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Effects of altered availability of Na+ on guinea-pig airway smooth muscle contractility
Pulmonary Pharmacology (1990) 3 121-127
0952-0600/90/0003-0121/$10.00
©l990 Longman Group UK Ltd
PULMONARY PHARMACOLOGY
Effects of Altered Availability of Na + on Guinea-pig Airway Smooth Muscle Contractility D . Raeburn Department of Pharmacology and Experimental Therapeutics, Louisiana State University Medical Centre, New Orleans, Louisiana, USA SUMMARY. The effects of altering the availability of sodium ions (Na + ) on contractility of the guinea-pig isolated trachealis was examined using regimens which are reported to inhibit Na + /K + ATPase activity (ouabain), Na + /H + exchange (amiloride, ammonium ion (NH 4 ')) or Na'/Ca2+ exchange (reduced extracellular Na+) . Inhibition of Na + /K + ATPase and reversal of Na'/Ca 2+ exchange resulted in increased "Ca uptake and contraction of the trachealis by voltage-sensitive and voltage-insensitive mechanisms respectively . When Na + /H+ exchange was inhibited by amiloride the tissues relaxed to below their baseline tension. The relaxation was not due to reduced Ca2 + influx. Treatment with NH 4 + produced a contractile response. Reduced extracellular Na + caused a transient contraction as a result of reversal of the normal Na '/Ca"+ exchange process leading to accumulation of Ca 2 + within the cell. Since the effects of amiloride and reduced extracellular sodium were different, it is unlikely that amiloride is acting primarily by inhibiting Na'/Ca 2+ exchange. Amiloride reduced tissue sensitivity to methacholine and KCI without affecting Ca 21 influx . This may involve a secondary stimulation of Na'/Ca 2+ exchange following changes in [Na + ]t . Ouabain also reduced tissue sensitivity to methacholine and KCI . These findings suggest that Na + are important in determining smooth muscle contractility . If NH 4 + is altering pH then, at the concentrations used, the changes in (H + ] were not sufficient to alter responses to the spasmogens .
and cell contraction following membrane depolarization (with Ca" influx) and reversal of the normal Na+/Ca2+ exchange . Na + pump activity has been demonstrated in airway smooth muscle where, as in other tissues, it is ouabain sensitive ." In several cell types a mechanism for Na+/H + exchange also exists where the efflux of H + is coupled to the influx of Na+,9 .1° Although ions can move in either direction, under physiological conditions the large inward Na+ gradient makes the exchanger function as a Na + uptake pathway . 10 Na+/H + exchange has a stoichiometry of one, is electrically neutral, is insensitive to ouabain but is blocked by the diuretic agent amiloride . Ammonium ions (NH4 ' ) have an affinity for the exchange mechanisms similar to Na+ (although the maximum rate of exchange is lower) suggesting that competition may occur between these cations to reduce the efficiency of the exchanger . 9,1o Blockade of the Na+/H+ exchanger will result in an increase in [H+] 1 (i .e . intracellular acidification) . In cardiac muscle, Kim et al 11 suggest that increased [H'] will reduce [Ca"] by a Ca"/H' interaction which results in a negative inotropic response . It is reasonable therefore to assume that reversal of normal Na'/Ca" exchange, inhibition of Na+/H+ exchange and inhibition of Na + /K + ATPase activity will ultimately alter [Ca2+ ] i and hence smooth muscle contractility . Accordingly, the purpose of the
INTRODUCTION Contraction of airway and other smooth muscle is dependent upon an increase in the concentration of free, ionized calcium ions ([Ca 2+ ] i ) in the cytoplasm following influx of extracellular Ca" through specific Ca 2 + channels or the release of Ca 2 + from internal stores . The role of Ca 2 + in the contraction of airway smooth muscle has been reviewed extensively ." There are several mechanisms controlling sodium ion (Na + ) and Ca 21 movements in smooth muscle cells and often the processes are linked either directly, e .g . Na'/Ca" exchange, or indirectly following the inhibition of Na + /H + exchange or Na + /K + pumping . Thus, processes which alter transmembrane Na+ movements will affect the concentration of Na+ inside and outside the cell and subsequently affect [Ca 2 +] I and hence contractility . Na'/Ca 2+ exchange, which normally removes Ca 2 + from the cell, can be reversed by bathing the tissues in a low Na + physiological solution to reduce [Na + ] a and cause the efflux of Na+ down its concentration gradient in exchange for Ca" .' - ' Inhibition of Na + /K + pumping results in increased [Na + ] I which in turn leads to an increase in [Ca 2+ ] 1 Address correspondence to : D . Raeburn, Biological Research, Dagenham Research Centre, Rhone-Poulenc Ltd, Rainham Road South, Dagenham, Essex RM10 7XS, England . 121
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present study was to examine the effects of these diverse ways of altering the availability of Na + on muscle cell contractility in the guinea-pig trachealis .
METHODS Male, Hartley guinea pigs (340-500 g ; HarlanSprague Dawley Inc ., Indianapolis, IN, USA) were killed by stunning and bleeding . The trachea was removed, placed in a modified Krebs-Henseleit solution (MKH) and dissected free of connective tissue . Tissues were prepared for the experimental protocols as follows . Tension studies The trachea was slit through the cartilage along its longitudinal axis and strips consisting of two adjacent rings of cartilage were cut . Some contractile agonists can stimulate the release of bronchoactive agents from the airway epithelium . 12-14 To exclude these indirect effects the epithelium was removed from each preparation . The tracheal strips were placed in water-jacketed tissue baths (37°C) containing MKH and gassed with 5% CO 2 in oxygen. Changes in the isometric force of contraction were measured using force-displacement transducers (FT03C, Grass Instruments, Quincy, MA, USA) . The tissues were equilibrated for 60 min under the optimum applied load of 2 g and washed at 15 min intervals with fresh MKH . The composition of the MKH was as follows (mM) : NaCl 113, KCl 4 .8, CaC1 2 2 .5, KH 2PO4 1 .2, MgSO4 1 .2, NaHCO 3 25 and glucose 5 .7 . A low Na-MKH was prepared by substituting NaCl with sucrose (246 mM) which is isosmolar with normal, Na + containing, MKH . The sucrose itself is without pharmacological effects . Concentration-response curves to methacholine, KCl, ouabain, amiloride and NH4' were constructed by the cumulative addition of the drug . In some experiments a response curve to methacholine or KCl (control curves) was constructed and when the maximum response had been achieved the tissues were washed with fresh MKH until responses had returned to baseline . Then ouabain (1 or 100 MM), amiloride (1 or 100 µM), NH4' (0 .1 or 1 mM) or vehicle was added to the tissue bath 30 min prior to the construction of a second curve in the presence of the test substance . The responses obtained were expressed as a percentage of their appropriate pretreatment control response . The effect of reducing [Na+] o on basal tone and on responses to amiloride, KCl and methacholine were examined by changing the bathing fluid (after equilibration in normal MKH) to the low NaMKH and the responses obtained were compared with control .
