Volume 114, Issue 6, Pages 1257-1267 (June 1998) Levamisole inhibits intestinal Cl− secretion via basolateral K+ channel blockade  Edward C. Mun, Julio M. Mayol, Martin Riegler, Timothy C. O'Brien, Omid C. Farokhzad, Jaekyung C. Song, Charalabos Pothoulakis, Bruce J. Hrnjez, Jeffrey B. Matthews  Gastroenterology  Volume 114, Issue 6, Pages 1257-1267 (June 1998) DOI: 10.1016/S0016-5085(98)70432-9 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Effects of phenylimidazothiazole on cAMP-elicited ISC response in T84 cells. (A) Cells were treated with phenylimidazothiazoles (10 mmol/L levamisole, 1.0 mmol/L L-bromotetramisole, and 1.0 mmol/L D-bromotetramisole) as indicated by the arrow (P), whereas the control monolayers were treated with an equal concentration (by volume) of dimethyl sulfoxide. After 30 minutes, monolayers were then treated with 10 μmol/L forskolin as indicated by arrow (F) at time 0 at room temperature, and time-dependent ISC measurements were continued for another 30 minutes. All three compounds significantly inhibited forskolin-elicited ISC response (P < 0.0001 for all three compounds by ANOVA). Values are means ± SEM of 6 inserts for each group. (B) All inserts were stimulated with 10 μmol/L forskolin at time 0 arrow [F]). At peak ISC response (time 30 minutes), 10 mmol/L levamisole and 1.0 mmol/L L- and D-bromotetramisole were added (arrow [P]). All three agents immediately inhibited the ISC response (P < 0.0001 for all three by ANOVA; n = 6). Dose-dependent inhibition of forskolin-evoked ISC response by levamisole and D-bromotetramisole. Monolayers were pretreated at room temperature with graded concentrations of levamisole or D-bromotetramisole for 15 minutes. Forskolin, 10 μmol/L, was then added, and ISC measurements were performed after 20 minutes of incubation. For the given concentration of phenylimidazothiazole compound, ISC response is expressed as percentage of maximal ISC obtained in the absence of phenylimidazothiazoles. Values are means ± SEM (each n = 4). Gastroenterology 1998 114, 1257-1267DOI: (10.1016/S0016-5085(98)70432-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Effects of phenylimidazothiazole on cAMP-elicited ISC response in T84 cells. (A) Cells were treated with phenylimidazothiazoles (10 mmol/L levamisole, 1.0 mmol/L L-bromotetramisole, and 1.0 mmol/L D-bromotetramisole) as indicated by the arrow (P), whereas the control monolayers were treated with an equal concentration (by volume) of dimethyl sulfoxide. After 30 minutes, monolayers were then treated with 10 μmol/L forskolin as indicated by arrow (F) at time 0 at room temperature, and time-dependent ISC measurements were continued for another 30 minutes. All three compounds significantly inhibited forskolin-elicited ISC response (P < 0.0001 for all three compounds by ANOVA). Values are means ± SEM of 6 inserts for each group. (B) All inserts were stimulated with 10 μmol/L forskolin at time 0 arrow [F]). At peak ISC response (time 30 minutes), 10 mmol/L levamisole and 1.0 mmol/L L- and D-bromotetramisole were added (arrow [P]). All three agents immediately inhibited the ISC response (P < 0.0001 for all three by ANOVA; n = 6). Dose-dependent inhibition of forskolin-evoked ISC response by levamisole and D-bromotetramisole. Monolayers were pretreated at room temperature with graded concentrations of levamisole or D-bromotetramisole for 15 minutes. Forskolin, 10 μmol/L, was then added, and ISC measurements were performed after 20 minutes of incubation. For the given concentration of phenylimidazothiazole compound, ISC response is expressed as percentage of maximal ISC obtained in the absence of phenylimidazothiazoles. Values are means ± SEM (each n = 4). Gastroenterology 1998 114, 1257-1267DOI: (10.1016/S0016-5085(98)70432-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Effects of phenylimidazothiazole on cAMP-elicited ISC response in T84 cells. (A) Cells were treated with phenylimidazothiazoles (10 mmol/L levamisole, 1.0 mmol/L L-bromotetramisole, and 1.0 mmol/L D-bromotetramisole) as indicated by the arrow (P), whereas the control monolayers were treated with an equal concentration (by volume) of dimethyl sulfoxide. After 30 minutes, monolayers were then treated with 10 