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Effects of acetazolamide and 4-aminoprydine on the responses of deflationary slowly adapting pulmonary stretch receptors to CO2 inhalation in the rat
Life Sciences 72 (2003) 1757 – 1771 www.elsevier.com/locate/lifescie
Effects of acetazolamide and 4-aminoprydine on the responses of deflationary slowly adapting pulmonary stretch receptors to CO2 inhalation in the rat S. Matsumoto *, M. Ikeda, T. Nishikawa, S. Yoshida, J. Kadoi, T. Tanimoto, C. Saiki, M. Takeda Department of Physiology, Nippon Dental University, School of Dentistry at Tokyo, 1-9-20 Fujimi, Chiyoda-ku, Tokyo 102-8159, Japan Received 22 May 2002; accepted 8 November 2002
Abstract The inhibitory effect of CO2 on deflationary slowly adapting pulmonary stretch receptors (deflationary SARs) was investigated before and after administration of acetazolamide, a carbonic anhydrase (CA) inhibitor, or 4aminopyridine (4-AP), a K+ channel blocker, in anesthetized, artificially ventilated rats after unilateral vagotomy. CO2 inhalation (maximum tracheal CO2 concentration ranging from 9 to 12%) for approximately 60 s decreased the impulse activity of deflationary SARs but had no significant effect on tracheal pressure (PT) as an index of bronchomotor tone. Acetazolamide treatment (20 mg/kg) diminished the inhibitory response of deflationary SARs to CO2 inhalation. 4-AP (0.7 and 2.0 mg/kg) dose-dependently attenuated the decrease in deflationary SAR activity induced by CO2 inhalation. When comparing the maximum attenuation due to 4-AP (2.0 mg/kg) and acetazolamide (20 mg/kg) in CO2-induced deflationary SAR inhibition, blockade of K+ channels had a more pronounced effect. These results suggest that inhibition of deflationary SARs by CO2 inhalation may be largely mediated by the stimulating action of 4-AP-sensitive K+ currents in the nerve terminals of the receptors. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Deflationary slowly adapting pulmonary stretch receptor; CO2 inhalation; Inhibition; CA inhibitor; K+ channel blocker
* Corresponding author. Tel.: +81-3-3261-8706; fax: +81-3-3261-8740. E-mail address: [email protected] (S. Matsumoto). 0024-3205/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved. doi:10.1016/S0024-3205(02)02508-0
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Introduction Three categories of vagal afferent receptors have been identified in the mammalian lung: slowly adapting pulmonary stretch receptors (SARs), rapidly adapting pulmonary stretch receptors (RARs) and vagal C fiber endings. There are few studies investigating the afferent properties of vagal sensory receptors in the rat lung. For example, Tsubone [1] reported three different types of pulmonary sensory receptors, i. e. pulmonary stretch receptors, deflation-sensitive receptors and irritant-like receptors. But neither vagal C fibers nor RARs have been described in that study. In the same species, Bergren and Peterson [2] have found that RARs were extremely rare, and that excitation of the vagal C fiber activity after i. v. administration of capsaicin did not occur as a result of the mechanical change. They also suggested that all the three different types of vagal afferent receptors described by Tsubone [1] belong to the same category of SARs. The four subtypes of SARs described by Bergren and Peterson [2] were as follows: inflationary (I), most inflationary (MI), deflationary (D) and most deflationary (MD). These subtypes are consistent with four categories of receptors in rabbits, cats and monkeys (i. e. phasic and tonic inspiratory units and phasic and tonic expiratory units) [3]. Deflationary SARs (21% of the SARs) fire during the deflation phase of the ventilatory cycle and the discharge of receptors is stimulated by deflation of the lungs [2,4]. Tsubone [1] reported that 20 out of 69 vagal afferent fibers recorded were deflationary SARs. Although the function and reflex effects of deflationary SARs remain to be determined, it is possible that they mediate the Hering-Breuer deflation reflex [1,5,6] and monitor lung compliance [2]. Because the airway smooth muscle thickens during lung deflation, the lengthening effect of the deflationary SARs oriented perpendicular to the axis of the airway smooth muscle is thought to be an effective stimulus [2]. This implies that the transduction properties of deflationary SARs are quite different from those of the inspiratory SARs oriented in series with the airway smooth muscle. In a number of mammalian species, inhalation of CO2 gas mixtures is known to inhibit SAR activity [7–12]. At present there are known to be two main mechanisms of CO2-induced SAR inhibition, including a direct effect of CO2 mediated by an increase in the H+ concentration [10,13,14] and a hyperpolarizing action responsible for the activation of 4-aminopyridine (4-AP)sensitive K+ channels [11]. We hypothesized that CO2 inhalation exerts an inhibitory effect on the activity of deflationary SARs through the activation of 4-AP-sensitive K+ channels rather than that of CO2 hydration, but no studies have examined this hypothesis. To determine whether there is a difference between an increase in the H+ concentration and a hyperpolarizing action due to K+ channel activation on inhibition of the deflationary SAR activity associated with CO2 inhalation, we performed two study series in anesthetized, artificially ventilated rats after unilateral vagotomy. First, the response of SARs to CO2 inhalation was examined before and after administration of acetazolamide, a carbonic anhydrase (CA) inhibitor. Second, the changes in SAR activity in response to CO2 inhalation were compared before and after administration of 4-AP, a well known K+ channel blocker. Materials and methods Animal preparation The experiments were performed on 14 Wistar rats (300–350 g). All experimental protocols used in this study were approved by the Animal Use and Care Committe of Nippon Dental University. Each rat
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was first anesthetized with sodium pentobarbital (45–50 mg/kg) intraperitoneally. The trachea was reached through a midline incision in the neck and cannulated below the larynx. The trachea and esophagus were dissected free and retracted rostrally to obtain sufficient space for liquid paraffin. Tracheal pressure (PT) was measured by connecting a polyethylene catheter inserted into the tracheal tube to a pressure transducer. The right carotid artery was cannulated for continuous monitoring blood pressure (BP). A polyethylene catheter was also inserted into the right jugular vein for administration of drugs or 0.9% NaCl solution. Then the left vagus nerve was exposed and sectioned, whereas the right vagus nerve was left intact. The animals were paralyzed with intravenous administration of gallamine (5–10 mg/kg), and additional doses (3–5 mg/kg) of gallamine were administered to avoid spontaneous respiratory movements, as required. The level of anesthesia was monitored by assessing the pressor response to tail pinch and supplemental doses of sodium pentobarbital (9–10 mg/kg/h) through a cannula inserted into the jugular vein were administered as required. The stroke volume of the respirator was set at 10 ml/kg and its frequency ranged from 50 to 60 cycle/min. Measurement of deflationary slowly adapting pulmonary stretch receptors The peripheral end of the cut left vagus nerve was desheathed. To record the single unit activity of deflationary SARs, the thin strands containing afferent nerve fibers were separated, placed on unipolar silver electrodes and submerged in a pool of warm liquid paraffin (37–38 jC). The deflationary SARs were identified on the basis of their characteristic firing patterns, as follows: (1) The deflationary SARs fired during the deflationary phase only. (2) The deflationary SAR discharge was inhibited by inflation of the lungs. (3) The deflationary SARs discharge continued as long as the respirator was stopped. (4) The increase in the receptor discharge occurred after forced deflation. We determined the adaptation index (AI) of deflationary SARs at stopping the respirator in the deflationary phase as well as during the deflation and inflation of the lungs as the peak frequency of the receptors during the experimental procedures minus the average frequency during the second s of the procedures and then divided this by the peak frequency, by applying the method of Knowlton and Larrabee [5] or Widdicombe [15]. The value for AI of deflationary SARs was below 50%, as reported by Tsubone [1] and Bergren and Peterson [2], particularly when lung deflation was maintained for over 5 s. The deflationary SAR activity was amplified with a preamplifier, and the individual receptor amplitude was selected with a window discriminator and fed into a counter. The number of spike discharges was counted with a spike counter (bin width: 100 ms). Experimental procedures for deflation and inflation of the lungs During artificial ventilation the respirator was turned off at expiration. In that case the PT fell to 0 cmH2O. After occluding the inspiratory line of the respirator, the lungs were then subjected to a negative pressure of approximately 15 and 25 cmH2O for over 5 s. In order to reverse atelectasis, subsequently hyperinflation (inflation volume = 3 tidal volumes) was performed by means of a syringe connected to the expiratory line of the respirator. The negative pressure was generated from the inlet port of a vacuum motor, changing the variable level, and connected to the expiratory line of the respirator. Concerning the inflation of the lungs, the respirator was first turned off at expiration. Positive pressure (approximately + 10 and + 15 cmH2O) was then applied for over 5 s by increasing lung volume through a syringe connected to the expiratory line of the respirator.
