V. A. SNETKOV* and J. P. T. WARD

Transcription

1 Experimental Physiology (1999), 84, Printed in Great Britain ION CURRENTS IN SMOOTH MUSCLE CELLS FROM HUMAN SMALL BRONCHIOLES: PRESENCE OF AN INWARD RECTIFIER K CURRENT AND THREE TYPES OF LARGE CONDUCTANCE K CHANNEL V. A. SNETKOV* and J. P. T. WARD Department of Respiratory Medicine and Allergy, Guy s, King s and St Thomas School of Medicine, King s College London, St Thomas Campus, London SE1 7EH, UK (MANUSCRIPT RECEIVED 2 MARCH 1999, ACCEPTED 23 JUNE 1999) summary Bronchoconstriction of small bronchioles plays a major role in the increase in airway resistance following agonist challenge. There is evidence that the airway smooth muscle (ASM) of small bronchioles differs functionally from that in larger airways. Little is known however about the electrophysiology of small bronchioles. Ion currents were therefore studied in airway smooth muscle cells freshly dissociated from human intralobular bronchioles, with a diameter between 0 3 and 1 0 mm. As previously reported for human large airways, the major outward current in these cells was due to activity of large conductance K (BK) channels, with a relatively minor component due to a voltage-gated delayed rectifier current (IDR), which was only observed in 30% of cells. Three distinct types of iberiotoxin- and TEA-sensitive large conductance K channel contributed to large conductance K current (IBK). These included a highly voltage- and Ca -sensitive 200pS channel previously reported in human large airways, and two smaller channels of 150 and 100 ps previously seen only in human fetal or cultured ASM. In contrast to large airways, ASM cells from bronchioles also demonstrated a voltage-gated inward rectifier current (IIR). IIR was activated by hyperpolarisation below the K equilibrium potential and could be blocked by submillimolar concentrations of Cs or Ba, and partially by physiological concentrations of Na. Corresponding single channels with a conductance of 17 ps could also be recorded in the cell-attached configuration. A small voltage-independent current was also observed which was resistant to classic K and Cl channel blockers but which could be abolished by replacement of Na with the impermeant cation N-methyl-ª_glucamine (NMDG ). Corresponding non-selective single channels of 20 ps could be seen in inside-out mode. These results demonstrate that ASM from small bronchioles differs in terms of ion currents and channels from ASM derived from large airways, with possible implications for function. introduction It has been predicted that much of the resistance to airflow seen during agonist challenge is due to contraction of small (< 2 mm i.d.) airways (Moreno et al. 1986). However there have been few in vitro studies on these functionally significant small bronchioles, particularly in man, and most previous reports have been on tissue derived from trachea or main bronchus. Even so, there are some studies that have reported significant pharmacological and mechanical differences between airways from different parts of the airway tree in a variety of species, including rabbit (Fleisch & Calkins, 1976; Fujiwara et al. 1988), preterm lambs (Gauthier et al. 1992), sheep (Mustafa et al. 1994), and rat (Chopra et al. 1994, 1997). This apparently parallels the well-known variation in function that occurs between arteries of different calibre and position within the vascular system (e.g. Leach et al. 1992; Archer, 1996). * Corresponding author: vladimir.snetkov@kcl.ac.uk 1887