μmol/L forskolin as indicated by arrow (F) at time 0 at room temperature, and time-dependent ISC measurements were continued for another 30 minutes. All three compounds significantly inhibited forskolin-elicited ISC response (P < 0.0001 for all three compounds by ANOVA). Values are means ± SEM of 6 inserts for each group. (B) All inserts were stimulated with 10 μmol/L forskolin at time 0 arrow [F]). At peak ISC response (time 30 minutes), 10 mmol/L levamisole and 1.0 mmol/L L- and D-bromotetramisole were added (arrow [P]). All three agents immediately inhibited the ISC response (P < 0.0001 for all three by ANOVA; n = 6). Dose-dependent inhibition of forskolin-evoked ISC response by levamisole and D-bromotetramisole. Monolayers were pretreated at room temperature with graded concentrations of levamisole or D-bromotetramisole for 15 minutes. Forskolin, 10 μmol/L, was then added, and ISC measurements were performed after 20 minutes of incubation. For the given concentration of phenylimidazothiazole compound, ISC response is expressed as percentage of maximal ISC obtained in the absence of phenylimidazothiazoles. Values are means ± SEM (each n = 4). Gastroenterology 1998 114, 1257-1267DOI: (10.1016/S0016-5085(98)70432-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Effect of levamisole on cAMP-independent ISC responses in T84 cells. (A) Effect of levamisole on carbachol-elicited ISC response. After equilibration in HPBR, monolayers were further incubated for 10 minutes in the absence (control) and presence of 10 mmol/L levamisole, as noted by the arrow (L). Carbachol, 100 μmol/L, was then added to both groups as indicated by the arrow (C). Levamisole blunted subsequent carbachol-evoked current increase (P < 0.0001 by ANOVA). Values are means ± SEM (each n = 4). (B) Effect of levamisole on genistein-evoked ISC response. Genistein, 50 μmol/L, was added to the monolayers at time 0 as indicated by the arrow (G). After 20 minutes, one group received 10 mmol/L levamisole while the control group received an equal volume of dimethyl sulfoxide. Levamisole significantly inhibited genistein-activated ISC response (P < 0.0001 by ANOVA). Data are means ± SEM (n = 6). (C) Dose-dependent inhibition by levamisole on ISC responses activated by independent secretory agonists. ISC responses to 10 μmol/L forskolin, 100 μmol/L carbachol, and 50 μmol/L genistein were measured in the presence of varying concentrations of levamisole as described in Materials and Methods. Percent response of the peak ISC evoked in the absence of levamisole is plotted against concentration of levamisole. Carbachol ISC response was more sensitive (IC50, ~0.3 mmol/L) than that of forskolin or genistein (IC50, ~2.5 mmol/L) to inhibition by levamisole. Values are means ± SEM of 6 inserts in each group. Gastroenterology 1998 114, 1257-1267DOI: (10.1016/S0016-5085(98)70432-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Effect of levamisole on cAMP-independent ISC responses in T84 cells. (A) Effect of levamisole on carbachol-elicited ISC response. After equilibration in HPBR, monolayers were further incubated for 10 minutes in the absence (control) and presence of 10 mmol/L levamisole, as noted by the arrow (L). Carbachol, 100 μmol/L, was then added to both groups as indicated by the arrow (C). Levamisole blunted subsequent carbachol-evoked current increase (P < 0.0001 by ANOVA). Values are means ± SEM (each n = 4). (B) Effect of levamisole on genistein-evoked ISC response. Genistein, 50 μmol/L, was added to the monolayers at time 0 as indicated by the arrow (G). After 20 minutes, one group received 10 mmol/L levamisole while the control group received an equal volume of dimethyl sulfoxide. Levamisole significantly inhibited genistein-activated ISC response (P < 0.0001 by ANOVA). Data are means ± SEM (n = 6). (C) Dose-dependent inhibition by levamisole on ISC responses activated by independent secretory agonists. ISC responses to 10 μmol/L forskolin, 100 μmol/L carbachol, and 50 μmol/L genistein were measured in the presence of varying concentrations of levamisole as described in Materials and Methods. Percent response of the peak ISC evoked in the absence of levamisole is plotted against concentration of levamisole. Carbachol