Membrane currents in canine bronchial artery and their regulation by excitatory agonists

Q. J. Li and L. J. Janssen

Asthma Research Group, Father Sean O'Sullivan Research Center, St. Joseph's Hospital, Department of Medicine, McMaster University, Hamilton, Ontario, Canada L8N 4A6


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The bronchial vasculature plays an important role in airway physiology and pathophysiology. We investigated the ion currents in canine bronchial smooth muscle cells using patch-clamp techniques. Sustained outward K+ current evoked by step depolarizations was significantly inhibited by tetraethylamonium (1 and 10 mM) or by charybdotoxin (10-6 M) but was not significantly affected by 4-aminopyridine (1 or 5 mM), suggesting that it was primarily a Ca2+-activated K+ current. Consistent with this, the K+ current was markedly increased by raising external Ca2+ to 4 mM but was decreased by nifedipine (10-6 M) or by removing external Ca2+. When K+ currents were blocked (by Cs+ in the pipette), step depolarizations evoked transient inward currents with characteristics of L-type Ca2+ current as follows: 1) activation that was voltage dependent (threshold and maximal at -50 and -10 mV, respectively); 2) inactivation that was time dependent and voltage dependent (voltage causing 50% maximal inactivation of -26 ± 22 mV); and 3) blockade by nifedipine (10-6 M). The thromboxane mimetic U-46619 (10-6 M) caused a marked augmentation of outward K+ current (as did 10 mM caffeine) lasting only 10-20 s; this was followed by significant suppression of the K+ current lasting several minutes. Phenylephrine (10-4 M) also suppressed the K+ current to a similar degree but did not cause the initial transient augmentation. None of these three agonists elicited inward current of any kind. We conclude that bronchial arterial smooth muscle expresses Ca2+-dependent K+ channels and voltage-dependent Ca2+ channels and that its excitation does not involve activation of Cl- channels.

potassium; calcium; chloride; adrenergic; U-46619


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE BRONCHIAL VASCULATURE plays a very important role in maintaining the normal function of the respiratory system: it nourishes the airway wall, conditions inspired air, and is involved in defense and clearance of the airways. Dysfunction of bronchial vasculature may contribute to asthma, particularly exercise- and cold/dry air-induced bronchoconstriction (22), and to edema formation in the lung occurring after acute lung injury with smoke inhalation and acid aspiration (1, 4, 17); also, adequate bronchial blood flow is now recognized to be vital to postoperative success after lung transplantation (12, 16).

Like most systemic vasculature, the bronchial circulation is regulated primarily via an excitatory adrenergic innervation and inhibitory nonadrenergic noncholinergic innervation (3, 25) and is also regulated by blood-borne autacoids, including inflammatory mediators; the primary constrictor autacoid among the latter is thromboxane A2 (3, 15).

Constriction and relaxation of vascular smooth muscle is mediated in part through changes in membrane conductances: in general, excitatory stimuli cause opening of voltage-dependent Ca2+ channels, usually via activation of Cl- channels and subsequent membrane depolarization, whereas vasodilators activate K+ channels, leading to membrane hyperpolarization and decreased Ca2+ influx (13). Although much work has been done to investigate the ion channels and their regulation in other systemic arteries, there have been no electrophysiological studies of the bronchial vasculature. The purpose of this study, then, was to classify the ion channels present in freshly dissociated canine bronchial arterial smooth muscle cells and their regulation by excitatory agonists.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Cell isolation. Adult mongrel dogs (15-30 kg; either sex) were killed by intravenous injection of pentobarbital sodium (30 mg/kg), and lobes of lung were excised. Bronchial vasculature (0.5-1 mm OD) on third- to fifth-order airways was exposed by removing overlying connective tissue and parenchyma. Bronchial veins were generally sparse or completely absent in these intraparenchymal airways (since the bronchial circulation drains into the pulmonary veins) and are easily distinguished from the bronchial arteries on the basis of diameter and relative wall thickness; we used only bronchial arteries in these studies.

Tissues were either used immediately or stored at 4°C for use the next day. We found no functional differences in tissues that were studied immediately compared with those used after 24 h of refrigeration. Tissues were transferred to digestion solution that contained collagenase and elastase (see composition in Solutions) and were incubated at 37°C for 1 h, after which they were gently triturated to liberate individual myocytes. These were allowed to settle and adhere to the bottom of a recording chamber (1 ml volume) and were superfused with standard Ringer solution at a rate of 2-3 ml/min; unless indicated otherwise, experiments were conducted at room temperature. The dissociated cells were studied within 8 h after dissociation. Electrophysiological responses were tested in cells that were phase dense and appeared relaxed.