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Experimental design The experiments were designed to compare the roles of generalized CO2 hydration and K+ channel activation in the deflationary SAR response to inhalation of CO2 gas mixtures (maximal tracheal CO2 concentration ranging from 9 to 12%) balanced with O2 (21%) and N2 (1) In seven deflationary SAR fibers in seven rats, the effect of CO2 inhalation for over 60 s on deflationary SAR activity and PT was determined. Twenty min after acetazolamide administration (20 mg/kg), the same sets of experiments were performed. The effectiveness of acetazolamide was determined by the presence of a slightly decreased tracheal CO2 concentration. (2) In seven deflationary SAR fibers in seven rats, the changes in deflationary SAR activity and PT in response to CO2 inhalation for over 60 s were examined. Ten min after administration of 4-AP (0.7 and 2.0 mg/kg), the same tests were repeated under the same conditions. The absence of 4-AP effects was confirmed by the restored activity of deflationary SARs. Lung compliance was restored to the control by inflating the lungs for several respiratory cycles with a volume of 30 ml/kg. Drugs Acetazolamide (Takeda Pharmaceutical Cooperation, Japan, 500 mg) was diluted with 0.9% NaCl (20 mg/ml). 4-AP was obtained from Sigma Chemical (St. Louis, MO, U.S.A.). 4-AP (10 mg) was dissolved in and diluted with 0.9% NaCl. Data analysis Under control conditions, the firing rates of deflationary SARs in each whole respiratory cycle were measured over five respiratory cycles and expressed as imp/s. The deflationary SAR responses to stopping the respirator for approximately 5 s, deflation (approximately 15 and 25 cmH2O) and inflation (approximately + 10 and + 15 cmH2O) of the lungs for 5–10 s were obtained by counting the firing rates of receptors during the mechanical changes in the lungs, and the average activities of deflationary SARs were calculated and expressed as imp/s. Similarly, the control values for PT were averaged over five respiratory cycles and expressed as cmH2O. The responses of PT to stopping the respirator and lung deflation and inflation were obtained by measuring the respiratory parameter (PT), as described above, and the average values for PT were expressed as cmH2O. Before CO2 inhalation, the average activities of deflationary SARs were measured and expressed as imp/s, as described above. Furthermore, the deflationary SAR response to CO2 inhalation was obtained by counting the firing rate of receptors at 10-s intervals and by performing the measurements over 120 s, and the average activities of deflation SARs during one whole respiratory cycle was expressed as imp/s. Similarly, control values for PT and mean BP (MBP) were averaged over five respiratory cycles and expressed as cmH2O and mmHg, respectively. The responses of PT and MBP to CO2 inhalation were obtained by measuring the respiratory parameter, as described above, and the average values for PT and MBP were expressed as cmH2O and mmHg, respectively. Concerning the three measured parameters (tracheal CO2 concentration, PT and deflationary SAR activity), the time-dependent differences for control and CO2-inhaled rats were compared by means of a one-way analysis of variance for repeated measurements. The statistical significance of the effects of acetazolamide (20 mg/kg) and 4-AP (0.7 and 2.0 mg/kg) on the response of deflationary SARs to CO2 inhalation was analyzed by means of a paired
S. Matsumoto et al. / Life Sciences 72 (2003) 1757–1771 Fig. 1. Responses in PT, deflationary SAR activity and BP to stopping the respirator at the deflation phase (A), lung deflation (B) and lung inflation (C and D).
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t–test. Furthermore, the comparison of acetazolamide and 4-AP treatments on the magnitude of maximum inhibition was made by means of an unpaired t–test. All values were expressed as mean F S. E. A P value less than 0.05 was considered statistically different.
Results The firing behavior of deflationary SAR Fig. 1 shows typical examples of the effects of stopping the respirator in the deflation phase, lung deflation and lung inflation on deflationary SAR activity, PT and BP. A train of impulses occurred after stopping the respirator and the response was associated with an increase in the pulse pressure of BP (Fig. 1A). The deflation (approximately 25 cmH2O) of the lungs caused vigorous stimulation of the deflationary SAR activity and the discharges continued in a slowly adapting fashion, and lung deflation caused a transient increase in the pulse pressure of BP, which was followed by a decrease in BP. (Fig. 1B). Conversely, the inflation (approximately + 10 cmH2O) of the lungs abolished the activity of deflationary SARs and reduced BP (Fig. 1C). Lung inflation at approximately + 15 cmH2O evoked a transient and burst activity, which adapted rapidly (Fig. 1D). The responses of deflationary SAR activity and deflationary SAR AI to the deflation and inflation of the lungs in 14 different deflationary SAR preparations in 14 rats are summarized in Fig. 2. The average discharges of deflationary SARs during two different negative pressures at 14.8 F 0.2 and 24.8 F 0.3 cmH2O were 1.5 and 2.2 fold increase in the average activity seen after stopping the respirator (0 cmH2O, respectively), and the average values for deflationary SAR AI during two negative pressures were almost the same
Fig. 2. Changes in deflationary SAR activity (A) and deflationary AI (B) in response to lung inflation and lung deflation. Values are mean F S. E. (n = 14).
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(approximately 35%) (Fig. 2B). In nine out of fourteen deflationary SARs, they revealed silent activity during lung inflation ranging from approximately + 10 to + 15 cmH2O. But the remaining five deflationary SARs had very little activity during lung inflation, which usually showed a rapidly adapting fashion (Fig. 2B). Effect of acetazolamide on response of deflationary SARs to CO2 inhalation Typical examples of the effect of acetazolamide on the response of deflationary SAR activity and PT to CO2 inhalation are shown in Fig. 3A,B. The decrease in deflationary SAR activity occurred
Fig. 3. Responses in PT and deflationary SAR activity to CO2 inhalation before (A) and after (B) administration of acetazolamide (20 mg/kg).
