INTRODUCTION

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THE LEVEL OF CONTRACTILE tone exhibited by smooth muscle cells is dependent on intrinsic (myogenic) and extrinsic (neuronal, epithelial, and hormonal) factors that regulate plasmalemmal ion channel activities, Ca²⁺ release from the endoplasmic reticulum, intracellular Ca²⁺ concentration, and the interaction of actin and myosin filaments, i.e., cross-bridge cycling (17). Dysfunctional control of smooth muscle tone contributes to a variety of pathological conditions. For example, asthma has been attributed to alterations in control of airway smooth muscle contractility; anaphylactic bronchospasm is associated with a marked hyperreactivity of airway smooth muscle to a variety of pharmacological and physical stimuli, including histamine (8, 33).

The level of resting membrane potential is important for the control of airway smooth muscle tone (13, 33). Action potentials are not observed in bronchial muscle. Rather, contractile tone is postulated to result from maintained, nonregenerative depolarizations that increase the open probability of voltage-dependent L-type Ca²⁺ channels (13, 14, 33). Sustained Ca²⁺ influx due to steady-state activation of L-type Ca²⁺ channels occurs over a narrow range of membrane potentials between 50 and 20 mV and is known to cause significant elevations in intracellular Ca²⁺ levels in isolated airway myocytes (13, 14). A role for 4-aminopyridine (4-AP)-sensitive delayed rectifier K⁺ channels (K_dr) in contributing to control of membrane potential, Ca²⁺ channel activity, and tone is indicated for the airways; exposure of intact tissues and/or isolated tracheal/bronchial myocytes of dogs, ferrets, and humans to 4-AP leads to depolarization and contraction (1, 15, 23). The biophysical properties of delayed rectifier K⁺ current [I_K(dr)] are well described for tracheal myocytes; however, little is known concerning this conductance in smaller-caliber airways. This is potentially significant since the site of airway dysfunction in pathological conditions, such as asthma, is either the central (2nd to 6th order) bronchi (immediate asthmatic response) or the peripheral bronchi (delayed asthmatic response; see Ref. 36).

Previous studies using a canine model of bronchial hyperreactivity demonstrated that tracheal smooth muscle of dogs sensitized with ovalbumin possessed an enhanced contractile response to histamine mediated by H₁ receptors, as well as spontaneous phasic contractile activity and an increased myogenic response (4, 5). Similar findings were subsequently obtained for airway smooth muscle from second- to sixth-order bronchi of ragweed-sensitized dogs, including an increased capacity and velocity of shortening of the smooth muscle and increased release of histamine to ragweed pollen antigen (8, 22). The second- to sixth-order bronchi were also shown to be more sensitive to ragweed pollen compared with tracheal smooth muscle by several orders of magnitude (39). The characteristics of bronchial smooth muscle of ragweed-sensitized dogs have been reviewed (43).

Protein kinase C (PKC) of airway and nonairway smooth muscles is activated by a variety of contractile agonists, including acetylcholine and histamine, and enhanced PKC activity has been implicated to contribute to the pathogenesis of asthma (17, 34, 35). We recently obtained evidence that I_K(dr) of vascular smooth muscle cells is suppressed by PKC (2, 10, 11). Whether I_K(dr) of bronchial myocytes is similarly regulated by PKC is unknown.

Accordingly, in this study, we employed freshly isolated myocytes from bronchi of age-matched, control, and ragweed pollen-sensitized dogs and the standard whole cell patch-clamp technique to 1) characterize the properties of I_K(dr) in cells isolated from a small-airway preparation, 2) determine whether the magnitude or properties of 4-AP-sensitive I_K(dr) were altered in myocytes from bronchial tissues from ragweed pollen-sensitized dogs, and 3) assess the effect on the magnitude and properties of I_K(dr) of activation of PKC.

	METHODS

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Animal sensitization. Dogs were housed, sensitized, and then killed with a lethal injection of pentobarbital sodium (30 mg/kg body wt iv) according to a research protocol consistent with the Canadian Council on Animal Care and approved by local animal care committees of the Medical Research Council of Canada. Newborn mongrel dogs were immunized within 24 h of birth by intraperitoneal injection of 500 µg of ragweed (Ambrosia elatior) pollen extract as previously described (24). Booster injections were given on a weekly basis for 8 wk and then at a biweekly interval for an additional 8 wk. Age-matched, littermate control dogs received a similar schedule of intraperitoneal injections with adjuvant alone. Sensitization to ragweed pollen extract was confirmed by homologous passive cutaneous anaphylaxis in normal adult dogs. For the experiments reported here, bronchial tissues were obtained from sensitized dogs with a serum immunoglobulin E anti-ragweed antibody titer of equal to or greater than 256 dilutions. We employed tissues from 9 sensitized and 10 control littermate dogs from five different litters. Tissues from three adult mongrel dogs were also employed in some of the preliminary experiments to characterize the outward K⁺ current components of isolated bronchial myocytes and to define the recording conditions appropriate for the study of I_K(dr) of bronchial myocytes with minimal contamination from other conductances.