Electrophysiological study. The majority of recordings were made using the nystatin perforated-patch configuration of the conventional patch-clamp recording technique. Pipette tip resistances ranged from 3 to 5 MOmega , and access resistances ranged from 9 to 39 MOmega (70-80% compensated). Membrane currents were filtered at 5 kHz and sampled at 2 Hz. Acquisition and analysis of data were accomplished using Axopatch 200B and pCLAMP8 software (Axon Instruments, Foster City, CA).

Solutions. Digestion solution contained collagenase (blend F; 0.9 U/ml; Sigma), elastase (type IV, 12.5 U/ml), and BSA (1 mg/ml) in Ca2+- and Mg2+-free Hanks' solution (pH 7.4).

For most of the recordings, we used standard Ringer solution containing the following (in mM): 130 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 20 HEPES, and 10 D-glucose (pH 7.4). Ca2+-free media was prepared by omitting CaCl2 and adding 1 mM EGTA. The pipette solution generally contained the following (in mM): 140 KCl, 1 MgCl2, 0.4 CaCl2, 20 HEPES, 1 EGTA, and 150 U/ml nystatin (pH 7.2).

For recordings of Ca2+ currents, on the other hand, we used Ringer solution with Ca2+ substituted by 5 mM Ba2+ and a modified pipette solution. When the perforated-patch configuration was employed, we used a pipette solution consisting of the following (in mM): 130 CsCl, 10 tetraethylammonium (TEA), 1 MgCl2, 20 HEPES, 5 EGTA, and 150 U/ml nystatin (pH 7.2). For whole cell recordings of Ca2+ current, this pipette solution was supplemented with 4 mM ATP (Na+ salt) and 0.3 mM GTP (Na+ salt).

Chemicals. All chemicals were obtained from Sigma Chemical. TEA, 4-aminopyridine, phenylephrine, and caffeine were prepared as aqueous stock solutions. Nifedipine and U-46619 were dissolved in absolute ethanol and then diluted with bathing medium; the final concentration of ethanol in the application pipette was 0.01%. Phenylephrine, caffeine, and U-46619 were applied by pressure ejection (Picospritzer II; General Valve, Fairfield, NJ) from micropipettes placed close to the cells, whereas ion channel blockers were applied directly via the bathing medium.

Statistics. Data are expressed as means ± SD. Statistical significance (P < 0.05) was determined using a two-tailed Student's t-test.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Outward K+ current. At resting membrane potentials, the canine bronchial arterial smooth muscle cells were quiescent, with an input resistance ranging from 1 to 10 GOmega and no spontaneous current activity whatsoever (Fig. 1A); none of the cells we studied (~100 cells from >25 animals) exhibited any spontaneous transient inward currents like those we have described previously in airway smooth muscle cells (8) or that others have found in vascular smooth muscle (21, 23). At more depolarized potentials, these cells generally exhibited spontaneous transient outward currents (STOCs; Fig. 1, B and C).


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Fig. 1.   Basal currents. A: at a holding potential (Vh) of -60 mV and in the absence of any exogenous agonist, canine bronchial smooth muscle cells exhibited no spontaneous currents of any kind. In two different cells held at 0 mV, however, the membrane current was quite "noisy" (B and C) and often accompanied by spike-like transient outward currents up to 100 pA in amplitude (C).

Stepwise depolarizing commands (20-mV increments) from a holding potential of -70 mV evoked large sustained outward currents often accompanied by STOCs (Fig. 1; n = 44). The average amplitude of the current at 70 mV was 49 ± 8 pA/pF (n = 44). This outward current exhibited little or no inactivation, decreasing <10% over the course of the depolarizing pulse.

Vascular smooth muscle cells generally exhibit two major types of K+ currents (Ca2+ activated and delayed rectifier) that can be distinguished on the basis of sensitivity to TEA, 4-aminopyridine, and charybdotoxin. We found that TEA significantly inhibited the sustained and transient outward currents, leaving only a small outward current devoid of any spike-like outward currents: currents at +70 mV were reduced to 50 ± 14 and 84 ± 3% of control in the presence of 1 mM TEA (6 cells; n = 5) or 10 mM TEA (7 cells; n = 6; Fig. 2A), respectively. These inhibitory effects of TEA were reversible upon washout of TEA. Likewise, charybdotoxin (10-6 M) also significantly inhibited the sustained outward current (67 ± 13% of control at +70 mV; 5 cells, n = 4) and abolished the large spike-like outward currents in a reversible fashion (Fig. 2C). 4-Aminopyridine, on the other hand, had no significant effect on the currents at bath concentrations of 1 mM (7 cells; n = 6) nor 5 mM (6 cells; n = 5; Fig. 2B).


