Introduction

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Nonspecific airway hyperresponsiveness (AHR) to inhaled stimuli such as histamine and methacholine is an important diagnostic feature of bronchial asthma (1). Bronchial asthma has been characterized as chronic airway inflammation associated with airway infiltration of various inflammatory cells, including eosinophils (2). Activated eosinophils are known to secrete cationic proteins, such as major basic protein (MBP) (3), that have been suggested to be involved in AHR in various ways (4). MBP is a highly cationic polypeptide (3) and the effect of MBP is mimicked by synthetic polycations, such as poly-L-arginine (pL-Arg) and poly-L-lysine (pL-Lys) (4, 6).

Chronic bronchitis is a clinical entity characterized by cough and mucus hypersecretion and is sometimes referred to as neutrophilic bronchitis, whereas bronchial asthma may be thought of as eosinophilic bronchitis. These different entities share some common features, especially AHR (9). Tracheal instillation of cathepsin G, one of the cationic proteins from neutrophils, has been reported to induce AHR without producing epithelial damage in rat in vivo (10). Neutrophils have also been shown to inhibit the arachidonate-induced relaxation of guinea-pig trachea, as do eosinophils (11). Moreover, a population-based study suggested that the leukocyte count, particularly that of neutrophils, paralleled the bronchial methacholine responsivity (12). In addition to MBP from eosinophils and cathepsin G from neutrophils, a number of cationic proteins have been identified in a variety of cell types, e.g., neutrophils (-defensins, cationic antimicrobial protein [CAP] 37, and CAP 57/BPI/BP) (13), platelet (platelet factor [PF] 4) (14), and airway epithelial cells (-defensins) (15). These cationic proteins have principally been considered in the context of defense mechanisms against bacterial and parasitic invasion, probably by alterating the membrane permeability, which ultimately leads to cell death (16). In addition to the antimicrobial action, these cationic proteins may play roles in the pathogenesis of AHR in chronic inflammatory airway diseases, including bronchial asthma and chronic bronchitis.

Although the cationic charge appears to be important in many of the biologic actions of cationic proteins (10, 17), the precise mechanism and site of action underlying their ability to increase airway responsiveness are unknown. MBP has been reported to augment agonist-elicited smooth-muscle contraction indirectly in an epithelial-dependent manner in guinea pig in vitro (5). In addition, MBP and synthetic polycations induced AHR in vivo with mechanisms mediated by the generation of bradykinin (6) or by stimulating the parasympathetic nervous system (7) in guinea pig. As well as these indirect mechanisms, synthetic polycations including pL-Arg and pL-Lys have been reported to contract isolated guinea-pig trachea directly in a concentration-dependent, epithelial-independent manner (8). In a recent investigation, this direct contractile action of synthetic polycations and MBP was also shown in bovine tracheal smooth-muscle strips (4). Moreover, the investigators found, using fura-2 Ca²⁺-indicator dye, that the cationic proteins increased the cellular Ca²⁺ concentration ([Ca²⁺]_i) and augmented the histamine- or bradykinin-stimulated [Ca²⁺]_i responses in cultured bovine tracheal smooth-muscle cells (4). These findings may explain the mechanism of the direct contractile action of polycations on airway smooth muscle that results in hyperresponsiveness. However, the mechanistic basis for the polycation-induced Ca²⁺ mobilization in airway smooth muscle still remained to be elucidated. To address this issue, we examined the effects of synthetic cationic polypeptides pL-Arg and pL-Lys on single smooth-muscle cells from bovine trachea using a patch-clamp technique. Here we demonstrate that the model polycations depolarized the tracheal smooth-muscle membrane and inhibited large-conductance Ca²⁺-dependent K⁺ channels (maxi-K channels) without affecting the Ca²⁺-influx pathway (L-type Ca²⁺ channels). The depolarization may activate the voltage-dependent Ca²⁺ channels on the plasma membrane, resulting in an increased Ca²⁺ influx from extracellular compartments, which will ultimately energize the intracellular contractile machinery. The suppression of maxi-K channels may further intensify the contractile effect by inhibiting the relaxation of airway smooth muscle.