Cell isolation. Canine lungs were quickly excised and placed in ice-cold Krebs-Henseleit physiological salt solution (see Solutions for composition). The bronchial trees were dissected free from the lung parenchyma and then placed in ice-cold nominally Ca²⁺-free Krebs-Henseleit solution. Bronchial branches of fourth- to sixth-order airways (which represent the airways involved in the hyperreactive response) were selected according to the method of Shioya et al. (37). The smooth muscle layer was dissected free of adherent epithelial cells and adventitia under a dissection microscope using fine iris scissors, placed in fresh ice-cold nominally Ca²⁺-free Krebs-Henseleit solution, and stored overnight until tissue digestion the next day. Results of preliminary experiments, performed using myocytes dispersed from tissues on the same day as they were obtained, were not different from those in which the tissues were stored overnight. Freshly dispersed, relaxed single myocytes were prepared from tissue pieces of ~3 mm² based on a method previously described (30). Briefly, bronchial smooth muscle tissues were placed in a low-Ca²⁺ physiological salt solution containing collagenase, protease, and elastase (for composition see below) and gently bubbled with 95% O₂-5% CO₂ at 33-35°C for 20-35 min. After removal from the digestion solution, the pieces were washed several times in low-Ca²⁺ solution and stored at 4°C until required (within 5 h). Myocytes were liberated from the digested tissue pieces by gentle trituration. Viable myocytes used in the patch-clamp experiments were spindle shaped, optically refractive, and relaxed, as is apparent for the representative myocyte shown in Fig. 1.

View larger version (106K): [in this window] [in a new window]   Fig. 1.   Light micrograph of a single bronchial myocyte isolated from a control dog. Scale bar indicates 20 µm.

Solutions. The Krebs-Henseleit solution for dissection and overnight storage of intact bronchial tissues contained (in mM) 115 NaCl, 25 NaHCO₃, 1.38 KH₂PO₄, 2.51 KCl, 2.46 MgCl₂, 1.91 CaCl₂, and 5.56 dextrose (pH 7.4 when gassed with 95% O₂-5% CO₂) with the exception that the storage solution lacked the added CaCl₂. The low-Ca²⁺ physiological salt solution used in cell isolation contained (in mM) 120 NaCl, 25 NaHCO₃, 4.2 KCl, 0.6 KH₂PO₄, 1.2 MgCl₂, and 0.01 CaCl₂ (pH 7.4 when bubbled with 95% O₂-5% CO₂ at room temperature, 20-22°C), supplemented with collagenase (564 U/ml Sigma type 1A; Sigma Chemical), protease (10 U/ml Sigma type XXVII), and elastase (81 U/ml Sigma type IV).

The standard bath solution employed in the whole cell voltage-clamp experiments contained the following (in mM): 120 NaCl, 3 NaHCO₃, 4.2 KCl, 1.2 KH₂PO₄, 0.5 MgCl₂, 10 glucose, 1.8 CaCl₂, and 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; pH 7.4 corrected with NaOH). The standard whole cell pipette solution contained the following (in mM): 110 potassium gluconate, 30 KCl, 0.5 MgCl₂, 5 HEPES, 5 Na₂ATP, and either 0.05 ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or 10 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), as stated in RESULTS, to produce varied levels of internal Ca²⁺ buffering (pH adjusted to 7.2 with KOH).

Electrophysiological recordings. Isolated myocytes were placed in a 300-µl bath and allowed to settle to the bottom of the chamber before a constant flow of bath solution (~0.5 ml/min) at room temperature (20-22°C) was initiated. A standard whole cell patch-clamp recording technique was employed in the experiments (16). Pipettes were prepared from capillary glass (7052 glass; Richland Glass) using a Sutter P-87 puller (Sutter Instruments) and MF-83 microforge (Narashige Scientific Instrument Laboratory). Tip resistances of 1-3 M were obtained when filled with pipette solution. Recordings were performed using either an Axopatch 1-D or 200A amplifier (Axon Instruments). Pipette potential and capacitance were nulled and a 5- to 15-G seal formed with the membrane. To correct for junction potential, 20 pipettes were nulled in pipette solution and then transferred to bath solution. A consistent value of 10 mV for the junction potential was obtained and employed to correct all voltage-clamp protocols and current recordings. Whole cell voltage-clamp protocols were applied using pClamp software (Axon Instruments). Data were filtered at 2 kHz by an on-board 8-pole Bessel filter before digitization with a Labmaster TL-1-125 or Digidata 1200 analog-to-digital converter (Axon Instruments) and stored on hard disk in a 486 PC clone. Data were displayed and analyzed off-line using pClamp (Axon Instruments) and Origin software (MicroCal Software).

Values for net and tail currents due to I_K(dr) were normalized for cell capacitance and expressed as picoamperes per picofarad. Cell capacitance was determined by integration of the capacity transient evoked by 20-mV command steps. Current-voltage (I-V) relations for net and tail currents were determined in the following manner: net current due to I_K(dr), with minimal contamination by other conductances, was measured at the end of 250-ms pulses and plotted as a function of command step voltage between 80 and +30 mV. Tail currents evoked by repolarization to 40 mV following steps to between 80 and +30 mV and due to deactivating K_dr exclusively were calculated as the difference between the peak amplitude of the tail and sustained current levels at 200 ms. Drug-sensitive difference currents were determined by digital subtraction of current traces obtained in the presence of drug from those recorded in its absence.

