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Journal of Physiology (2001), 536.3, pp. 693-701
© Copyright 2001 The Physiological Society
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ABSTRACT |
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INTRODUCTION |
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It is well accepted that fluid reabsorption from the term lung at birth is inhibited by luminal amiloride (Olver et al. 1986; O'Brodovich et al. 1990), suggesting that amiloride-sensitive Na+ channels may be rate-limiting in the process. The observation that transgenic mice which lack the epithelial sodium channel 
-subunit, ENaC, have fatally impaired lung fluid clearance (Hummler et al. 1996) further supports an important role for amiloride-sensitive pathways in the perinatal period. However, in the mature lung, amiloride-insensitive fluid reabsorption accounts for up to 70 % of the total clearance, with some variation among species (Ramsden et al. 1992; Norlin et al. 1998). Even though numerous studies have also reported large amiloride-insensitive whole-cell Na+ currents in adult alveolar epithelial type II cells (e.g. Haskell et al. 1994; Kemp et al. 1997), none has characterised this component and there still appears to be common consent that ENaC activity primarily underlies adult lung fluid homeostasis. That the cellular and in vivo/in situ data both support a role for amiloride-insensitive vectorial Na+ transport highlights the importance of investigating the nature of this component before the precise mechanism by which postnatal fluid homeostasis is maintained can be fully understood.
Recently, compelling direct evidence has been presented on the nature of this significant amiloride-insensitive component to adult lung fluid reabsorption. Junor et al. (1999), employing the well-characterised postnatal sheep in situ lung preparation, have convincingly demonstrated that a substantial component of adult lung fluid reabsorption is insensitive to the blocking effects of maximal concentrations of amiloride and that the remaining absorptive response is inhibited by either dichlorobenzamil or pimozide. Both of these latter drugs are relatively selective inhibitors of the cyclic nucleotide-gated non-selective cation channel. Interestingly, stimulated fetal sheep fluid reabsorption is unaffected by similar pharmacological manipulation (Junor et al. 2000), indicating that expression of this channel in the pulmonary epithelium may be developmentally regulated.
Cyclic nucleotide-gated cation channels were first described by Fesenko et al. (1985), and were subsequently cloned from retinal rods (Kaupp et al. 1989). The functional channel is heteromeric with a smaller six transmembrane domain -subunit representing the conductance (Bonigk et al. 1993) and a larger modulatory 
-subunit (Korschen et al. 1995). Since the initial molecular characterisation of the bovine rod photoreceptor channel (CNG1), a number of homologues of -subunits have been cloned by homology screening and PCR, including rat olfactory receptor CNG2 (Dhallan et al. 1990) and bovine cone photoreceptor CNG3 (Biel et al. 1994). Cloned -subunits include CNG4 (Biel et al. 1996) from cattle, CNG5 (Bradley et al. 1994) from rat and CNG6 from mouse (Gerstner et al. 2000). It is now known that CNG genes are widely expressed in a tissue-specific manner. Their mRNAs have been localised in both sensory and non-sensory tissues, including lung in general (Ding et al. 1997) and airway specifically (Schwiebert et al. 1997), while mRNA for all three -subunits (CNG1-3) and CNG are expressed in the pulmonary epithelial cell line A549 (Xu et al. 1999).
Expressed cyclic nucleotide-gated channels have a number of pharmacological and biophysical characteristics that distinguish them from ENaC or other amiloride-sensitive Na+ channels. As employed in the postnatal lung reabsorption study, they are selectively blocked by the dopamine D2 receptor antagonist pimozide (Junor et al. 1999). This is an important pharmacological tool since adult rat alveolar Na+ transport is not affected by another D2 receptor antagonist, S-sulpiride (Barnard et al. 1999), suggesting that pimozide action in lung is due to binding to cyclic nucleotide-gated cation channels. Characteristically, these channels are activated by micromolar concentrations of cGMP, or its membrane-permeable analogue 8Br-cGMP (Fesenko et al. 1985), are inhibited by di- and trivalent cations (Karpen et al. 1993), and show little or no preference for Na+ over K+ as the permeating ion, with GK often very slightly higher than GNa (Picones & Korenbrot, 1992).