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immediately after the tracheal CO2 concentration began to increase. The response was not associated with any significant change in PT, as an index of bronchomotor tone. After CO2 inhalation ceased, deflationary SARs returned to the control activity within 30 s (Fig. 3A). The inhibitory effect of CO2 inhalation on deflationary SAR activity was attenuated by pretreatment with acetazolamide (20 mg/kg), which elicited a slight increase in the receptor activity but which had no significant effect on the tracheal CO2 concentration (Fig. 3B). The responses of 7 different deflationary SAR fibers in 7 rats to CO2 inhalation before and after pretreatment with acetazolamide at 20 mg/kg were compared (Fig. 4A). In accordance with the duration of CO2 inhalation, CO2-induced deflationary SAR inhibition become more pronounced. At 60 s after CO2 inhalation, the reduction in deflationary SAR activity in the presence of acetazolamide reached 39.2% of the baseline activity, which was significantly smaller than that before acetazolamide treatment (63.4%). As shown in Fig. 4B,C, acetazolamide treatment (20 mg/kg) did not cause any significant change in the tracheal CO2 concentration or PT. The MBP values before and during CO2 inhalation in the absence of acetazolamide were 89.5 F 2.8 and 99.8 F 3.7 mmHg, respectively,
Fig. 4. Changes in tracheal CO2 concentration (A), PT (B) and deflationary SAR activity (C) in response to CO2 inhalation before (o) and after (.) pretreatment with acetazolamide (20 mg/kg). Values are mean F S. E. (n = 7). Control: before CO2 inhalation. *P < 0.05 for statistical significance from control values. #P < 0.05 for statistical significance from acetazolamide effects.
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and those values in the presence of acetazolamide (20 mg/kg) were 90.2 F 3.2 and 101.3 F 3.8 mmHg, respectively. Acetazolamide treatment had no significant effect on the maximum changes in MBP induced by CO2 inhalation. Effect of 4-AP on response of deflationary SARs to CO2 inhalation Inhalation of CO2 gas mixtures immediately inhibited deflationary SAR activity but had no significant effect on PT. After CO2 inhalation stopped, deflationary SARs returned to the control activity within 30 s (Fig. 5A). The inhibitory effect of CO2 inhalation on deflationary SAR activity was abolished by pretreatment with 4-AP (2.0 mg/kg), which produced a significant increase in deflationary SAR activity and had no significant effect on PT (Fig. 5B). The inhibition of deflationary SARs by CO2 became more
Fig. 5. Responses in PT and deflationary SAR activity to CO2 inhalation before (A) and after (B) administration of 4-AP (2.0 mg/kg).
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pronounced, in accordance with the duration of CO2 inhalation. At 60 s after CO2 inhalation, the responses of 7 different deflationary SAR fibers in 7 rats to CO2 inhalation before and after pretreatment with 4-AP at 0.7 and 2.0 mg/kg were compared (Fig. 6A). 4-AP (0.7 and 2.0 mg/kg) significantly attenuated the inhibitory effect of CO2 inhalation on deflationary SAR activity (percent inhibition: absence, 64.6; in the presence of 4-AP, 0.7 mg/kg, 41.6; 2.0 mg/kg, 6.7). The responses of tracheal CO2 concentration and PT to CO2 inhalation were not significantly altered by pretreatment with 4-AP at either dose (Fig. 6B,C). The administration of 4-AP caused an increase in BP, but this effect was short-lasting. The MBP values before and during CO2 inhalation in the absence of 4-AP were 86.4 F 2.9 and 98.5 F 3.2 mmHg, respectively, and those values in the presence of 4-AP at 0.7 mg/kg were 88.2 F 3.1 and 99.4 F 3.7 mmHg, respectively. After 4-AP treatment at 2.0 mg/kg, MBP values before and during CO2 inhalation were 88.2 F 3.1 and 101.4 F 3.5 mmHg, respectively. The maximum change in MBP in response to CO2 inhalation was not significantly influenced by 4AP treatment. As shown in Fig. 7, pretreatment with 4-AP (2.0 mg/kg) attenuated the maximum reduction in deflationary SAR activity produced by CO2 inhalation from 64.6 to 6.7% and acetazolamide (20 mg/kg) reduced the maximum inhibition of deflationary SAR activity during CO2 inhalation from
Fig. 6. Changes in tracheal CO2 concentration (A), PT (B) and deflationary SAR activity (C) in response to CO2 inhalation before (o) and after pretreatment with 4-AP at 0.7 (.) and 2.0 (E) mg/kg. Values are mean F S. E. (n = 7). Control: before CO2 inhalation. *P < 0.05 for statistical significance from control values. #P < 0.05 for statistical significance from 4-AP effects.
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Fig. 7. Group data for inhibitory responses of deflationary SAR activity to CO2 inhalation before (5) and after (n) pretreatment with acetazolamide and 4-AP. The inhibition was expressed as a percent change from the baseline deflationary SAR activity. Values are mean F S. E. for each seven animals. *P < 0.05, control versus acetazolamide and 4-AP. #P < 0.05, 4-AP (2.0 mg/kg)-treated versus acetazolamide (20 mg/kg)-and 4-AP (0.7 mg/kg)-treated.
63.4 to 39.6%. When comparing the maximum inhibitory action of 4-AP (2.0 mg/kg) and acetazolamide (20 mg/kg), blockade of K+ channels resulted in a greater attenuation of CO2-induced deflationary SAR inhibition.
Discussion In the present study we showed that CO2 inhalation inhibited deflationary SAR activity, and that this inhibitory effect was attenuated by either acetazolamide or 4-AP. Furthermore, we found that the magnitude of attenuation provoked by 4-AP (2.0 mg/kg) became more pronounced than that after acetazolamide treatment (20 mg/kg). Based on evidence that the dosage of acetazolamide of 20 mg/kg or less could inhibit 99.99% of the CA enzymatic activity and not have any nonspecific action [16], it is conceivable that the inhibitory effect of CO2 on deflationary SARs may be largely mediated by the activation of 4-AP-sensitive K+ channels in the receptor terminals. The generalized SARs, mediating the Hering–Breuer inflation reflex, have been classified into two different groups, ‘‘low-threshold’’ and ‘‘high-threshold’’ [17,18]. Such receptors are thought to be arranged in series for the airway smooth muscle. During lung inflation the mechanical deformation through stretch or distension of the airway smooth muscle can stimulate SARs [19], whereas the deflation of the lungs in the rat inhibits impulse activities of inflationary and most inflationary SARs [2]. In this study under normal inflation, the deflationary SARs fired during deflation only and their
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persistent discharges had train or bust impulses. The deflationary SARs proportionally increased their activity as the negative pressure of deflation was increased, and under these conditions the mean AI values of deflationary SARs were approximately 35%. From these observations, it is conceivable that the transduction properties of deflationary SARs are quite different from those of inflationary and most inflationary SARs. The firing behavior of deflationary SARs was different from that of the pulmonary deflation receptors responding to deflation due to an asthmatic attack in guinea-pigs [20,21] and the expiratory receptors responding to collapse, forced deflation and hyperinflation in the rabbit [6]. For this reason, all deflationary SARs had very little or no activity, particularly during inflation of the lungs. The SARs immediately decrease their activity after the onset of CO2 inhalation [10,11]. A similar effect of CO2 on deflationary SARs was observed in this study. This probably implies that the rapid reaction of deflationary SARs after CO2 inhalation is explained by the change in the bronchial lumen CO2 rather than the variation in the blood CO2 concentration. Matsumoto et al. [10] reported that administration of acetazolamide at a dose of 20 mg/kg sufficient to selectively block the enzymatic activity of CA [16] significantly attenuated CO2-induced SAR inhibition in the rabbit. In this study we showed that acetazolamide treatment at 20 mg/kg significantly suppressed