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Contrasting effects of linaclotide and lubiprostone on restitution of epithelial cell barrier properties and cellular homeostasis after exposure to cell stressors.

Cuppoletti J, Blikslager AT, Chakrabarti J, Nighot PK, Malinowska DH - BMC Pharmacol. (2012)

Bottom Line: Linaclotide, but not lubiprostone increased [cGMP]i as expected and [Ca(2+)]i and linaclotide depolarized while lubiprostone hyperpolarized the T84 plasma membrane potential suggesting that lubiprostone may lead to greater cellular stability compared to linaclotide.In T84 cells, as found with linaclotide but not with lubiprostone, transepithelial resistance was slightly but significantly decreased by guanylin, STa and 8-bromo cGMP and fluorescent dextran fluxes were increased by guanylin.However the physiological implications of these small but statistically significant changes remain unclear.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular & Cellular Physiology, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0576, USA. John.Cuppoletti@uc.edu

ABSTRACT

Background: Linaclotide has been proposed as a treatment for the same gastrointestinal indications for which lubiprostone has been approved, chronic idiopathic constipation and irritable bowel syndrome with constipation. Stressors damage the epithelial cell barrier and cellular homeostasis leading to loss of these functions. Effects of active linaclotide on repair of barrier and cell function in pig jejunum after ischemia and in T84 cells after treatment with proinflammatory cytokines, interferon-γ and tumor necrosis factor-α were examined. Comparison with effects of lubiprostone, known to promote repair of barrier function was carried out.

Results: In ischemia-damaged pig jejunum, using measurements of transepithelial resistance, (3)H-mannitol fluxes, short-circuit current (Cl(-) secretion) and occludin localization, active linaclotide failed to effectively promote repair of the epithelial barrier or recovery of short-circuit current, whereas lubiprostone promoted barrier repair and increased short-circuit current. In control pig jejunum, 1 μM linaclotide and 1 μM lubiprostone both caused similar increases in short-circuit current (Cl(-) secretion). In T84 cells, using measurements of transepithelial resistance, fluxes of fluorescent macromolecules, occludin and mitochondrial membrane potential, active linaclotide was virtually ineffective against damage caused by interferon-γ and tumor necrosis factor-α, while lubiprostone protected or promoted repair of epithelial barrier and cell function. Barrier protection/repair by lubiprostone was inhibited by methadone, a ClC-2 inhibitor. Linaclotide, but not lubiprostone increased [cGMP]i as expected and [Ca(2+)]i and linaclotide depolarized while lubiprostone hyperpolarized the T84 plasma membrane potential suggesting that lubiprostone may lead to greater cellular stability compared to linaclotide. In T84 cells, as found with linaclotide but not with lubiprostone, transepithelial resistance was slightly but significantly decreased by guanylin, STa and 8-bromo cGMP and fluorescent dextran fluxes were increased by guanylin. However the physiological implications of these small but statistically significant changes remain unclear.

Conclusions: Considering the physiological importance of epithelial barrier function and cell integrity and the known impact of stressors, the finding that lubiprostone, but not active linaclotide, exhibits the additional distinct property of effective protection or repair of the epithelial barrier and cell function after stress suggests potential clinical importance for patients with impaired or compromised barrier function such as might occur in IBS.