In the present paper, we have employed the patch-clamp technique to study amiloride-insensitive whole-cell cation currents and their regulation by cGMP, inorganic cations and pimozide in order to address the hypothesis that adult alveolar epithelial type II cells functionally express cyclic nucleotide-gated cation channels. Some of this work has appeared previously in abstract form (Kemp et al. 1998).
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METHODS |
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Solutions
All chemicals were of the highest grade available and were purchased from Sigma Chemical Co. (St Louis, MO, USA), unless otherwise stated. Solution I contained (mM): NaCl 135, KCl 5, MgCl2 1.2, Hepes 10, CaCl2 1.0, D-glucose 10; pH 7.4 with NaOH. Solution II contained (mM): NaCl 135, KCl 5, MgCl2 1.2, Hepes 10, EGTA 1.0, D-glucose 10; pH 7.4 with NaOH. Neutralisation solution contained (mM): NaCl 136, NaPO4 2.2, KCl 5.3, Hepes 10, D-glucose 5.6, EDTA 2; supplemented with 1 % bovine serum albumin and 0.1 % soybean trypsin inhibitor. Minimum completely defined serum-free (MDSF) medium contained 1:1 Dulbecco's modified Eagle's medium:Ham's F12 medium supplemented with 1.25 mg ml-1 bovine serum albumin, 10 mM Hepes, 0.1 mM non-essential amino acids, 2.0 mM L-glutamine, 100 U ml-1 sodium penicillin G and 100 µg ml-1 streptomycin. Patch-clamp bath and pipette solutions were essentially chloride-free and contained (mM): sodium isethionate 140, potassium isethionate 5, Hepes 10, CaSO4 1.0, MgSO4 1.2, D-glucose 5; pH 7.4. In the selectivity experiments, the K+-rich bath solution contained (mM): sodium isethionate 5, potassium isethionate 140, Hepes 10, CaSO4 1.0, MgSO4 1.2, D-glucose 5; pH 7.4. For all experiments, the bath ground electrode was a Ag-AgCl pellet connected to the bath solution via an agar bridge (3 % agar in 3 M KCl, w/v).
Cell isolation and culture
Adult male Sprague-Dawley rats were anaesthetised with sodium pentobarbital (2.5 mg kg-1, I.P.), and type II cells were isolated from the lungs by elastase digestion and differential adhesion (e.g. Dobbs et al. 1980; Kemp et al. 1994; Danto et al. 1998). Briefly, alveolar macrophages were removed by 10 
lavage with the Ca2+-free solution II, and the pulmonary vascular bed was cleared of blood by transcardial perfusion with phosphate-buffered saline. Lungs were removed and instilled, to slightly more than physiological volume, via a tracheal catheter, with solution I containing 2.0-2.5 U ml-1 elastase (Worthington Biochemical, Freehold, NJ, USA) and incubated in 5 % CO2-95 % air for 45 min at 37 °C. The lungs were then chopped finely in neutralisation solution, and filtered sequentially through filters of mesh sizes 100, 40 and 10 µm before being plated onto IgG-coated bacteriological plates. Contaminating cells were allowed to adhere to the plates for 1 h at 37 °C before the type II fraction was aspirated and spun at 150 g for 10 min. The cell pellet was then resuspended in MDSF medium and the cells seeded onto glass coverslips at a density of 2 105 cm-2, and cultured for up to 2 days in a humidified air:CO2 mixture (19:1) at 37 °C.
Patch-clamp studies
Coverslips were placed in a perfusion bath (maximum volume, 400 µl; flow, 7 ml min-1) mounted on the stage of a Nikon TM-D inverted microscope and viewed using phase-contrast optics. Only those cells containing the granular inclusions typical of type II cell morphology were chosen for study. Pipettes were manufactured from thin-walled, filamented borosilicate glass (World Precision Instruments, Sarasota, FL, USA) using a two-stage puller (Narishige PB-9) and had resistances of 4 M
. Voltage clamp was achieved using an Axopatch 2B amplifier (Axon Instruments, Foster City, CA, USA) in resistive-feedback mode. Voltage protocols were generated and current recording/analysis were achieved using the pCLAMP 7 suite of software (Axon Instruments).
Whole-cell Na+ currents were recorded at ambient temperature in symmetrical sodium isethionate solutions. Cells were voltage clamped at a holding potential of 0 mV, and were either ramped (duration, 1 s) from -80 to +80 mV at 0.2 Hz or stepped in 20 mV increments from -80 to +80 mV (1 Hz).
