PT to normal inßation and hyperinßation before and after administration of 4-AP (2 mg/kg). Pretreatment with 4-AP caused an increase in the SAR activity during deßation but did not signiÞcantly alter PT. In addition, excitatory responses of SAR activity to hyperinßation were not signiÞcantly inßuenced by 4-AP treatment. The effects of 4-AP (2 mg/kg) on the responses of SAR activity to hyperinßation in 5 rabbits are summarized in Fig. 3C. The SAR activities during control and after 4-AP treatment were 43.6 6 4.4 and 50.8 6 4.2 imp/s, respectively, and, during hyperinßation in the absence and presence of 4-AP, were 84.1 6 3.7 and 92.2 6 6.3 imp/s, respectively. The excitatory effect of hyperinßation on SAR activity was not signiÞcantly inßuenced by the K1 channel blocker 4-AP (2 mg/kg) (percent excitation: absence, 88.4 6 7.4, n55; in the presence of 4-AP, 2 mg/kg, 83.4 6 7.6, n55, P.0.05). 4-AP treatment had no signiÞcant effect on the responses of PT to normal inßation and hyperinßation. Discussion With the exception of rapidly adapting pulmonary stretch receptors (RARs) and vagal C Þbers, three different types of afferent receptors originating in the rat lungs have been demon-
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strated by Tsubone [16] who termed pulmonary stretch receptors, deßation-sensitive receptors and irritant-like receptors. Similar types of the units in the same species are observed in the study of Bergren and Peterson [17]; they stated that all of these receptors belonged to the same category of SARs. Concerning the Þring patterns of SARs, Bergren and Peterson [17] also classiÞed four subtypes of receptors such as inßationary units, most inßationary units, most deßationary units and deßationary units. The SARs of this study corresponded with the most inßationary SARs that increased their activity during inßation and decreased their discharge during deßation in the control, and these receptors increased their activity linearly as inßation volumes increased. Veratridine results in slowing of the Na1 channel inactivation and also maintains the channels at the opening state [18], indicating the concomitant increase in Na1 inßux occurs in the condition in which the membrane is not under voltage control [8,19]. Intravenous administration of veratridine stimulated SAR activity but the response was not associated an increase in PT as a global index of bronchomotor tone. These results of this study are consistent with those obtained in the rabbit, demonstrating that opening of voltage-dependent Na1 channels by veratridine does not elicit the release of acetylcholine (ACh) from parasympathetic nerve endings [20]. Although the right vagus nerve was left intact in this study, at a dose of veratridine ranging 50 mg/kg it did not facilitate either ACh release or smooth muscle contraction due to efferent innervation of the right vagus nerve. In addition, the similarity between ßecainide block of veratridine-induced SAR stimulation in the present experiments and its action on the rabbit [13] supports the idea that there are no functional differences concerning the antagonizing effect of ßecainide. Flecainide treatment (6 mg/kg) that blocked 50 mg/kg veratridine-induced SAR stimulation had no signiÞcant effect on the excitatory responses of SAR activity to hyperinßation. Therefore, it is conceivable that the excitatory mechanism of hyperinßation on SAR activity may not be related to the function of ßecainide-sensitive Na1 channels. Presumably, the different types of Na1 channels that are insensitive to a Na1 channel blocker ßecainide may exist on the SAR endings. Concerning the sensitivity of SAR activity to ßecainide, it should consider a number of factors in addition to Na1 channel subtypes, such as the spatial distribution and the density of Na1 channels. In addition, subsequent administration of 3 mg/kg ßecainide (a total dose 5 9 mg/kg), which reduced the discharge of SARs in normal ventilation, greatly attenuated hyperinßation-induced SAR stimulation, indicating that ßecainide administration up to 9 mg/kg would inhibit the function of Na1 channel subtypes that are insensitive to ßecainide. Further studies are needed to clarify the functional coupling between Na1 channel subtypes and SA channels on the SAR endings. In the myelinated axons of the rat sciatic nerve Þbers, 4-AP-sensitive K1 channels are related to action potential repolarization [15] and 4-AP can elicit both membrane depolarization and repetitive Þring [21,22]. In this study, administration of 4-AP in the control ventilation increased SAR activity and caused a pressor effect as described in previous studies [23, 24], but the latter effect was very short lasting. The broad spike of action potentials in myelinated axons of the rat and frog occurs after external application of 4-AP [15,25], but we could not observe such an effect because we measured extracellular action potentials of SARs. In comparison with the action between ßecainide and 4-AP, they were quite opposite effects on SAR activity in the control condition, indicating that the effect of ßecainide on SAR activity is not explained by blockade of Ito. The fact that 4-AP treatment had no signif-
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icant effect on the excitatory response of SAR activity to hyperinßation suggests that 4-APsensitive K1 currents would not play a signiÞcant role in the hyperinßation-induced excitation of SAR activity.