	Materials and Methods

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Cell Preparation

Fresh bovine tracheae were obtained from a local abattoir and immediately transferred into an ice-cold transportation solution containing (in mM) 140 NaCl, 4.7 KCl, 1.13 MgCl₂, 10 glucose, and 10 N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid (Hepes). The smooth-muscle layer was isolated from the posterior membranous portion of the trachea, the surrounding connective tissue and epithelium were removed, and the isolated smooth-muscle strips were cut into small pieces. These smooth-muscle strips were then dispersed enzymatically into single cells with collagenase (100 U/ml), papain (1 mg/ml), and DL-dithiothreitol (2 mM) for 40 min at 37°C. After incubation, the tissue pieces were gently agitated and single smooth-muscle cells were resuspended in the extracellular solution.

Electrical Recordings

Standard patch-clamp recording techniques were used. Ionic currents were measured with a patch-clamp amplifier (EPC9; HEKA Electronic, Lambrecht/Pfalz, Germany), low-pass filtered at 1 kHz and monitored on both a built-in software oscilloscope and a thermal pen recorder (RECTI-HORIZ-8K; Nippondenki Sanei, Tokyo, Japan) with a bandwidth of 0 to 300 Hz. Patch pipettes were made of glass capillary (Drummond Scientific Co., Broonall, PA) with an outer diameter of 1.5 mm using a vertical puller (PP-83; Narishige Scientific Instruments, Tokyo, Japan), and had a tip resistance of 4 to 6 M. The junction potential between the patch-pipette and bath solution was nulled by the amplifier circuitry. After establishing a high-resistance (> 2 G) tight seal, the whole-cell configuration was obtained by rupturing the patch membrane with negative pressure applied to the pipette tip. The estimated leakage currents were subtracted on the basis of the responses to depolarizing or hyperpolarizing pulses, and the capacitance current and series resistance were compensated using the amplifier circuitry. The outside-out patch was obtained after the establishment of the whole-cell configuration by raising the patch electrode to the air-water interface. In the excised patch experiments, channel signals were low-pass filtered at 30 kHz, stored by a digital-audio tape recorder (TEAC RD-125T; TEAC, Tokyo, Japan), and then analyzed with Patch Analyst Pro software (TM Corp., Tokyo, Japan). In analyzing the open-state probabilities (nP_os) and unitary current amplitudes, current signals stored in the digital audiotape were digitized at 4 kHz and low-pass filtered at 1.0 kHz. When reproducing the channel currents as a figure, we used a digital thermal recorder (Thermal Arraycorder WR7700; Graphtec, Tokyo, Japan) with a bandwidth of 5 kHz, directly connected to the digital audiotape recorder. The solutions used were of the following composition (in mM): extracellular (bath) solution, 140 NaCl, 4.7 KCl, 1.13 MgCl₂, 1.2 CaCl₂, 10 glucose, and 10 Hepes; and intracellular (pipette) solution, 140 KCl, 1.13 MgCl₂, 10 Hepes, 10 glucose, 0.5 ethyl glycol-bis(-aminoethyl ether)- N,N,N',N'-tetraacetic acid (EGTA), and 1 Na₂-adenosine triphosphate (ATP), adjusted to pH 7.2 with NaOH (bath solution) or KOH (pipette solution). In the experiments isolating the delayed-rectifier K⁺ current, the solutions used were of the following composition (in mM): the extracellular, 110 tetraethyl ammonium (TEA)-Cl, 1.13 MgCl₂, 10 Hepes, 10 glucose, and 1.2 CaCl₂; and the intracellular, 140 KCl, 1.13 MgCl₂, 10 Hepes, 10 EGTA, 10 glucose, and 1 Na₂ATP. In the experiments measuring the inward Ca²⁺ current, the solutions used were (in mM): the extracellular, 110 TEA-Cl, 1.13 MgCl₂, 10 Hepes, 10 glucose, and 20 CaCl₂; and the intracellular, 130 CsCl₂, 1.13 MgCl₂, 10 Hepes, 0.5 EGTA, and 10 glucose; and the pH was adjusted to 7.2 by Tris [hydroxymethyl] aminomethane. All experiments were carried out at room temperature, 20 to 25°C. The fluids were superfused over the cell by hydrostatic pressure-driven application (40 to 50 cm H₂O) through polyethylene tubes (0.5 mm inner diameter).