Statistical analysis
P < 0.05 was taken as significant. For comparisons of two data sets, Student's paired or unpaired t tests were employed as appropriate. In the text and figures, values are quoted as the mean ± S.E.M.
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RESULTS |
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Whole-cell cation currents
Amiloride sensitivity. In cells studied ~2 days after isolation, either voltage-clamp protocol elicited whole-cell Na+ currents which were essentially linear with a conductance of 802.5 ± 123.3 pS (n = 9; Fig. 1A and B). Consistent with our previous report (Kemp et al. 1999), 10 µM amiloride only moderately, but significantly (P < 0.05), inhibited the currents by about 25 % (Fig. 1A), indicating expression of both amiloride-sensitive and amiloride-insensitive components of the cation current.
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Figure 1. Amiloride- and Zn2+-sensitive currents in isolated rat alveolar type II pneumocytes A, whole-cell currents elicited by 50 ms voltage steps to -80 mV from a holding potential of 0 mV employing symmetrical sodium isethionate solutions. The upper panel shows the currents recorded during sequential addition of 1 mM ZnCl2 and 10 µM amiloride. The lower panel shows the current response when the compounds were added in reverse order. B, whole-cell current elicited by a 500 ms voltage ramp from -80 to +80 mV (holding potential, 0 mV) before (Control), during (1 mM Zn2+) and after (Wash) ZnCl2 addition to the bath solution. C, mean time course of Zn2+ inhibition of inward and outward currents. D, concentration-response data for Zn2+ inhibition of whole-cell Na+ currents. | ||
Zn2+ sensitivity. Zn2+ is a known inhibitor of non-selective cation channels and K+ channels, but is an ineffective blocker of recombinant trimeric ENaC (Schild et al. 1997). ZnSO4 (or ZnCl2) at 1 mM significantly (P < 0.0005), rapidly (maximal inhibition achieved within 60 s) and reversibly inhibited both inward and outward cation currents (Fig. 1B and C) such that whole-cell cation conductance was reduced to 438.0 ± 68.7 pS (n = 9). Following a 90 s wash-out, conductances were not significantly different (P = 0.17) from those before Zn2+ addition (at 838.8 ± 136.7 pS). The inhibitory effect of Zn2+ was concentration dependent, with a calculated IC50 of 134 ± 37 µM (n = 17 cells; Fig. 1D). In addition, the effects of Zn2+ and amiloride were additive and independent of the order of blocker addition (Fig. 1A).
Cation inhibition profile. Figure 2 shows the effects of Zn2+ plus three additional multivalent cations on alveolar type II whole-cell cation currents. Figure 2A shows a typical time course of inhibition, with exemplar I-V curves shown in Fig. 2B (derived from the ramp protocol). Figure 2C shows the mean effects of each cation inhibitor. Gd3+, Zn2+ and La3+ (10 mM) were all effective at rapidly, reversibly and significantly blocking whole-cell cation currents by about 60 %. Whole-cell conductances were reduced from 857.0 ± 157.0 pS (control; n = 12) to 218.4 ± 23.7 pS by Gd2+ (n = 12; P < 0.001), 221.1 ± 30.7 pS by Zn2+ (n = 10; P < 0.001) and 267.0 ± 30.3 pS by La3+ (n = 9; P < 0.001). In contrast, Ni2+ was a very weak inhibitor of these currents, and at 10 mM, was only able to block about 30 %, reversibly but non-significantly reducing the conductance to 658.8 ± 165.0 pS (n = 5). Final wash-out (as shown in Fig. 2A) restored the conductance to 744.2 ± 220.0 pS, a level not significantly different from control.
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Figure 2. Multivalent cation inhibition of whole-cell Na+ currents A, typical time course of inhibition of inward Na+ current by 10 mM of the multivalent cation indicated. The duration of application is shown by the horizontal bars. B, exemplar ramp currents (from a holding potential of 0 mV) in the absence (Control) and presence of cations. C, mean inhibition caused by each of the multivalent cations. | ||
Pimozide blockade. Superfusing alveolar epithelial type II cells with 10 µM pimozide resulted in significant (P < 0.005) inhibition of both inward and outward whole-cell cation currents which was complete within 2 min (Fig. 3A and B). This concentration of pimozide reduced whole-cell conductance from 662 ± 139 pS to 247 ± 48 pS (n = 9), a mean reduction of 55 %. The inhibitory effect of pimozide was concentration dependent, with an IC50 of ca 1 µM, and a maximal effective dose of between 10 and 100 µM (Fig. 3C).