Acknowledgment The authors thank Eizai Pharmaceutical Co. Ltd. for the gift of flecainide.
References 1. Sachs F. Baroreceptor mechanisms at the cellular level. Federation Proceeding 1987; 46 (1): 12Ð6. 2. Morris CE. Mechanosensitive ion channels. Journal of Membrane Biology 1990; 113 (2): 93Ð107. 3. Diamond J, Gray JAB, Inman DR. The relationship between receptor potentials and the concentration of sodium ions. Journal of Physiology (London) 1958; 142: 382Ð94. 4. Rydqvist B, Purali N. Transducer properties of the rapidly adapting stretch receptor neurone in the crayÞsh (Pacifastacus leniusculus). Journal of Physiology (London) 1993; 469: 193Ð211. 5. Juusola M, Seyfarth E-A, French AA. Sodium-dependent receptor current in a new mechanoreceptor preparation. Journal of Neuroplysiology 1994; 72 (6) : 3026Ð8. 6. Hšger U, Torkkeli PH, Seyfarth E-A, French AA. Ionic selectivity of mechanically activated channels in spider neurons. Journal of Neurophysiology 1997; 78 (4): 2079Ð85. 7. Gatteral WA. Cellular and molecular biology of voltage-gated sodium channels. Physiological Review 1992; 72: S 15Ð8. 8. Narahashi T. Chemical as tools in the study of excitable membranes. Physiological Review 1974; 54 (4): 813Ð89. 9. Rose CR, Ransom BR. Regulation of intracellular sodium in cultured rat hippocampal neurones. Journal of Physiology (London) 1997; 499 (3): 573Ð87. 10. Banitt EH, Bronn WR, Coyne WE, Achmid JR. Antiarrhythmic. 2. Systenthesis and antiarrhythmic activity of N-(piperidylalkyl) trißuoroethoxybenzamides. Journal of Medical Chemistry. 1977; 20 (6): 821Ð6. 11. Campbell TJ, Williams EMV. Voltage-and time-dependent depression of maximum rate of depolarization of guinea-pig ventricular action potentials by two new antiarrhythmic drugs, ßecainide and lorcainide. Cardiovascular Research 1983; 17 (5): 251Ð8. 12. Matsumoto S, Shimizu T. Flecainide blocks the stimulatory effect of veratridine on slowly adapting pulmonary stretch receptors in anaesthetized rabbits without changing lung mechanics. Acta Physiological Scandinavia 1995; 155 (3): 297Ð302. 13. Strassman AM, Raymond SA. Electrophysiological evidence for tetrodotoxin-resistant sodium channels in slowly conducting dural sensory Þbers. Journal of Neurophysiology 1999; 81 (2): 413Ð24. 14. Wang Z, Fermini B, Nattel S. Effects of ßecainide, quidine, and 4-aminoprydine on transient outward and ultrarapid delayed rectiÞer currents in human atrial myocytes. Journal of Pharmacology and Experimental Therapeutics. 1995; 272 (1): 184Ð96. 15. Kocsis JD, Eng DL, Gordon TR, Waxman SG. Functional differences between 4-aminoprydine and tetraethylammonium-sensitive potassium channels in myelinated axons. Neuroscience Letters 1987; 75 (2): 193Ð8. 16. Tubone H. Characteristics of vagal afferent activity in rats: three types of pulmonary receptors respondings to collapse, inßation, and deßation of the lung. Experimental Neurology 1986; 92: 541Ð52. 17. Bergren DR, Peterson DF. IdentiÞcation of vagal sensory receptors in the rat lung: are there subtypes of slowly adapting receptors? Journal of Physiology (London) 1993; 464: 681Ð98. 18. Strichartz G, Rando T, Wang GK. An integrated view of the molecular toxicology of sodium channel gating in excitable cells. Annual Review of Neuroscience 1987; 10: 237Ð67.