Chemicals

Hepes was purchased from Dojin Co. Ltd., Kumamoto, Japan. Collagenase was from Wako Pure Chemicals, Osaka, Japan. Other chemicals and reagents, including pL-Arg (average molecular weight [mw] = 12,100) and pL-Lys (average mw = 26,500), were all purchased from Sigma Chemical Co., St. Louis, MO.

Statistical Analysis

The data are expressed as means ± standard error of the mean. For mean comparisons, two-tailed paired or unpaired Student's t test was used. A P < 0.05 was considered statistically significant. n denotes the number of experiments on different cells.

	Results

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Effects of Polycations on the Whole-Cell Membrane Potential

The membrane potential (Mp) was recorded with a whole-cell current-clamp configuration. The mean resting Mp was 39.7 ± 1.1 mV (n = 54). Both pL-Arg and pL-Lys depolarized the Mp rapidly, causing it reach a plateau level within a few seconds after the introduction of either polycation. The depolarizing effect persisted as long as the polycation was present (up to 15 min in the present study) and the effect was rapidly reversible with removal of the agent. As the membrane depolarized, the Mp often exhibited transient, spiky, sporadic hyperpolarizations (Figure 1A). As shown in Figure 1B, both of the agents significantly depolarized the membrane in a concentration- dependent fashion with a similar potency. That is, in the presence of pL-Lys, the Mp was depolarized from a baseline of 40.1 ± 1.5 to 25.1 ± 5.0 mV (P < 0.05; n = 9) with 10 µg/ml and from 43.6 ± 3.9 to 25.1 ± 4.5 mV (P < 0.05; n = 6) with 100 µg/ml. Similarly, pL-Arg depolarized it from 39.3 ± 1.7 to 25.6 ± 6.1 mV (P < 0.05; n = 11) with 10 µg/ml and from 38.0 ± 3.3 to 21.0 ± 4.3 mV (P < 0.05; n = 6) with 100 µg/ml.

View larger version (23K): [in this window] [in a new window]   Figure 1.   Effects of polycationic peptides on the membrane potential in single bovine tracheal myocytes. (A) A representative recording of the membrane potential in the whole-cell current-clamp condition. pL-Arg (10 µg/ml) rapidly and significantly depolarized the membrane. The mean resting membrane potential was about 40 mV, which was depolarized to about 20 mV in the presence of the polycationic peptides. The membrane often showed spontaneous transient hyperpolarizations as the membrane depolarized. (B) Summarized data of changes in membrane potentials expressed by  values from the prior baseline values. Both pL-Lys and pL-Arg depolarized the membrane in a concentration-dependent manner (n = 4-11 for each group). The measurements were done at about 1 to 2 min after the introduction of the polycation. *P < 0.05 compared with baseline values.

Effects of Polycationic Peptides on Whole-Cell Delayed-Rectifier K⁺ Current

It has been reported that the resting Mp of tracheal myocytes is determined largely by the delayed rectifier K⁺ channel (K_DR) (18). Therefore, we examined the effects of the polycationic peptides on whole-cell K_DR-current. The K_DR current was isolated under conditions of very low internal Ca²⁺ and external high TEA (see MATERIALS AND METHODS). As shown in Figure 2, both pL-Arg and pL-Lys (100 µg each) suppressed the currents significantly with a similar potency. In the presence of the polycations, the peak currents at the respective step voltage were attenuated to around 20% of the pretreatment control values, e.g., from 4.08 ± 1.21 to 0.77 ± 0.25 picoampere/picofarad (pA/pF) at 0 mV Mp and from 10.53 ± 3.00 to 2.73 ± 0.56 at +40 mV (P < 0.05; n = 5) (see Figure 2B).