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Figure 3. Pimozide inhibition of whole-cell currents A, typical time course of pimozide inhibition. The period of application is shown by the horizontal bar. B, exemplar ramp currents in the period before (Control), during (Pimozide) and following (Wash) the application of 10 µM pimozide to the bath solution. C, concentration-response data for pimozide inhibition of whole-cell Na+ currents. | ||
In a separate series of experiments (n = 6), sequential addition of Zn2+ and pimozide resulted in significant reductions in amiloride-insensitive whole-cell conductance from 484.4 ± 67.9 pS (10 µM amiloride) to 253.1 ± 34.5 pS (5 mM Zn2+; P < 0.01) and 213.0 ± 31.5 pS (5 mM Zn2+ followed by 10 µM pimozide; P < 0.005). The values obtained with Zn2+ alone or with Zn2+ plus pimozide were not significantly different (P > 0.4) from each other. Addition of these blockers in reverse order also revealed no overlapping inhibitory effect and supported the notion that the Zn2+- and pimozide-sensitive currents are identical.
cGMP activation
Cell superperfusion with the membrane-permeable analogue of cGMP 8Br-cGMP (100 µM) evoked a significant (P < 0.05), robust activation of whole-cell cation current to about 220 % of control in all cells tested (n = 5). This activation was apparent in either the presence (Fig. 4A) or absence (Fig. 4B) of 10 µM amiloride. Furthermore, in addition to the Zn2+ sensitivity of the basal, unstimulated currents (to 50 % of control; n = 5), Zn2+ was able to inhibit almost completely the cGMP-activated currents by 80 % of control (Fig. 4A and C). The conductances achieved following Zn2+ inhibition before or during cGMP activation were not significantly different (P > 0.2) from each other.
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Figure 4. Cyclic nucleotide activation of whole-cell Na+ currents A, typical time course of activation by 100 µM 8Br-cGMP of the Zn2+-sensitive inward current in the presence of 10 µM amiloride. Periods of application are indicated by horizontal bars. B, typical time course of activation by 100 µM 8Br-cGMP of the Zn2+-sensitive inward current in the absence of 10 µM amiloride. Periods of application are indicated by horizontal bars. C, mean activation/inhibition in the absence of amiloride by Zn2+, cGMP and the two agents added together. | ||
Further evidence that the cGMP-stimulated current is Zn2+ sensitive is shown in Fig. 5. In the continued presence of 1 mM Zn2+, application of 100 µM 8Br-cGMP was unable to activate currents (Fig. 5A and B). The mean conductance level achieved during cGMP perfusion in the presence of Zn2+ (54 % of control) was not significantly different (P > 0.3) from that seen in the presence of Zn2+ alone (50 % of control).
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Figure 5. Effect of cyclic nucleotide addition in the presence of Zn2+ A, typical time course showing lack of activation of amiloride-insensitive Na+ currents by 100 µM 8Br-cGMP in the presence of 1 mM Zn2+. Periods of application are indicated by horizontal bars. B, mean inhibition by Zn2+ and the lack of activation by cGMP. W/O, washout. | ||
Cation selectivity
Consistent with the contribution of non-selective cation channels to alveolar epithelial type II amiloride-insensitive current are the selectivity data shown in Fig. 6. Figure 6A shows an exemplar time course of the effects of 1 mM Zn2+ on inward cation currents (at -60 mV) in the presence of either extracellular Na+-rich or extracellular K+-rich solutions. Inward currents are increased and Zn2+-sensitive inward current is augmented by K+ perfusion; currents return to control levels when extracellular Na+-rich solution is reinstated. The current-voltage relationships shown in Fig. 6B support the conclusion that Zn2+-sensitive currents select mildly for K+ over Na+, since the reversal potential is shifted to the right with K+-rich extracellular solution. In symmetrical Na+ solutions, Zn2+-inhibitable currents (obtained by subtracting control currents from those in the presence of 1 mM Zn2+) were linear and reversed at zero potential (Fig. 6C). Following exchange of the bath solution for one containing 140 mM K+, Zn2+-inhibitable currents reversed at 7.94 ± 0.71 mV (following empirical calculation and subtraction of the liquid junction potential: 3.66 ± 0.04 mV, n = 5) and showed weak inward rectification, consistent with a slightly higher permeability for K+ than for Na+ (Fig. 6C). Use of the reversal potential differences measured in bi-ionic solutions allowed calculation of PNa/PK as 0.73 ± 0.02 (n = 5).