the inhibitory effect of CO2 inhalation on deflationary SARs, so that CO2-induced deflationary SAR inhibition may be related to the presence of the CA enzyme in the lungs. Although CA activity of myelinated afferent fibers has been found in rat peripheral nerves [22,23], no similar localization of the CA enzyme has been identified in rat airway afferent nerves. Neverthless, the exact mechanism by which an increase in the H+ concentration caused by CO2 hydration attenuates deflationary SAR activity, as suggested by many investigators [10,12–14], remains to be determined. Coates et al. [24] demonstrated that an increase in baseline discharge in laryngeal CO2 receptors occurred after administration of acetazolamide, which had a suppressive effect on CO2-induced largngeal CO2 receptor inhibition. This seems to suggest the induction of a rise in the receptor tissue pH after acetazolamide administration but the inhibition of CA activity after acetazolamide treatment is considered to elicit a disequilibrium in the CO2 transport system, which leads to increased CO2 tension and decreased pH levels in the tissue. When considering evidence that acetazolamide has an additional effect in decreasing the availability of protons for Na+ –H+ exchange, taken together, it is possible to speculate the idea that blockade of CA-dependent CO2 hydration produces a further decrease in pH on the SAR endings as well as in the vicinity of the endings. Perhaps, we cannot completely rule out the possibility that the difference in the receptor or tissue pH produced by a CA inhibitor acetazolamide determines the baseline receptor discharge of deflationary SARs. In this study we found that administration of acetazolamide had no significant effect on the baseline activity; a slight increase in the basal discharge of deflationary SARs was seen in three receptors but the remaining four receptors had decreased basal activity. These findings are similar to the observation that acetazolamide did not significantly alter the average basal activity of SARs in rabbits [10] and in rats [12]. Several different types of K+ channels have been identified on the basis of electrophysiological and pharmacological properties. These are: Kv1, Kv2, Kv3, Kv4 [25,26] and a calcium-activated K+ channel current (IKCa) [27]. The timing of action potential formation and the repetitive firing pattern of neuronal cells are controlled by both a fast-inactivating A-type current (IA, Kv1 and Kv4) and a sustained K+ current of delayed-rectifier type (IK, Kv2 and Kv3) [28,29]. In this study, administration of 4-AP dose-dependently increased deflationary SAR activity during normal ventilation and caused a
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pressor effect reported by other investigators [30,31], and the latter effect was short-lasting and the response was associated with a slight increase in heart rate. Because some deflationary SARs exhibited discharges apparently to the cardiac rhythm during normal inflation, the increase in basaline receptor discharges may partly involve such an effect. 4-aminoprydine-sensitive K+ channels are known to be related to action potential repolarization in the myelinated axons of rat sciatic nerve fibers [32] and the application of 4-AP can elicit a broad spike of action potentials [32,33]. But we could not obtain the latter effect because we were measuring extracellular action potentials. The excitatory effect of 4-AP on the baseline receptor discharge is explained by the fact that this K+ channel blocker results in both membrane depolarization and repetitive firing in squid axons [34,35]. Indeed, McAlexander and Undem [36] demonstrated that 4-AP application (100 AM–1 mM) caused repetitive firing of nodose ganglion-derived afferent fibers innervating the isolated guinea-pig trachea and bronchus, and that the conduction velocity of these nodose fibers was in the A y range. In this study 4-AP at a dose of 0.7 mg/kg significantly attenuated the inhibitory response of deflationary SAR activity to CO2 inhalation, and administration of 4-AP up to 2.0 mg/kg abolished CO2-induced deflationary SAR inhibition. Because prior treatment with a CA inhibitor could attenuate CO2-induced deflationary inhibition and this attenuation was much less than the inhibition seen after 4-AP treatment (2.0 mg/kg), an increase in the H+ concentration at the receptor site may promote the activation of IA on deflationary SAR terminals, which is the major current responsible for terminal repolarization after a spike. We further obtained that in isolated rat nodose ganglion (NG) neurons sensitive to tetrodotoxin (TTX), IA currents were blocked by the application of 4-AP and partially attenuated by acetazolamide application (unpublished observations). Thus, the stimulating action of K+ efflux therefore inhibits the repolarization of the membrane potential of deflationary SAR terminals and, as a result, decreases the number of action potentials during CO2 inhalation. In other neuronal structures, hyperpolarization in accordance with an increase in K+ conductance is observed after a decrease in the extracellular pH caused by CO2 [37]. In conclusion, we have shown that administration of acetazolamide or 4-AP resulted in attenuation of CO2-induced deflationary SAR inhibition, and that the magnitude of inhibition caused by 4-AP (2.0 mg/kg) was much greater than that seen after acetazolamide administration (20 mg/kg). The results suggest that inhibition of deflationary SARs caused by CO2 inhalation may be largely mediated by the stimulating action of 4-AP -sensitive K+ currents in the nerve terminals of the receptors.
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[6] Luck JC. Afferent vagal fibres with an expiratory discharge in the rabbit. Journal of Physiology 1970;211:63 – 71. [7] Bartlett Jr D, Sant’Ambrogio G. Effect of local and systemic hypercapnia on the discharge of stretch receptors in the airways of the dog. Respiration Physiology 1976;26:91 – 9. [8] Kunz AL, Kawashiro T, Sheid P. Study of CO2 sensitive vagal afferents in cat lung. Respiration Physiology 1976;27: 347 – 55. [9] Coleridge HM, Coleridge JCG, Banzett RB. Effects of CO2 on afferent vagal endings in the canine lung. Respiration Physiology 1978;34:135 – 41. [10] Matsumoto S, Okamura H, Suzuki K, Sugai N, Shimizu T. Inhibitory mechanism of CO2 inhalation on slowly adapting pulmonary stretch receptors in the anesthetized rabbit. Journal of Pharmacology and Experimental Therapeutics 1996;279:402 – 9. [11] Matsumoto S, Takahashi T, Tanimoto T, Saiki C, Takeda M. Effects of potassium channel blockers on CO2-induced slowly adapting pulmonary stretch receptor inhibition. Journal of Pharmacology Experimental Therapeutics 1999;290: 974 – 9. [12] Lai CJ, Kou YR. Inhibitory effect of inhaled wood smoke on the discharge of pulmonary stretch receptors in rats. Journal of Applied Physiology 1998;84:1138 – 43. [13] Mustafa MEKY, Purves MJ. The effect of CO2 upon discharge from slowly adapting stretch receptors in the lungs of rabbits. Respiration Physiology 1972;16:197 – 212. [14] Sant’Ambrogoio G, Miserocchi G, Mortola J. Transient responses of pulmonary stretch receptors in the dog to inhalation of carbon dixoide. Respiration Physiology 1974;22:191 – 7. [15] Widdicombe JG. The site of pulmonary stretch receptors in the cat. Journal of Physiology 1954;125:336 – 51. [16] Maren TH. Use of inhibitors in physiological studies of carbonic anhydrase. American Journal of Physiology 1977;232: F291 – 7. [17] Sant’Ambrogio G. Information arising from the tracheobronchial tree of mammals. Physiological Review 1982;62: 531 – 69. [18] Ravi K. Distribution and location of slowly adapting pulmonary stretch receptors in the airways of cats. Journal of Autonomic Nervous System 1986;15:205 – 16. [19] Matsumoto S, Ikeda M, Nishikawa T. Effects of sodium and potassium channel blockers on hyperinflation-induced slowly adapting pulmonary stretch receptor stimulation in the rat. Life Sciences 2000;67:2167 – 75. [20] Koller EA. Afferent vagal impulses in anaphylactic bronchial asthma. Acta Neurobiologiae Experimentalis 1973;33:51 – 