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19. Ulbrich W. The effect of veratridine on excitable membranes of nerve and muscle. Ergebnisse der Physiologie, Biologischen Chemie und Experimentellen Pharmakologie 1969; 61: 18Ð71. 20. Matsumoto S, Takahashi T, Tanimoto T, Saiki C, Takeda M, Ojima K. Excitatory mechanism of veratridine on slowly adapting pulmonary stretch receptors in anesthetized rabbits. Life Sciences 1998; 63 (16): 1431Ð7. 21. Yeh JZ, Oxford GS, Wu CH, Narahashi T. Interactions of aminoprydines with potassium channels of squid axon membranes. Biophysics Journal 1976; 16 (1): 77Ð81. 22. Yeh JZ, Oxford GS, Wu CH, Narahashi T. Dynamics of aminoprydine block of potassium channels in squid axon membrane. Journal of General Physiology 1976; 65 (5): 519Ð35. 23. Yanagisawa T, Taira N. Positive inotropic effect of 4-aminoprydine on dog ventricular muscle. NaunynSchmiedebergÕs Archives of Pharmacology 1979; 307 (3): 207Ð12. 24. Chang F-CT, Bauer RM, Benton BJ, Kellen SA, Capacio BR. 4-aminoprydine antagonizes saxitoxin-and tetrodotoxin-induced cardiorespiratory depression. Toxicon 1996; 34 (6): 671Ð90. 25. Poulter MO, Padjen AL. Different voltage-dependent potassium couductances regulate action potential repolarization and excitabilty in frog myelinated axon. Neuroscience 1995; 68 (2): 497Ð504.
Effects of sodium and potassium channel blockers on hyperinßation-induced slowly adapting pulmonary stretch receptor stimulation in the rat S. Matsumoto*, M. Ikeda, T. Nishikawa Department of Physiology, Nippon Dental University, School of Dentistry at Tokyo, 1-9-20 Fujimi, Chiyoda-ku, Tokyo 102-8159, Japan Received 14 October 1999; accepted 3 February 2000
Abstract The excitatory responses of slowly adapting pulmonary stretch receptor (SAR) activity to hyperinßation (inßation volume 5 3 tidal volumes) for approximately 10 respiratory cycles were examined before and after administration of ßecainide, a Na1 channel blocker, and 4-aminoprydine (4-AP), a K1 channel blocker. The experiments were performed in anesthetized, artiÞcially ventilated rats after unilateral vagotomy. During hyperinßation the SARs increased their activity during inßation and decreased their discharge during deßation. The magnitude of increased SAR activity during inßation became more prominent as compared to that of decreased receptor activity during deßation. Flecainide treatment (6 mg/kg) that was sufÞcient to block veratridine (50 mg/kg)-induced SAR stimulation did not signiÞcantly alter the excitatory response of SAR activity to hyperinßation. Subsequent administration of 3 mg/kg ßecainide (a total dose, 9 mg/kg) resulted in a greater inhibition of hyperinßationinduced SAR stimulation. Although administration of 4-AP (2 mg/kg) usually stimulated SAR activity, particularly in the deßation phase, in the control ventilation, 4-AP treatment had no signiÞcant effect on hyperinßation-induced SAR stimulation. These results suggest that the excitatory effect of hyperinßation on SAR activity may not be involved in the activation of either ßecainide-sensitive Na1 channels or 4-AP-sensitive K1 channels. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Slowly adapting pulmonary stretch receptor; Hyperinßation; Na1 channel; K1 channel; Rat
Introduction As a generalized phenomenon the mechanical deformation through stretch or distension of the airway smooth muscle is thought to stimulate the activity of slowly adapting pulmonary stretch receptors (SARs). The stretch-activated (SA) channels have been identiÞed in a * Corresponding author. Tel.: 81-3-3261-8706; fax: 81-3-3261-8740. 0024-3205/00/$ Ð see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 0 )0 0 8 1 3 -4