View larger version (22K): [in this window] [in a new window]   Figure 2.   Effects of synthetic polycations on delayed rectifier K+ current. The intracellular Ca2+ was removed by adding 10 mM EGTA and external NaCl was replaced with TEA-Cl, thus excluding contamination by Ca2+-dependent whole-cell currents (see MATERIALS AND METHODS). The voltage pulse protocol was as follows: the membrane potential was held at 70 mV from which step voltages of 1.5-s duration were applied in the range of 40 to +40 mV Mp in 20-mV steps. Leak currents were subtracted and capacitance currents were compensated with the amplifier circuitry. The summarized data from five experiments are shown in B. The magnitudes of the currents, in the absence (open squares) or presence (closed diamonds) of the polycations, were normalized by cell surface area and expressed as pA/pF on the vertical axis. *P < 0.05 as compared with the values before the treatments with the polycations.

Effects of Polycationic Peptides on Whole-Cell K⁺ Current

In voltage-clamp whole-cell experiments, we often observed spontaneous transient outward currents (STOCs) (Figure 3). STOCs have been characterized in many systems including vascular (21), intestinal (22), and tracheal (23) smooth-muscle cells, and are believed to represent the activation of Ca²⁺-dependent K⁺ currents due to spontaneous and sporadic release of internally sequestered Ca²⁺. As exemplified in Figure 3A, both pL-Arg (10 µg/ml; n = 6) and pL-Lys (10 µg/ml; n = 4) acutely suppressed the amplitude of STOCs in an immediate manner that was reversible with the withdrawal of the polycations. We analyzed the effects of the polycations on STOC activity by alterations in the frequency and amplitude compared with the pretreatment control values. Both parameters were significantly suppressed in the presence of either agent, i.e., the frequency was suppressed to 57.7 ± 13.6% of the preceding control values without the polycations (P < 0.01; n = 7) and the amplitude was decreased to 77.1 ± 9.8% of the controls (P < 0.05; n = 7) (Figure 3B).

View larger version (61K): [in this window] [in a new window]   Figure 3.   (A) A representative recording showing the effect of pL-Arg on the STOCs. The single tracheal myocyte was voltage-clamped at 0 mV in the whole-cell configuration. pL-Arg and pL-Lys (10 µg/ml; n = 6 and n = 4, respectively) suppressed the activity of STOCs reversibly. The interrupted line indicates the level of the baseline current. The apparent baseline shift to the upward direction indicates the activation of the currents before returning to the baseline value. (B) Summarized data of the effects of the polycations on the activity of STOCs. An upward current over 15 pA was identified as STOC, and we analyzed the changes in frequency and amplitude with the pretreatment control values as 100%. Data from pL-Arg and pL-Lys were combined (n = 7). *P < 0.05 and **P < 0.01 compared with values without the polycations.

Effects of Polycationic Peptides on Large Conductance K⁺ Channels on Excised Outside-Out Patches

Because STOCs are believed to be carried by K⁺ effluxed through large-conductance Ca²⁺-sensitive K⁺ channels (maxi-K channels) (21) and because it was the maxi-K channel that was the most frequently and easily identifiable channel on excised patch membranes (18, 24), we examined whether the cationic polypeptides affect this type of channel on outside-out membrane patches in a quasi-physiologic electrolyte environment. A representative recording of maxi-K channel activity is shown in Figure 4A. This was identified as a maxi-K channel because (1) the slope conductance at 0 mV Mp was about 130 pS; (2) the estimated reversal potential was about 80 mV, which was close to the equilibrium for K⁺ under the present electrolyte compositions (Figure 4B); and (3) the channel activity was totally abolished by TEA (10 mM) and charybdotoxin (100 nM) but was insensitive to 4-aminopyridine (4AP) (2 mM) (data not shown). As shown in Figure 4A, the introduction of either pL-Arg or pL-Lys attenuated both the nP_o and the amplitude of the unitary current of the channel. The current-voltage (I-V) relationship of the channel demonstrated that the channel had a slope conductance of 124.0 ± 7.9 pS at 0 mV Mp in the standard extra- and intracellular solutions (n = 18), which was decreased to 66.6 ± 3.9 pS in the presence of the cationic polypeptides (n = 14; about 54% of control, P < 0.0001). The nP_o was also decreased significantly to 21.8 ± 20.3% (P < 0.05; n = 6) and 26.5 ± 9.5% (P < 0.005; n = 4) of the pretreatment control values by pL-Arg and pL-Lys, respectively (Figure 4C). These suppressing effects of pL-Arg on nP_o and amplitude were abolished in the presence of heparin, a negatively charged macromolecule. Heparin has been reported to neutralize the positive charge of polycations and to abolish their effects (8, 10, 17). Using 5 U/ml of heparin, we tested the outside-out maxi-K⁺ channel activity. Heparin alone exhibited no effect on the activities of the channel. However, the nP_o and amplitude attenuated by pL-Arg (10 µg/ml) regained their preceding baseline values with the additional heparin (n = 3).