Drugs. 4-Phorbol 12,13-dibutyrate (PDBu), Na₂ATP, EGTA, BAPTA, tetraethylammonium chloride (TEA⁺), and 4-AP were obtained from Sigma Chemical; 1,2- and 1,3-isomers of dioctanoyl-sn-glycerol (diC₈) were obtained from Serdary Research Laboratories. A stock solution of PDBu (10 mM) was prepared in dimethyl sulfoxide. Diacylglycerol (DAG) analogs were dissolved in hexane. All were added directly to the bath solution at the desired concentration on the day of use. Vehicle controls had no effect on currents recorded. 4-AP was dissolved in bath solution, and the pH was readjusted to 7.4.

Statistics. All values in the text and Figs. 1-9 are presented as means ± SE. Data were compared by paired or unpaired Student's t-test for single comparisons. Individual data points in the I-V relations for myocytes exposed to single treatments were compared with a paired Student's t-test, and in experiments where multiple treatments were applied to single cells, the data points were compared by repeated-measures ANOVA followed by Bonferroni's test for multiple comparisons. A level of P < 0.05 was considered to be statistically significant.

	RESULTS

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Morphological studies. Bronchial myocytes isolated from control and sensitized dogs and employed in the whole cell patch-clamp studies were identical to the representative cell shown in Fig. 1. Myocytes from control dogs had an average length of 228 ± 36 µm (range 155-307 µm, n = 59 from three separate cell isolations). There was no difference in the capacitive cell surface area of myocytes from control and sensitized dogs: values of 4,210 ± 290 (n = 16) and 3,710 ± 338 (n = 13) µm² (P > 0.05) were obtained based on a specific membrane capacitance of 1 µF/cm² and average cell capacitances of 42.1 ± 2.9 and 37.1 ± 3.4 pF (P > 0.05), respectively. The cells were relaxed on isolation and were capable of repeated contractions to elevated external K⁺ or histamine (data not shown).

Identification of macroscopic K⁺ currents. The identity and properties of macroscopic K⁺ currents of canine bronchial myocytes have not been characterized in detail. For this reason, we conducted a series of preliminary experiments to identify conditions that would permit selective recordings of I_K(dr). These initial experiments demonstrated the presence of 1) inward L-type Ca²⁺ currents that showed rundown within the first 2-3 min of recording and 2) two dominant components of macroscopic outward K⁺ current in bronchial myocytes of adult and young control or ragweed pollen-sensitized dogs. In the presence of minimal intracellular Ca²⁺ chelation with 0.05 mM EGTA in the pipette solution, command steps from a holding potential of 60 mV to between 80 and +30 mV every 10 s evoked net outward K⁺ current positive to 40 mV, which displayed voltage- and time-dependent activation and was "noisy" positive to 0 mV (Fig. 2A). Outward current evoked during voltage steps increased exponentially with applied voltage and on average reached a density of 58 ± 4 pA/pF at +30 mV (Fig. 2B).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Macroscopic currents of canine bronchial myocytes recorded with minimal internal Ca2+ chelation (0.05 mM EGTA in pipette solution). A: representative family of whole cell currents recorded from a bronchial myocyte of a control dog during 250-ms pulses to between 80 and +30 mV in 10-mV intervals followed by repolarizing steps to 40 mV applied every 10 s. Arrow indicates end-pulse current amplitude used in current-voltage (I-V) plot of B. Dashed line in this and other figures represents zero current. B: average I-V relation for macroscopic current in four bronchial myocytes from control dogs recorded in the presence of minimal internal Ca2+ chelation. Data points are mean end-pulse current amplitudes normalized to cell capacitance ± SE. C: representative quasi-steady-state macroscopic current recorded during a 16-s voltage-ramp protocol applied between 90 and +30 mV.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Inhibition of noisy macroscopic current due to large-conductance Ca2+-activated K+ channels with tetraethylammonium chloride (TEA+). A: representative quasi-steady-state macroscopic currents evoked by an 8-s voltage-ramp protocol (to increase amplitude of noisy current) between 90 and +30 mV in the absence [control (Con)] and presence of 0.1 mM TEA+. B: TEA+-sensitive difference current obtained by digital subtraction of current trace recorded in the presence of TEA+ from control trace.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Inhibition of macroscopic current of bronchial myocytes recorded with high internal Ca2+ chelation (10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid in pipette solution) by 4-aminopyridine (4-AP). A: representative families of whole cell currents recorded from a single bronchial myocyte during 250-ms pulses to between 80 and +30 mV in 10-mV intervals and repolarizing steps to 40 mV before (Con) and during treatment with 10 mM 4-AP. 4-AP-sensitive difference currents are also shown (Con-4-AP). Pulses were applied at 10-s intervals. B: average I-V relations for end-pulse (Inet) and tail (Itail) current due to delayed rectifier K+ current [IK(dr] in 3 bronchial myocytes from control dogs in the absence and presence of 4-AP. Data points are mean end-pulse and peak tail current amplitudes normalized to cell capacitance ± SE.