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number of vertebrate tissues, invertebrates and plants [1,2]. The mechanism mediating the mechanosensitivity of SARs has not yet been determined, but those receptors are believed to have SA channels on the endings. In the mechanoreceptor cells and spider mechanoreceptor neurons, ion channels responsible for the mechanically activated currents are mainly selective for Na1 [3Ð6]. In view of the action potential generation, Na1 conductance plays an important role in depolarization of the nerve cell membranes. However, the relationship between the activation of SA channels on SAR endings and the inßux of Na 1 remains to be determined. Veratridine modiÞcation of Na1 channels in the nerve cell is TTX-sensitive [7, 8], and TTX can block veratridine-induced increase in the intracellular Na1 concentration ([Na1]i) of cultured rat hippocampal neurons [9]. Based on evidence that the Na1 channel blocker ßecainide [10,11] abolished veratridine-induced SAR stimulation [12], it is possible that stimulation of SARs by veratridine is mediated by the fast, TTX-sensitive Na1 channels, but not by the slow, TTX-insensitive Na1 channels. A recent study has demonstrated that a tetrodotoxin (TTX)-resistant channel subtype is expressed in the terminal axonal branches of many of the more slowly conducting dural afferent nerve Þbers such as slow Ad and C Þbers [13]. However, it has not been established whether the function of SA channels on the SAR endings is related to an increase in Na1 inßux via TTX-sensitive Na1 channels. In the present study the activation of SA channels of SARs due to hyperinßation was evaluated by an increase in SAR activity. We therefore investigated whether augmentation of SAR activity seen during hyperinßation is altered by pretreatment with ßecainide in a sufÞcient dose to abolish 50 mg/kg veratridine-induced SAR stimulation. On the other hand, ßecainide in human atrial myocytes is known to block the transient outward K1 current (Ito) [14]. In other series of experiments, responses of SARs to hyperinßation were compared before and after administration of 4-aminoprydine (4-AP) that blocks the Ito related to the action potential repolarization in the myelinated axons of isolated rat sciatic nerve [15]. The experiments were performed in anesthetized, artiÞcially ventilated rats after unilateral vagotomy. In this study, we selected SARs that responded to hyperinßation. Materials and methods Animal preparation Thirteen Wistar rats, weighing 280 to 360 g, were anesthetized with sodium pentobarbital (45Ð50 mg/kg, i.p.). The trachea was exposed through a middle incision in the neck and cannulated below the larynx. In order to obtain a wide space for liquid parafÞn, the trachea and esophagus were dissected free and retracted rostrally. Tracheal pressure (PT) was measured by connecting a polyethylene catheter inserted into the tracheal tube to a pressure transducer. The right carotid artery and jugular vein were cannulated for continuous monitoring of blood pressure (BP) and for administration of drugs or a 0.9% NaCl solution, respectively. During the course of experiments, supplemental doses (9Ð10 mg/kg, i.v.) of sodium pentobarbital were administered to maintain the level of anesthesia, abolishing the corneal reßex and pain reßexes induced by tail pinch. Then the left vagus nerve was exposed and sectioned. The right vagus nerve was left intact. The rectal temperature was maintained at around 378C by
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means of a heating lamp. The animals were paralyzed with an intravenous administration of gallamine (5Ð10 mg/kg) for 30Ð60 min and additional doses (3Ð5 mg/kg) of gallamine were administered to eliminate spontaneous respiratory movements as needed. The stroke volume of the respirator was set at 10 ml/kg and its frequency ranged from 50 to 60 cycles/min. Measurement of slowly adapting pulmonary stretch receptors The peripheral end of the cut left vagus nerve was desheathed. To record the single unit activity of SARs, thin strands containing afferent nerve Þbers were separated, placed on a unipolar silver electrode and submerged in a pool with warm liquid parafÞn (37Ð388C). The SARs were identiÞed, on the basis of their Þring behavior during inßation, as follows: 1) The SARs (low-threshold) increased their activity during inßation and decreased their discharge during deßation. 