2 836 V. A. SNETKOV AND J. P. T. WARD It is now well documented that smooth muscle cells derived from the walls of conduit and resistance arteries have different electrophysiological characteristics (Nelson & Quayle, 1995; Archer, 1996). However, virtually nothing is known about the electrophysiology of smooth muscle cells from different parts of the airway tree, and there is also a relative paucity of data for human airway smooth muscle (ASM) of any type. This has potentially important implications, as we have previously found that ASM from human large airways differs significantly in terms of its electrophysiological characteristics from that of several other species commonly used for the study of ASM function (Snetkov et al. 1995). The aim of this study was therefore to examine the major ion currents in ASM derived from human small intralobular bronchioles (< 1 mm i.d.). We found that these bronchioles differ significantly from larger airways, in that they exhibit an inward rectifier K current, and show the presence of two additional types of large conductance K channel that are not normally found in human adult large airways, but which have been described in human fetal ASM and human cultured ASM (Snetkov et al. 1996, 1998). methods Freshly dissociated cells Healthy lung tissue was obtained from 11 patients following lobectomy. None of the patients had asthma. The study was approved by the Ethics Committee of the West Lambeth Health Authority. Intralobular bronchioles with internal diameter mm were carefully dissected from tissue specimens within 2 h of surgery. Epithelium-denuded pieces were incubated for 20 min at 36 C in 2 ml Ca -free physiological salt solution (PSS; see below) containing 1 mg ml collagenase (Sigma type XI), 0 5 mg ml papain, 2 mg ml bovine serum albumin and 2 mò dithiothreitol. The tissue was triturated gently with a flame-polished Pasteur pipette, and cells were spun down and resuspended in 0 2 ml of fresh Ca -free PSS. Five to ten minutes before use a drop of the cell suspension was placed on a cleaned glass coverslip. This procedure provided a sufficient number of well-attached spindle-shaped smooth muscle cells. Recording and analysis Single cells were studied with standard patch-clamp techniques using an Axopatch_1C amplifier (Axon Instruments, Inc., Foster City, CA, USA) The experiments were carried out at room temperature. The bath ( 0 5 ml) was continuously superfused (1 2 ml min ) with external PSS. Whole-cell and single channel currents were filtered at 5 or 1 khz respectively ( 3 db, low-pass 80 db decade, Bessel-type built-in filter) and digitised with a 20 or 5 khz sampling frequency using a CED 1401 interface and PATCH v.6 software (Cambridge Electronic Design, Cambridge, UK). Current voltage relationships were obtained using a voltage ramp protocol. The holding potential was maintained at either 60 mv (normal PSS) or 0 mv (high K PSS), and a 1 s ramp from 100 to +100 mv was applied every 10 s. Cell capacitance was measured using the amplifier s built-in circuit. Solutions and reagents The solution routinely used as intracellular solution in whole-cell recording, and both as extra- and intracellular solution in the inside-out patch configuration contained (mò): 140 KCl, 2 MgClµ, 5 ethylene glycol-bis-(â-aminoethyl ether)-n,n,n',n'-tetraacetic acid (EGTA), 10 N-2-hydroxyethyl-piperazine-N'- 2-ethanesulphonic acid (Hepes), adjusted to ph 7 2 with KOH. The PSS used as the bath solution in whole-cell recordings contained (mò): 135 NaCl, 5 KCl, 2 MgClµ, 2 CaClµ, 11 glucose, 10 Hepes, adjusted to ph 7 4 with NaOH. Ca -free PSS used for tissue dispersion had the same composition except that CaClµ was omitted and 0 1 mò EGTA added. In experiments where various concentrations of K were used, NaCl was replaced by KCl on an equimolar basis. Charybdotoxin (ChTX) and iberiotoxin (IbTX) were obtained from Peninsula Laboratories Europe (Merseyside, UK). All other reagents were purchased from Sigma Chemical Company (Poole, UK). Data are presented as means ± s.e.m. unless otherwise stated; n refers to the number of cells or patches examined.