Characteristics of I_K(dr) in bronchial myocytes from control dogs. The kinetics and voltage dependence of activation, deactivation, and inactivation of bronchial smooth muscle I_K(dr) were determined. Activation during a 250-ms test pulse to +20 mV and subsequent deactivation at 40 mV were both best fit with two exponentials (Table 1). Slow inactivation of I_K(dr) was apparent during command pulses to potentials positive to 0 mV. Longer-duration, 4- to 10-s steps to +20 mV were applied to bronchial myocytes to examine the time dependence of inactivation. I_K(dr) decayed relatively rapidly during the first 4 s of the command pulses and thereafter exhibited only a very slow decline in amplitude. A biexponential function was found to best describe the inactivation kinetics, and, at +20 mV, the average values for the fast and slow time constants (Table 1) were similar to those previously reported for other smooth muscle cells (e.g., 0.14 and 1.1 s at +5 mV for canine tracheal myocytes; see Ref. 7). The voltage dependence of I_K(dr) activation and inactivation was studied. Figure 5 illustrates representative examples of 1) tail currents recorded at 40 mV after steps to between 80 and +30 mV (Fig. 5A) and 2) whole cell currents evoked by a double-pulse protocol that employed 4-s conditioning pulses to between 110 and +20 mV followed by command steps of 200-ms duration to +20 mV (Fig. 5B). Figure 5C shows the average values for normalized peak tail current versus voltage and normalized I_K(dr) availability as a function of command and conditioning step voltages, respectively. Peak tail current amplitude at 40 mV after command steps to between 80 and +30 mV was measured (i.e., peak minus sustained amplitude after 200 ms), normalized to the maximal amplitude, and plotted as a function of command step voltage. Availability after a 4-s conditioning pulse was determined from the amplitude of current present at the end of 200-ms pulses to +20 mV and plotted as a function of conditioning pulse voltage. The plots of normalized current versus voltage of the command step or conditioning pulse were best fit with single Boltzmann functions (Fig. 5). Values for half-maximal potential (V_0.5) and slope for activation and inactivation curves from 15 and 9 myocytes, respectively, were averaged and are given in Table 1. Similar values for these parameters were also obtained in two myocytes using 10-s conditioning pulses (not shown).

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Voltage dependence of activation and inactivation of IK(dr) in bronchial myocytes from control and ragweed pollen-sensitized dogs. A: representative tail currents recorded during 200-ms repolarizing steps to 40 mV after test pulses to between 80 and +30 mV (voltage of step indicated at left of each trace) from a bronchial myocyte of a control dog. B: representative family of whole cell currents evoked by a double-pulse protocol to assess availability of IK(dr) in a bronchial myocyte from a control dog. Prepulse steps of 4-s duration to between 110 and +20 mV in 10-mV intervals were applied from holding potential of 60 mV every 15 s and followed by a brief 5-ms step to 130 mV and a constant 200-ms depolarizing test pulse to +20 mV. C: activation (circles) and inactivation (squares) for myocytes from control (open symbols) and ragweed pollen-sensitized (closed symbols) dogs. Each point is the mean ± SE of n = 9-15 myocytes. Activation data were obtained from protocols as in A; peak tail amplitudes were normalized to maximal amplitude and plotted against the voltage of the test pulse. Inactivation data were obtained in protocols as in B; the fraction of inactivating current was plotted against the voltage of the 4-s conditioning step. Solid lines passing through the activation and inactivation data points are the best fits of Boltzmann distribution functions obtained by a Leven-Marquardt nonlinear least squares fitting algorithm: activation Y = {1 + exp[(V0.5  V)/k]}1, where the voltage at half-maximal activation (V0.5) and slope (k) were 15.7 and 7.8 mV versus 17.7 and 7.6 mV in myocytes from control and sensitized dogs, respectively, and V is the voltage of the prepulse step. Inactivation curves Y = {1 + exp[(V  V0.5)/k]}1, where V0.5 and k were 26.0 and 6.3 mV versus 29.9 and 5.9 mV in myocytes from control and sensitized dogs, respectively. * Value in myocytes from sensitized animals was significantly different (P < 0.05) from that in control dogs by unpaired Student's t-test.

Comparison of I_K(dr) in bronchial myocytes from control and sensitized dogs. Whether the properties of I_K(dr) were altered in myocytes obtained from ragweed pollen-sensitized dogs was determined by applying similar voltage-clamp protocols under identical recording conditions to those described for cells from control dogs. Representative families of I_K(dr) of bronchial myocytes of control and ragweed pollen-sensitized dogs and average I-V relations for end-pulse and tail current amplitude of 15 and 13 control and sensitized myocytes, respectively, are shown in Fig. 6. Plots of average normalized current versus voltage for activation and inactivation of I_K(dr) in myocytes from sensitized dogs are shown in Fig. 5. Average values for time constants of activation, deactivation, and inactivation, as well as the voltages for half-maximal activation and inactivation, and values for slope factors are given in Table 1. No statistically significant difference was noted for 1) end-pulse and tail current densities (Fig. 6), 2) voltage dependence of activation (Fig. 5), or 3) kinetics of activation, inactivation, or deactivation (Table 1) of I_K(dr) between myocytes isolated from the bronchi of sensitized versus control dogs.