2) The increase in SAR activity was proportional to that in the inßation volume of the respirator. 3) The discharge of SARs continued as long as the tracheal tube was occluded in a hyperinßated condition. The SAR activity was ampliÞed and selected by means of a window discriminator for counting the number of impulses. Experimental design The experiments were designed to test the roles of generalized Na1 and K1 channels in the responses of SARs to hyperinßation. 1) In 8 SAR Þbers in 8 rats, changes in SAR activity and PT in response to hyperinßation (inßation volume 5 3 VT) for about 10 respiratory cycles were determined. Ten minutes after intravenous administration of ßecainide (6 mg/kg), a Na1 channel blocker, sufÞcient for blocking the excitatory response of SAR activity to the Na1 channel opener veratridine (50 mg/kg, i.v.), the same sets of experiments were repeated. After subsequent administration of ßecainide (3 mg/kg, a total dose 5 9 mg/kg), the experiments were performed under the same conditions. 2) In 5 SAR Þbers in 5 rats, responses of SAR activity and PT to hyperinßation for about 10 respiratory cycles were examined before and after intravenous administration of 4-AP (2 mg/kg). Drugs The drugs used in this study were veratridine (Sigma Chemical, St Louis, MO), ßecainide (Eizai Pharmaceutical Co. Ltd. Tokyo, Japan) and 4-AP (Sigma). Veratridine (10 mg) was dissolved in a small amount of weak HCl and diluted with a 0.9% NaCl solution. Flecainide (10 mg) was dissolved in a 5% glucose solution. 4-AP (20 mg) was dissolved in a 0.9% NaCl solution. Statistical analysis During control condition, the Þring rate of SARs during one whole respiratory cycle was measured over several respiratory cycles and expressed as imp/s. The SAR responses to hyperinßation (inßation volume 53 VT) for approximately ten respiratory cycles and to veratridine administration were obtained by counting the Þring rates of receptors during hyperinßation and between onset of the increased receptor activity and recovery to the control level, respectively, and the average activities of SARs during one whole respiratory cycle were expressed as imp/s. Similarly, control values for PT were averaged over several respiratory cy-
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cles and expressed as cmH2O. The responses of PT to hyperinßation and veratridine administration were obtained by measuring the respiratory parameter, as described above, and the average values of PT were expressed as cmH2O. The statistical signiÞcance of the effects of ßecainide (a total dose, 6 and 9 mg/kg) and 4-AP (2 mg/kg) on the responses of SAR activity and PT to hyperinßation was calculated by a two-way ANOVA. All values were expressed as mean 6 S.E. A value of P,0.05 was considered statistically signiÞcant. Results Administration of veratridine (50 mg/kg) caused excitation of the SAR activity during both inßation and deßation, and this excitation lasted for about 150 s. Under these conditions, the value of PT did not change signiÞcantly (Fig. 1A). Flecainide administration (6 mg/ kg) usually inhibited SAR activity during deßation, and 10 min later the receptor discharge showed a decrease in the SAR activity during both inßation and deßation but the response was not associated with any signiÞcant change in PT. The veratridine-induced SAR stimulation was blocked by pretreatment with ßecainide (Fig. 1B). In a ßecainide (6 mg/kg)-treated animal, as shown in Fig. 2A and 2B, this Na1 channel blocker caused a small inhibition on the responses of SAR activity to normal inßation and hyperinßation. The effects of ßecainide (6 mg/kg) on the responses of SAR activity to hyperinßation in 8 rats are summarized in Fig.