"Ca uptake studies The La" resistant 45 Ca content was measured as previously described for this preparation 15 as modified by Raeburn et al ." The epithelium was removed as for the tension studies . Briefly, cartilage-free strips of trachealis were equilibrated in MKH for 30 min then transferred to MKH or low Na-MKH to which had been added 45 Ca (0.5 µCi/ml) . The tissues were incubated in the radioactive `loading' solution for 10 min (methacholine) or 20 min (low Na, ouabain, KCl) at 37°C . Where appropriate, ouabain, methacholine or KCl was added 5 min after the addition of 45 Ca. Tissues were incubated for 30 min with amiloride in the loading solution . At the end of the experiment the tissues were rinsed (5 s) in Trisbuffered MKH 16 to prevent precipitation of phosphate or bicarbonate prior to incubation for 60 min (3 washes) at 0 .5°C in Ca t+ free Tris-MKH containing La" (80 mM) and gassed with 100% oxygen . Liquid scintillation counting was used to determine the radioactivity present in each tissue sample . 45 Ca uptake was expressed in nmol/mg of protein using the Lowry method .' 7 Statistical analysis The results are presented as mean + SEM. pD 2 (- log EC50 M) values were calculated from linear regression analysis of probit transformed data . Differences between values for control and treated tissues were assessed for significance using a one-way analysis of variance (ANOVA), p<0 .05 was accepted as significant . Drugs The following drugs were used : methacholine chloride, amiloride hydrochloride, verapamil hydrochloride, ouabain and lanthanum chloride (Sigma Chemical Co ., St Louis, MO, USA) and ammonium chloride (Baker Chemical Co ., Phillipsburg, NJ, USA) . Drug solutions were prepared in distilled, de-ionised water on a daily basis . Radiolabelled Ca t+ (45 CaC1 2) was obtained from New England Nuclear, Boston, MA, USA . The scintillant used was `Ready Gel' (Beckman, Fullerton, CA, USA) .
RESULTS Effects of amiloride Amiloride (0 .1-100µM) produced a concentrationdependent relaxation of guinea-pig trachealis which was maximal at 100 µM (Figure 1, Table 1) . The cumulative addition of methacholine (10 nM100 µM) or KCI (4-100 mM) produced con-
Sodium and Airway Contractility
Table 1 . The effects of amiloride or ouabain on the contractility of guinea-pig trachealis in vitro
100-+ C 0 O-
80 -
x ro
60 -
a- 40 0
123
Agonist
n
pD2
Max . response (g)
Amiloride Ouabain
7 7
4.60+_0.03 6.35+0.21
-0 .234+0 .06 +0 .538±0 .18
20 0I 7
I
1
1
6
5
4
maximum force of contraction (Table 2) . The higher concentration of ouabain (100,W) substantially reduced the maximum response to KCl and prevented calculation of a pD 2 (Table 2).
-log [amiloride ] (M) Effects of reduced [Na +],, Fig. 1-Concentration-response curve to the cumulative addition of amiloride (0 .1-100 pM) plotted as percentage maximum relaxation of the guinea-pig trachealis, n = 7.
centration-dependent increases in the force of contraction . The drug vehicle was without effect on tissue reactivity . Amiloride (100 µM, 30 min pretreatment) reduced tone and tissue sensitivity to methacholine and KCl compared with the control (Table 2) . A lower concentration of amiloride (1 µM) was without effect . A small increase in the maximum force of contraction to methacholine and KCl was seen following amiloride treatment but this probably reflects the reduction in basal tone produced by amiloride (Table 2) . Effects of ouabain Ouabain (0 .01-100 pM) produced a concentrationdependent increase in the force of contraction of the trachealis (Fig . 2, Table 1) . No further contractility was seen on increasing the concentration of ouabain . Ouabain (1 or 100 µM, 30 min pretreatment) significantly reduced tissue sensitivity to methacholine without affecting the maximum response (Table 2) . Tissue sensitivity to KCl was slightly reduced by ouabain (1 pM, 30 min pretreatment) without affecting the
r 8
I
I
I
7 6
5
4
1
-log [ouabain I (M) Fig . 2-Concentration-response curve to the cumulative addition of ouabain (0 .01-100 pM) plotted as percentage maximum contraction of the guinea-pig trachealis, n = 7 .
Transferring the tissues from the regular MKH to the low Na-MKH produced a biphasic response . An immediate contraction (405 ± 53 mg, lasting 333 ± 34 s, n = 16) was seen which was generally followed by a sustained relaxation to below the original baseline (Fig . 3A) . In some tissues the secondary relaxation developed into a tonic contracture which was of lesser magnitude than the initial phase of the response (Fig . 3B) . The re-addition of Na + (NaCl, 100 mM) to tissues incubated in low Na-MKH resulted in a further small relaxation, the original level of tone was not restored until the tissues were washed in normal MKH . Amiloride (100 pM, 30 min pretreatment) and verapamil (1 uM, 30 min) had little or no effect on the magnitude of the immediate response (75± 15% and 82± 14% respectively of control) or on the time course (n=5) . Ouabain (100µM, 30 min) markedly depressed the contraction to low Na-MKH to 38±3% of control (n=3) . Amiloride (1-100µM, n=5) relaxed tissues in the presence of reduced [Na+] .. The pD 2 value of 6.07±0 .13 for amiloride in reduced [Na+] o indicated increased sensitivity of the tissue to amiloride in the low Na-MKH . The maximum relaxation to amiloride (178+31 mg) obtained in low Na-MKH was not different from the normal MKH control (Table 1) . In reduced [Na +]. the responses to methacholine (5 µM, approximately EC75) and KCl (75 mM, EC BO) were markedly depressed (Fig . 4) . The overall shape and time course of the response to methacholine was not altered but the maximum force of contraction only achieved 22 ± 3 of the regular MKH control, n = 6 . In the low NaMKH the response to KCl in some tissues more clearly exhibited distinct phasic and tonic components and achieved only 59+3% of control, n=6 . The re-addition of Na + produced a small relaxation followed by an abrupt contraction returning the tissue to its pretreatment control level of tone in the case of tissues contracted with methacholine . In KCl-contracted tissues the re-addition of Na + did not produce relaxation but resulted in a slow, steady return of the level of tone approaching that seen in control tissues in regular MKH .
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Pulmonary Pharmacology
Table 2. The effects of amiloride or ouabain on responses to methacholine or KCI in guinea-pig trachealis in vitro Methacholine max . (g)
n
pD 2
Control 1 µM 100 MM
13 7 6
6 .22±0 .06 6 .06±0 .17 5 .84±0 .07*
Control 1 pM 100µM
10 5 5
6 .14±0 .07 5 .87±0 .31* 5 .54±0 .12*
Amiloride 1 .16±0 .17 1 .50±0.19 1 .53±0 .27 Ouabain 1 .01±0.10 0.72±0.09 1 .08±0.36
KCI pD 2
max . (g)
9
1 .67+0 .02 n.d . 1 .40±0 .02*
1 .04+0 .14 n .d . 1 .20±0.16
12 6 6
1 .77+ _ 0 .09 1 .53+ _ 0 .06* n.d .
0 .84+_0 .12 0.85+_0.05 0 .20±0 .06
n 9
* p < 0 .05 vs control n .d . not determined A
A
W
• Low Na'
K CI
• Na'
B
MCh 5i4
•Low Na'
• (ow Na+ MKH Fig . 3-Representative traces showing the effects of changing the bathing fluid to a reduced Na+ solution in the guinea-pig trachealis . Panel A: Initial transient contractile response commonly followed by a secondary relaxation to below the original baseline . Panel B : A response showing the occasional development of a secondary contracture of reduced magnitude .
• Na'
• MCh
10 min
Fig . 4-The effects of low Na bathing solution on responses to KCI (Panel A) and methacholine (MCh, Panel B) in the guineapig trachealis . Tissues were contracted in normal MKH, washed back to baseline (W) then the bathing fluid changed to the low Na-MKH . When the responses stabilised the tissues were rechallenged with KCI or MCh . At the peak of the second response (in low Na+) Na + were added back .
100-1 Effects of NH,' The addition of NH4 + (0 .1-5 mM) produced concentration-dependent increases in the force of contraction to a maximum of 486±62 mg (Fig . 5) . Since concentrations of NH, + greater than 10 mM produced responses which were complex in nature where, for any given concentration of the ion, contraction-relaxation cycles were seen, it was thought reasonable to assume that the maximum contractile event occurred with NH4' (5 mM) . Pretreatment of the tissues with NH,' (0 .1 or 1 mM) did not affect responses to methacholine or KCI (Table 3) .
0 80.m 60C_ .6...
0 40U
200I
4
I
I
I
3 -tog [NH4+ ] (M)
Fig. 5-Concentration-response curve to the cumulative addition of NH,+ ion (0 .1-5 mM) plotted as percentage maximum contraction in the guinea-pig trachealis, n = 6 .
"'Ca uptake studies Amiloride (100 µM, 30 min pretreatment) did not affect "Ca uptake into the trachealis . Transferring the tissues to the low Na-MKH, treatment with KCl (40 mM) or ouabain (100,uM) increased significantly "Ca uptake over basal (Table 4) . It was not possible
to assess the effects of low Na-MKH or ouabain on KCl-induced 4S Ca uptake since both treatments on their own produced greater uptake than was seen with KCI (Table 4) . Methacholine did not stimulate 4S Ca uptake confirming previous reports (data not shown) .
Sodium and Airway Contractility Table 3. The effects of NH,' on responses to methacholine or KCl in the guinea-pig trachealis in vitro n
Max . response (g)
pD2
Control NH4+(0 .1 MM) (1 .0 MM)
10 5 5
Control NH4+(0.1 MM) (1 .0 MM)
6 3 3
Methacholine 6.55±0 .07 6 .63±0 .05 6 .50±0 .10 KCl 1 .69±0 .04 1 .67±0 .04 1 .74±0 .05
1 .16±0 .17 1 .09±0 .08 1 .19±0 .12 0 .85±0 .10 0 .95±0 .13 0 .87±0 .12
Table 4 . The effects of various treatments on 45 Ca uptake into the guinea-pig trachealis in vitro Treatment
n
Basal KC1 (40 mM) Amiloride (100 pM) Ouabain (100 pM) Low [Na+l o
5 5 5 4 4
45 Ca content (n mol/mg protein)
74+12 127± 13* 75±6 937±83* 214±44*
* p < 0 .05 vs basal
DISCUSSION The first conclusion to be drawn from this study is that the various treatments used to modulate Na + movements across the cell membrane produce different effects depending on the Na + transport process under investigation . Inhibition of Na + /K + pumping or Na + /Ca' + exchange results in contraction of the trachealis via a voltage dependent and a voltage independent mechanism . Inhibition of Na + /H + exchange with amiloride on the other hand produces relaxation . Treatment with NH,' was not equivalent to treatment with amiloride suggesting a mechanism of action other than interference with Na+/H+ exchange by NH,' . It is possible that this ion produced its effects by directly increasing [H + ] i in turn leading to an increase in [Ca 2+] i as the cell attempts to return to its basal pH . The different effects seen with amiloride and the reduction in [Na+] o suggests that, under the present circumstances, amiloride is acting primarily on Na+/H + exchange with lesser effects on Na+/Ca' + exchange, an effect which has been reported to occur at higher concentrations of amiloride . t e The increase in contractility following inhibition of Na + /K + ATPase and Na'/Ca" exchange is accompanied by the influx of extracellular Ca" as indicated by the radiolabelled Ca` data . These findings are similar to those reported by Van Breemen et al 19 for intestinal and vascular smooth muscle . In the case of Na'/Ca" exchange the present findings confirm those of Bullock et al' in bovine trachealis and Kawanishi et al20 in the guinea pig . In the present study, the influx of Ca l ' causing contraction in reduced (Na + ],, is probably not via voltage-dependent Cal ' channels since it is relatively unaffected by verapamil. This confirms the findings of Johannson
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and Hellstrand 2i in rat portal vein . Cal' influx is involved since extracellular Ca t+ is required for the contractile response to reduced [Nal]o .21 Conversely, ouabain-stimulated Ca t+ influx occurs via voltage-gated channels since the contraction is significantly inhibited by nifedipine and verapamil . 22 Amiloride, at the highest concentration examined, reduced tissue sensitivity both to KCl and methacholine without affecting maximum responses . This suggests (since tissue sensitivity is assessed during the sustained, tonic phase in cumulative concentrationresponse studies) that amiloride is reducing the availability of the activator Cat + required for this phase of contraction i .e . extracellular Ca 2+ .23 This action of the relaxant is not due to a reduced Ca l ' influx through voltage sensitive channels (since there was no effect on 45 Ca uptake) but may be the result of the reduced [Na + ]; stimulating Na'/Ca 2+ exchange to reduce [Ca 2+]1 as [Na+]i again is increased . The effects, if any, of amiloride on Ca l ' efflux in smooth muscle are unknown . It has been suggested (see Introduction) that inhibition of the Na+/H + exchanger produces intracellular acidification due to a retention of H+ and that the protons could compete with Ca" for intracellular cationic binding sites . More Ca 21 would thus be subject to the Ca 21 removal mechanisms and in cardiac muscle a negative inotropic response would be seen,"t A reduced contractility in airway smooth muscle following amiloride treatment could be explained by this effect . It has been suggested previously that amiloride can directly inhibit myosin light chain kinase activity in intestinal muscle but has no effect on calmodulin activation . 24 This action of the relaxant cannot be ruled out in the present study . Like amiloride, ouabain reduced tissue sensitivity to methacholine and KCl and, at the highest concentration, reduced the magnitude of the response to KCl . This is also the case in guinea-pig ileum ."9 Why inhibition of Na + /K + ATPase activity should have this effect is unclear . Van Breemen et a1 19 speculate that the inhibition may be due to blockade of Ca" influx (not the case in the present study) or sequestration of cytosolic Ca 2 + away from the myofilaments . Another possible explanation may be that the already elevated [Ca 2+ ] i produced by inhibiting Na + /K + pumping is preventing further Ca 2 + influx by stimulating Na'/Ca 2+ exchange . It is also possible that refilling of the cytosolic Ca 2 + stores is blocked . In visceral afferent neurones in the rabbit 25 ouabain can induce membrane hyperpolarization due to increased K + conductance . Were this effect to occur in smooth muscle a reduced contractility would be expected . Exposure to NH 4 + , while in itself causing contraction, did not affect tissue sensitivity or maximum responses to methacholine or KC1 . This suggests that NH4' is not affecting Ca 21 influx . The contractile response to NH4' is unlikely to be a result of
1 26 Pulmonary Pharmacology
intracellular acidification but more likely due to less efficient Na'/Ca" exchange (due to competition for the mechanism) leading to increased [Ca 2+]1 NH4 ' has been reported to be a weak inhibitor of Na + /K + ATPase . 26 This action cannot be ruled out . In conditions of reduced [Na'],, several interesting phenomena were observed . Tissue sensitivity to amiloride was substantially increased which would reasonably be expected to be the case . The contraction following the reduction in [Na']., while not affected by Cat+ entry blockers or amiloride, was inhibited by ouabain. This probably reflects ouabain causing an increase in [Na + ]i in turn stimulating Ca" loss . The overall reduced effect reflecting the stronger inward Na + driving force (due to concentration gradient effects) occurring on blockade of the Na + /K + pump overcoming the reduction in [Na'].. Inhibition of Na'/Ca" exchange by reducing [Na+] o markedly reduced tissue responsiveness to methacholine but had a lesser effect on KC1 . Brading et a1 4 suggest that reducing [Na + ] o prevents refilling of the intracellular Ca t+ store in intestinal muscle thereby suppressing the activity of carbachol . This is presumed to occur since filling of the intracellular Ca" store is by a Na'/Ca 2+ exchange process . If [Na+] in the store falls then Ca" cannot enter . Since it is established that the response to muscarinic agonists is less dependent on the influx of extracellular Cat+ than the response to KCI' , ' the results suggest that inhibition of Na'/Ca" exchange has a greater effect on intracellular Ca" mobilization . Support for this view comes from the lack of change to the overall shape and time course but reduced maximum of the response to methacholine in reduced [Na'].. Since Na'/Ca 2+ exchange affected Ca t+ influx by a voltage-independent mechanism while the sustained phase of a response is blocked by calcium antagonists, it would be expected that the tonic component of the response to methacholine would have been less affected . This suggests utilisation of more than one pathway for extracellular activator Cat + being involved in response maintenance . It must be acknowledged, however, that although less depression of the maximum response was seen, the time to half maximum was substantially increased for KCl which may reflect slow entry of Ca" via the `leak pathway' . Brading et al' also suggest that reduced [Na']. may alter the membrane potential (encourage repolarization) and render depolarization more difficult . In summary, Na + play an important role in the development and maintenance of tone in the guineapig trachealis in vitro . The various Na'/Ca 2 + interactions are both direct, and more commonly, indirect and very difficult to delineate accurately . Clearly this is an important area for further study and, with time, more informative data will emerge . As stated in the introduction, for the sake of simplicity the influence of the epithelium was not examined in this study . Since
the epithelium is a site of active Na + transport in many organ systems including the respiratory tract, these indirect effects may also have important influences on the underlying airway smooth muscle . This aspect has been studied recently' where it was found that modulation of the activity of the epithelial Na + /K + pump can modulate airway responsiveness to some contractile stimuli. References 1 . Rodger I W . Excitation-contraction coupling and uncoupling in airway smooth muscle. Br J Clin Pharmacol 1985 ; 20: 255S-266S . 2 . Rodger I W . Calcium ions and the contraction of airway smooth muscle . In : Kay A B (ed) . Asthma : Clinical Pharmacology and therapeutic progress . Blackwell, Oxford, 1986 pp 114-127 . 3 . Reuter H, Blaustein M P, Haeusler G . Na-Ca exchange and tension development in arterial smooth muscle . Phil Trans R Soc B 1973 ; 265 : 78-94 . 4 . Brading A F, Burnett M, Sneddon P . The effect of sodium removal on the contractile response of the guinea-pig taenia coli to carbachol . J Physiol 1980 ; 306 :411-420 . 5 . Bullock C G, Fettes J J F, Kirkpatrick C T . Tracheal smooth muscle-second thoughts on sodium-calcium exchange. J Physiol 1981 ; 318 : 46 . 6 . Souhrada M, Souhrada J F . The direct effect of temperature on airway smooth muscle . Resp Physiol 1981 ; 44 : 311-323 . 7 . Chideckel E W, Frost J L, Mike P, Fedan J S . The effect of ouabain on tension in isolated respiratory tract muscle of humans and other species . Br J Pharmacol 1987; 92: 609-614 . 8 . Raeburn D, Fedan J S . The effects of alterations in electrogenic Na'/K+ pumping in guinea-pig isolated trachealis: Their modulation by the epithelium . Br J Pharmacol 1989 ; 98 : 343-350 . 9 . Mahnensmith R L, Aronson P S . The plasma membrane sodium-hydrogen exchanger and its role in physiological and pathological processes. Circ Res 1985 ; 56 : 773-788 . 10 . Frelin C, Vigne P, Barbry P, Lazdunski M . Molecular properties of amiloride action and of its Na+ transporting targets . Kidney Intl 1987 ; 32 : 785-793 . 11 . Kim D, Cragoe E J, Smith T W . Relations among sodium pump inhibition, Na-Ca and Na-H exchange activities and Ca-H interaction in cultured chick heart cells . Circ Res 1987 ; 60 : 185-193 . 12 . Fedan J S, Hay D W P, Farmer S G, Raeburn D . Epithelial cells: Modulation of airway smooth muscle reactivity . In : Rodger I W, Barnes P J, Thompson N C (eds) . Asthma : Basic mechanisms and clinical management . Academic Press, New York, 1988 pp 143-159 . 13 . Vanhoutte P M . Epithelium derived relaxing factor : Myth or reality . Thorax 1988; 43 : 665-668 . 14 . Raeburn D . Epithelium, eicosanoids and airway reactivity . Gen Pharmacol 1990 ; 21 : 11-16. 15 . Raeburn D, Rodger I W . Lack of effect of leukotriene D4 on Ca-uptake in airway smooth muscle . Br J Pharmacol 1984 ; 83: 499-504 . 16 . Raeburn D, Hay D W P, Fedan J S . Calcium uptake into guinea-pig trachealis : The effect of epithelium removal . Cell Calcium 1987 ; 8 : 429-436. 17 . Lowry 0 H, Rosebrough N J, Farr A L, Randell R J . Protein measurement with Folin phenol reagent . J Biol Chem 1951 ; 193 : 265-275 . 18 . Siegl P K, Cragoe E J, Trumble M J, Kaczorowski G J . Inhibition of Na'/Ca2 + exchange in membrane vesicle and papillary muscle preparations from guinea-
Sodium and Airway Contractility pig heart by analogues of amiloride . Proc Natl Acad Sci 1984 ; 81 : 3238-3242 . 19. Van Breemen C, Aaronson P, Loutzenhiser R . Sodium-calcium interactions in mammalian smooth muscle . Pharmacol Rev 1979; 30: 167-208 . 20 . Kawanishi M, Baba K, Tomita T . Effects of Na removal and readmission on the mechanical response in the guinea-pig tracheal smooth muscle . Jap J Physiol 1984; 34: 127-139 . 21 . Johansson B, Hellstrand P . Contractures induced by reversed Na'/Ca" exchange in rat portal vein : Effects of calcium antagonists . J Cardiovasc Pharmacol 10 1987; (suppl 1) : S75-S81 . 22 . Sweetland J, Raeburn D. Effects of potassium channel openers on Cat+ influx, Na + /Ca" exchange and Na+/K + pumping in guinea-pig trachea in vitro . Br J Pharmacol 1989; 98: 882 . 23 . Ahmed F, Foster R W, Small R C, Weston A H . Some features of the spasmogenic actions of
acetylcholine and histamine in guinea-pig isolated trachealis . Br J Pharmacol 1984 ; 83 : 227-233 . 24. Ozaki H, Kojima T, Moriyama T, Karaki H, Urukawa N, Kohama K, Nonomura Y . Inhibition by amiloride of contractile elements in smooth muscle of guinea-pig taenia cecum and chicken gizzard . J Pharmacol Exp Ther 1987; 243 : 370-377 . 25 . Higashi H, Katayama Y, Morita K, North R A . Ouabain augments calcium-dependent potassium conductance in visceral primary afferent neurones of the rabbit. J Physiol 1987 ; 389 : 629-645 . 26. Granges G, Martin B C, Favre H . Comparative effects of ouabain, natriuretic factor and ammonium chloride in the toad urinary bladder . Experentia 1986 ; 42 : 1235-1238 . Date received : 28 July 1989 Date revised : 18 September 1989 Date accepted : 3 November 1989
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0952-0600/90/0003-0121/$10.00
©l990 Longman Group UK Ltd
PULMONARY PHARMACOLOGY
Effects of Altered Availability of Na + on Guinea-pig Airway Smooth Muscle Contractility D . Raeburn Department of Pharmacology and Experimental Therapeutics, Louisiana State University Medical Centre, New Orleans, Louisiana, USA SUMMARY. The effects of altering the availability of sodium ions (Na + ) on contractility of the guinea-pig isolated trachealis was examined using regimens which are reported to inhibit Na + /K + ATPase activity (ouabain), Na + /H + exchange (amiloride, ammonium ion (NH 4 ')) or Na'/Ca2+ exchange (reduced extracellular Na+) . Inhibition of Na + /K + ATPase and reversal of Na'/Ca 2+ exchange resulted in increased "Ca uptake and contraction of the trachealis by voltage-sensitive and voltage-insensitive mechanisms respectively . When Na + /H+ exchange was inhibited by amiloride the tissues relaxed to below their baseline tension. The relaxation was not due to reduced Ca2 + influx. Treatment with NH 4 + produced a contractile response. Reduced extracellular Na + caused a transient contraction as a result of reversal of the normal Na '/Ca"+ exchange process leading to accumulation of Ca 2 + within the cell. Since the effects of amiloride and reduced extracellular sodium were different, it is unlikely that amiloride is acting primarily by inhibiting Na'/Ca 2+ exchange. Amiloride reduced tissue sensitivity to methacholine and KCI without affecting Ca 21 influx . This may involve a secondary stimulation of Na'/Ca 2+ exchange following changes in [Na + ]t . Ouabain also reduced tissue sensitivity to methacholine and KCI . These findings suggest that Na + are important in determining smooth muscle contractility . If NH 4 + is altering pH then, at the concentrations used, the changes in (H + ] were not sufficient to alter responses to the spasmogens .
and cell contraction following membrane depolarization (with Ca" influx) and reversal of the normal Na+/Ca2+ exchange . Na + pump activity has been demonstrated in airway smooth muscle where, as in other tissues, it is ouabain sensitive ." In several cell types a mechanism for Na+/H + exchange also exists where the efflux of H + is coupled to the influx of Na+,9 .1° Although ions can move in either direction, under physiological conditions the large inward Na+ gradient makes the exchanger function as a Na + uptake pathway . 10 Na+/H + exchange has a stoichiometry of one, is electrically neutral, is insensitive to ouabain but is blocked by the diuretic agent amiloride . Ammonium ions (NH4 ' ) have an affinity for the exchange mechanisms similar to Na+ (although the maximum rate of exchange is lower) suggesting that competition may occur between these cations to reduce the efficiency of the exchanger . 9,1o Blockade of the Na+/H+ exchanger will result in an increase in [H+] 1 (i .e . intracellular acidification) . In cardiac muscle, Kim et al 11 suggest that increased [H'] will reduce [Ca"] by a Ca"/H' interaction which results in a negative inotropic response . It is reasonable therefore to assume that reversal of normal Na'/Ca" exchange, inhibition of Na+/H+ exchange and inhibition of Na + /K + ATPase activity will ultimately alter [Ca2+ ] i and hence smooth muscle contractility . Accordingly, the purpose of the
INTRODUCTION Contraction of airway and other smooth muscle is dependent upon an increase in the concentration of free, ionized calcium ions ([Ca 2+ ] i ) in the cytoplasm following influx of extracellular Ca" through specific Ca 2 + channels or the release of Ca 2 + from internal stores . The role of Ca 2 + in the contraction of airway smooth muscle has been reviewed extensively ." There are several mechanisms controlling sodium ion (Na + ) and Ca 21 movements in smooth muscle cells and often the processes are linked either directly, e .g . Na'/Ca" exchange, or indirectly following the inhibition of Na + /H + exchange or Na + /K + pumping . Thus, processes which alter transmembrane Na+ movements will affect the concentration of Na+ inside and outside the cell and subsequently affect [Ca 2 +] I and hence contractility . Na'/Ca 2+ exchange, which normally removes Ca 2 + from the cell, can be reversed by bathing the tissues in a low Na + physiological solution to reduce [Na + ] a and cause the efflux of Na+ down its concentration gradient in exchange for Ca" .' - ' Inhibition of Na + /K + pumping results in increased [Na + ] I which in turn leads to an increase in [Ca 2+ ] 1 Address correspondence to : D . Raeburn, Biological Research, Dagenham Research Centre, Rhone-Poulenc Ltd, Rainham Road South, Dagenham, Essex RM10 7XS, England . 121
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Pulmonary Pharmacology
present study was to examine the effects of these diverse ways of altering the availability of Na + on muscle cell contractility in the guinea-pig trachealis .
METHODS Male, Hartley guinea pigs (340-500 g ; HarlanSprague Dawley Inc ., Indianapolis, IN, USA) were killed by stunning and bleeding . The trachea was removed, placed in a modified Krebs-Henseleit solution (MKH) and dissected free of connective tissue . Tissues were prepared for the experimental protocols as follows . Tension studies The trachea was slit through the cartilage along its longitudinal axis and strips consisting of two adjacent rings of cartilage were cut . Some contractile agonists can stimulate the release of bronchoactive agents from the airway epithelium . 12-14 To exclude these indirect effects the epithelium was removed from each preparation . The tracheal strips were placed in water-jacketed tissue baths (37°C) containing MKH and gassed with 5% CO 2 in oxygen. Changes in the isometric force of contraction were measured using force-displacement transducers (FT03C, Grass Instruments, Quincy, MA, USA) . The tissues were equilibrated for 60 min under the optimum applied load of 2 g and washed at 15 min intervals with fresh MKH . The composition of the MKH was as follows (mM) : NaCl 113, KCl 4 .8, CaC1 2 2 .5, KH 2PO4 1 .2, MgSO4 1 .2, NaHCO 3 25 and glucose 5 .7 . A low Na-MKH was prepared by substituting NaCl with sucrose (246 mM) which is isosmolar with normal, Na + containing, MKH . The sucrose itself is without pharmacological effects . Concentration-response curves to methacholine, KCl, ouabain, amiloride and NH4' were constructed by the cumulative addition of the drug . In some experiments a response curve to methacholine or KCl (control curves) was constructed and when the maximum response had been achieved the tissues were washed with fresh MKH until responses had returned to baseline . Then ouabain (1 or 100 MM), amiloride (1 or 100 µM), NH4' (0 .1 or 1 mM) or vehicle was added to the tissue bath 30 min prior to the construction of a second curve in the presence of the test substance . The responses obtained were expressed as a percentage of their appropriate pretreatment control response . The effect of reducing [Na+] o on basal tone and on responses to amiloride, KCl and methacholine were examined by changing the bathing fluid (after equilibration in normal MKH) to the low NaMKH and the responses obtained were compared with control .