6. [21] Koller EA, Ferrer P. Studies on the role of lung deflation reflex. Respiration Physiology 1970;10:172 – 83. [22] Cammer W, Tansey FA. Immunocytochemical localization of carbonic anhydrase in myelinated fibers in peripheral nerves of rat and mouse. Journal of Histochemistry and Cytochemistry 1987;35:865 – 70. [23] Szaboks M, Kopp M, Schaden GE. Carbonic anhydrase activity in the peripheral nervous system of rats: The enzyme as a maker of muscle afferents. Brain Resarch 1989;492:129 – 38. [24] Coates EL, Knuth SL, Bartlett Jr D. Laryngeal CO2 receptors: influence of systemic PCO2 and carbonic anhydrase inhibition. Respiration Physiology 1996;104:53 – 61. [25] Butlen A, Wei A, Baker K, Salkoff L. A family of putative potassium channel genes in Drosophila. Science 1989;243: 943 – 7. [26] Wei A, Covarrubias M, Butler A, Baker K, Pak M, Salkoff L. K+ current diversity is produced by an extended gene family conserved in Drosophila and mouse. Science 1990;248:599 – 603. [27] Meech RW, Standen NB. Potassium activation in Helix aspersa neurons under voltage clamp: A component mediated by calcium influx. Journal of Physiology 1975;249:211 – 39. [28] Dekin MS, Getting PA. In vitro characterization of neurons in the ventral part of the nucleus tractus solitarius. II. Ionic basis for repetitive firing patterns. Journal of Neurophysiology 1987;58:215 – 29. [29] Spigelman I, Zwang L, Carlen PL. Patch – clamp study of postnatal development of CA1 neurons in rat hippocampal slices: membrane excitability and K+ currents. Journal of Neurophysiology 1992;68:55 – 69. [30] Yanagisawa T, Taira N. Positive inotropic effect of 4-aminopyridine on dog ventricular muscle. Naunyn – Schmiedeberg’s Archives of Pharmacology 1979;307:207 – 12. [31] Chang F-CT, Bauer RM, Benton BJ, Kedder SA, Capacio BR. 4-Aminopyridine antagonizes saxtoxin- and tetrodotoxininduced cardiorespiratory depression. Toxicon 1996;34:671 – 90. [32] Kocsis JD, Eng DL, Gordon TR, Waxman SG. Functional differences between 4-aminopyridine and tetraethylammoniumsensitive potassium channels in myelinated axons. Neuroscience Letters 1987;75:193 – 8.
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[33] Poulter MO, Padjen AL. Different voltage-dependent potassium conductances regulate action potential repolarization and excitability in frog myelinated axon. Neuroscience 1995;68:494 – 504. [34] Yeh JZ, Oxtord GS, Wu CH, Narahashi T. Interactions of aminoprydines with potassium channels of squid axon membranes. Biophysics Journal 1976;16:77 – 81. [35] Yeh JZ, Oxford GS, Wu CH, Narahashi T. Dynamics of aminopyridine block of potassium channels in squid axon membrane. Journal of General Physiology 1976;68:519 – 35. [36] McAlexander MA, Undem BJ. Potassium channel blockade induces action potential generation in guinea-pig airway vagal afferent neurons. Journal of Autonomic Nervous System 2000;78:158 – 64. [37] Brown AM. Effects of CO2 and pH on neuronal membranes. Federation Proceeding 1972;31:1399 – 403.
Effects of acetazolamide and 4-aminoprydine on the responses of deflationary slowly adapting pulmonary stretch receptors to CO2 inhalation in the rat S. Matsumoto *, M. Ikeda, T. Nishikawa, S. Yoshida, J. Kadoi, T. Tanimoto, C. Saiki, M. Takeda Department of Physiology, Nippon Dental University, School of Dentistry at Tokyo, 1-9-20 Fujimi, Chiyoda-ku, Tokyo 102-8159, Japan Received 22 May 2002; accepted 8 November 2002
Abstract The inhibitory effect of CO2 on deflationary slowly adapting pulmonary stretch receptors (deflationary SARs) was investigated before and after administration of acetazolamide, a carbonic anhydrase (CA) inhibitor, or 4aminopyridine (4-AP), a K+ channel blocker, in anesthetized, artificially ventilated rats after unilateral vagotomy. CO2 inhalation (maximum tracheal CO2 concentration ranging from 9 to 12%) for approximately 60 s decreased the impulse activity of deflationary SARs but had no significant effect on tracheal pressure (PT) as an index of bronchomotor tone. Acetazolamide treatment (20 mg/kg) diminished the inhibitory response of deflationary SARs to CO2 inhalation. 4-AP (0.7 and 2.0 mg/kg) dose-dependently attenuated the decrease in deflationary SAR activity induced by CO2 inhalation. When comparing the maximum attenuation due to 4-AP (2.0 mg/kg) and acetazolamide (20 mg/kg) in CO2-induced deflationary SAR inhibition, blockade of K+ channels had a more pronounced effect. These results suggest that inhibition of deflationary SARs by CO2 inhalation may be largely mediated by the stimulating action of 4-AP-sensitive K+ currents in the nerve terminals of the receptors. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Deflationary slowly adapting pulmonary stretch receptor; CO2 inhalation; Inhibition; CA inhibitor; K+ channel blocker
* Corresponding author. Tel.: +81-3-3261-8706; fax: +81-3-3261-8740. E-mail address: [email protected] (S. Matsumoto). 0024-3205/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved. doi:10.1016/S0024-3205(02)02508-0
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Introduction Three categories of vagal afferent receptors have been identified in the mammalian lung: slowly adapting pulmonary stretch receptors (SARs), rapidly adapting pulmonary stretch receptors (RARs) and vagal C fiber endings. There are few studies investigating the afferent properties of vagal sensory receptors in the rat lung. For example, Tsubone [1] reported three different types of pulmonary sensory receptors, i. e. pulmonary stretch receptors, deflation-sensitive receptors and irritant-like receptors. But neither vagal C fibers nor RARs have been described in that study. In the same species, Bergren and Peterson [2] have found that RARs were extremely rare, and that excitation of the vagal C fiber activity after i. v. administration of capsaicin did not occur as a result of the mechanical change. They also suggested that all the three different types of vagal afferent receptors described by Tsubone [1] belong to the same category of SARs. The four subtypes of SARs described by Bergren and Peterson [2] were as follows: inflationary (I), most inflationary (MI), deflationary (D) and most deflationary (MD). These subtypes are consistent with four categories of receptors in rabbits, cats and monkeys (i. e. phasic and tonic inspiratory units and phasic and tonic expiratory units) [3]. Deflationary SARs (21% of the SARs) fire during the deflation phase of the ventilatory cycle and the discharge of receptors is stimulated by deflation of the lungs [2,4]. Tsubone [1] reported that 20 out of 69 vagal afferent fibers recorded were deflationary SARs. Although the function and reflex effects of deflationary SARs remain to be determined, it is possible that they mediate the Hering-Breuer deflation reflex [1,5,6] and monitor lung compliance [2]. Because the airway smooth muscle thickens during lung deflation, the lengthening effect of the deflationary SARs oriented perpendicular to the axis of the airway smooth muscle is thought to be an effective stimulus [2]. This implies that the transduction properties of deflationary SARs are quite different from those of the inspiratory SARs oriented in series with the airway smooth muscle. In a number of mammalian species, inhalation of CO2 gas mixtures is known to inhibit SAR activity [7–12]. At present there are known to be two main mechanisms of CO2-induced SAR inhibition, including a direct effect of CO2 mediated by an increase in the H+ concentration [10,13,14] and a hyperpolarizing action responsible for the activation of 4-aminopyridine (4-AP)sensitive K+ channels [11]. We hypothesized that CO2 inhalation exerts an inhibitory effect on the activity of deflationary SARs through the activation of 4-AP-sensitive K+ channels rather than that of CO2 hydration, but no studies have examined this hypothesis. To determine whether there is a difference between an increase in the H+ concentration and a hyperpolarizing action due to K+ channel activation on inhibition of the deflationary SAR activity associated with CO2 inhalation, we performed two study series in anesthetized, artificially ventilated rats after unilateral vagotomy. First, the response of SARs to CO2 inhalation was examined before and after administration of acetazolamide, a carbonic anhydrase (CA) inhibitor. Second, the changes in SAR activity in response to CO2 inhalation were compared before and after administration of 4-AP, a well known K+ channel blocker. Materials and methods Animal preparation The experiments were performed on 14 Wistar rats (300–350 g). All experimental protocols used in this study were approved by the Animal Use and Care Committe of Nippon Dental University. Each rat