View larger version (29K): [in this window] [in a new window]   Fig. 6.   Comparison of IK(dr) density in bronchial myocytes from control versus sensitized dogs. A: representative families of macroscopic currents of myocytes from control (Con) and ragweed pollen-sensitized (Sens) dogs evoked by 250-ms pulses from a holding potential of 60 mV to between 80 and +30 mV followed by repolarization to 40 mV. Pulses were applied at 10-s intervals. B: average I-V relations for end-pulse (Inet) and tail (Itail) current amplitude in 15 and 13 myocytes from control (open symbols) and sensitized (closed symbols) dogs, respectively. Data points are means of current amplitude normalized to cell capacitance ± SE. No data points were significantly different from control when analyzed by Student's t-test.

Inhibition of I_K(dr) by PKC activation. Activation of PKC is thought to play an important role in the signal transduction pathway of histamine, which contracts airway smooth muscle, and alterations in the activity of PKC isoenzymes have been implicated in the pathogenesis of asthma and bronchospasm of hyperresponsive airways of sensitized animals (4, 17, 35). Accordingly, we determined the effects on I_K(dr) of direct PKC activation with an analog of DAG, 1,2-diC₈, and the phorbol ester PDBu. Eleven myocytes from control and sensitized dogs were used (6 and 5 myocytes, respectively), and a similar change in I_K(dr) during PKC activation was observed in all cells tested. Figure 7 shows the effects of the inactive DAG analog 1,3-diC₈ followed by 1,2-diC₈ on I_K(dr) amplitude. To monitor the time course of effects of the DAG analogs on I_K(dr) amplitude, depolarizing steps to +10 mV followed by a brief step to 40 mV were applied every 15 s. Figure 7A shows a representative example of the change in normalized end-pulse current amplitude under control conditions and during sequential exposure to the inactive DAG analog 1,3-diC₈ (10 µM) followed by active DAG analog 1,2-diC₈ (1 and 10 µM). The representative current recordings shown in Fig. 7, insets, were obtained at the four time points indicated by the arrows. No effect on I_K(dr) amplitude was evident with 1,3-diC₈, but end-pulse amplitude slowly declined in a concentration-dependent fashion upon exposure to 1,2-diC₈. Figure 7B shows superimposed, representative difference current traces obtained by digital subtraction of traces recorded at 30-s intervals during exposure to 1,2-diC₈. The onset of inhibition of I_K(dr) required ~60 s, so the initial difference currents showed no change. However, the amplitude of the outward 1,2-diC₈-sensitive difference current increased with time, reaching a peak sustained level within 3-5 min. The difference currents at peak inhibition by 1,2-diC₈ were very similar to those determined for 4-AP inhibition (Fig. 4).

View larger version (31K): [in this window] [in a new window]   Fig. 7.   Time course of effect of diacylglycerol analogs on IK(dr). A: representative changes in end-pulse current amplitude (I) evoked by 250-ms pulses from 60 to +10 mV and normalized to the amplitude under control conditions at time 0 (I0). Pulses were applied at 15-s intervals under control conditions and during sequential exposure to 10 µM 1,3-dioctanoyl-sn-glycerol (diC8) and 1 and 10 µM 1,2-diC8. Insets: representative traces of current recordings in the absence of drug and in the presence of either 1,3-diC8 or 1,2-diC8 at 1 and 10 µM as indicated by arrows. B: superimposed, 1,2-diC8-sensitive difference currents obtained by digital subtraction of macroscopic current evoked by a similar protocol as in A applied at 30-s intervals during exposure to 1,2-diC8 (10 µM) from current recorded in the absence of drug. Open and closed circles indicate difference currents for pulses applied immediately after switching to bath solution containing diacylglycerol analog and during peak, sustained effect of the drug, respectively.

View larger version (25K): [in this window] [in a new window]   Fig. 8.   Protein kinase C (PKC) activation with diacylglycerol analog reversibly inhibits bronchial smooth muscle IK(dr). A: representative families of whole cell currents evoked by 250-ms pulses to between 80 and +30 mV in 10-mV steps followed by repolarization to 40 mV during sequential exposure of a single bronchial myocyte to control bath solution (Control), 10 µM 1,3-diC8, 10 µM 1,2-diC8, and wash with control bath solution (Wash). B: average I-V relations for end-pulse (Inet) and tail (Itail) current amplitude normalized to cell capacitance in the absence of diacylglycerol analog (Con; open square; n = 7), presence of 1,3-diC8 (open circle; n = 5) or 1,2-diC8 (closed circle; n = 7), and after 10-15 min wash in control solution (open diamond; n = 3). Note: 7 myocytes were exposed to active analog, but only 5 of these had prior exposure to inactive analog, and only 3 myocytes were held for a sufficient period to exhibit washout. * Significantly different from the value determined for control condition (P < 0.05) by repeated measures ANOVA followed by Bonferroni's test for comparisons of individual groups.

View larger version (27K): [in this window] [in a new window]   Fig. 9.   PKC activation with the phorbol ester 4-phorbol 12,13-dibutyrate (PDBu; 100 nM) inhibits bronchial smooth muscle IK(dr). A: representative families of whole cell currents evoked by 250-ms pulses to between 80 and +30 mV in 10-mV steps followed by repolarization to 40 mV during sequential exposure of a single bronchial myocyte to control bath solution (Control) and 100 nM PDBu. B: average I-V relations for end-pulse (Inet; circles) and tail (Itail; squares) current amplitude normalized to cell capacitance in the absence of PDBu (Con; open symbols; n = 4) or presence of PDBu (closed symbols; n = 4). * Significantly different from the value determined for control condition (P < 0.05) by paired Student's t-test.

	DISCUSSION

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In this study, the whole cell voltage-clamp technique was used to characterize the properties of I_K(dr), as well as to determine the effect of ragweed pollen sensitization and direct activation of PKC with a DAG analog or phorbol ester on this conductance of isolated canine bronchial myocytes. We show for the first time that 1) the voltage dependence of inactivation for I_K(dr) in bronchial myocytes is different from that in the trachea, 2) inactivation is shifted to more negative potentials in myocytes from sensitized compared with age-matched control dogs, and 3) PKC activation reduces I_K(dr) amplitude. Reduced I_K(dr) due to decreased availability and/or inhibition by PKC activation may have implications for altered control of airway smooth muscle tone in asthma.

Two K⁺ channels, BK_Ca and K_dr, were shown to contribute to macroscopic currents of canine bronchial myocytes on the basis of pharmacology, Ca²⁺ sensitivity, and kinetic behavior. Noisy macroscopic current carried by BK_Ca was present positive to 20 mV with minimal intracellular Ca²⁺ chelation and was selectively inhibited with 0.1 mM TEA⁺. In contrast, macroscopic current due to K_dr was insensitive to low TEA⁺ and high levels of intracellular Ca²⁺ chelation, but it was readily blocked by 4-AP and displayed slow, time-dependent inactivation. The presence of these two channel types in bronchial myocytes is consistent with previous studies of airway myocytes of dog, ferret, pig (7, 26, 29), and humans (1, 18, 41). I_K(dr) of canine bronchial myocytes was studied using 10 mM BAPTA in the pipette to provide for substantial chelation of intracellular Ca²⁺ and minimal contamination of whole cell currents by Ca²⁺-activated conductances, such as BK_Ca, Ca²⁺-activated Cl, and Ca²⁺-dependent nonselective cation currents. Additionally, depolarizing voltage-clamp steps were limited to potentials of +30 mV or less to avoid voltage-dependent activation of BK_Ca. The ability of 4-AP to substantially inhibit tail currents produced during deactivation of K_dr at 40 mV as well as end-pulse current amplitude is consistent with minimal contamination by non-K_dr channel activity. The presence of I_K(dr) in all canine bronchial myocytes studied here differs from that reported for human myocytes in which the current was only present in 17% of cells employed (41). However, Janssen (18) described I_K(dr) in human bronchial smooth muscle that was sensitive to 4-AP, and Adda et al. (1) described the presence of mRNA encoding the voltage-gated K⁺ channels Kv1.1, Kv1.2, and Kv1.5 in human airway muscle.

The fittings of the kinetics of inactivation and deactivation are consistent with other smooth muscle preparations, both being best described by biexponential functions with two time constants on the order of 0.2-0.5 and 2-5 s (inactivation) and 10 and 60 ms (deactivation; see Refs. 2, 3, 7, 26, 28, 40, 46). However, few studies have quoted values for activation time constants due to the overlap with activation of transient outward currents. We did not observe evidence in any cell for the presence of "A"-type outward current (note the transient current in the presence of 1,2-diC₈ resulted from the change in I_K(dr); see Refs. 2 and 10) indicating its absence or complete inactivation at the holding potential (60 mV) employed. A dual-exponential fit of I_K(dr) activation in bronchial myocytes is consistent with that of cloned rabbit vascular Kv1.5 (O. Clément-Chomienne, K. Ishii, M. Walsh, and W. Cole, unpublished observation). This study shows that I_K(dr) of canine bronchial myocytes is activated at voltages positive to approximately 45 mV and that it displays time- and voltage-dependent inactivation. The overlap of voltage ranges for activation and inactivation suggests that steady-state K_dr window current may be present between approximately 45 and 0 mV, with peak outward I_K(dr) at approximately 25 mV. These properties are similar to those previously reported for I_K(dr) of ferret tracheal myocytes; K_dr window current was observed between approximately 50 and 10 mV at 37°C (15). This more negative activation voltage of 50 mV compared with the present study is likely a function of the bath temperature used to record the ferret tracheal I_K(dr); activation, but not inactivation, of I_K(dr) of smooth muscle cells is known to occur over a more negative range at higher temperatures (e.g., see Ref. 10). It is significant that the voltage for half-maximal availability of I_K(dr) of bronchial myocytes was less negative compared with the value reported for canine and porcine tracheal cells: 26 mV versus 46 and 53 mV for whole cell and for single K_dr channel currents in the trachea (7). It would seem unlikely that differences in recording conditions in the two studies can explain the divergent values for V_0.5 of inactivation (only slightly different pipette solutions were employed, and both were done at room temperature). Moreover, under identical recording conditions (temperature, bath and pipette solutions, protocol) to those employed in this study, V_0.5 for inactivation of I_K(dr) of rabbit portal vein myocytes was also more negative (approximately 40 mV) than that observed in the present study, even though the V_0.5 for activation was similar: approximately 16 and 11 mV in bronchial and portal vein myocytes, respectively (2, 10). The physiological implication of the less negative voltage dependence of inactivation is that I_K(dr) of bronchial myocytes will contribute steady-state current over a wider range of membrane potentials and, therefore, its importance to control of membrane potential and contractile tone may be greater than in the trachea. The role of I_K(dr) was similarly concluded to be enhanced in small versus large pulmonary arterial vessels (6). In this case, however, smaller vessels were found to possess more cells with I_K(dr) (6).