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Fig. 2.   Pharmacological sensitivity of outward currents. Step depolarizations (20-mV increments) from a holding potential of -70 mV evoked large sustained outward currents upon which spike-like transient outward currents were often superimposed (see A, B, and C, left). The sustained and transient outward currents were markedly suppressed by tetraethylammonium (TEA; A) or by charybdotoxin (B) but not by 4-aminopyridine (C). Left and middle, representative examples of effects; right, mean effects of TEA (n = 6), 4-aminopyridine (4-AP; n = 5), and charybdotoxin (ChTX; n = 4).

These data suggest that a substantial portion of the outward current evoked by depolarizing pulses is a Ca2+-dependent K+ current, with little or no contribution from voltage-dependent delayed-rectifier K+ currents.

Role of Ca2+ influx in K+ current activation. Given that the outward currents triggered by membrane-depolarizing pulses are predominantly Ca2+ dependent, we next investigated the contribution of external Ca2+ to their activation.

The magnitudes of the outward K+ currents were significantly reduced, but not abolished, when the external bathing medium was replaced with a Ca2+-free buffer and were restored to control levels by reintroduction of external Ca2+ (Fig. 3A). On the other hand, their magnitudes were markedly increased when external Ca2+ concentration was increased to 4 mM (Fig. 3A). On average, the magnitude of the current evoked by a depolarizing pulse to +70 mV was inhibited 61 ± 8% in Ca2+-free media (n = 6; P < 0.05) and augmented slightly (but not significantly) to 116 ± 68% of control in 4 mM Ca2+-containing buffer (7 cells from 6 dogs; Fig. 3C).


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Fig. 3.   Role of external Ca2+ in outward K+ currents. A: K+ currents evoked using step depolarizations (see legend for Fig. 2) were markedly reduced in size when external Ca2+ was omitted, markedly increased in size when Ca2+ concentration ([Ca2+]) was increased to 4 mM, and then restored to normal amplitudes when [Ca2+] was returned to 1 mM. B: application of nifedipine (10-6 M) during step depolarizations also markedly decreased the amplitudes of the outward currents in a reversible fashion. Mean amplitudes of K+ currents recorded under these conditions are given in C (n = 4-6). [Ca2+]ext, external [Ca2+].

These data clearly indicate that external Ca2+ can markedly influence the magnitude of the K+ currents evoked by depolarizing pulses. We therefore investigated the effect of nifedipine, a selective blocker of voltage-dependent Ca2+ channels, on these outward K+ currents. Nifedipine (10-6 M) also markedly reduced K+ currents in a reversible fashion (Fig. 3B); on average, K+ currents evoked by step depolarizations to +70 mV were inhibited by 72 ± 4% (n = 4; P < 0.05; Fig. 3D).

Voltage-dependent Ca2+ current. The data presented above provide indirect evidence for voltage-dependent Ca2+ channels in these cells. We examined the Ca2+ currents directly by replacing K+ in the pipette solution with Cs+ to block K+ currents (TEA was also present in the pipette solution, for the same purpose) and by replacing Ca2+ in the bathing medium with Ba2+ (5 mM) to augment the inward current.

Depolarizing step commands (10-mV increments) from a holding potential of -70 mV evoked transient inward currents (Fig. 4). Activation of these currents was time dependent (peak activation occurring within 24 ± 13 ms; n = 13) and voltage dependent (threshold approximately equal to -30 mV, half-maximal at -3 ± 7 mV, and maximal at +20 mV; Fig. 4). The currents also inactivated in a fashion that was time dependent; mean tau  for inactivation at -10 mV was 206 ± 53 ms. Repolarization to the holding potential did not evoke slowly decaying tail currents reminiscent of Ca2+-dependent Cl- current (10) nor of T-type Ca2+ currents (6).