Fig. 1. Responses in PT and SAR activity to veratridine (. 50 mg/kg) before (A) and after (B) administration of ßecainide (6 mg/kg). 100 s indicates the elapsed time between recordings.
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Fig. 2. Responses in PT and SAR activity to control inßation (inßation volume 5 1 VT) and hyperinßation (inßation volume 5 3 VT) before (A) and after administration of ßecainide at a total dose of 6 (B) and 9 mg/kg (C). D: Changes in SAR activity and PT in response to hyperinßation (inßation volume 5 3 VT) before (h) and after pretreatment with ßecainide at a total dose of 6 (h) and 9 mg/kg (j). Vertical bars show mean 6 standard error (n58). * Statistically signiÞcant different from control values (P,0.05). # Statistically signiÞcant different from the values before pretreatment of ßecainide (P,0.05).
2D. The discharges of SARs during control and after ßecainide treatment were 49.2 6 4.6 and 46.2 6 5.1 imp/s, respectively, and, during hyperinßation in the absence and presence of ßecainide, were 83.4 6 6.2 and 79.4 6 4.9 imp/s, respectively. However, the Na1 channel blocker ßecainide (6 mg/kg) did not signiÞcantly alter the excitatory effect of hyperinßation on SAR activity (percent excitation: absence, 77.4 6 6.9, n58; in the presence of ßecainide, 6 mg/kg, 78.1 6 7.2, n58, P.0.05). Subsequent administration of ßecainide (3 mg/kg) caused a greater inhibition on the SAR responses to both normal ventilation and hyperinßation (Fig. 2A and 2C). Fig. 2D summarizes the effect of ßecainide at a total dose of 9 mg/kg on the SAR response to hyperinßation. The discharges of SARs during control and after ßecainide administration (a total dose, 9 mg/kg) were 49.2 6 4.6 and 38.2 6 3.5 imp/s, respectively, and, during hyperinßation in the absence and presence of ßecainide were 83.4 6 6.2 and 42.2 6 3.4 imp/s, respectively. Flecainide at a total dose of 9 mg/kg greatly inhibited hyperinßation-induced SAR stimulation (percent excitation: absence, 77.4 6 6.9, n58; in the presence of ßecainide, a total dose of 9 mg/kg, 10.4 6 2.8, n58, P,0.05). The changes in PT in responses to normal ventilation and hyperinßation were not signiÞcantly altered by administration of ßecainide at any dose (Fig. 2D).
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Fig. 3. Responses in PT and SAR activity to control inßation (inßation volume 5 1 VT) and hyperinßation (inßation volume 5 3 VT) before (A) and after (B) the administration of 4-AP (2 mg/kg). C: Changes in SAR activity and PT in response to hyperinßation (inßation volume 5 3 VT) before (h) and after (j) pretreatment with 4-AP. Vertical bars show mean 6 standard error (n55). * Statistically signiÞcant difference from control values (P,0.05). # Statistically signiÞcant difference from the values before pretreatment with 4-AP.