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was first anesthetized with sodium pentobarbital (45–50 mg/kg) intraperitoneally. The trachea was reached through a midline incision in the neck and cannulated below the larynx. The trachea and esophagus were dissected free and retracted rostrally to obtain sufficient space for liquid paraffin. Tracheal pressure (PT) was measured by connecting a polyethylene catheter inserted into the tracheal tube to a pressure transducer. The right carotid artery was cannulated for continuous monitoring blood pressure (BP). A polyethylene catheter was also inserted into the right jugular vein for administration of drugs or 0.9% NaCl solution. Then the left vagus nerve was exposed and sectioned, whereas the right vagus nerve was left intact. The animals were paralyzed with intravenous administration of gallamine (5–10 mg/kg), and additional doses (3–5 mg/kg) of gallamine were administered to avoid spontaneous respiratory movements, as required. The level of anesthesia was monitored by assessing the pressor response to tail pinch and supplemental doses of sodium pentobarbital (9–10 mg/kg/h) through a cannula inserted into the jugular vein were administered as required. The stroke volume of the respirator was set at 10 ml/kg and its frequency ranged from 50 to 60 cycle/min. Measurement of deflationary slowly adapting pulmonary stretch receptors The peripheral end of the cut left vagus nerve was desheathed. To record the single unit activity of deflationary SARs, the thin strands containing afferent nerve fibers were separated, placed on unipolar silver electrodes and submerged in a pool of warm liquid paraffin (37–38 jC). The deflationary SARs were identified on the basis of their characteristic firing patterns, as follows: (1) The deflationary SARs fired during the deflationary phase only. (2) The deflationary SAR discharge was inhibited by inflation of the lungs. (3) The deflationary SARs discharge continued as long as the respirator was stopped. (4) The increase in the receptor discharge occurred after forced deflation. We determined the adaptation index (AI) of deflationary SARs at stopping the respirator in the deflationary phase as well as during the deflation and inflation of the lungs as the peak frequency of the receptors during the experimental procedures minus the average frequency during the second s of the procedures and then divided this by the peak frequency, by applying the method of Knowlton and Larrabee [5] or Widdicombe [15]. The value for AI of deflationary SARs was below 50%, as reported by Tsubone [1] and Bergren and Peterson [2], particularly when lung deflation was maintained for over 5 s. The deflationary SAR activity was amplified with a preamplifier, and the individual receptor amplitude was selected with a window discriminator and fed into a counter. The number of spike discharges was counted with a spike counter (bin width: 100 ms). Experimental procedures for deflation and inflation of the lungs During artificial ventilation the respirator was turned off at expiration. In that case the PT fell to 0 cmH2O. After occluding the inspiratory line of the respirator, the lungs were then subjected to a negative pressure of approximately 15 and 25 cmH2O for over 5 s. In order to reverse atelectasis, subsequently hyperinflation (inflation volume = 3 tidal volumes) was performed by means of a syringe connected to the expiratory line of the respirator. The negative pressure was generated from the inlet port of a vacuum motor, changing the variable level, and connected to the expiratory line of the respirator. Concerning the inflation of the lungs, the respirator was first turned off at expiration. Positive pressure (approximately + 10 and + 15 cmH2O) was then applied for over 5 s by increasing lung volume through a syringe connected to the expiratory line of the respirator.
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Experimental design The experiments were designed to compare the roles of generalized CO2 hydration and K+ channel activation in the deflationary SAR response to inhalation of CO2 gas mixtures (maximal tracheal CO2 concentration ranging from 9 to 12%) balanced with O2 (21%) and N2 (1) In seven deflationary SAR fibers in seven rats, the effect of CO2 inhalation for over 60 s on deflationary SAR activity and PT was determined. Twenty min after acetazolamide administration (20 mg/kg), the same sets of experiments were performed. The effectiveness of acetazolamide was determined by the presence of a slightly decreased tracheal CO2 concentration. (2) In seven deflationary SAR fibers in seven rats, the changes in deflationary SAR activity and PT in response to CO2 inhalation for over 60 s were examined. Ten min after administration of 4-AP (0.7 and 2.0 mg/kg), the same tests were repeated under the same conditions. The absence of 4-AP effects was confirmed by the restored activity of deflationary SARs. Lung compliance was restored to the control by inflating the lungs for several respiratory cycles with a volume of 30 ml/kg. Drugs Acetazolamide (Takeda Pharmaceutical Cooperation, Japan, 500 mg) was diluted with 0.9% NaCl (20 mg/ml). 4-AP was obtained from Sigma Chemical (St. Louis, MO, U.S.A.). 4-AP (10 mg) was dissolved in and diluted with 0.9% NaCl. Data analysis Under control conditions, the firing rates of deflationary SARs in each whole respiratory cycle were measured over five respiratory cycles and expressed as imp/s. The deflationary SAR responses to stopping the respirator for approximately 5 s, deflation (approximately 15 and 25 cmH2O) and inflation (approximately + 10 and + 15 cmH2O) of the lungs for 5–10 s were obtained by counting the firing rates of receptors during the mechanical changes in the lungs, and the average activities of deflationary SARs were calculated and expressed as imp/s. Similarly, the control values for PT were averaged over five respiratory cycles and expressed as cmH2O. The responses of PT to stopping the respirator and lung deflation and inflation were obtained by measuring the respiratory parameter (PT), as described above, and the average values for PT were expressed as cmH2O. Before CO2 inhalation, the average activities of deflationary SARs were measured and expressed as imp/s, as described above. Furthermore, the deflationary SAR response to CO2 inhalation was obtained by counting the firing rate of receptors at 10-s intervals and by performing the measurements over 120 s, and the average activities of deflation SARs during one whole respiratory cycle was expressed as imp/s. Similarly, control values for PT and mean BP (MBP) were averaged over five respiratory cycles and expressed as cmH2O and mmHg, respectively. The responses of PT and MBP to CO2 inhalation were obtained by measuring the respiratory parameter, as described above, and the average values for PT and MBP were expressed as cmH2O and mmHg, respectively. Concerning the three measured parameters (tracheal CO2 concentration, PT and deflationary SAR activity), the time-dependent differences for control and CO2-inhaled rats were compared by means of a one-way analysis of variance for repeated measurements. The statistical significance of the effects of acetazolamide (20 mg/kg) and 4-AP (0.7 and 2.0 mg/kg) on the response of deflationary SARs to CO2 inhalation was analyzed by means of a paired
S. Matsumoto et al. / Life Sciences 72 (2003) 1757–1771 Fig. 1. Responses in PT, deflationary SAR activity and BP to stopping the respirator at the deflation phase (A), lung deflation (B) and lung inflation (C and D).