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Fig. 4.   Voltage-dependent Ca2+ currents. A: in a cell dialyzed with Cs+-containing pipette solution and held at -70 mV, depolarizing steps evoked inward currents that were reversibly abolished by nifedipine (10-6 M). B: mean peak amplitudes of these inward currents plotted against membrane voltage. C: same cell represented in A was held under voltage clamp for 5 s at conditioning potentials ranging from -100 to +30 mV, after which inward Ca2+ currents were evoked by a step to +10 mV. D: Boltzmann analysis of voltage dependence of activation () and inactivation () in one cell reveals incomplete voltage-dependent inactivation and substantial "window current" at voltages more positive than -30 mV. I/Imax, ratio of current to maximal current.

We used a different voltage protocol to examine the voltage dependence of inactivation as follows: cells were held under voltage clamp at potentials ranging from -100 to +30 mV (10-mV increments) for 4.9 s ("conditioning pulses"), followed by return to the holding potential (of -70 mV) for 54 ms and then a test pulse to +10 mV (1 s duration). A typical plot of the magnitude of the test pulses against voltage of the conditioning pulses is shown in Fig. 4D. Voltage-dependent inactivation was incomplete, and Boltzmann analysis of the data yielded a mean voltage causing 50% maximal inactivation of -26 ± 2 mV (n = 5).

Effects of caffeine on membrane currents. In addition to Ca2+ influx pathways, intracellular Ca2+ concentration ([Ca2+]i) can also be elevated by release of internally sequestered Ca2+ (2, 20). Millimolar concentrations of caffeine trigger release of internal Ca2+ by enhancing the opening of ryanodine receptors on the sarcoplasmic reticulum. This release is transient, however, so it is generally not possible to characterize the effects of caffeine on membrane currents evoked by a series of depolarizing step commands. Instead, we examined the effects of caffeine while holding the membrane potential constant at various levels. When the cells were held under voltage clamp at 0 mV, caffeine (10 mM) evoked a substantial outward current that was transient in nature, rising to a mean peak value of 688 ± 248 pA and then decaying to baseline over the course of 2-3 s even though caffeine continued to be applied (Fig. 5A). Caffeine also evoked contractions (data not shown). When the same cells were held at -60 mV, however, caffeine had no discernible effect on membrane current (Fig. 5B).


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Fig. 5.   Membrane currents evoked by caffeine. A: in a cell held under voltage clamp at 0 mV, caffeine (10 mM; 1 s application from puffer pipette) evoked large transient outward current that peaked within 2 s and decayed completely back to baseline. B: at a holding potential of -60 mV, however, this same cell showed no response to caffeine. C: in another cell, ramp depolarizations (from -70 to +50 mV) were used to explore the entire current-voltage relationship of the caffeine responses; although caffeine (10 mM) did cause cell contraction (data not shown) and substantial augmentation of outward current, it did not evoke any inward current whatsoever.

We also investigated the effects of caffeine using ramp depolarizing commands from a holding potential of -70 up to +50 mV at a rate of 120 mV/s. In this way, it is possible to capture the complete current-voltage relationship of the caffeine-evoked currents, although it must be understood that both [Ca2+]i (and thus the degree of current enhancement) and membrane potential are changing at the same time but at different rates and/or in different directions. Before application of caffeine, ramp depolarizations evoked only outward current with a similar current-voltage relationship as the K+ currents described above (compare Fig. 1 with Fig. 5C); no inward currents were seen in any cell held at negative membrane potentials (17 cells; n = 9). Upon application of caffeine (10 mM), the outward currents were increased 499 ± 40% (8 cells, n = 5; P < 0.01), as shown in Fig. 5C. However, there was still no indication whatsoever of any inward (i.e., Cl-) current in any of the cells tested (Fig. 5C).

Effect of phenylephrine and thromboxane mimetic on membrane currents. In general, the adrenergic innervation, through its actions on alpha -adrenoceptors, represents the primary excitatory neural input for the bronchial vasculature (25). Thromboxane A2 is also a potent spasmogen in many vascular beds (15). We therefore tested the effects of the alpha -adrenoceptor agonist phenylephrine and the thromboxane mimetic U-46619 on membrane currents using the same protocol described above for caffeine-evoked responses.

Although phenylephrine (10-4 M) did cause the cells to contract (data not shown), it did not evoke any inward current during maintained voltage clamp at -60 mV or when a range of voltages was tested using ramp depolarizations or incrementing step commands (n = 6; data not shown). Also, although adrenergic agonists cause release of internally sequestered Ca2+ in these cells (7), we did not observe a transient augmentation of K+ currents upon application of phenylephrine during step commands (Fig. 6A) or ramp depolarizations. To the contrary, we noted a delayed but prolonged suppression of K+ currents: for example, Fig. 6A shows the magnitude of K+ currents evoked in one cell using depolarizing step commands to +10 mV before and after 2 min of exposure to phenylephrine (10-4 M). On average, K+ currents were suppressed to 65 ± 13% of control by phenylephrine when bath temperature was 37°C (n = 4); at room temperature, however, phenylephrine had very little effect on the currents, reducing them to only 94 ± 8% (n = 4).