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Figure 6. Sodium and potassium permeability of Zn2+-sensitive currents A, exemplar time course of the effects of 1 mM Zn2+ on inward cation currents (at -60 mV) in the presence of either extracellular Na+-rich or extracellular K+-rich solution. The horizontal bars show the duration and composition of the extracellular solutions. B, mean current-voltage relationships for the experimental conditions shown in Fig. 6A. Each line represents the mean of five voltage ramps. C, current-voltage relationships for the Zn2+-sensitive components of the currents recorded in Na2+-rich (Zn2+-sens(Nao)) or K+-rich (Zn2+-sens(Ko)) extracellular solutions. These reversal potential data allowed, following empirical calculation and subtraction of the liquid junction potential of 3.66 ± 0.04 mV (n = 5), calculation of the mean PNa/PK as 0.73 ± 0.02. | ||
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DISCUSSION |
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The data presented herein provide strong evidence that an important component of adult rat alveolar epithelial type II pneumocyte whole-cell Na+ conductance is amiloride insensitive. This observation is in general agreement with numerous studies showing significant amiloride-insensitive postnatal lung fluid reabsorption in vivo or in situ (e.g. Norlin et al. 1998; Junor et al. 1999). Similar permeability to both Na+ and K+, the pattern of inhibition by multivalent cations (including Zn2+), and the effects of pimozide implicate cyclic nucleotide-gated cation channels as effectors for the observed amiloride-insensitive current. Pimozide action was essentially irreversible. The irreversibility of the actions of this drug in both cellular (Nicol, 1993) and in situ preparations (Junor et al. 1999) has not previously been reported. Although there is no clear explanation for the irreversibility that we observed, the pimozide IC50 of ~1 µM is similar to that previously reported for inhibition of the rod photoceptor CNG channel (Nicol, 1993), which suggests that the pimozide effect that we report here is likely to be a specific action on CGN channels. The in situ observations of Junor et al. (1999) are suggestive of the involvement of cyclic nucleotide-gated non-selective cation channels. However, these investigators did not present evidence that absorption could be activated by cGMP and based their conclusions solely on inhibitor data. Our inhibitor studies agree strongly with the data obtained from the sheep lung model. Furthermore, in the present paper, we directly demonstrate an activation of whole-cell cation current by 8Br-cGMP which is abolished by Zn2+ both in the presence and in the absence of amiloride. These findings correlate well with, and extend the scope of, recent observations in situ that this channel type is important in postnatal lung fluid reabsorption (Junor et al. 1999).
The electrophysiological profile of the amiloride-insensitive cation conductance is consistent with expression of functional cyclic nucleotide-gated cation channels in alveolar epithelial type II cells. These channels are characterised by their insensitivity to amiloride, relative non-selectivity for Na+ over K+, inhibition by pimozide, and rapid and reversible blockade by multivalent cations (including Zn2+) with relatively high affinity (IC50 of 134 µM for Zn2+). They are different from ENaC, which is insensitive to Zn2+ at the concentrations employed here (Schild et al. 1997). Concentration-dependent pimozide blockade similar in magnitude (but not additive) to the observed Zn2+-inhibitable conductance, together with inhibition by Zn2+ of both basal and cGMP-activated currents, support the notion that a pimozide-blockable cation channel underlies a large component of alveolar epithelial type II cell cation conductance. Pimozide is a dopamine D2 receptor antagonist. Since postnatal lung fluid clearance is unaffected by another D2 antagoinst, S-sulpiride (Barnard et al. 1999), it seems likely that the action of pimozide is through direct binding to cyclic nucleotide-gated cation channels.
In the adult lung, there is a growing body of evidence to suggest that a substantial component of active Na+ absorption across alveolar epithelium is amiloride insensitive and independent of ENaC activity. In the sheep, amiloride sensitivity of basal and adrenaline-evoked fluid reabsorption wanes as a function of postnatal age (Ramsden et al. 1992). In guinea-pig lungs, amiloride-blockable fluid reabsorption also falls dramatically from fetus to adult (Norlin et al. 1998). This decrease in amiloride sensitivity correlates well with ENaC steady-state mRNA levels (Finley et al. 1998). Furthermore, in adult rats of the same species and age as were used as the source of cells in the current study, more than half of the basal and stimulated lung fluid clearance was found to be insensitive to the inhibitory effects of luminal amiloride (Barnard et al. 1997). Consistent with these findings, Junor et al. (1999) demonstrated that a substantial component of lung liquid absorption is amiloride insensitive in sheep at 6 months of age. Inhibition of amiloride-insensitive lung liquid absorption by dichlorobenzamil and pimozide, selective inhibitors of cyclic nucleotide-gated cation channels, led to the suggestion that cyclic nucleotide-gated cation channels play a major role in the amiloride-insensitive component of lung liquid absorption in postnatal lungs (Junor et al. 1999), consistent with results in the present study. The present study suggests that the amiloride-insensitive component of the Na+ conductance in adult alveolar type II cells cultured short term (and on impermeable supports) is quantitatively more important than that which can be blocked by amiloride. This is different from previous observations made in monolayers cultured long term on permeable substrata (e.g. Cheek et al. 1989). This discrepancy may be due to a number of different factors which include, most notably, induction of a type I phenotype in cells grown to monolayer in longer-term culture. Thus, it might be argued that data from alveolar epithelial monolayer cultures often reflect more closely type I than type II cell function. At present, this controversy cannot be resolved, but it is interesting to note that our recent studies on isolated alveolar type I cells have demonstrated that the amiloride-blockable Na+ conductance is larger than the amiloride-insensitive flux (unpublished data). Another interesting difference between data obtained from monolayers and isolated type II cells is the observation that monolayer Na+-dependent short-circuit current does not appear to be sensitive to the actions of cGMP in a manner comparable to that reported herein (Cott et al. 1986). Again, although there is no clear explanation for this difference, a recent in vivo study in rat (the same species as employed in this investigation) has shown activation of amiloride-insensitive fluid reabsorption with 8Br-cGMP (Norlin et al. 2001) and suggests that cGMP insensitivity may be a property of alveolar epithelial cells only when maintained in longer-term culture on permeable supports.
By in situ hybridisation, mRNA for CNG1 has been localised to rat airway epithelia, bronchi, bronchioles, and alveolar epithelial type I and type II cells, suggesting a potential role for these channels in Na+ entry into epithelial cells from the airspaces of the lung (Ding et al. 1997). Functional evidence for the presence of cyclic nucleotide-gated channels has been demonstrated in rat tracheal epithelial cells in which both basal and cGMP-stimulated Na+ transport are inhibited by known blockers of nucleotide-gated cation channels (Schwiebert et al. 1997). Using the whole-cell patch-clamp technique, 8Br-cGMP-stimulated currents that are inhibited by l-cis-diltiazem and dichlorobenzamil have also been demonstrated in the pulmonary epithelial A549 cell line, consistent with RT-PCR data indicating the presence of both CNG1-3 and CNG in these cells (Xu et al. 1999).
In summary, we have presented data indicating that cyclic nucleotide-gated cation channels are functionally expressed in adult rat alveolar epithelial type II cells. Additive effects of selective inhibitors of these channels on whole-cell currents indicate that cyclic nucleotide-gated cation channels probably mediate a major part of the amiloride-insensitive component of cation conductance in alveolar epithelial cells. In conjunction with previous in vivo studies implicating these channels in amiloride-insensitive fluid absorption, these findings suggest that cyclic nucleotide-gated cation channels located in the alveolar epithelium of the distal lung make an important contribution to fluid absorption in the adult lung.
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Acknowledgements
We note with appreciation the expert technical support of Martha Jean Foster and Suzie Parra. This work was supported in part by the American Heart Association, National Institutes of Health Research grants HL38578, HL38621, HL38658, HL62569 and HL64635, the Baxter Foundation, the Hastings Foundation, the British Heart Foundation and the Wellcome Trust. E.D.C. is Hastings Professor of Medicine and Kenneth T. Norris Jr Chair of Medicine.
Corresponding author
P. J. Kemp: School of Biomedical Sciences, Worsely Medical and Dental Building, University of Leeds, Leeds LS2 9JT, UK.
Email: p.z.kemp{at}leeds.ac.uk
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