Figs. 3A and 3B show the responses of SAR activity and PT to normal inßation and hyperinßation before and after administration of 4-AP (2 mg/kg). Pretreatment with 4-AP caused an increase in the SAR activity during deßation but did not signiÞcantly alter PT. In addition, excitatory responses of SAR activity to hyperinßation were not signiÞcantly inßuenced by 4-AP treatment. The effects of 4-AP (2 mg/kg) on the responses of SAR activity to hyperinßation in 5 rabbits are summarized in Fig. 3C. The SAR activities during control and after 4-AP treatment were 43.6 6 4.4 and 50.8 6 4.2 imp/s, respectively, and, during hyperinßation in the absence and presence of 4-AP, were 84.1 6 3.7 and 92.2 6 6.3 imp/s, respectively. The excitatory effect of hyperinßation on SAR activity was not signiÞcantly inßuenced by the K1 channel blocker 4-AP (2 mg/kg) (percent excitation: absence, 88.4 6 7.4, n55; in the presence of 4-AP, 2 mg/kg, 83.4 6 7.6, n55, P.0.05). 4-AP treatment had no signiÞcant effect on the responses of PT to normal inßation and hyperinßation. Discussion With the exception of rapidly adapting pulmonary stretch receptors (RARs) and vagal C Þbers, three different types of afferent receptors originating in the rat lungs have been demon-
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strated by Tsubone [16] who termed pulmonary stretch receptors, deßation-sensitive receptors and irritant-like receptors. Similar types of the units in the same species are observed in the study of Bergren and Peterson [17]; they stated that all of these receptors belonged to the same category of SARs. Concerning the Þring patterns of SARs, Bergren and Peterson [17] also classiÞed four subtypes of receptors such as inßationary units, most inßationary units, most deßationary units and deßationary units. The SARs of this study corresponded with the most inßationary SARs that increased their activity during inßation and decreased their discharge during deßation in the control, and these receptors increased their activity linearly as inßation volumes increased. Veratridine results in slowing of the Na1 channel inactivation and also maintains the channels at the opening state [18], indicating the concomitant increase in Na1 inßux occurs in the condition in which the membrane is not under voltage control [8,19]. Intravenous administration of veratridine stimulated SAR activity but the response was not associated an increase in PT as a global index of bronchomotor tone. These results of this study are consistent with those obtained in the rabbit, demonstrating that opening of voltage-dependent Na1 channels by veratridine does not elicit the release of acetylcholine (ACh) from parasympathetic nerve endings [20]. Although the right vagus nerve was left intact in this study, at a dose of veratridine ranging 50 mg/kg it did not facilitate either ACh release or smooth muscle contraction due to efferent innervation of the right vagus nerve. In addition, the similarity between ßecainide block of veratridine-induced SAR stimulation in the present experiments and its action on the rabbit [13] supports the idea that there are no functional differences concerning the antagonizing effect of ßecainide. Flecainide treatment (6 mg/kg) that blocked 50 mg/kg veratridine-induced SAR stimulation had no signiÞcant effect on the excitatory responses of SAR activity to hyperinßation. Therefore, it is conceivable that the excitatory mechanism of hyperinßation on SAR activity may not be related to the function of ßecainide-sensitive Na1 channels. Presumably, the different types of Na1 channels that are insensitive to a Na1 channel blocker ßecainide may exist on the SAR endings. Concerning the sensitivity of SAR activity to ßecainide, it should consider a number of factors in addition to Na1 channel subtypes, such as the spatial distribution and the density of Na1 channels. In addition, subsequent administration of 3 mg/kg ßecainide (a total dose 5 9 mg/kg), which reduced the discharge of SARs in normal ventilation, greatly attenuated hyperinßation-induced SAR stimulation, indicating that ßecainide administration up to 9 mg/kg would inhibit the function of Na1 channel subtypes that are insensitive to ßecainide. Further studies are needed to clarify the functional coupling between Na1 channel subtypes and SA channels on the SAR endings. In the myelinated axons of the rat sciatic nerve Þbers, 4-AP-sensitive K1 channels are related to action potential repolarization [15] and 4-AP can elicit both membrane depolarization and repetitive Þring [21,22]. In this study, administration of 4-AP in the control ventilation increased SAR activity and caused a pressor effect as described in previous studies [23, 24], but the latter effect was very short lasting. The broad spike of action potentials in myelinated axons of the rat and frog occurs after external application of 4-AP [15,25], but we could not observe such an effect because we measured extracellular action potentials of SARs. In comparison with the action between ßecainide and 4-AP, they were quite opposite effects on SAR activity in the control condition, indicating that the effect of ßecainide on SAR activity is not explained by blockade of Ito. The fact that 4-AP treatment had no signif-
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icant effect on the excitatory response of SAR activity to hyperinßation suggests that 4-APsensitive K1 currents would not play a signiÞcant role in the hyperinßation-induced excitation of SAR activity.
Acknowledgment The authors thank Eizai Pharmaceutical Co. Ltd. for the gift of flecainide.
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