3 ION CHANNELS IN HUMAN BRONCHIOLES 837 Fig. 1. A, representative large TEA- and ChTX-sensitive currents observed in the majority of cells isolated from human small bronchioles. Control, current observed in normal PSS in the absence of TEA and ChTX. B, 4_AP-sensitive component (delayed rectifier) present in some cells. results The isolation protocol used yielded a number of phase-contrast dense cells that varied in terms of shape and size. All experiments were performed on spindle-shaped smooth muscle cells. The cells were significantly smaller as reflected by cell capacitance (7 8 ± 1 3 pf, n = 69), than those previously reported for human main bronchus (16 1 ± 0 4 pf, n = 41; P<0 01; Snetkov et al. 1998). Three distinct types of K current could be discriminated using the whole-cell configuration in smooth muscle cells derived from human small bronchioles, though not every type was present in all cells. However, the relative distribution was not apparently different between patients. In the large majority of cells (62 of 69) depolarisation in whole-cell configuration with <1 nò Ca in the pipette induced a large outward current that was noisy, did not have a welldefined activation threshold and exhibited no noticeable inactivation. The outward current was substantially reduced by tetraethylammonium (TEA, 2 mò; Fig. 1A), ChTX (100nÒ; Fig. 1A) or IbTX (50 nò; not shown), suggesting that it was due to activation of large conductance Ca -activated K (BK) channels. Corresponding large conductance single channels ( 200 ps) could be recorded in whole-cell mode even at 60 mv and more positive potentials. In contrast, 4_aminopyridine (4_AP, 2 mò) only caused significant inhibition of the depolarisation-induced outward current in 22 of 69 cells (Fig. 1 B), and even in these cells the 4_AP-sensitive component was relatively small, with only five cells showing a current >100 pa at 0 mv. The 4_APsensitive current activated upon depolarisation toward 30 mv and more positive, inactivated substantially during prolonged depolarisation, and was hence identified as a delayed rectifier (IDR). In cell-attached and inside-out membrane patches large conductance K channels were observed, with the majority of patches exhibiting multiple openings, indicating a high channel density. The channel characteristics were examined in more detail in 15 inside-out patches containing only a few channels each, so as to allow more accurate determination of conductance. Single channel recordings revealed the presence of three types of large conductance K channel,

4 838 V. A. SNETKOV AND J. P. T. WARD Fig. 2. A, two types of IbTX-sensitive large conductance currents recorded in an outside-out patch. Pipette solution contained 140 mò KCl, 0 Ca, 5 mò EGTA and 100 ìò GTP. Bath solution contained 135 mò NaCl and 5 mò KCl. Vm = 20 mv. B, voltage dependence of open state probability (Npï) for channels with different conductances (1, 189 ps; 0, 156 ps; 8, 112 ps). Inside-out configuration, symmetrical 140 mò KCl, intracellular solution containing 0 Ca and 5 mò EGTA. Fig. 3. Inwardly rectifying whole-cell currents induced by 50 ms hyperpolarising voltage pulses, 140 mò K. A, external solution containing 135 mò Na and 5 mò K ; steps from a holding potential of 60 mv to 40, 80, 100, 120, 140, 160 and 180 mv. B, external solution containing 0 mò Na and 140 mò K ; steps from a holding potential of 0 mv to +20, 20, 40, 60, 80 and 100 mv. all of which were blocked by IbTX (50 nò; Fig. 2 A) or TEA(1 mò; not shown). Of 15 patches, six contained channels with a conductance of 189±8pS (n = 6) in symmetrical [KCl] (140 mò), which were active at negative potentials (< 50 mv and more positive) even with a very low cytosolic [Ca ] (< 1 nò). These channels were identical to those that we have previously described in ASM cells freshly dissociated from human large bronchi (Snetkov et al. 1995). Two other large conductance K channel types were also observed. Six of 15 patches contained channels with a conductance of 156 ± 10 ps (n = 6), which were active only at a membrane potential (Vm) of +50 mv or more positive with low cytosolic [Ca ], and

5 ION CHANNELS IN HUMAN BRONCHIOLES 839 Fig. 4. Whole-cell current voltage relationships obtained using 0 5 s voltage ramps from 100 to +100 mv in solutions with different external K concentrations (5 140mÒ). Na was replaced by equimolar K. Intracellular [K ] was 140 mò. four patches contained channels of 112 ± 16 ps (n =4)(Fig. 2 B). The latter had no apparent voltage dependence, and ran down rapidly after patch excision; they usually disappeared completely after 3 5 min in inside-out configuration. GTP (100 ìò) or GTPãS (10 ìò) applied from the cytosolic side restored the activity of the channels without affecting either the 189 or 156 ps channels. Following irreversible activation by GTPãS the 112 ps channels could be recorded with a high open state probability (Pï) at any potential from 80 to +80 mv.in contrast, both the 189 and 156 ps channels increased open state probability with depolarisation andïor an increase in [Ca ]é. Individual patches with multiple channels could exhibit different combinations of the three channels. The 112 and 156 ps channels were identical to those of similar conductance previously identified in cultured growing human adult ASM, and freshly dissociated human fetal ASM (Snetkov et al. 1996, 1998). In addition to IBK and IDR, 24 of 69 cells exhibited an inward rectifier current (IIR) following hyperpolarisation towards potentials negative to the K equilibrium potential. When normal PSS was used as a bath solution, this hyperpolarisation-induced inward current was noisy and inactivated substantially over a 50 ms pulse (Fig. 3 A); no measurable decline was observed when extracellular Na was replaced with K (Fig. 3 B). During gradual replacement of Na with K the reversal potential of IIR followed the calculated K equilibrium potential (Fig. 4). Extracellular Cs or Ba blocked IIR in a concentration- and voltage-dependent manner, the Cs block being notably faster (Fig. 5). The EC50 for Cs block at equilibrium was 57±3ìÒ at 100 mv (n = 3). Ba caused the current to decay, the rate of decay increasing with Ba concentration (Fig. 5 B). The channel blocking rate constant for Ba, calculated from the slope of the relationship between the time constant for this decay and Ba concentration was (2 3 ± 0 3) ² 10Ç Ò s at 100 mv (n = 3). The current response to voltage ramps demonstrated strong voltage dependence of cation block (Fig. 6). In cell-attached configuration