We also found that the availability of I_K(dr) between 50 and 20 mV was decreased in tissues from sensitized compared with control dogs because of a negative shift in voltage dependence of inactivation. Although small in magnitude, this hyperpolarizing shift in availability of I_K(dr) is of potential significance to the electrical behavior of the hyperresponsive airways of ragweed pollen-sensitized dogs. The shift in the inactivation curve predicts that the amplitude of steady-state I_K(dr) will be reduced over the critical range of membrane potentials between 50 and 20 mV over which the L-type Ca²⁺ window current also occurs (13, 14). Such small changes in current amplitude will have large effects on membrane potential in cells with a large input resistance, as is the case for smooth muscle cells (1-15 G; see Ref. 31).

It is possible that the difference in V_0.5 for inactivation between tracheal and bronchial myocytes, as well as the negative shift in availability of K_dr channels in the myocytes of sensitized dogs, may arise from the expression of different K_dr channels in the two tissues. Voltage-gated K⁺ channels consist of pore-forming - and modulatory -subunits. The voltage dependence of inactivation of voltage-gated K⁺ channels is known to be affected by association with some -subunits. For example, Kv1.5, which is the dominant Kv present in human bronchi (1), displays a more negative voltage dependence of inactivation but no change in kinetics when expressed in the presence of Kv2.1 (45). Alternatively, the formation of heterotetramers between different -subunits of the Kv1 family, which possess distinct inactivation properties, can give rise to channels with novel, intermediate properties, including differences in voltage dependence of inactivation (32). Therefore, differences in the expression of a -subunit and/or increased formation of heteromultimers of Kv1.5 and Kv1.1, which are expressed in human airways (1), could be the cellular basis for the difference in inactivation of I_K(dr) in myocytes from trachea and bronchi, as well as the negative shift in availability of I_K(dr) in myocytes of sensitized compared with control dogs. Further studies will be required to address these issues.

In this study, we employed two structurally different analogs of DAG and a phorbol ester to determine the effect of activation of PKC on I_K(dr) of bronchial smooth muscle. The active DAG analog 1,2-diC₈ and the phorbol ester PDBu were found to cause a decrease in the amplitude of I_K(dr), but the inactive analog 1,3-diC₈ was without effect. This indicates that nonspecific effects of 1,2-diC₈, for example, changes in membrane fluidity or direct channel block, were not involved. The activity of BK_Ca, L-type Ca²⁺, ATP-sensitive K⁺, and nonselective cation channels in smooth muscle were previously reported to be affected by PKC activation (see Ref. 27 for a review). A change in these conductances also cannot account for the present data for the following reasons: 1) the effects of PKC activation on K_dr were determined under conditions that minimized the contribution of BK_Ca and Ca²⁺-activated Cl current to macroscopic current (high intracellular Ca²⁺ chelation with 10 mM BAPTA and command potentials negative to +30 mV); 2) 1,2-diC₈ had no effect on time-independent current under the recording conditions employed, suggesting that changes in ATP-sensitive K⁺ and nonselective cation currents were not involved; and 3) activation of PKC was associated with a decline in K_dr tail current amplitude at 40 mV where little contamination of macroscopic current by Ca²⁺ current would be expected.