ISC response was more sensitive (IC50, ~0.3 mmol/L) than that of forskolin or genistein (IC50, ~2.5 mmol/L) to inhibition by levamisole. Values are means ± SEM of 6 inserts in each group. Gastroenterology 1998 114, 1257-1267DOI: (10.1016/S0016-5085(98)70432-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Effect of levamisole on cAMP-independent ISC responses in T84 cells. (A) Effect of levamisole on carbachol-elicited ISC response. After equilibration in HPBR, monolayers were further incubated for 10 minutes in the absence (control) and presence of 10 mmol/L levamisole, as noted by the arrow (L). Carbachol, 100 μmol/L, was then added to both groups as indicated by the arrow (C). Levamisole blunted subsequent carbachol-evoked current increase (P < 0.0001 by ANOVA). Values are means ± SEM (each n = 4). (B) Effect of levamisole on genistein-evoked ISC response. Genistein, 50 μmol/L, was added to the monolayers at time 0 as indicated by the arrow (G). After 20 minutes, one group received 10 mmol/L levamisole while the control group received an equal volume of dimethyl sulfoxide. Levamisole significantly inhibited genistein-activated ISC response (P < 0.0001 by ANOVA). Data are means ± SEM (n = 6). (C) Dose-dependent inhibition by levamisole on ISC responses activated by independent secretory agonists. ISC responses to 10 μmol/L forskolin, 100 μmol/L carbachol, and 50 μmol/L genistein were measured in the presence of varying concentrations of levamisole as described in Materials and Methods. Percent response of the peak ISC evoked in the absence of levamisole is plotted against concentration of levamisole. Carbachol ISC response was more sensitive (IC50, ~0.3 mmol/L) than that of forskolin or genistein (IC50, ~2.5 mmol/L) to inhibition by levamisole. Values are means ± SEM of 6 inserts in each group. Gastroenterology 1998 114, 1257-1267DOI: (10.1016/S0016-5085(98)70432-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Effect of phenylimidazothiazoles on forskolin-evoked ISC response in isolated human colon. Isolated human colonic mucosal sheets were mounted in Ussing chambers filled with a nutrient buffer as described in Materials and Methods. After a period of equilibration, forskolin (10 μmol/L), added at arrow (F), evoked a sustained current response. (A) Levamisole, 2 mmol/L, was then added to basolateral buffer (at L) and promptly inhibited forskolin-elicited ISC (n = 3 mucosal sheets each; P < 0.0001 by ANOVA) almost identically to its inhibition in the T84 cell model in Figure 1B. (B) Similarly, 0.2 mmol/L L-bromotetramisole and D-bromotetramisole added at arrow(B) also inhibited forskolin-activated ISC (each n = 3; P < 0.0001 and P < 0.0002 by ANOVA for L- and D-bromotetramisole, respectively). Values are means ± SEM. Gastroenterology 1998 114, 1257-1267DOI: (10.1016/S0016-5085(98)70432-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Effects of phenylimidazothiazoles on carbachol-evoked ISC reponse in isolated human colon. (A) Human colonic mucosal sheets were preincubated in nutrient buffer in the presence or absence of 2.0 mmol/L levamisole (added at arrow [L]) for 10 minutes. Subsequent basolateral addition of 100 μmol/L carbachol (C) elicited a transient ISC response in control monolayers similar in pattern to that seen in T84 cells (Figure 2A), whereas levamisole pretreatment significantly blunted this response (each n = 3 mucosal sheets; P < 0.0001 by ANOVA). (B) Both 0.2 mmol/L L-bromotetramisole and D-bromotetramisole also completely prevented carbachol-elicited ISC (each n = 3; P < 0.0001 by ANOVA). Values are means ± SEM. Gastroenterology 1998 114, 1257-1267DOI: (10.1016/S0016-5085(98)70432-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 (A) Effect of levamisole on apical membrane Cl− conductance (ICl) in T84 monolayers. After equilibration in low-Cl− buffer, high-basolateral (150 mmol/L) to low-apical (0 mmol/L) chloride ion gradient was applied, as indicated by arrow (Cl), followed by basolateral addition of nystatin (500 U/mL) at arrow (N). After basal ICl transients subsided, 10 mmol/L levamisole was added apically (L). A relatively small but sizable apical current (11.6 ± 1.81 μA/cm2; n = 5) was generated by levamisole. L- and