6 840 V. A. SNETKOV AND J. P. T. WARD Fig. 5. Block of IIR with varying concentrations of Cs (A) and Ba (B). Currents were induced by 50 ms hyperpolarising pulses from a holding potential of 0 to 100 mv. Internal and external [K ] were both 140 mò. Fig. 6. Voltage dependence of cation block of IIR: A, 500 ìò Cs ; B, 500 ìò Ba. Currents were induced by 0 5 s voltage ramps from 100 to +100 mv. Internal and external K were both 140 mò. inwardly rectifying single channels could be recorded with a conductance of 17 ± 4 ps(n = 5). In most cases the membrane patch contained only one channel with high open state probability (Fig. 7). In the large majority of cells we also found a small linear current which was resistant to K channel blockers (TEA, ChTX, IbTX, 4_AP and glibenclamide) and the Cl channel blockers niflumic acid (100 ìò), SITS (100 ìò), and DIDS (100 ìò). The current was most easily observed in seven of 69 cells with a small (< 100 pa) outward current, but could be uncovered in other cells by blocking IBK and IDR with TEA or IbTX and 4_AP. The current could be abolished by substitution of extracellular Na with the non-permeable cation N-methyl- ª_glucamine (NMDG ). Corresponding ion channels with a conductance of 20±5pS(n =7) were observed in excised inside-out patches, and could also be seen in some cases in cell attached and whole-cell configurations (Fig. 8). These channels were active at a wide range of

7 ION CHANNELS IN HUMAN BRONCHIOLES 841 Fig. 7. Inward rectifier single channel activity in a cell-attached patch. Both pipette and bath solution contained 140 mò K. A, channel activity at membrane potentials from 0 to 160 mv; c denotes the closed state and o the open state. B, corresponding current voltage relationship, showing a slope conductance of 18 ps. Fig. 8. A, whole-cell current response to a voltage ramp from 100 to +100mV, physiological ion gradient. A small linear TEA-resistant current could be seen at negative membrane potentials. B, TEA-resistant single channel activity at 60 mv membrane potential, recorded in whole-cell configuration from the same cell. Activity was abolished by complete replacement of Na by NMDG. holding potentials, resistant to K and Cl channel blockers, and had a reversal potential close to 0 mv with both a quasi-physiological ion gradient (5 mò KºÏ140 mò K ) and symmetrical 140 mò K. This activity disappeared reversibly upon the replacement of extracellular Na in physiological solution with NMDG (Fig. 8 B). The results suggest that these channels were relatively non-selective between K and Na ions (i.e. non-selective cation channels). Although of similar conductance, these non-selective cation channels could readily be distinguished