The ability of PKC activation to suppress I_K(dr) of bronchial myocytes is consistent with our previous observations on rabbit portal vein (2, 10). A family of at least 11 related PKC isoforms possessing different properties has been characterized, and at least five isoforms are known to be expressed in smooth muscle tissues (9, 17). The effects of 1,2-diC₈ and PDBu on I_K(dr) of bronchial myocytes can be attributed to PKC, , or  for the following reasons. PKC_I, _II, , , , and  were identified in immunoblots using isoform-specific antibodies from canine bronchial preparations (12). The low intracellular Ca²⁺ level under our recording conditions (due to dialysis with pipette solution containing 10 mM BAPTA) likely precludes a role for the Ca²⁺-dependent isoforms in the suppression of I_K(dr) in our experiments, and the Ca²⁺-independent isoform PKC is insensitive to DAG and phorbol esters (9). In rabbit portal vein, the effect of PKC on I_K(dr) was attributed to PKC (10). Inhibition of I_K(dr) due to activation of PKC may contribute to dysfunctional control of membrane potential in asthma. This view is based on the following. 1) Histamine release is elevated in bronchi of sensitized animals (8). 2) Histamine and other contractile agonists are known to stimulate DAG production, leading to PKC activation in airway smooth muscle (35). 3) Activation of PKC causes a tonic increase in tone in human airway tissue; 100 nM PDBu produces a sustained contraction that is ~40% of the maximal response to acetylcholine (34). A role for changes in membrane potential and activation of voltage-gated Ca²⁺ channels is suggested by the fact that phorbol ester-induced contractions are dependent on extracellular Ca²⁺ influx and inhibited by treatment with dihydropyridines (34). 4) PKC activation has been implicated in the pathogenesis of airway smooth muscle hyperreactivity (33, 35). In light of the ability of DAG analog and phorbol ester to depress I_K(dr), the lack of a difference in current density in bronchial myocytes of sensitized versus control dogs argues against an intrinsic difference in basal PKC activity in the two groups. However, as noted above, the bronchi of the sensitized dog are known to release more histamine in response to antigen challenge compared with control animals (8). This difference in histamine release may be expected to produce 1) an extrinsic change in PKC activity and 2) an enhanced depression of I_K(dr) in situ that would not be observed in the isolated myocytes in the absence of antigen, mast cells, and/or histamine.

The changes in I_K(dr) of bronchial myocytes associated with ragweed pollen sensitization or PKC activation identified in this study may be two contributing factors to the mechanism(s) responsible for dysfunctional airway smooth muscle contractility in asthma. This view is based on compelling evidence that K_dr contributes to the control of membrane potential and the level of depolarization of smooth muscle during exposure to contractile or relaxant stimuli (1, 10, 13, 25, 28, 31, 38). Previous studies on intact airways from ferrets and humans employing 4-AP and charybdotoxin to inhibit K_dr and BK_Ca, respectively, indicated that I_K(dr) contributes to resting K⁺ conductance but that BK_Ca is not as important under basal conditions (1, 15). Treatment with 4-AP (in the presence of tetrodotoxin and atropine) caused depolarization of tracheal smooth muscle membrane potential by at least 5 mV and produced tonic contraction, the latter being sensitive to inhibition of L-type Ca²⁺ channels with dihydropyrines (1, 15, 23). Tonic contractions due to steady-state Ca²⁺ influx in airway smooth muscle are possible because of the presence of a small L-type Ca²⁺ window current, over the same voltage range as that of K_dr window current, which is capable of producing marked increases in intracellular Ca²⁺ concentration (13). I_K(dr) activation has been postulated to function as a voltage-dependent, feedback mechanism for control of Ca²⁺ influx in smooth muscle (11, 31); depolarization due to activation of inward currents in bronchial myocytes carried by T- or L-type Ca²⁺, Cl, and/or nonselective cation channels (13, 19, 20, 21) is offset by the activation of outward K⁺ current. In cerebral resistance arteries, inhibition of I_K(dr) with 4-AP causes enhanced myogenic reactivity to increased intraluminal pressure (25). K_dr may function in parallel with a Ca²⁺-dependent feedback mechanism involving BK_Ca activation in response to changes in intracellular Ca²⁺ concentration (31). Precise changes in the amplitude of outward current via these voltage- and Ca²⁺-dependent feedback mechanisms would prevent regenerative Ca²⁺ influx and permit the graded depolarizations and tone development observed in bronchi. Further studies are required to determine whether alterations in BK_Ca channels also contribute to airway hyperresponsiveness.

This study identifies two different mechanisms by which the contribution of K_dr to control of membrane potential may be reduced in airway smooth muscle in asthma: 1) a negative shift in voltage dependence of inactivation resulting in reduced steady-state I_K(dr) between 40 and 20 mV and 2) inhibition by PKC activation. Because the level of membrane potential is determined by a balance between inward and outward currents in smooth muscle (31), a decline in steady-state I_K(dr) due to a change in availability or suppression by PKC activation would result in depolarization and may contribute to the reported shift in membrane potential to more positive potentials in sensitized canine airway tissues (42). A reduction in steady-state I_K(dr) may also contribute to the development of airway hyperreactivity that is observed in asthma; contractile agonists are known to activate inward conductances, for example, L-type Ca²⁺ (44), Cl, and nonselective cation currents (20, 21), and depolarize tracheal myocytes. A decline in the voltage-dependent feedback control of membrane potential may therefore result in enhanced depolarization, greater L-type Ca²⁺ channel activation, and an increased rise in intracellular Ca²⁺ concentration during exposure to contractile agonists. The net result would be greater development of tone and increased peak force, which are typical of hyperreactive airway tissues (4, 8, 33, 35). Further experiments on bronchial smooth muscle are required to assess 1) the role of I_K(dr) in depolarization of intact bronchial tissues to specific contractile agonists and 2) whether activation of phospholipase C-coupled receptors can produce a PKC-dependent decline in K_dr channel activity in bronchial myocytes.