View larger version (32K): [in this window] [in a new window]   Figure 4.   (A) A representative experiment showing the effect of pL-Lys (100 µg/ml) on the activity of a large-conductance, calcium-activated K+ (BK) channel in an outside-out excised patch clamped at +20 mV. K+ concentrations were 140 and 4.7 mM in the pipette and bath solution, respectively. The channel signals were stored in a digital-audio tape filtered at 30 kHz, and were analyzed with 4 kHz digitization low-pass filtered at 1 kHz. The recorder used for reproducing the channel signals had a bandwidth of 5 kHz. The nPo (shown under the channel recording) as well as the unitary current of the channel (see also inset) were attenuated in the presence of pL-Lys, and were restored after washout of the polycationic peptide. In the inset, tracings are shown with an expanded time scale extracted from the points indicated by asterisks (*) on the current recording. (B) The current-voltage relationship of the K+ channel on excised outside-out patches from single myocytes of bovine trachea. The channel had a slope conductance of about 124 pS at 0 mV (control, open squares). In the presence of either pL-Arg or pL-Lys (closed diamonds), the conductance of the channel decreased to 67 pS (about 54% of control) at 0 mV. (C) Inhibitory effect of polycations on the K+ channel activity in outside-out patches at +20 mV Mp. Both pL-Arg (10 µg/ml) and pL-Lys (10 µg/ml) significantly decreased the nPo of the channel (n = 4-6). *P < 0.05, **P < 0.005 as compared with the values before the treatments with the polycations.

Effects of Polycationic Peptides on Whole-Cell Ca²⁺ Current

In a recent report, synthetic polycationic peptides were found to directly mobilize Ca²⁺ and to increase the basal tension in bovine tracheal smooth muscle (4). To examine the possibility of polycations acting directly on Ca²⁺ channels to cause Ca²⁺ influx, we investigated the effects of the polycations on the depolarization-activated whole-cell Ca²⁺ current. The methods of identifying Ca²⁺ current largely conformed to a previous publication (25), as described in MATERIALS AND METHODS. As shown in Figure 5A, we recognized inward currents stimulated by the membrane depolarization that were completely abolished in the presence of nifedipine, an inhibitor of L-type Ca²⁺ channels. The electric properties of the present inward currents, including the I-V relationship (Figure 5B), were qualitatively identical to the Ca²⁺-channel currents reported in canine tracheal myocytes (25, 26) and others (18). The polycations were without effect on the Ca²⁺-channel current in bovine tracheal myocytes (Figure 5B).