8 842 V. A. SNETKOV AND J. P. T. WARD Fig. 9. Whole-cell current responses to a voltage ramp from 100 to +100 mv in an ASM cell isolated from a bronchiole of less than 200 ìm i.d., with 5 mò (top trace) and 140 mò K (bottom trace) in the external solution. IIR is apparent at negative potentials, and single, large conductance channel openings at very positive potentials. from inwardly rectifying channels by their specific characteristics, including lack of rectification, different equilibrium potential, and much shorter open time (see Figs 7A and 8B). For comparison, some experiments were also performed on smooth muscle cells derived from smaller bronchioles than those used in the main part of the study. Cells from the smallest bronchioles technically available ( ìm i.d.) exhibited IIR, but only a few large conductance K currents per cell (Fig. 9), and therefore demonstrated a small outward current (n = 12). There was no discernable IDR in any of these cells, although non-selective cation channels were observed in whole-cell configuration. discussion We have described for the first time the electrophysiology of smooth muscle cells derived from human small bronchioles (< 1 mm i.d.), and have shown that there are notable differences in the ion currents and associated single channels compared with ASM cells from main bronchus. It is now generally accepted that bronchoconstriction of such small bronchioles plays an important role in the increase in airway resistance that is elicited by agonists (Moreno et al. 1986), and by inference also in asthma. However, although some animal studies have suggested that there may be significant differences in the pharmacological and mechanical function of airways from different parts of the bronchial tree (Fleisch & Calkins, 1976; Fujiwara et al. 1988; Gauthier et al. 1992; Mustafa et al. 1994; Chopra et al. 1994, 1997), there have been very few in vitro studies of any type on human small bronchioles. Moreover data obtained from non-human species may not necessarily reflect the situation in human ASM, as we have previously described significant differences between the electrophysiology of ASM from human airways and that of other species (Snetkov et al. 1995, 1996, 1998). ASM cells derived from human small bronchioles were significantly smaller than those derived from main bronchus or trachea, and had approximately half the capacitance ( 8 vs. 16 pf), although they were larger than cells derived from human fetal ASM ( 4 pf; Snetkov et al. 1998). There was also a greater degree of heterogeneity in size. In common with our previous studies on freshly dissociated ASM cells from human adult (Snetkov et al. 1995) and fetal airways (Snetkov et al. 1998), and cultured human ASM from main bronchus (Snetkov et al. 1996), cells from small bronchioles exhibited an outward current on depolarisation that was largely due to activation of BK channels, with only a small proportion of cells exhibiting any significant IDR. However, a significantly greater proportion of these cells exhibited a measurable IDR compared with cells from fresh or cultured main bronchus (bronchioles: 32%; bronchus:

9 ION CHANNELS IN HUMAN BRONCHIOLES %, from Snetkov et al. 1995; cultured: 14%, from Snetkov et al. 1996), though the contribution of IDR to total outward current remained small. We have previously demonstrated that IBK is a main determinant of resting Vm in human ASM, with little contribution from IDR (Snetkov et al. 1995). This is in direct contrast to studies on most other species, where IDR is the major component of outward current, and plays the major role in regulation of resting Vm (Boyle et al. 1992; Kotlikoff, 1993; Fleischmann et al. 1993). Large conductance K channels Three distinct types of large conductance K channel were observed in ASM cells from small bronchioles, with conductances of 200, 150 and 100 ps. Although all were sensitive to TEA, ChTX and IbTX, they could be distinguished clearly in terms of unitary conductance, voltage and Ca sensitivity, and were indistinguishable from the three channel types of similar conductances that we have previously described in human fetal and human ASM cultured from large airways (Snetkov et al. 1996, 1998). The 200 ps channel had a high voltage sensitivity, and showed activity at negative potentials (< 50 mv) even with a low intracellular [Ca ] (< 1 nò). The 150 ps channel was also stimulated by increasing [Ca ], but activated at much more positive potentials, whereas the 100 ps channel showed no apparent voltage sensitivity, and ran down rapidly on patch excision in the absence of GTP (see also Snetkov et al. 1996). These results contrast sharply with those from ASM cells freshly isolated from adult large bronchi and trachealis, where only the 200 ps channel was observed in significant numbers (Snetkov et al. 1996, 1998). It is interesting to note that with the exception of ASM from large airways, the relative proportions of the three channel types were approximately the same in small bronchioles, fetal ASM and cultured ASM (present study; Snetkov et al. 1996, 1998). Large conductance K channels with conductances around 100 and 200 ps have also been reported to coexist in smooth muscle cells isolated from pig trachea (Saunders & Farley, 1991) and canine renal artery (Gelband & Hume, 1992), and two channels with similar conductances but different Ca and voltage sensitivities have been demonstrated in bovine mesenteric artery (Sansom & Stockand, 1994). It is very unlikely that these variants of the large conductance K channel are derived from different genes, and it has been suggested that their high functional variability is due to alternatively spliced variants (Lagrutta et al. 1994; Pallanck & Ganetzky, 1994). It is interesting to note that the 200 and 150 ps channels that we have described here and previously in human ASM show many common features with the A1 (200 ps) and A3 (150 ps) variants of the Slowpoke channel (Lagrutta et al. 1994). We have previously suggested that the presence of the 150 and 100 ps channels may be related to a more proliferative or secretory ASM phenotype (Snetkov et al. 1998), and expression of large conductance K channels has been linked to the cell cycle (Day et al. 1993). We can speculate that the small bronchioles may have a higher cell turnover and thus rate of proliferation than main bronchus, though this needs to be investigated using markers for cell growth and proliferation. Be that as it may, our results clearly show that there are significant differences between large and small airways in terms of the K channels that are responsible for regulating the resting Vm of ASM, which could have implications for excitability both during rest, and following a rise in intracellular [Ca ] following agonist stimulation. Non-selective cation channels The large majority of ASM cells from small bronchioles exhibited a small linear current that was voltage insensitive and unaffected by classical K or Cl channel blockers. This was not a

10 844 V. A. SNETKOV AND J. P. T. WARD leak current, as it could be abolished by substitution of Na with the impermeant cation NMDG, suggesting that it was carried by non-selective cation channels. Corresponding single channels of 20 ps conductance could also be recorded, and these relatively non-selective cation channels were also unaffected by K channel blockers, but were again abolished by replacement of extracellular Na with NMDG. These channels seem to be present in all parts of the airway tree, and to be conserved independent of contractile or proliferative phenotype, as we have observed a similar current in human fetal and adult bronchus ASM, and ASM growing in culture (Snetkov et al. 1998, and authors unpublished observations). Non-selective cation channels have been implicated as a putative Ca -influx pathway in the response to muscarinic agonists in tracheal myocytes of dog, guinea-pig and horse (Janssen & Sims, 1992; Fleischmann et al. 1997), and in preliminary experiments we have found that the activity of these nonselective cation channels is increased in cells from human small bronchioles during stimulation with methacholine or leukotriene DÚ (authors unpublished observations). Inward rectifier K channels About 35% of ASM cells isolated from small bronchioles expressed an inwardly rectifying K current. This current could be activated by hyperpolarisation, had a reversal potential close to the K equilibrium potential, and was sensitive to the blocking action of extracellular Cs, Ba and, to a lesser extent, Na. The corresponding single channels had a conductance of 17 ps. This current therefore shared the most typical features of IIR found elsewhere (reviewed in Quayle et al. 1997). It also had a current density not dissimilar to that reported for cerebral arteries, with the same Ba blocking rate at 100 mv ( 2 ² 10Ç Ò s ; Quayle et al. 1993). The density of channels was, however, lower than that in endothelial cells which typically contain many inward rectifier channels per membrane patch (Hoyer et al. 1991). The conductance of 17 ps is within the range of typical values for inward rectifier channels, and similar to that shown in endothelial cells (e.g. Takeda et al. 1987; Hoyer et al. 1991). IDR was initially described in skeletal muscle and cardiac cells, and was later found in tunicate eggs and neurons (reviewed in Hille, 1992), as well as in cultured (Takeda et al. 1987) and freshly dissociated (Hoyer et al. 1991) endothelial cells. IIR is not regarded as a current typical for smooth muscle cells, and has so far only been described in smooth muscle from small arteries and arterioles in the cerebral and coronary circulations (Quayle et al. 1993, 1997; Johnson et al. 1998), although a low density of IIR has recently been reported in small renal arteries (Prior et al. 1998). IIR has not previously been described in ASM except in cultured cells (Snetkov et al. 1996), and was not present in freshly isolated ASM from human large airways (Snetkov et al. 1995). There is therefore a distinct parallel between the airways and vascular system, in that IIR can apparently only be observed in small bronchioles, arteries and arterioles, but not in larger conduit airways or arteries. The possible function of IIR in bronchioles is unclear. It has been suggested that it may play a major role in the regulation or setting of resting Vm in arterioles and small arteries, and be involved in the vasodilatation seen in some small arteries in response to a small rise in [K ] (Quayle et al. 1997). This is consistent with a recent report that IIR is important for both setting basal tone and K -induced vasodilatation in pressurised cerebral arteries of the rat (Johnson et al. 1998). However increases in external [K ] do not cause relaxation of human small bronchioles (authors unpublished observations), and a potential physiological role for IIR in bronchioles is further undermined by the apparent heterogeneity of ASM cells from this tissue, in that only 35% seemed to express significant IIR. It is still possible, however, that

11 ION CHANNELS IN HUMAN BRONCHIOLES 845 IIR does contribute to the maintenance of resting Vm in small bronchioles, but further studies are required before the actual function of IIR in human bronchioles, if any, can be ascertained. The data obtained from the smallest bronchioles examined was in many ways similar to a subset of cells derived from human fetal ASM, with low total outward current, only very few large conductance channels per cell, absent or very small IDR, but conserved non-selective cation channels (Snetkov et al. 1998). It is possible either that these cells are of a phenotype more strongly biased towards secretion or proliferation, or that they are not true smooth muscle cells at all. It is also not clear whether bronchioles of this size (< 300 ìm i.d.) are able to constrict to any significant amount. Nevertheless, these results do emphasise the relationship between airway calibre and changes in function. In summary, we have found that human ASM cells from small bronchioles differ significantly in terms of ion currents and channel distribution compared with those from large airways. The major differences include the presence of IIK in bronchioles, and a more fetal or proliferative distribution of large conductance K channel types. The physiological effects of these differences are currently unclear, although they are bound to have an impact to some extent on ASM excitability and function. Although small bronchioles may be of prime importance in the response of airway resistance to agonist challenge, the large majority of studies on ASM function and response to drugs is currently performed on large airway preparations from nonhuman species. In view of our present results and those from the small number of other reports that have related changes in airway function to calibre, translation of data from large airways to the clinical environment should be treated with some caution. Supported by the Wellcome Trust, grant number (V.A.S). references Archer, S. L. (1996). Diversity of phenotype and function of vascular smooth muscle cells. Journal of Laboratory and Clinical Medicine 127, Boyle, J. P., Tomasic, M. & Kotlikoff, M. I. (1992). Delayed rectifier potassium channels in canine and porcine airway smooth muscle cells. Journal of Physiology 447, Chopra, L. C., Hucks, D., Twort, C. H. & Ward, J. P. (1997). Effects of protein tyrosine kinase inhibitors on contractility of isolated bronchioles of the rat. American Journal of Respiratory Cell and Molecular Biology 16, Chopra, L. C., Twort, C. H. C. & Ward, J. P. T. (1994). Differences in sensitivity to the specific protein kinase C inhibitor Ro between small and large bronchioles of the rat. British Journal of Pharmacology 113, Day, M. L., Pickering, S. J., Johnson, M. H. & Cook, D. I. (1993). Cell-cycle control of a largeconductance K channel in mouse early embryos. Nature 365, Fleisch, J. H. & Calkins, P. J. (1976). Comparison of drug-induced responses of rabbit trachea and bronchus. Journal of Applied Physiology 41, Fleischmann, B. K., Wang, Y. X. & Kotlikoff, M. I. (1997). Muscarinic activation and calcium permeation of non-selective cation currents in airway myocytes. American Journal of Physiology 272, C Fleischmann, B. K., Washabau, R. J. & Kotlikoff, M. I. (1993). Control of resting membrane potential by delayed rectifier potassium currents in ferret airway smooth muscle cells. Journal of Physiology 469, Fujiwara, T., Itoh, T. & Kuriyama, H. (1988). Regional differences in the mechanical properties of rabbit airway smooth muscle. British Journal of Pharmacology 94, Gauthier, S. P., Wolfson, M. R., Deoras, K. R. & Shaffer, T. H. (1992). Structure-function of airway generations 0 to 4 in the preterm lambs. Pediatric Research 31,

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