D-bromotetramisole (0.1 mmol/L) similarly activated apical ICl (10.8 ± 1.48 and 12.3 ± 3.74 μA/cm2, respectively; n = 5). A representative tracing is shown. (B) Effect of levamisole on forskolin-elicited apical ICl in T84 monolayers. Forskolin, 10 μmol/L (added at F), elicited a large current (ICl, 78.3 ± 8.3 mA/cm2; n = 6) but was not further activated or inhibited by subsequent addition of 10 mmol/L levamisole (L). Similarly, 1.0 mmol/L bromotetramisoles did not affect the forskolin-stimulated ICl response. A representative tracing is shown. Gastroenterology 1998 114, 1257-1267DOI: (10.1016/S0016-5085(98)70432-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 Effects of levamisole on basolateral 86Rb+ uptake in T84 cells. Monolayers grown on permeable supports (1 cm2) were preincubated in HPBR in the absence (2) or presence (■) of 10 mmol/L levamisole for 20 minutes at room temperature. Each group was then further incubated in the presence or absence of 20 μmol/L bumetanide. Some of the inserts were stimulated with basolateral forskolin (10 μmol/L). Monolayers were then transferred to wells containing 1 μCi/mL 86Rb+ for 3 minutes. (A) Bumetanide-sensitive 86Rb+ uptake was the same in control and levamisole groups under both unstimulated (each n = 3; P = 0.95) and forskolin-stimulated (P = 0.95) conditions. (B) Bumetanide-insensitive 86Rb+ uptake was similarly unaffected by levamisole in both basal (n = 3; P = 0.07) and forskolin-stimulated (n = 3; P > 0.30) conditions. Data are means ± SEM. Gastroenterology 1998 114, 1257-1267DOI: (10.1016/S0016-5085(98)70432-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 7 Effect of phenylimidazothiazoles on basolateral K+ conductance (IK) in T84 cells. (A) After equilibration in low-K+ buffer, monolayers were subjected to high-apical (140 mmol/L) to low-basolateral (5 mmol/L) potassium ion gradient, indicated by arrow (K). Subsequent apical nystatin (500 U/mL) treatment (N) uncovers resting K+ current (ΔIK = 3.1 ± 0.2 μA/cm2; n = 12) through constitutively open apical Cl− channels. Levamisole, 10 mmol/L, was added (L) to one of the monolayers (dotted line) before subsequent treatment with basolateral 100 μmol/L carbachol (C) to both control (solid line) and levamisole-treated (dotted line). Levamisole decreased the resting IK to prepermeabilization level (ΔIK = −3.6 ± 0.4 μA/cm2; n = 6). Additionally, typical carbachol-elicited IK response was completely abolished by levamisole pretreatment. L- and D-bromotetramisoles similarly inhibited the resting IK (ΔIK = −5.67 ± 2.33 and −5.83 ± 1.83 μA/cm2, respectively; each n = 3) as well as carbachol-elicited IK response. Data represent typical IK tracings of permeabilized T84 monolayer with or without levamisole treatment. (B) Effects of barium on basolateral K+ conductance (IK) in T84 cells. In this representative IK tracing, 3 mmol/L barium (added at Ba) completely inhibited resting IK similar to levamisole as in A. However, subsequent carbachol (100 μmol/L)-elicited IK response was not affected by pretreatment with Ba2+. Ba2+ dose-dependently inhibited only the resting IK (IC50, 0.70 + 0.26 mmol/L; n = 3). (C) Dose-dependent inhibition of resting K+ conductance by phenylimidazothiazoles. After the IK transients subsided after apical permeabilization as in A and B, small increments of phenylimidazothiazole compounds were added to the basolateral buffer, and IK changes were recorded accordingly. L-Bromotetramisoles (2) and D-bromotetramisoles (▴) were significantly more potent than levamisole (•) in resting IK inhibition (IC50, ~0.50, 0.45, and 6.9 mmol/L for L-bromotetramisole, D-bromotetramisole, and levamisole, respectively; each n = 3). Ba2+ similarly inhibited this resting IK in a dose-dependent fashion (n = 3; not shown). Data are means ± SEM. Gastroenterology 1998 114, 1257-1267DOI: (10.1016/S0016-5085(98)70432-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 7 Effect of phenylimidazothiazoles on basolateral K+ conductance (IK) in T84 cells. (A) After equilibration in low-K+ buffer, monolayers were subjected to high-apical (140 mmol/L) to low-basolateral (5 mmol/L) potassium ion gradient, indicated by arrow (K). Subsequent apical nystatin (500 U/mL) treatment (N) uncovers resting