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Fig. 6.   Effects of phenylephrine (PE) on membrane currents. Step depolarizations to +10 mV (from a holding potential of -70 mV) delivered to a bronchial arterial smooth muscle cell at 10-s intervals evoked outward K+ currents. A: representative tracings. B: mean amplitude of currents in B. Application of PE (10-4 M in puffer pipette; 1 s application) did not evoke any inward current but did cause a slowly developing, long-lasting, and reversible suppression of the outward K+ currents (A and B).

U-46619 (10-6 M) also did not evoke any inward current whatsoever in any cells tested (e.g., Fig. 7A) but did cause a marked enhancement of K+ currents in many but not all of the cells (5 of 11 cells studied, lasting 10-20 s), followed by a marked suppression of the same (e.g., Fig. 7, A and B). On average, K+ currents were reduced to 63 ± 11% (n = 4); this electrophysiological response was accompanied by contraction of the cells (data not shown). We did not compare the effect of temperature on these responses to U-46619.


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Fig. 7.   Effects of U-46619 on membrane currents. A: representative tracing showing repeated ramp depolarizations (from -70 to +50 mV) and outwardly rectifying K+ currents accompanied by spontaneous transient outward currents that they evoked. B: from data shown in A, certain consecutive membrane current responses were averaged (indicated by open boxes in A labeled a-c), decimated (i.e., only every 10th sample taken), and then superimposed. U-46619 (10-6 M in application pipette; 1-s applications), applied two times at different distances from the cell, caused these currents to be first augmented (b) and then suppressed (c) compared with control (a).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The bronchial circulation plays an important role in many aspects of airway physiology and pathophysiology. Thus a thorough understanding of the mechanisms underlying excitation-contraction coupling in this vascular bed would be highly clinically valuable. In this study, we provide the first electrophysiological description of bronchial arterial smooth muscle cells.

K+ currents. We found the K+ current evoked by depolarizing voltage commands in the bronchial artery to be predominantly Ca2+ dependent in nature, since it was 1) markedly and reversibly inhibited by TEA or charybdotoxin but not by 4-aminopyridine; 2) reduced substantially by interfering with Ca2+ influx using nifedipine or by removing external Ca2+ but increased by raising external Ca2+; and 3) enhanced by releasing internally sequestered Ca2+ using caffeine. Voltage-dependent K+ current, on the other hand, appears to make little or no contribution, since 4-aminopyridine had very little effect on the depolarization-evoked currents. In general, Ca2+-dependent K+ currents play an important role in the regulation of many vascular smooth muscles, as discussed in more detail below (13, 18, 19).

Ca2+ currents. The Ca2+ currents that we recorded in these cells exhibited many characteristics of "L-type" current, including: 1) threshold and peak activation at approximately -30 and +20 mV, respectively; 2) half-maximal inactivation at approximately -25 mV; 3) tau  for inactivation of ~200 ms; 4) absence of slowly decaying tail currents upon repolarization; and 5) sensitivity to dihydropyridines. Inactivation of these currents was incomplete, leaving substantial "window current" at all voltages more positive than -30 mV. This has tremendous physiological importance, since it allows for sustained influx of Ca2+ during agonist stimulation, which leads to membrane depolarization. Activation of these Ca2+ currents is sufficient to evoke contraction, as indicated by the substantial contractions that we have previously described in intact tissues exposed to KCl (7).

Cl- currents. Although many vascular smooth muscle cells exhibit Ca2+-dependent Cl- currents spontaneously and/or when internal [Ca2+]i is elevated by various stimuli (14, 18, 23), some appear not to (18). Interestingly, we found no evidence for inward current of any kind in these bronchial arterial smooth muscle cells, neither spontaneously nor in response to voltage-dependent Ca2+ influx (Fig. 4) or release of internally sequestered Ca2+ induced by caffeine (Fig. 5), phenylephrine (Fig. 6), or U-46619 (Fig. 7), even though these stimuli are sufficient to cause cell shortening and substantial augmentation of K+ current (except for phenylephrine). Thus we conclude that these tissues do not express functional Cl- channels.