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t–test. Furthermore, the comparison of acetazolamide and 4-AP treatments on the magnitude of maximum inhibition was made by means of an unpaired t–test. All values were expressed as mean F S. E. A P value less than 0.05 was considered statistically different.
Results The firing behavior of deflationary SAR Fig. 1 shows typical examples of the effects of stopping the respirator in the deflation phase, lung deflation and lung inflation on deflationary SAR activity, PT and BP. A train of impulses occurred after stopping the respirator and the response was associated with an increase in the pulse pressure of BP (Fig. 1A). The deflation (approximately 25 cmH2O) of the lungs caused vigorous stimulation of the deflationary SAR activity and the discharges continued in a slowly adapting fashion, and lung deflation caused a transient increase in the pulse pressure of BP, which was followed by a decrease in BP. (Fig. 1B). Conversely, the inflation (approximately + 10 cmH2O) of the lungs abolished the activity of deflationary SARs and reduced BP (Fig. 1C). Lung inflation at approximately + 15 cmH2O evoked a transient and burst activity, which adapted rapidly (Fig. 1D). The responses of deflationary SAR activity and deflationary SAR AI to the deflation and inflation of the lungs in 14 different deflationary SAR preparations in 14 rats are summarized in Fig. 2. The average discharges of deflationary SARs during two different negative pressures at 14.8 F 0.2 and 24.8 F 0.3 cmH2O were 1.5 and 2.2 fold increase in the average activity seen after stopping the respirator (0 cmH2O, respectively), and the average values for deflationary SAR AI during two negative pressures were almost the same
Fig. 2. Changes in deflationary SAR activity (A) and deflationary AI (B) in response to lung inflation and lung deflation. Values are mean F S. E. (n = 14).
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(approximately 35%) (Fig. 2B). In nine out of fourteen deflationary SARs, they revealed silent activity during lung inflation ranging from approximately + 10 to + 15 cmH2O. But the remaining five deflationary SARs had very little activity during lung inflation, which usually showed a rapidly adapting fashion (Fig. 2B). Effect of acetazolamide on response of deflationary SARs to CO2 inhalation Typical examples of the effect of acetazolamide on the response of deflationary SAR activity and PT to CO2 inhalation are shown in Fig. 3A,B. The decrease in deflationary SAR activity occurred
Fig. 3. Responses in PT and deflationary SAR activity to CO2 inhalation before (A) and after (B) administration of acetazolamide (20 mg/kg).
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immediately after the tracheal CO2 concentration began to increase. The response was not associated with any significant change in PT, as an index of bronchomotor tone. After CO2 inhalation ceased, deflationary SARs returned to the control activity within 30 s (Fig. 3A). The inhibitory effect of CO2 inhalation on deflationary SAR activity was attenuated by pretreatment with acetazolamide (20 mg/kg), which elicited a slight increase in the receptor activity but which had no significant effect on the tracheal CO2 concentration (Fig. 3B). The responses of 7 different deflationary SAR fibers in 7 rats to CO2 inhalation before and after pretreatment with acetazolamide at 20 mg/kg were compared (Fig. 4A). In accordance with the duration of CO2 inhalation, CO2-induced deflationary SAR inhibition become more pronounced. At 60 s after CO2 inhalation, the reduction in deflationary SAR activity in the presence of acetazolamide reached 39.2% of the baseline activity, which was significantly smaller than that before acetazolamide treatment (63.4%). As shown in Fig. 4B,C, acetazolamide treatment (20 mg/kg) did not cause any significant change in the tracheal CO2 concentration or PT. The MBP values before and during CO2 inhalation in the absence of acetazolamide were 89.5 F 2.8 and 99.8 F 3.7 mmHg, respectively,
Fig. 4. Changes in tracheal CO2 concentration (A), PT (B) and deflationary SAR activity (C) in response to CO2 inhalation before (o) and after (.) pretreatment with acetazolamide (20 mg/kg). Values are mean F S. E. (n = 7). Control: before CO2 inhalation. *P < 0.05 for statistical significance from control values. #P < 0.05 for statistical significance from acetazolamide effects.
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and those values in the presence of acetazolamide (20 mg/kg) were 90.2 F 3.2 and 101.3 F 3.8 mmHg, respectively. Acetazolamide treatment had no significant effect on the maximum changes in MBP induced by CO2 inhalation. Effect of 4-AP on response of deflationary SARs to CO2 inhalation Inhalation of CO2 gas mixtures immediately inhibited deflationary SAR activity but had no significant effect on PT. After CO2 inhalation stopped, deflationary SARs returned to the control activity within 30 s (Fig. 5A). The inhibitory effect of CO2 inhalation on deflationary SAR activity was abolished by pretreatment with 4-AP (2.0 mg/kg), which produced a significant increase in deflationary SAR activity and had no significant effect on PT (Fig. 5B). The inhibition of deflationary SARs by CO2 became more
Fig. 5. Responses in PT and deflationary SAR activity to CO2 inhalation before (A) and after (B) administration of 4-AP (2.0 mg/kg).
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pronounced, in accordance with the duration of CO2 inhalation. At 60 s after CO2 inhalation, the responses of 7 different deflationary SAR fibers in 7 rats to CO2 inhalation before and after pretreatment with 4-AP at 0.7 and 2.0 mg/kg were compared (Fig. 6A). 4-AP (0.7 and 2.0 mg/kg) significantly attenuated the inhibitory effect of CO2 inhalation on deflationary SAR activity (percent inhibition: absence, 64.6; in the presence of 4-AP, 0.7 mg/kg, 41.6; 2.0 mg/kg, 6.7). The responses of tracheal CO2 concentration and PT to CO2 inhalation were not significantly altered by pretreatment with 4-AP at either dose (Fig. 6B,C). The administration of 4-AP caused an increase in BP, but this effect was short-lasting. The MBP values before and during CO2 inhalation in the absence of 4-AP were 86.4 F 2.9 and 98.5 F 3.2 mmHg, respectively, and those values in the presence of 4-AP at 0.7 mg/kg were 88.2 F 3.1 and 99.4 F 3.7 mmHg, respectively. After 4-AP treatment at 2.0 mg/kg, MBP values before and during CO2 inhalation were 88.2 F 3.1 and 101.4 F 3.5 mmHg, respectively. The maximum change in MBP in response to CO2 inhalation was not significantly influenced by 4AP treatment. As shown in Fig. 7, pretreatment with 4-AP (2.0 mg/kg) attenuated the maximum reduction in deflationary SAR activity produced by CO2 inhalation from 64.6 to 6.7% and acetazolamide (20 mg/kg) reduced the maximum inhibition of deflationary SAR activity during CO2 inhalation from
Fig. 6. Changes in tracheal CO2 concentration (A), PT (B) and deflationary SAR activity (C) in response to CO2 inhalation before (o) and after pretreatment with 4-AP at 0.7 (.) and 2.0 (E) mg/kg. Values are mean F S. E. (n = 7). Control: before CO2 inhalation. *P < 0.05 for statistical significance from control values. #P < 0.05 for statistical significance from 4-AP effects.
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Fig. 7. Group data for inhibitory responses of deflationary SAR activity to CO2 inhalation before (5) and after (n) pretreatment with acetazolamide and 4-AP. The inhibition was expressed as a percent change from the baseline deflationary SAR activity. Values are mean F S. E. for each seven animals. *P < 0.05, control versus acetazolamide and 4-AP. #P < 0.05, 4-AP (2.0 mg/kg)-treated versus acetazolamide (20 mg/kg)-and 4-AP (0.7 mg/kg)-treated.