K+ current (ΔIK = 3.1 ± 0.2 μA/cm2; n = 12) through constitutively open apical Cl− channels. Levamisole, 10 mmol/L, was added (L) to one of the monolayers (dotted line) before subsequent treatment with basolateral 100 μmol/L carbachol (C) to both control (solid line) and levamisole-treated (dotted line). Levamisole decreased the resting IK to prepermeabilization level (ΔIK = −3.6 ± 0.4 μA/cm2; n = 6). Additionally, typical carbachol-elicited IK response was completely abolished by levamisole pretreatment. L- and D-bromotetramisoles similarly inhibited the resting IK (ΔIK = −5.67 ± 2.33 and −5.83 ± 1.83 μA/cm2, respectively; each n = 3) as well as carbachol-elicited IK response. Data represent typical IK tracings of permeabilized T84 monolayer with or without levamisole treatment. (B) Effects of barium on basolateral K+ conductance (IK) in T84 cells. In this representative IK tracing, 3 mmol/L barium (added at Ba) completely inhibited resting IK similar to levamisole as in A. However, subsequent carbachol (100 μmol/L)-elicited IK response was not affected by pretreatment with Ba2+. Ba2+ dose-dependently inhibited only the resting IK (IC50, 0.70 + 0.26 mmol/L; n = 3). (C) Dose-dependent inhibition of resting K+ conductance by phenylimidazothiazoles. After the IK transients subsided after apical permeabilization as in A and B, small increments of phenylimidazothiazole compounds were added to the basolateral buffer, and IK changes were recorded accordingly. L-Bromotetramisoles (2) and D-bromotetramisoles (▴) were significantly more potent than levamisole (•) in resting IK inhibition (IC50, ~0.50, 0.45, and 6.9 mmol/L for L-bromotetramisole, D-bromotetramisole, and levamisole, respectively; each n = 3). Ba2+ similarly inhibited this resting IK in a dose-dependent fashion (n = 3; not shown). Data are means ± SEM. Gastroenterology 1998 114, 1257-1267DOI: (10.1016/S0016-5085(98)70432-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 7 Effect of phenylimidazothiazoles on basolateral K+ conductance (IK) in T84 cells. (A) After equilibration in low-K+ buffer, monolayers were subjected to high-apical (140 mmol/L) to low-basolateral (5 mmol/L) potassium ion gradient, indicated by arrow (K). Subsequent apical nystatin (500 U/mL) treatment (N) uncovers resting K+ current (ΔIK = 3.1 ± 0.2 μA/cm2; n = 12) through constitutively open apical Cl− channels. Levamisole, 10 mmol/L, was added (L) to one of the monolayers (dotted line) before subsequent treatment with basolateral 100 μmol/L carbachol (C) to both control (solid line) and levamisole-treated (dotted line). Levamisole decreased the resting IK to prepermeabilization level (ΔIK = −3.6 ± 0.4 μA/cm2; n = 6). Additionally, typical carbachol-elicited IK response was completely abolished by levamisole pretreatment. L- and D-bromotetramisoles similarly inhibited the resting IK (ΔIK = −5.67 ± 2.33 and −5.83 ± 1.83 μA/cm2, respectively; each n = 3) as well as carbachol-elicited IK response. Data represent typical IK tracings of permeabilized T84 monolayer with or without levamisole treatment. (B) Effects of barium on basolateral K+ conductance (IK) in T84 cells. In this representative IK tracing, 3 mmol/L barium (added at Ba) completely inhibited resting IK similar to levamisole as in A. However, subsequent carbachol (100 μmol/L)-elicited IK response was not affected by pretreatment with Ba2+. Ba2+ dose-dependently inhibited only the resting IK (IC50, 0.70 + 0.26 mmol/L; n = 3). (C) Dose-dependent inhibition of resting K+ conductance by phenylimidazothiazoles. After the IK transients subsided after apical permeabilization as in A and B, small increments of phenylimidazothiazole compounds were added to the basolateral buffer, and IK changes were recorded accordingly. L-Bromotetramisoles (2) and D-bromotetramisoles (▴) were significantly more potent than levamisole (•) in resting IK inhibition (IC50, ~0.50, 0.45, and 6.9 mmol/L for L-bromotetramisole, D-bromotetramisole, and levamisole, respectively; each n = 3). Ba2+ similarly inhibited this resting IK in a dose-dependent fashion (n = 3; not shown). Data are means ± SEM. Gastroenterology 1998 114, 1257-1267DOI: (10.1016/S0016-5085(98)70432-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions