Clinical Science (1992) 83, 331-336 (Printed in Great Britain)
331
Effects of Ihydroxytryptamine and Shydroxytryptamine receptor agonists on ion transport across mammalian airway epithelia A. GRAHAM,
E. W. F. W. ALTON
and D. M. GEDDES
Ion Transport laboratory, National H e a r t and Lung Institute, London, U.K. (Received 13 March/l5 May 1992; accepted 27 May 1992)
1. IHydroxytryptamine has been suggested as a candidate for an endogenous inhibitor of airway sodium transport. Amiloride, an inhibitor of epithelial sodium channels, has therapeutic potential in disorders of airway ion transport such as cystic fibrosis, but its duration of action in vivo is short. 5-Hydroxytryptamine and related compounds have been studied to investigate whether any might be a useful alternative to amiloride for clinical use, and to further assess the possible physiological role of 5-hydroxytryptamine in the regulation of airway ion transport. 2. Sheep tracheal epithelium was mounted in Ussing chambers under short-circuit conditions. Mucosal application of 5-hydroxytryptamine resulted in an immediate, reversible, concentration-related decrease in the short-circuit current, maximal with 38% inhibition of the short-circuit current at 25 mmol/l. This response was completely inhibited by pretreatment of tissues with mucosal amiloride (100 pmol/l). These features are consistent with a direct effect of 5-hydroxytryptamine on amiloride-sensitive sodium channels. Similar results were obtained in a limited number of studies using human bronchial epithelium. 3. The effects of mucosal addition of a range of 5-hydroxytryptamine agonists were studied to determine if any was a more potent blocker of amiloridesensitive sodium transport than 5-hydroxytryptamine. The 5-HT3 agonist 2-methyl-5-hydroxytryptamine had no effect on the short-circuit current at concentrations of up to 5mmol/l. The 5-HT,, agonist sumatriptan had no effect at concentrations below 5 mmol/l and at 5 mmol/l had only a transient effect. The 5-HT,, agonists buspirone and 8-hydroxy-2(di-n-propy1amino)tetralin and the 5-HT2 agonist a-methyl-5-hydroxytryptamine were all more potent inhibitors of the short-circuit current than 5-hydroxytryptamine, but, although their effects were reduced by pretreatment of tissues with mucosal amiloride (lOOpmol/l), none had a specific effect on the amiloride-sensitive sodium current. The effect of buspirone on the short-circuit current was also studied
after mucosa. sodium substitutm, and although its effect was again reduced, significant inhibition of the short-circuit current still occurred, indicating that ion transport processes other than sodium absorption were being affected. 4. Mucosal application of ondansetron, an antagonist a t the 5-HT3 receptor (an ion channel), also produced a dose-related inhibition of the short-circuit current that was not mediated via the amiloride-sensitive sodium current. Pretreatment of tissues with ordansetron had no effect on the subsequent response to 5-h ydrox ytr yptamine. 5. We conclude that mucosally applied 5-hydroxytryptamine specifically inhibits amiloride-sensitive sodium transport in airway epithelia, but with a median inhibitory concentration too high for it to be therapeutically useful. The high median inhibitory concentration also indicates that 5-hydroxytryptamine is unlikely to be a physiological regulator of sodium channels. Screening a number of 5-hydroxytryptamine receptor agonists has failed to identify a more potent inhibitor of sodium transport which may have had therapeutic potential. INTRODUCTION
Active ion transport across airway epithelia is likely to be a major factor determining the depth and composition of airway surface liquid (ASL) [l]. The dominant ion-transport process in the conducting airways of several species including man is sodium absorption [2-4]. This is mediated by both amiloride-sensitive and amiloride-insensitive pathways [S, 61. Alterations in the volume of the ASL resulting from changes in transepithelial ion transport may influence mucociliary clearance [MCC]. In cystic fibrosis (CF), in which sodium absorption is increased two- to three-fold [7], MCC can be improved with nebulized amiloride in uiuo [8]. Pilot studies using nebulized amiloride in patients with CF suggest that it may delay the progression of lung damage and reduce sputum viscosity [9]. How-
Key words: airway, Ehydroxytryptamine, ion transport. Abbreviations: ASL, airway surface liquid: CF, cystic fibrosis, G, conductance; SHT, Ehydroxytryptamine; I,,, short-circuit current; IC,,, median inhibitory concentration; MCC, mucociliary clearance; M H - D P A T , 8-hydroxy-2jdi-n-propy1amino)tetralin; PD, potential difference. Correspondence: Dr A. Graham, Ion Transport Laboratory, National Heart and Lung Institute, Manresa Road, London SW3 6LR, U.K.
332
A. Graham et al.
ever, the duration of action of inhaled amiloride is short, the effects on MCC lasting up to 40min [8] and on airway potential difference less than 30min [101. Frequent administration is therefore required and good compliance is thus difficult to achieve. A longer-acting compound with similar effects would have useful therapeutic potential. One approach to identifying such an agent is to investigate endogenous compounds which may have a physiological role in the regulation of airway epithelial sodium transport. 5-Hydroxytryptamine (5-HT) has been suggested as such a candidate after the demonstration that it inhibited amiloridesensitive sodium transport in the baboon bronchus [113. Furthermore, 5-HT-containing cells are numerous within the upper airways of a number of larger mammals, including the sheep and monkey [l2]. The pharmacology of 5-HT has been extensively studied and several agonists and antagonists to the different receptor sites, some of which are now in clinical use, have been developed [13-151. The effects of 5-HT are mediated through the protein kinase A (5-HT1,, 5-HT1,, 5-HT1, and 5-HT4 receptors) and protein kinase C (5-HTIc and 5-HT, receptors) pathways via G proteins [16, 171 and also by direct activation of ion channels (5-HT3 receptor) [18]. Inhibition of sodium transport by stimulation of protein kinase C and elevation of intracellular calcium levels has been demonstrated in mammalian airways [19, 201. Thus, 5-HT might influence sodium transport by both direct channeland receptor-mediated mechanisms. In this study the effects of 5-HT, 5-HT receptor agonists and a 5-HT receptor antagonist on airway ion transport have been investigated using sheep tracheal epithelium, a predominantly sodiumabsorbing epithelium [4, 211, the ion transport characteristics of which are comparable with those of human bronchial epithelium [6]. A limited number of experiments have been performed using human bronchial epithelium, when available, for comparison. METHODS Ussing chamber studies
Sheep trachea were obtained fresh from a local abbatoir and were immersed immediately in cold (4°C) Krebs-Henseleit solution of composition (mmol/l): Na+, 145.0; C1-, 126.0; K + , 5.9; Ca2+,2.5; MgZ+, 1.2; HCO;, 26.0; PO:-, 1.2; SO:-, 1.2; glucose, 5.6. The epithelium was dissected free of underlying tissues and was mounted in Ussing chambers of area 1.28cm2. Human bronchi were obtained from whole diseased lungs (primary pulmonary hypertension and sarcoidosis) removed at transplantation and were also immersed immediately in cold Krebs-Henseleit solution:. For the main bronchi the epithelium was partially dissected from underlying tissues, but the smaller bronchi
were simply opened longitudinally before mounting. Both were mounted in Ussing chambers of area 0.28 cm2, All tissues were bathed with KrebsHenseleit solution at 37°C (pH 7.4) and bubbled through gas lifts with a 95% 0,/5% COz mixture. The potential difference (PD) across the tissue was measured using lmol/l KC1/2% agar bridges connected to a DVC-1000 voltage clamp (World Precision Instruments) by calomel electrodes (Russell pH Limited). The offset of the electrodes was measured before tissue mounting and suitable adjustments were made to recorded values. Short-circuit current (Z,J was measured using 0.9% NaC1/2% agar bridges connected to the clamp by Ag/AgCl electrodes. The tissues were maintained continuously under short-circuit conditions except for brief (15 s) intervals to measure PD. Conductance ( C ) was calculated from Ohm's law. Tissues with a conductance of greater than 10mS/cm2 (sheep trachea) and 14mS/cm2 (human bronchus) [3] were excluded. Once tissues were electrically stable, defined by a rate of rise in I,, of less than 3pA/h (sheep trachea) and 0.4 pA/h (human bronchus), experimental interventions were commenced. Drugs and chemicals
In sodium substitution experiments, sodium chloride and sodium bicarbonate were substituted with equimolar choline chloride and choline bicarbonate, respectively, in the Krebs-Henseleit solution. Amiloride was a gift from Merck, Sharp and Dohme, and sumatriptan and ondansetron were gifts from Glaxo Group Research (Greenford, Middx., U.K.). 5-HT creatinine sulphate and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OHDPAT) were obtained from Sigma Chemical Co. (Poole, Dorset, U.K.). Buspirone, a-methyl-5-HT maleate and 2-methyl-5-HT maleate were obtained from Semat Technical (U.K.) Ltd. Amiloride was dissolved in Krebs-Henseleit solution and was applied in a dilution of 1 in 10. 5-HT was dissolved in Krebs-Henseleit solution at 37°C and was applied in a range of dilutions from 1 to 100 to 1 in 1 (i.e. the Krebs solution bathing one side of the tissue was substituted with that containing 5-HT). 8OH-DPAT was dissolved in Krebs-Henseleit solution and was applied in dilutions of 1 in 500 to 1 in 5. a-Methyl-5-HT, 2-methyl-5-HT and ondansetron were dissolved in distilled water and were applied in dilutions of 1 in 1000 and 1 in 100. Buspirone and sumatriptan were dissolved in distilled water and were applied in dilutions of 1 in 2000 to 1 in 20. Experimental protocols
In dose-response studies control tissues were treated with diluent or by substitution of the appropriate volume of Krebs-Henseleit solution. In experiments in which tissues were pretreated with amiloride this was added to the mucosal surface in
Wydroxytryptamine and airway ion transport
a concentration of 100pmol/l 5 min before the drug under investigation which was then added to both the amiloride-pretreated tissue and to a paired nonpretreated tissue from the same animal. In experiments using mucosal sodium substitution the tissues were first mounted in standard Krebs-Henseleit solution until bioelectrically stable. The mucosal surface was then rinsed several times with sodiumfree Krebs-Henseleit solution, which was then used to replace the mucosal bathing fluid. The 0.9% NaC1/2% agar current bridges were replaced with 0.9% choline chloride/2% agar bridges at this time. Bioelectric properties were stable after a further 15-20 min at which time the drug under investigation was added.
1
2o V
'
10-5
I
lo-'
10-3 5-HT concn. (mol/l)
lo-'
10-1
100
Statistics
The Mann-Whitney U-test was used to compare means. The Wilcoxon rank sum test was used to compare the effects of an intervention against baseline. Results are expressed as means with SEM in parentheses for convenience.
333
L*
80
--.4
I
5 60
-?I % M n
W
RESULTS Baseline values
Baseline bioelectric properties for sheep tracheal epithelium (n=267 tissues, 68 sheep) were: PD, -9.6(0.3) mV; I,,, 51 (2) pA/cm2; G, 5.8(0.1)mS/cm2. For human bronchial epithelium (n= 11 tissues, two individuals) the corresponding values were: PD, - 3.8 (0.5)mV; I,, 30(8) pA/cm2; and G, 8.0(0.7) mS/cm2. Effects of tissue pretreatment protocols
Addition of amiloride (100 pmol/l, n = 32) in sheep trachea resulted in a fall in I,, of 20(2)pA/cm2 [38% of the baseline current of 53(4)pA/cm2 in these tissues] and a fall in G of 0.3(0.1)mS/cmz (5% of the baseline G of 5.8(0.3)mS/cm2]. In human bronchial epithelium muscosal amiloride (100 pmol/l, n=2) produced falls in I,, of 8 and 21 pA/cm2 from 32 and 44 pA/cm2, respectively, with no change in G. In sheep trachea mucosal sodium substitution resulted in a fall in I,, of 21 (3) pA/cm2 (n= 10) [45% of the baseline current of 47 (8) pA/cm2 in these tissues] and a fall in G of 1.2(0.2)mS/cm2 [21% of the baseline G of 5.6 (0.4) mS/cm2]. Effects of 5-HT on biolelectric properties in sheep tracheal epithelium
Mucosal addition of 5-HT resulted in an immediate, concentration-related decrease in I,, which was maximal with 38(7)% (n=6) inhibition of I,, at 25 mmol/l and a median inhibitory concentration (IC5,,) of 5mmol/l (Fig. la). This was associated with a concentration-related decrease in G of up to
20
0 I
I
-5
10-4
I
10-3
10-2
10-1
5-HT concn. (mol/l)
Fig. 1. Doseresponse curves showing the effect of mucosal 5-HT on ( a ) /sc and (b) G in sheep tracheal epithelium. Tissue numbers are indicated at each point. Error bars indicate SEM and where not shown fall within the symbol. All results have been corrected for the effects of controls. Statistical significance: *P
331
Effects of Ihydroxytryptamine and Shydroxytryptamine receptor agonists on ion transport across mammalian airway epithelia A. GRAHAM,
E. W. F. W. ALTON
and D. M. GEDDES
Ion Transport laboratory, National H e a r t and Lung Institute, London, U.K. (Received 13 March/l5 May 1992; accepted 27 May 1992)
1. IHydroxytryptamine has been suggested as a candidate for an endogenous inhibitor of airway sodium transport. Amiloride, an inhibitor of epithelial sodium channels, has therapeutic potential in disorders of airway ion transport such as cystic fibrosis, but its duration of action in vivo is short. 5-Hydroxytryptamine and related compounds have been studied to investigate whether any might be a useful alternative to amiloride for clinical use, and to further assess the possible physiological role of 5-hydroxytryptamine in the regulation of airway ion transport. 2. Sheep tracheal epithelium was mounted in Ussing chambers under short-circuit conditions. Mucosal application of 5-hydroxytryptamine resulted in an immediate, reversible, concentration-related decrease in the short-circuit current, maximal with 38% inhibition of the short-circuit current at 25 mmol/l. This response was completely inhibited by pretreatment of tissues with mucosal amiloride (100 pmol/l). These features are consistent with a direct effect of 5-hydroxytryptamine on amiloride-sensitive sodium channels. Similar results were obtained in a limited number of studies using human bronchial epithelium. 3. The effects of mucosal addition of a range of 5-hydroxytryptamine agonists were studied to determine if any was a more potent blocker of amiloridesensitive sodium transport than 5-hydroxytryptamine. The 5-HT3 agonist 2-methyl-5-hydroxytryptamine had no effect on the short-circuit current at concentrations of up to 5mmol/l. The 5-HT,, agonist sumatriptan had no effect at concentrations below 5 mmol/l and at 5 mmol/l had only a transient effect. The 5-HT,, agonists buspirone and 8-hydroxy-2(di-n-propy1amino)tetralin and the 5-HT2 agonist a-methyl-5-hydroxytryptamine were all more potent inhibitors of the short-circuit current than 5-hydroxytryptamine, but, although their effects were reduced by pretreatment of tissues with mucosal amiloride (lOOpmol/l), none had a specific effect on the amiloride-sensitive sodium current. The effect of buspirone on the short-circuit current was also studied
after mucosa. sodium substitutm, and although its effect was again reduced, significant inhibition of the short-circuit current still occurred, indicating that ion transport processes other than sodium absorption were being affected. 4. Mucosal application of ondansetron, an antagonist a t the 5-HT3 receptor (an ion channel), also produced a dose-related inhibition of the short-circuit current that was not mediated via the amiloride-sensitive sodium current. Pretreatment of tissues with ordansetron had no effect on the subsequent response to 5-h ydrox ytr yptamine. 5. We conclude that mucosally applied 5-hydroxytryptamine specifically inhibits amiloride-sensitive sodium transport in airway epithelia, but with a median inhibitory concentration too high for it to be therapeutically useful. The high median inhibitory concentration also indicates that 5-hydroxytryptamine is unlikely to be a physiological regulator of sodium channels. Screening a number of 5-hydroxytryptamine receptor agonists has failed to identify a more potent inhibitor of sodium transport which may have had therapeutic potential. INTRODUCTION
Active ion transport across airway epithelia is likely to be a major factor determining the depth and composition of airway surface liquid (ASL) [l]. The dominant ion-transport process in the conducting airways of several species including man is sodium absorption [2-4]. This is mediated by both amiloride-sensitive and amiloride-insensitive pathways [S, 61. Alterations in the volume of the ASL resulting from changes in transepithelial ion transport may influence mucociliary clearance [MCC]. In cystic fibrosis (CF), in which sodium absorption is increased two- to three-fold [7], MCC can be improved with nebulized amiloride in uiuo [8]. Pilot studies using nebulized amiloride in patients with CF suggest that it may delay the progression of lung damage and reduce sputum viscosity [9]. How-
Key words: airway, Ehydroxytryptamine, ion transport. Abbreviations: ASL, airway surface liquid: CF, cystic fibrosis, G, conductance; SHT, Ehydroxytryptamine; I,,, short-circuit current; IC,,, median inhibitory concentration; MCC, mucociliary clearance; M H - D P A T , 8-hydroxy-2jdi-n-propy1amino)tetralin; PD, potential difference. Correspondence: Dr A. Graham, Ion Transport Laboratory, National Heart and Lung Institute, Manresa Road, London SW3 6LR, U.K.
332
A. Graham et al.
ever, the duration of action of inhaled amiloride is short, the effects on MCC lasting up to 40min [8] and on airway potential difference less than 30min [101. Frequent administration is therefore required and good compliance is thus difficult to achieve. A longer-acting compound with similar effects would have useful therapeutic potential. One approach to identifying such an agent is to investigate endogenous compounds which may have a physiological role in the regulation of airway epithelial sodium transport. 5-Hydroxytryptamine (5-HT) has been suggested as such a candidate after the demonstration that it inhibited amiloridesensitive sodium transport in the baboon bronchus [113. Furthermore, 5-HT-containing cells are numerous within the upper airways of a number of larger mammals, including the sheep and monkey [l2]. The pharmacology of 5-HT has been extensively studied and several agonists and antagonists to the different receptor sites, some of which are now in clinical use, have been developed [13-151. The effects of 5-HT are mediated through the protein kinase A (5-HT1,, 5-HT1,, 5-HT1, and 5-HT4 receptors) and protein kinase C (5-HTIc and 5-HT, receptors) pathways via G proteins [16, 171 and also by direct activation of ion channels (5-HT3 receptor) [18]. Inhibition of sodium transport by stimulation of protein kinase C and elevation of intracellular calcium levels has been demonstrated in mammalian airways [19, 201. Thus, 5-HT might influence sodium transport by both direct channeland receptor-mediated mechanisms. In this study the effects of 5-HT, 5-HT receptor agonists and a 5-HT receptor antagonist on airway ion transport have been investigated using sheep tracheal epithelium, a predominantly sodiumabsorbing epithelium [4, 211, the ion transport characteristics of which are comparable with those of human bronchial epithelium [6]. A limited number of experiments have been performed using human bronchial epithelium, when available, for comparison. METHODS Ussing chamber studies
Sheep trachea were obtained fresh from a local abbatoir and were immersed immediately in cold (4°C) Krebs-Henseleit solution of composition (mmol/l): Na+, 145.0; C1-, 126.0; K + , 5.9; Ca2+,2.5; MgZ+, 1.2; HCO;, 26.0; PO:-, 1.2; SO:-, 1.2; glucose, 5.6. The epithelium was dissected free of underlying tissues and was mounted in Ussing chambers of area 1.28cm2. Human bronchi were obtained from whole diseased lungs (primary pulmonary hypertension and sarcoidosis) removed at transplantation and were also immersed immediately in cold Krebs-Henseleit solution:. For the main bronchi the epithelium was partially dissected from underlying tissues, but the smaller bronchi
were simply opened longitudinally before mounting. Both were mounted in Ussing chambers of area 0.28 cm2, All tissues were bathed with KrebsHenseleit solution at 37°C (pH 7.4) and bubbled through gas lifts with a 95% 0,/5% COz mixture. The potential difference (PD) across the tissue was measured using lmol/l KC1/2% agar bridges connected to a DVC-1000 voltage clamp (World Precision Instruments) by calomel electrodes (Russell pH Limited). The offset of the electrodes was measured before tissue mounting and suitable adjustments were made to recorded values. Short-circuit current (Z,J was measured using 0.9% NaC1/2% agar bridges connected to the clamp by Ag/AgCl electrodes. The tissues were maintained continuously under short-circuit conditions except for brief (15 s) intervals to measure PD. Conductance ( C ) was calculated from Ohm's law. Tissues with a conductance of greater than 10mS/cm2 (sheep trachea) and 14mS/cm2 (human bronchus) [3] were excluded. Once tissues were electrically stable, defined by a rate of rise in I,, of less than 3pA/h (sheep trachea) and 0.4 pA/h (human bronchus), experimental interventions were commenced. Drugs and chemicals
In sodium substitution experiments, sodium chloride and sodium bicarbonate were substituted with equimolar choline chloride and choline bicarbonate, respectively, in the Krebs-Henseleit solution. Amiloride was a gift from Merck, Sharp and Dohme, and sumatriptan and ondansetron were gifts from Glaxo Group Research (Greenford, Middx., U.K.). 5-HT creatinine sulphate and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OHDPAT) were obtained from Sigma Chemical Co. (Poole, Dorset, U.K.). Buspirone, a-methyl-5-HT maleate and 2-methyl-5-HT maleate were obtained from Semat Technical (U.K.) Ltd. Amiloride was dissolved in Krebs-Henseleit solution and was applied in a dilution of 1 in 10. 5-HT was dissolved in Krebs-Henseleit solution at 37°C and was applied in a range of dilutions from 1 to 100 to 1 in 1 (i.e. the Krebs solution bathing one side of the tissue was substituted with that containing 5-HT). 8OH-DPAT was dissolved in Krebs-Henseleit solution and was applied in dilutions of 1 in 500 to 1 in 5. a-Methyl-5-HT, 2-methyl-5-HT and ondansetron were dissolved in distilled water and were applied in dilutions of 1 in 1000 and 1 in 100. Buspirone and sumatriptan were dissolved in distilled water and were applied in dilutions of 1 in 2000 to 1 in 20. Experimental protocols
In dose-response studies control tissues were treated with diluent or by substitution of the appropriate volume of Krebs-Henseleit solution. In experiments in which tissues were pretreated with amiloride this was added to the mucosal surface in
Wydroxytryptamine and airway ion transport
a concentration of 100pmol/l 5 min before the drug under investigation which was then added to both the amiloride-pretreated tissue and to a paired nonpretreated tissue from the same animal. In experiments using mucosal sodium substitution the tissues were first mounted in standard Krebs-Henseleit solution until bioelectrically stable. The mucosal surface was then rinsed several times with sodiumfree Krebs-Henseleit solution, which was then used to replace the mucosal bathing fluid. The 0.9% NaC1/2% agar current bridges were replaced with 0.9% choline chloride/2% agar bridges at this time. Bioelectric properties were stable after a further 15-20 min at which time the drug under investigation was added.
1
2o V
'
10-5
I
lo-'
10-3 5-HT concn. (mol/l)
lo-'
10-1
100
Statistics
The Mann-Whitney U-test was used to compare means. The Wilcoxon rank sum test was used to compare the effects of an intervention against baseline. Results are expressed as means with SEM in parentheses for convenience.
333
L*
80
--.4
I
5 60
-?I % M n
W
RESULTS Baseline values
Baseline bioelectric properties for sheep tracheal epithelium (n=267 tissues, 68 sheep) were: PD, -9.6(0.3) mV; I,,, 51 (2) pA/cm2; G, 5.8(0.1)mS/cm2. For human bronchial epithelium (n= 11 tissues, two individuals) the corresponding values were: PD, - 3.8 (0.5)mV; I,, 30(8) pA/cm2; and G, 8.0(0.7) mS/cm2. Effects of tissue pretreatment protocols
Addition of amiloride (100 pmol/l, n = 32) in sheep trachea resulted in a fall in I,, of 20(2)pA/cm2 [38% of the baseline current of 53(4)pA/cm2 in these tissues] and a fall in G of 0.3(0.1)mS/cmz (5% of the baseline G of 5.8(0.3)mS/cm2]. In human bronchial epithelium muscosal amiloride (100 pmol/l, n=2) produced falls in I,, of 8 and 21 pA/cm2 from 32 and 44 pA/cm2, respectively, with no change in G. In sheep trachea mucosal sodium substitution resulted in a fall in I,, of 21 (3) pA/cm2 (n= 10) [45% of the baseline current of 47 (8) pA/cm2 in these tissues] and a fall in G of 1.2(0.2)mS/cm2 [21% of the baseline G of 5.6 (0.4) mS/cm2]. Effects of 5-HT on biolelectric properties in sheep tracheal epithelium
Mucosal addition of 5-HT resulted in an immediate, concentration-related decrease in I,, which was maximal with 38(7)% (n=6) inhibition of I,, at 25 mmol/l and a median inhibitory concentration (IC5,,) of 5mmol/l (Fig. la). This was associated with a concentration-related decrease in G of up to
20
0 I
I
-5
10-4
I
10-3
10-2
10-1
5-HT concn. (mol/l)
Fig. 1. Doseresponse curves showing the effect of mucosal 5-HT on ( a ) /sc and (b) G in sheep tracheal epithelium. Tissue numbers are indicated at each point. Error bars indicate SEM and where not shown fall within the symbol. All results have been corrected for the effects of controls. Statistical significance: *P