63.4 to 39.6%. When comparing the maximum inhibitory action of 4-AP (2.0 mg/kg) and acetazolamide (20 mg/kg), blockade of K+ channels resulted in a greater attenuation of CO2-induced deflationary SAR inhibition.
Discussion In the present study we showed that CO2 inhalation inhibited deflationary SAR activity, and that this inhibitory effect was attenuated by either acetazolamide or 4-AP. Furthermore, we found that the magnitude of attenuation provoked by 4-AP (2.0 mg/kg) became more pronounced than that after acetazolamide treatment (20 mg/kg). Based on evidence that the dosage of acetazolamide of 20 mg/kg or less could inhibit 99.99% of the CA enzymatic activity and not have any nonspecific action [16], it is conceivable that the inhibitory effect of CO2 on deflationary SARs may be largely mediated by the activation of 4-AP-sensitive K+ channels in the receptor terminals. The generalized SARs, mediating the Hering–Breuer inflation reflex, have been classified into two different groups, ‘‘low-threshold’’ and ‘‘high-threshold’’ [17,18]. Such receptors are thought to be arranged in series for the airway smooth muscle. During lung inflation the mechanical deformation through stretch or distension of the airway smooth muscle can stimulate SARs [19], whereas the deflation of the lungs in the rat inhibits impulse activities of inflationary and most inflationary SARs [2]. In this study under normal inflation, the deflationary SARs fired during deflation only and their
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persistent discharges had train or bust impulses. The deflationary SARs proportionally increased their activity as the negative pressure of deflation was increased, and under these conditions the mean AI values of deflationary SARs were approximately 35%. From these observations, it is conceivable that the transduction properties of deflationary SARs are quite different from those of inflationary and most inflationary SARs. The firing behavior of deflationary SARs was different from that of the pulmonary deflation receptors responding to deflation due to an asthmatic attack in guinea-pigs [20,21] and the expiratory receptors responding to collapse, forced deflation and hyperinflation in the rabbit [6]. For this reason, all deflationary SARs had very little or no activity, particularly during inflation of the lungs. The SARs immediately decrease their activity after the onset of CO2 inhalation [10,11]. A similar effect of CO2 on deflationary SARs was observed in this study. This probably implies that the rapid reaction of deflationary SARs after CO2 inhalation is explained by the change in the bronchial lumen CO2 rather than the variation in the blood CO2 concentration. Matsumoto et al. [10] reported that administration of acetazolamide at a dose of 20 mg/kg sufficient to selectively block the enzymatic activity of CA [16] significantly attenuated CO2-induced SAR inhibition in the rabbit. In this study we showed that acetazolamide treatment at 20 mg/kg significantly suppressed the inhibitory effect of CO2 inhalation on deflationary SARs, so that CO2-induced deflationary SAR inhibition may be related to the presence of the CA enzyme in the lungs. Although CA activity of myelinated afferent fibers has been found in rat peripheral nerves [22,23], no similar localization of the CA enzyme has been identified in rat airway afferent nerves. Neverthless, the exact mechanism by which an increase in the H+ concentration caused by CO2 hydration attenuates deflationary SAR activity, as suggested by many investigators [10,12–14], remains to be determined. Coates et al. [24] demonstrated that an increase in baseline discharge in laryngeal CO2 receptors occurred after administration of acetazolamide, which had a suppressive effect on CO2-induced largngeal CO2 receptor inhibition. This seems to suggest the induction of a rise in the receptor tissue pH after acetazolamide administration but the inhibition of CA activity after acetazolamide treatment is considered to elicit a disequilibrium in the CO2 transport system, which leads to increased CO2 tension and decreased pH levels in the tissue. When considering evidence that acetazolamide has an additional effect in decreasing the availability of protons for Na+ –H+ exchange, taken together, it is possible to speculate the idea that blockade of CA-dependent CO2 hydration produces a further decrease in pH on the SAR endings as well as in the vicinity of the endings. Perhaps, we cannot completely rule out the possibility that the difference in the receptor or tissue pH produced by a CA inhibitor acetazolamide determines the baseline receptor discharge of deflationary SARs. In this study we found that administration of acetazolamide had no significant effect on the baseline activity; a slight increase in the basal discharge of deflationary SARs was seen in three receptors but the remaining four receptors had decreased basal activity. These findings are similar to the observation that acetazolamide did not significantly alter the average basal activity of SARs in rabbits [10] and in rats [12]. Several different types of K+ channels have been identified on the basis of electrophysiological and pharmacological properties. These are: Kv1, Kv2, Kv3, Kv4 [25,26] and a calcium-activated K+ channel current (IKCa) [27]. The timing of action potential formation and the repetitive firing pattern of neuronal cells are controlled by both a fast-inactivating A-type current (IA, Kv1 and Kv4) and a sustained K+ current of delayed-rectifier type (IK, Kv2 and Kv3) [28,29]. In this study, administration of 4-AP dose-dependently increased deflationary SAR activity during normal ventilation and caused a
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pressor effect reported by other investigators [30,31], and the latter effect was short-lasting and the response was associated with a slight increase in heart rate. Because some deflationary SARs exhibited discharges apparently to the cardiac rhythm during normal inflation, the increase in basaline receptor discharges may partly involve such an effect. 4-aminoprydine-sensitive K+ channels are known to be related to action potential repolarization in the myelinated axons of rat sciatic nerve fibers [32] and the application of 4-AP can elicit a broad spike of action potentials [32,33]. But we could not obtain the latter effect because we were measuring extracellular action potentials. The excitatory effect of 4-AP on the baseline receptor discharge is explained by the fact that this K+ channel blocker results in both membrane depolarization and repetitive firing in squid axons [34,35]. Indeed, McAlexander and Undem [36] demonstrated that 4-AP application (100 AM–1 mM) caused repetitive firing of nodose ganglion-derived afferent fibers innervating the isolated guinea-pig trachea and bronchus, and that the conduction velocity of these nodose fibers was in the A y range. In this study 4-AP at a dose of 0.7 mg/kg significantly attenuated the inhibitory response of deflationary SAR activity to CO2 inhalation, and administration of 4-AP up to 2.0 mg/kg abolished CO2-induced deflationary SAR inhibition. Because prior treatment with a CA inhibitor could attenuate CO2-induced deflationary inhibition and this attenuation was much less than the inhibition seen after 4-AP treatment (2.0 mg/kg), an increase in the H+ concentration at the receptor site may promote the activation of IA on deflationary SAR terminals, which is the major current responsible for terminal repolarization after a spike. We further obtained that in isolated rat nodose ganglion (NG) neurons sensitive to tetrodotoxin (TTX), IA currents were blocked by the application of 4-AP and partially attenuated by acetazolamide application (unpublished observations). Thus, the stimulating action of K+ efflux therefore inhibits the repolarization of the membrane potential of deflationary SAR terminals and, as a result, decreases the number of action potentials during CO2 inhalation. In other neuronal structures, hyperpolarization in accordance with an increase in K+ conductance is observed after a decrease in the extracellular pH caused by CO2 [37]. In conclusion, we have shown that administration of acetazolamide or 4-AP resulted in attenuation of CO2-induced deflationary SAR inhibition, and that the magnitude of inhibition caused by 4-AP (2.0 mg/kg) was much greater than that seen after acetazolamide administration (20 mg/kg). The results suggest that inhibition of deflationary SARs caused by CO2 inhalation may be largely mediated by the stimulating action of 4-AP -sensitive K+ currents in the nerve terminals of the receptors.
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