"Ca uptake studies The La" resistant 45 Ca content was measured as previously described for this preparation 15 as modified by Raeburn et al ." The epithelium was removed as for the tension studies . Briefly, cartilage-free strips of trachealis were equilibrated in MKH for 30 min then transferred to MKH or low Na-MKH to which had been added 45 Ca (0.5 µCi/ml) . The tissues were incubated in the radioactive `loading' solution for 10 min (methacholine) or 20 min (low Na, ouabain, KCl) at 37°C . Where appropriate, ouabain, methacholine or KCl was added 5 min after the addition of 45 Ca. Tissues were incubated for 30 min with amiloride in the loading solution . At the end of the experiment the tissues were rinsed (5 s) in Trisbuffered MKH 16 to prevent precipitation of phosphate or bicarbonate prior to incubation for 60 min (3 washes) at 0 .5°C in Ca t+ free Tris-MKH containing La" (80 mM) and gassed with 100% oxygen . Liquid scintillation counting was used to determine the radioactivity present in each tissue sample . 45 Ca uptake was expressed in nmol/mg of protein using the Lowry method .' 7 Statistical analysis The results are presented as mean + SEM. pD 2 (- log EC50 M) values were calculated from linear regression analysis of probit transformed data . Differences between values for control and treated tissues were assessed for significance using a one-way analysis of variance (ANOVA), p<0 .05 was accepted as significant . Drugs The following drugs were used : methacholine chloride, amiloride hydrochloride, verapamil hydrochloride, ouabain and lanthanum chloride (Sigma Chemical Co ., St Louis, MO, USA) and ammonium chloride (Baker Chemical Co ., Phillipsburg, NJ, USA) . Drug solutions were prepared in distilled, de-ionised water on a daily basis . Radiolabelled Ca t+ (45 CaC1 2) was obtained from New England Nuclear, Boston, MA, USA . The scintillant used was `Ready Gel' (Beckman, Fullerton, CA, USA) .
RESULTS Effects of amiloride Amiloride (0 .1-100µM) produced a concentrationdependent relaxation of guinea-pig trachealis which was maximal at 100 µM (Figure 1, Table 1) . The cumulative addition of methacholine (10 nM100 µM) or KCI (4-100 mM) produced con-
Sodium and Airway Contractility
Table 1 . The effects of amiloride or ouabain on the contractility of guinea-pig trachealis in vitro
100-+ C 0 O-
80 -
x ro
60 -
a- 40 0
123
Agonist
n
pD2
Max . response (g)
Amiloride Ouabain
7 7
4.60+_0.03 6.35+0.21
-0 .234+0 .06 +0 .538±0 .18
20 0I 7
I
1
1
6
5
4
maximum force of contraction (Table 2) . The higher concentration of ouabain (100,W) substantially reduced the maximum response to KCl and prevented calculation of a pD 2 (Table 2).
-log [amiloride ] (M) Effects of reduced [Na +],, Fig. 1-Concentration-response curve to the cumulative addition of amiloride (0 .1-100 pM) plotted as percentage maximum relaxation of the guinea-pig trachealis, n = 7.
centration-dependent increases in the force of contraction . The drug vehicle was without effect on tissue reactivity . Amiloride (100 µM, 30 min pretreatment) reduced tone and tissue sensitivity to methacholine and KCl compared with the control (Table 2) . A lower concentration of amiloride (1 µM) was without effect . A small increase in the maximum force of contraction to methacholine and KCl was seen following amiloride treatment but this probably reflects the reduction in basal tone produced by amiloride (Table 2) . Effects of ouabain Ouabain (0 .01-100 pM) produced a concentrationdependent increase in the force of contraction of the trachealis (Fig . 2, Table 1) . No further contractility was seen on increasing the concentration of ouabain . Ouabain (1 or 100 µM, 30 min pretreatment) significantly reduced tissue sensitivity to methacholine without affecting the maximum response (Table 2) . Tissue sensitivity to KCl was slightly reduced by ouabain (1 pM, 30 min pretreatment) without affecting the
r 8
I
I
I
7 6
5
4
1
-log [ouabain I (M) Fig . 2-Concentration-response curve to the cumulative addition of ouabain (0 .01-100 pM) plotted as percentage maximum contraction of the guinea-pig trachealis, n = 7 .
Transferring the tissues from the regular MKH to the low Na-MKH produced a biphasic response . An immediate contraction (405 ± 53 mg, lasting 333 ± 34 s, n = 16) was seen which was generally followed by a sustained relaxation to below the original baseline (Fig . 3A) . In some tissues the secondary relaxation developed into a tonic contracture which was of lesser magnitude than the initial phase of the response (Fig . 3B) . The re-addition of Na + (NaCl, 100 mM) to tissues incubated in low Na-MKH resulted in a further small relaxation, the original level of tone was not restored until the tissues were washed in normal MKH . Amiloride (100 pM, 30 min pretreatment) and verapamil (1 uM, 30 min) had little or no effect on the magnitude of the immediate response (75± 15% and 82± 14% respectively of control) or on the time course (n=5) . Ouabain (100µM, 30 min) markedly depressed the contraction to low Na-MKH to 38±3% of control (n=3) . Amiloride (1-100µM, n=5) relaxed tissues in the presence of reduced [Na+] .. The pD 2 value of 6.07±0 .13 for amiloride in reduced [Na+] o indicated increased sensitivity of the tissue to amiloride in the low Na-MKH . The maximum relaxation to amiloride (178+31 mg) obtained in low Na-MKH was not different from the normal MKH control (Table 1) . In reduced [Na +]. the responses to methacholine (5 µM, approximately EC75) and KCl (75 mM, EC BO) were markedly depressed (Fig . 4) . The overall shape and time course of the response to methacholine was not altered but the maximum force of contraction only achieved 22 ± 3 of the regular MKH control, n = 6 . In the low NaMKH the response to KCl in some tissues more clearly exhibited distinct phasic and tonic components and achieved only 59+3% of control, n=6 . The re-addition of Na + produced a small relaxation followed by an abrupt contraction returning the tissue to its pretreatment control level of tone in the case of tissues contracted with methacholine . In KCl-contracted tissues the re-addition of Na + did not produce relaxation but resulted in a slow, steady return of the level of tone approaching that seen in control tissues in regular MKH .
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Pulmonary Pharmacology
Table 2. The effects of amiloride or ouabain on responses to methacholine or KCI in guinea-pig trachealis in vitro Methacholine max . (g)
n
pD 2
Control 1 µM 100 MM
13 7 6
6 .22±0 .06 6 .06±0 .17 5 .84±0 .07*
Control 1 pM 100µM
10 5 5
6 .14±0 .07 5 .87±0 .31* 5 .54±0 .12*
Amiloride 1 .16±0 .17 1 .50±0.19 1 .53±0 .27 Ouabain 1 .01±0.10 0.72±0.09 1 .08±0.36
KCI pD 2
max . (g)
9
1 .67+0 .02 n.d . 1 .40±0 .02*
1 .04+0 .14 n .d . 1 .20±0.16
12 6 6
1 .77+ _ 0 .09 1 .53+ _ 0 .06* n.d .
0 .84+_0 .12 0.85+_0.05 0 .20±0 .06
n 9
* p < 0 .05 vs control n .d . not determined A
A
W
• Low Na'
K CI
• Na'
B
MCh 5i4
•Low Na'
• (ow Na+ MKH Fig . 3-Representative traces showing the effects of changing the bathing fluid to a reduced Na+ solution in the guinea-pig trachealis . Panel A: Initial transient contractile response commonly followed by a secondary relaxation to below the original baseline . Panel B : A response showing the occasional development of a secondary contracture of reduced magnitude .
• Na'
• MCh
10 min
Fig . 4-The effects of low Na bathing solution on responses to KCI (Panel A) and methacholine (MCh, Panel B) in the guineapig trachealis . Tissues were contracted in normal MKH, washed back to baseline (W) then the bathing fluid changed to the low Na-MKH . When the responses stabilised the tissues were rechallenged with KCI or MCh . At the peak of the second response (in low Na+) Na + were added back .