Spasmogen-evoked changes in membrane currents. In most arterial preparations, agonist-evoked contractions involve activation of inward Cl- (14) and/or nonselective cation currents (11), leading to membrane depolarization and subsequent opening of voltage-dependent Ca2+ channels such as the ones described in this tissue. It is for this reason that Ca2+ channel blockers are so effective in the treatment of hypertension.

Electromechanical coupling is also sufficient to produce contractions in the bronchial artery, as indicated by the robust contractions evoked by high-millimolar concentrations of KCl (7). However, in the present study, we were unable to identify any spontaneous or agonist-stimulated inward currents in these cells, although they are present in other vascular beds (11, 14) and we have been able to demonstrate them in airway smooth muscle cells using identical experimental techniques (5, 8, 9). Another mechanism by which excitatory autacoids can evoke membrane depolarization in vascular smooth muscle (other than activation of an inward current) is suppression of outward current (19, 24). The resting membrane potential in the cells that we studied appears to be as low as -50 mV, as indicated by the current-voltage relationships in Fig. 2. It is clear, then, that there is a physiologically relevant K+ conductance active at rest, since only this type of conductance can hyperpolarize the membrane to this degree; the equilibrium potentials for all other ions are much more positive than this (those for Cl- and Mg2+ are both approx 0 mV, whereas those for Na+ and Ca2+ are both very positive). As such, any decrease in the membrane permeability to K+ will lead to depolarization; in preliminary experiments, we have found that TEA (5 mM) does evoke substantial contraction in intact tissues (data not shown). In fact, we did observe substantial suppression of outward K+ current after application of either phenylephrine or U-46619 (although the latter often briefly enhanced the K+ current). This suppression lasted several minutes, long after application of the agonists had ended. Moreover, adrenergic suppression was essentially abolished at room temperature, whereas the effect of U-46619 was not; this parallels our previous finding that adrenergic contractions were essentially abolished by cooling to room temperature, whereas those evoked by U-46619 were hardly affected (7). The mechanism underlying this suppression was not investigated in the present study. However, the inability of caffeine to suppress K+ currents suggests that this is not related to changes in Ca2+ concentration (since caffeine, phenylephrine, and U-46619 all release internal Ca2+). Instead, it likely involves G proteins (which are not normally activated by caffeine); these may act directly on the channels or stimulate downstream events such as activation of phospholipases and various kinases (20). The effects of caffeine on membrane current and mechanical activity are not secondary to inhibition of phosphodiesterase activity, since they were very transient, resolving to baseline within 20 s after application of caffeine; this time course mirrors that of caffeine-induced release of internally sequestered Ca2+.

Thus it appears that excitation-contraction coupling in bronchial smooth muscle involves membrane depolarization (via suppression of outward current) with voltage-dependent Ca2+-influx and nonelectromechanical coupling mechanisms (15). The latter mechanism may be more important than the former, particularly at lower temperatures (which are also physiologically relevant given the cooling of the airway during accelerated ventilation), since we have previously shown that excitatory mechanical responses in this tissue involve increased Ca2+ sensitivity of the contractile apparatus much more than elevation of [Ca2+]i per se (7).

In conclusion, canine bronchial artery smooth muscle cells exhibit Ca2+-dependent K+- and voltage-dependent (L-type) Ca2+ currents, with little or no evidence of voltage-dependent "delayed-rectifier" K+ currents or Cl- currents. The thromboxane mimetic U-46619 caused a transient increase of outward K+ currents, followed by marked suppression of the same that lasted several minutes. Adrenergic stimulation only produced the sustained suppression of K+ currents (not their enhancement), and only at a physiological temperature. Neither agonist activates inward (i.e., Cl-) current at all in this tissue. These electrophysiological data explain the mechanical effects produced by these agonists.


    ACKNOWLEDGEMENTS

These studies were supported by operating funds from the Canadian Institutes of Health Research and a Scientist Award from the Medical Research Council of Canada.


    FOOTNOTES

Address for reprint requests and other correspondence: L. J. Janssen, Dept. of Medicine, McMaster Univ., 50 Charlton Ave. E., Hamilton, Ontario, Canada L8N 4A6 (E-mail: janssenl{at}mcmaster.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published January 11, 2002;10.1152/ajplung.00421.2001

Received 30 October 2001; accepted in final form 10 January 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Lung Cell Mol Physiol 282(6):L1358-L1365
1040-0605/02 $5.00 Copyright © 2002 the American Physiological Society




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