View larger version (19K): [in this window] [in a new window]   Figure 5.   (A) Representative whole-cell recordings showing a calcium current evoked by depolarizing voltage pulses. The voltage pulse protocol is shown in the lower panel: the membrane potential was held at 70 mV from which step voltages of 200-ms duration were applied in the range of 45 to +60 mV Mp in 15-mV steps. The bath solution contained 20 mM Ca2+ and the pipette solution contained 0.5 mM EGTA with no added Ca2+. The upper panel shows the inward currents induced by the depolarizing voltage steps (control) which were totally abolished in the presence of nifedipine, an L-type Ca2+ channel blocker (lower panel). This clearly demonstrates that the inward currents shown in the upper panel were carried by Ca2+ influxed through dihydropyridine-sensitive Ca2+ channels. Note that small outward currents are visible after the abolition of the Ca2+ currents. This was thought to be due to a remnant KDR current as suggested by the activation/inactivation kinetics and voltage dependency, although it was small in amplitude (about 2 to 3% of normally activated KDR current in these cells). (B) The relationship between the Ca2+ current and the depolarized Mps in the presence (open diamonds) or absence (open squares) of the polycations. The experimental conditions were the same as in A. The peak inward currents (Ipeak) induced by the respective step pulses were plotted against the membrane potential. A maximal Ca2+ current was observed when the cell was stepped to a test potential of +30 mV. The polycations had no effect on the Ca2+ current.

	Discussion

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The contraction/relaxation in airway smooth muscle is regulated by complex pharmaco- and electromechanical processes. In the present study we investigated the effects of synthetic polycations on the electrophysiologic aspects of single tracheal myocytes. We chose the synthetic polycationic peptides pL-Arg and pL-Lys as model cationic proteins not only because they share a similar molecular weight and charge density with MBP, but also because they reportedly mimicked the biologic actions of endogenous cationic proteins including MBP, PF4, and cathepsin G (4, 8, 10, 17). The magnitude of the membrane depolarization induced by the synthetic polycations in the present study was from about 40 to 20 mV. A sustained depolarization around 20 mV has been observed to be sufficient for a sustained rise in [Ca²⁺]_i and resulting smooth-muscle contraction (18, 27). The Mp of bronchial smooth muscle is one of the major determinants of the bronchial tone and is regulated mainly by K⁺ channels (18, 19, 24). The opening of K⁺ channels hyperpolarizes and the closing depolarizes the membrane, which correlate to a certain extent with the relaxation and contraction of bronchial smooth muscle, respectively (28). Several types of K⁺ channels have been identified in airway myocytes, e.g., a large-conductance, voltage-dependent, Ca²⁺-activated K⁺ channel (referred to as a maxi-K, BK, or K_Ca channel) (18, 23, 24, 29, 30); a voltage-dependent, delayed-rectifying K⁺ channel (K_DR channel) (18, 19, 20); and an ATP-sensitive K⁺ channel (K_ATP channel) (31). Although the physiologic roles of the respective K⁺ channels in controlling smooth-muscle tone have not yet been unequivocally established, it appears likely that the resting membrane potential is determined largely by K_DR channels (18). It has been reported that the resting Mp of airway myocytes from ferret trachea (20) and human airway (19) was depolarized in the presence of 4AP, an inhibitor specific to K_DR channels, whereas charybdotoxin and glybenclamide, inhibitors specific to maxi-K channels and K_ATP channels, respectively, were without effect. In the present study, synthetic polycations mimicked the K_DR inhibitor and depolarized the resting Mp of bovine tracheal myocytes in a concentration-dependent fashion (Figure 1). This suggests that the synthetic polycations suppressed the activity of K_DR channels in bovine tracheal myocytes, thereby causing membrane depolarization. This idea was confirmed by the voltage-clamp experiments at very low intracellular Ca²⁺ (Figure 2). The slow-activating and time-dependent inactivating kinetics and the voltage dependency of the current were quite similar to those of the reported K_DR current in airway myocytes (18). These currents were significantly attenuated in the presence of the synthetic polycations.

Wylam and coworkers found that both MBP and model polycations induced an acute transient increase and a subsequent sustained elevation in basal [Ca²⁺]_i in cultured myocytes (4). There seems to be a general agreement concerning cellular Ca²⁺ mobilization that the initial transient increase in [Ca²⁺]_i is likely to have an intracellular origin and the subsequent sustained one comes from outside the cell. In the present electrophysiology we showed the latter pathway alone, that is, polycationic proteins can activate the Ca²⁺ entry via membrane depolarization, showing only a monophasic pattern, in tracheal myocytes. Of course, other mechanisms, e.g., a pharmacomechanical one, along with the present electromechanical pathway, can underlie the direct contractile action of polycations. However, we could not detect an electrophysiologic event corresponding to the initial transient increase in cellular Ca²⁺ observed in cultured myocytes (4). Thus, we found one aspect of the complex process by which polycationic peptides activate smooth-muscle cells. Another point that should be discussed concerns the apparent difference between the time courses in the reported tension development and in the present membrane depolarization. The present membrane depolarization showed an acute onset with the introduction of the polycations and was rapidly reversible, but isometric tension began to increase after 5 min of polycation treatment and was apparently irreversible by three rinses (4). We have no appropriate explanations for this difference, however differences in the tissue preparations might be considered. We used an isolated single tracheal myocyte, whereas 2-mm-wide by 10-mm-long tracheal wall smooth-muscle strips were used in Wylam's tension experiment. It may require a certain interval for 10 kD or more of the charged peptides to penetrate or to be washed out from the macroscopic mass of smooth muscle.

As is discernible in the Mp recording shown in Figure 1A, frequent transient hyperpolarizations were observed as depolarization progressed. Presumably, this is a reciprocal expression of the sporadic activation of Ca²⁺-activated K⁺ channels, i.e., STOCs. STOCs have been well characterized in smooth-muscle cells, including vascular (21), intestinal (22), and tracheal (23) myocytes, and are known to be carried by K⁺ via maxi-K channels (21). STOCs are also known to be activated by membrane depolarization (21), which seemed to be the cause of the appearance of the present spontaneous hyperpolarizations. Activations of maxi-K⁺ channels by adrenergic agonists relax airway smooth muscle, probably by sensitizing the channels to Ca²⁺ via phosphorylating the channel protein (29, 30). Thus, maxi-K channels and STOCs have been considered important regulators of smooth-muscle relaxation. In the present study, the synthetic polycations suppressed the STOCs recorded with the whole-cell configuration (Figure 3). This was most probably due to the direct effect of the polycations on maxi-K channels, as supported by the excised patch experiments. That is, maxi-K channels were inactivated in the presence of polycations both in the conductance and nP_os (Figure 4). It is possible that the decreased single-channel amplitude results from a decrease in the channel open time, producing an apparent decrease in conductance. To avoid this, we employed a 5-kHz bandwidth for the channel recording, differing from that in the whole-cell experiments (see MATERIALS AND METHODS). We found that the conductance of the maxi-K channel was decreased to about 60% of the control value in the presence of the polycations. Interestingly, this mimicked one of the "subconductance states" of the maxi-K channel reported in canine trachealis (32), which was 63% of the full conductance. Although it is beyond the scope of the present investigation, a detailed kinetic study may give some insights into the mode of action of polycations on the channel gating. However, the possibility still remains that the polycations affect the channel gating indirectly, e.g., due to arachidonic acid metabolites generated from lipids in the excised patch membrane.

When considering the physiologic significance of the present study, the results obtained from the Ca²⁺-current experiment seem to be important because the ineffectiveness of synthetic polycations on Ca²⁺ currents supports the notion of depolarization-induced Ca²⁺ influx. This also indicates that the action of polycations has some specificity that depends on the channel species. In some cells, small outward currents were observed after the abolition of the inward Ca²⁺ current by nifedipine (see Figure 5A, lower panel). Although small in amplitude, the current kinetics were reminiscent of the delayed rectifier K⁺ currents shown in Figure 2. We thought that, in spite of the complete replacement of K⁺ in the experimental solutions, potassium ions residual in the cell interior that escaped dialysis might be responsible for the small currents. A similar observation has been reported with regard to the rates of diffusional exchange, and this can occur especially in larger cells (33).

In the present study we demonstrated that the polycationic peptides depolarized the membrane and inhibited the maxi-K channel in bovine tracheal myocytes directly without affecting the Ca²⁺-channel current. The present investigation uncovered for the first time the mechanism underlying the direct contractile and Ca²⁺-mobilizing effects of polycations on airway smooth muscle (4, 8). The polycationic proteins released from a variety of cells, including eosinophils, neutrophils, and platelet and airway epithelial cells, may play some part in the pathogenesis of AHR in chronic inflammatory airway diseases.