100-1 Effects of NH,' The addition of NH4 + (0 .1-5 mM) produced concentration-dependent increases in the force of contraction to a maximum of 486±62 mg (Fig . 5) . Since concentrations of NH, + greater than 10 mM produced responses which were complex in nature where, for any given concentration of the ion, contraction-relaxation cycles were seen, it was thought reasonable to assume that the maximum contractile event occurred with NH4' (5 mM) . Pretreatment of the tissues with NH,' (0 .1 or 1 mM) did not affect responses to methacholine or KCI (Table 3) .
0 80.m 60C_ .6...
0 40U
200I
4
I
I
I
3 -tog [NH4+ ] (M)
Fig. 5-Concentration-response curve to the cumulative addition of NH,+ ion (0 .1-5 mM) plotted as percentage maximum contraction in the guinea-pig trachealis, n = 6 .
"'Ca uptake studies Amiloride (100 µM, 30 min pretreatment) did not affect "Ca uptake into the trachealis . Transferring the tissues to the low Na-MKH, treatment with KCl (40 mM) or ouabain (100,uM) increased significantly "Ca uptake over basal (Table 4) . It was not possible
to assess the effects of low Na-MKH or ouabain on KCl-induced 4S Ca uptake since both treatments on their own produced greater uptake than was seen with KCI (Table 4) . Methacholine did not stimulate 4S Ca uptake confirming previous reports (data not shown) .
Sodium and Airway Contractility Table 3. The effects of NH,' on responses to methacholine or KCl in the guinea-pig trachealis in vitro n
Max . response (g)
pD2
Control NH4+(0 .1 MM) (1 .0 MM)
10 5 5
Control NH4+(0.1 MM) (1 .0 MM)
6 3 3
Methacholine 6.55±0 .07 6 .63±0 .05 6 .50±0 .10 KCl 1 .69±0 .04 1 .67±0 .04 1 .74±0 .05
1 .16±0 .17 1 .09±0 .08 1 .19±0 .12 0 .85±0 .10 0 .95±0 .13 0 .87±0 .12
Table 4 . The effects of various treatments on 45 Ca uptake into the guinea-pig trachealis in vitro Treatment
n
Basal KC1 (40 mM) Amiloride (100 pM) Ouabain (100 pM) Low [Na+l o
5 5 5 4 4
45 Ca content (n mol/mg protein)
74+12 127± 13* 75±6 937±83* 214±44*
* p < 0 .05 vs basal
DISCUSSION The first conclusion to be drawn from this study is that the various treatments used to modulate Na + movements across the cell membrane produce different effects depending on the Na + transport process under investigation . Inhibition of Na + /K + pumping or Na + /Ca' + exchange results in contraction of the trachealis via a voltage dependent and a voltage independent mechanism . Inhibition of Na + /H + exchange with amiloride on the other hand produces relaxation . Treatment with NH,' was not equivalent to treatment with amiloride suggesting a mechanism of action other than interference with Na+/H+ exchange by NH,' . It is possible that this ion produced its effects by directly increasing [H + ] i in turn leading to an increase in [Ca 2+] i as the cell attempts to return to its basal pH . The different effects seen with amiloride and the reduction in [Na+] o suggests that, under the present circumstances, amiloride is acting primarily on Na+/H + exchange with lesser effects on Na+/Ca' + exchange, an effect which has been reported to occur at higher concentrations of amiloride . t e The increase in contractility following inhibition of Na + /K + ATPase and Na'/Ca" exchange is accompanied by the influx of extracellular Ca" as indicated by the radiolabelled Ca` data . These findings are similar to those reported by Van Breemen et al 19 for intestinal and vascular smooth muscle . In the case of Na'/Ca" exchange the present findings confirm those of Bullock et al' in bovine trachealis and Kawanishi et al20 in the guinea pig . In the present study, the influx of Ca l ' causing contraction in reduced (Na + ],, is probably not via voltage-dependent Cal ' channels since it is relatively unaffected by verapamil. This confirms the findings of Johannson
125
and Hellstrand 2i in rat portal vein . Cal' influx is involved since extracellular Ca t+ is required for the contractile response to reduced [Nal]o .21 Conversely, ouabain-stimulated Ca t+ influx occurs via voltage-gated channels since the contraction is significantly inhibited by nifedipine and verapamil . 22 Amiloride, at the highest concentration examined, reduced tissue sensitivity both to KCl and methacholine without affecting maximum responses . This suggests (since tissue sensitivity is assessed during the sustained, tonic phase in cumulative concentrationresponse studies) that amiloride is reducing the availability of the activator Cat + required for this phase of contraction i .e . extracellular Ca 2+ .23 This action of the relaxant is not due to a reduced Ca l ' influx through voltage sensitive channels (since there was no effect on 45 Ca uptake) but may be the result of the reduced [Na + ]; stimulating Na'/Ca 2+ exchange to reduce [Ca 2+]1 as [Na+]i again is increased . The effects, if any, of amiloride on Ca l ' efflux in smooth muscle are unknown . It has been suggested (see Introduction) that inhibition of the Na+/H + exchanger produces intracellular acidification due to a retention of H+ and that the protons could compete with Ca" for intracellular cationic binding sites . More Ca 21 would thus be subject to the Ca 21 removal mechanisms and in cardiac muscle a negative inotropic response would be seen,"t A reduced contractility in airway smooth muscle following amiloride treatment could be explained by this effect . It has been suggested previously that amiloride can directly inhibit myosin light chain kinase activity in intestinal muscle but has no effect on calmodulin activation . 24 This action of the relaxant cannot be ruled out in the present study . Like amiloride, ouabain reduced tissue sensitivity to methacholine and KCl and, at the highest concentration, reduced the magnitude of the response to KCl . This is also the case in guinea-pig ileum ."9 Why inhibition of Na + /K + ATPase activity should have this effect is unclear . Van Breemen et a1 19 speculate that the inhibition may be due to blockade of Ca" influx (not the case in the present study) or sequestration of cytosolic Ca 2 + away from the myofilaments . Another possible explanation may be that the already elevated [Ca 2+ ] i produced by inhibiting Na + /K + pumping is preventing further Ca 2 + influx by stimulating Na'/Ca 2+ exchange . It is also possible that refilling of the cytosolic Ca 2 + stores is blocked . In visceral afferent neurones in the rabbit 25 ouabain can induce membrane hyperpolarization due to increased K + conductance . Were this effect to occur in smooth muscle a reduced contractility would be expected . Exposure to NH 4 + , while in itself causing contraction, did not affect tissue sensitivity or maximum responses to methacholine or KC1 . This suggests that NH4' is not affecting Ca 21 influx . The contractile response to NH4' is unlikely to be a result of
1 26 Pulmonary Pharmacology
intracellular acidification but more likely due to less efficient Na'/Ca" exchange (due to competition for the mechanism) leading to increased [Ca 2+]1 NH4 ' has been reported to be a weak inhibitor of Na + /K + ATPase . 26 This action cannot be ruled out . In conditions of reduced [Na'],, several interesting phenomena were observed . Tissue sensitivity to amiloride was substantially increased which would reasonably be expected to be the case . The contraction following the reduction in [Na']., while not affected by Cat+ entry blockers or amiloride, was inhibited by ouabain. This probably reflects ouabain causing an increase in [Na + ]i in turn stimulating Ca" loss . The overall reduced effect reflecting the stronger inward Na + driving force (due to concentration gradient effects) occurring on blockade of the Na + /K + pump overcoming the reduction in [Na'].. Inhibition of Na'/Ca" exchange by reducing [Na+] o markedly reduced tissue responsiveness to methacholine but had a lesser effect on KC1 . Brading et a1 4 suggest that reducing [Na + ] o prevents refilling of the intracellular Ca t+ store in intestinal muscle thereby suppressing the activity of carbachol . This is presumed to occur since filling of the intracellular Ca" store is by a Na'/Ca 2+ exchange process . If [Na+] in the store falls then Ca" cannot enter . Since it is established that the response to muscarinic agonists is less dependent on the influx of extracellular Cat+ than the response to KCI' , ' the results suggest that inhibition of Na'/Ca" exchange has a greater effect on intracellular Ca" mobilization . Support for this view comes from the lack of change to the overall shape and time course but reduced maximum of the response to methacholine in reduced [Na'].. Since Na'/Ca 2+ exchange affected Ca t+ influx by a voltage-independent mechanism while the sustained phase of a response is blocked by calcium antagonists, it would be expected that the tonic component of the response to methacholine would have been less affected . This suggests utilisation of more than one pathway for extracellular activator Cat + being involved in response maintenance . It must be acknowledged, however, that although less depression of the maximum response was seen, the time to half maximum was substantially increased for KCl which may reflect slow entry of Ca" via the `leak pathway' . Brading et al' also suggest that reduced [Na']. may alter the membrane potential (encourage repolarization) and render depolarization more difficult . In summary, Na + play an important role in the development and maintenance of tone in the guineapig trachealis in vitro . The various Na'/Ca 2 + interactions are both direct, and more commonly, indirect and very difficult to delineate accurately . Clearly this is an important area for further study and, with time, more informative data will emerge . As stated in the introduction, for the sake of simplicity the influence of the epithelium was not examined in this study . Since
the epithelium is a site of active Na + transport in many organ systems including the respiratory tract, these indirect effects may also have important influences on the underlying airway smooth muscle . This aspect has been studied recently' where it was found that modulation of the activity of the epithelial Na + /K + pump can modulate airway responsiveness to some contractile stimuli. References 1 . Rodger I W . Excitation-contraction coupling and uncoupling in airway smooth muscle. Br J Clin Pharmacol 1985 ; 20: 255S-266S . 2 . Rodger I W . Calcium ions and the contraction of airway smooth muscle . In : Kay A B (ed) . Asthma : Clinical Pharmacology and therapeutic progress . Blackwell, Oxford, 1986 pp 114-127 . 3 . Reuter H, Blaustein M P, Haeusler G . Na-Ca exchange and tension development in arterial smooth muscle . Phil Trans R Soc B 1973 ; 265 : 78-94 . 4 . Brading A F, Burnett M, Sneddon P . The effect of sodium removal on the contractile response of the guinea-pig taenia coli to carbachol . J Physiol 1980 ; 306 :411-420 . 5 . Bullock C G, Fettes J J F, Kirkpatrick C T . Tracheal smooth muscle-second thoughts on sodium-calcium exchange. J Physiol 1981 ; 318 : 46 . 6 . Souhrada M, Souhrada J F . The direct effect of temperature on airway smooth muscle . Resp Physiol 1981 ; 44 : 311-323 . 7 . Chideckel E W, Frost J L, Mike P, Fedan J S . The effect of ouabain on tension in isolated respiratory tract muscle of humans and other species . Br J Pharmacol 1987; 92: 609-614 . 8 . Raeburn D, Fedan J S . The effects of alterations in electrogenic Na'/K+ pumping in guinea-pig isolated trachealis: Their modulation by the epithelium . Br J Pharmacol 1989 ; 98 : 343-350 . 9 . Mahnensmith R L, Aronson P S . The plasma membrane sodium-hydrogen exchanger and its role in physiological and pathological processes. Circ Res 1985 ; 56 : 773-788 . 10 . Frelin C, Vigne P, Barbry P, Lazdunski M . Molecular properties of amiloride action and of its Na+ transporting targets . Kidney Intl 1987 ; 32 : 785-793 . 11 . Kim D, Cragoe E J, Smith T W . Relations among sodium pump inhibition, Na-Ca and Na-H exchange activities and Ca-H interaction in cultured chick heart cells . Circ Res 1987 ; 60 : 185-193 . 12 . Fedan J S, Hay D W P, Farmer S G, Raeburn D . Epithelial cells: Modulation of airway smooth muscle reactivity . In : Rodger I W, Barnes P J, Thompson N C (eds) . Asthma : Basic mechanisms and clinical management . Academic Press, New York, 1988 pp 143-159 . 13 . Vanhoutte P M . Epithelium derived relaxing factor : Myth or reality . Thorax 1988; 43 : 665-668 . 14 . Raeburn D . Epithelium, eicosanoids and airway reactivity . Gen Pharmacol 1990 ; 21 : 11-16. 15 . Raeburn D, Rodger I W . Lack of effect of leukotriene D4 on Ca-uptake in airway smooth muscle . Br J Pharmacol 1984 ; 83: 499-504 . 16 . Raeburn D, Hay D W P, Fedan J S . Calcium uptake into guinea-pig trachealis : The effect of epithelium removal . Cell Calcium 1987 ; 8 : 429-436. 17 . Lowry 0 H, Rosebrough N J, Farr A L, Randell R J . Protein measurement with Folin phenol reagent . J Biol Chem 1951 ; 193 : 265-275 . 18 . Siegl P K, Cragoe E J, Trumble M J, Kaczorowski G J . Inhibition of Na'/Ca2 + exchange in membrane vesicle and papillary muscle preparations from guinea-
Sodium and Airway Contractility pig heart by analogues of amiloride . Proc Natl Acad Sci 1984 ; 81 : 3238-3242 . 19. Van Breemen C, Aaronson P, Loutzenhiser R . Sodium-calcium interactions in mammalian smooth muscle . Pharmacol Rev 1979; 30: 167-208 . 20 . Kawanishi M, Baba K, Tomita T . Effects of Na removal and readmission on the mechanical response in the guinea-pig tracheal smooth muscle . Jap J Physiol 1984; 34: 127-139 . 21 . Johansson B, Hellstrand P . Contractures induced by reversed Na'/Ca" exchange in rat portal vein : Effects of calcium antagonists . J Cardiovasc Pharmacol 10 1987; (suppl 1) : S75-S81 . 22 . Sweetland J, Raeburn D. Effects of potassium channel openers on Cat+ influx, Na + /Ca" exchange and Na+/K + pumping in guinea-pig trachea in vitro . Br J Pharmacol 1989; 98: 882 . 23 . Ahmed F, Foster R W, Small R C, Weston A H . Some features of the spasmogenic actions of
acetylcholine and histamine in guinea-pig isolated trachealis . Br J Pharmacol 1984 ; 83 : 227-233 . 24. Ozaki H, Kojima T, Moriyama T, Karaki H, Urukawa N, Kohama K, Nonomura Y . Inhibition by amiloride of contractile elements in smooth muscle of guinea-pig taenia cecum and chicken gizzard . J Pharmacol Exp Ther 1987; 243 : 370-377 . 25 . Higashi H, Katayama Y, Morita K, North R A . Ouabain augments calcium-dependent potassium conductance in visceral primary afferent neurones of the rabbit. J Physiol 1987 ; 389 : 629-645 . 26. Granges G, Martin B C, Favre H . Comparative effects of ouabain, natriuretic factor and ammonium chloride in the toad urinary bladder . Experentia 1986 ; 42 : 1235-1238 . Date received : 28 July 1989 Date revised : 18 September 1989 Date accepted : 3 November 1989
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