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Comparison of effects of active linaclotide and lubiprostone on T84 cell homeostasis: (A) Δ[cGMP]i; (B) Δ[Ca2+]i; (C) Δplasma membrane potential. 200 nM active linaclotide and 100 nM lubiprostone were used. Data is plotted as mean ± S.E. (A) Δ[cGMP]i: n = 5, p < 0.0005 lin vs lubi. 1 μM NaNP was the positive control. Basal [cGMP]is =1.7 ± 0.001, 3.1 ± 0.01 and 23.9 ± 0.002 pmol/105T84 cells for lin, lubi and PGE1 respectively. (B) Δ[Ca2+]i: n = 3, *p < 0.01, **p < 0.0005, #p < 0.02, ##p < 0.0025, xp < 0.001 all wrt 1 nM drug. Basal [Ca2+]is = 175.5 ± 7.1, 117.6 ± 6.4 and 80.8 ± 2.4 nM for lin, lubi and PGE1 respectively. (C) Δplasma membrane potential: n = 5, ##p < 0.001, *p < 0.02 all wrt 1 nM drug. PGE1 was used as positive control.
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Figure 3: Comparison of effects of active linaclotide and lubiprostone on T84 cell homeostasis: (A) Δ[cGMP]i; (B) Δ[Ca2+]i; (C) Δplasma membrane potential. 200 nM active linaclotide and 100 nM lubiprostone were used. Data is plotted as mean ± S.E. (A) Δ[cGMP]i: n = 5, p < 0.0005 lin vs lubi. 1 μM NaNP was the positive control. Basal [cGMP]is =1.7 ± 0.001, 3.1 ± 0.01 and 23.9 ± 0.002 pmol/105T84 cells for lin, lubi and PGE1 respectively. (B) Δ[Ca2+]i: n = 3, *p < 0.01, **p < 0.0005, #p < 0.02, ##p < 0.0025, xp < 0.001 all wrt 1 nM drug. Basal [Ca2+]is = 175.5 ± 7.1, 117.6 ± 6.4 and 80.8 ± 2.4 nM for lin, lubi and PGE1 respectively. (C) Δplasma membrane potential: n = 5, ##p < 0.001, *p < 0.02 all wrt 1 nM drug. PGE1 was used as positive control.

Mentions: Increases in [cGMP]i and [Ca2+i have been shown with linaclotide and guanylin and changes in occludin occur with breakdown of barrier function [8,33,35,36,40]. Therefore active linaclotide effects on [cGMP]i, [Ca2+i and plasma membrane potential were investigated and compared with effects of lubiprostone. The results are shown in Figure 3 as changes that occurred compared to vehicle controls (basal levels are given in the legend). Active linaclotide significantly increased [cGMP]i while lubiprostone significantly decreased [cGMP]i (Figure 3A, p < 0.0005). Active linaclotide and PGE1 significantly increased [Ca2+i, while lubiprostone significantly decreased [Ca2+i (Figure 3B, p values are given in the figure legend). Figure 3C shows that active linaclotide significantly depolarized the plasma membrane potential, while lubiprostone significantly hyperpolarized the plasma membrane potential (p values are given in the figure legend). Thus active linaclotide, but not lubiprostone, resulted in increased [cGMP]i as expected and increased [Ca2+i. Effects on the plasma membrane potential suggest that lubiprostone, but not linaclotide, leads to cell stabilization, that helps maintain cellular homeostasis.


Contrasting effects of linaclotide and lubiprostone on restitution of epithelial cell barrier properties and cellular homeostasis after exposure to cell stressors.

Cuppoletti J, Blikslager AT, Chakrabarti J, Nighot PK, Malinowska DH - BMC Pharmacol. (2012)

Comparison of effects of active linaclotide and lubiprostone on T84 cell homeostasis: (A) Δ[cGMP]i; (B) Δ[Ca2+]i; (C) Δplasma membrane potential. 200 nM active linaclotide and 100 nM lubiprostone were used. Data is plotted as mean ± S.E. (A) Δ[cGMP]i: n = 5, p < 0.0005 lin vs lubi. 1 μM NaNP was the positive control. Basal [cGMP]is =1.7 ± 0.001, 3.1 ± 0.01 and 23.9 ± 0.002 pmol/105T84 cells for lin, lubi and PGE1 respectively. (B) Δ[Ca2+]i: n = 3, *p < 0.01, **p < 0.0005, #p < 0.02, ##p < 0.0025, xp < 0.001 all wrt 1 nM drug. Basal [Ca2+]is = 175.5 ± 7.1, 117.6 ± 6.4 and 80.8 ± 2.4 nM for lin, lubi and PGE1 respectively. (C) Δplasma membrane potential: n = 5, ##p < 0.001, *p < 0.02 all wrt 1 nM drug. PGE1 was used as positive control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Comparison of effects of active linaclotide and lubiprostone on T84 cell homeostasis: (A) Δ[cGMP]i; (B) Δ[Ca2+]i; (C) Δplasma membrane potential. 200 nM active linaclotide and 100 nM lubiprostone were used. Data is plotted as mean ± S.E. (A) Δ[cGMP]i: n = 5, p < 0.0005 lin vs lubi. 1 μM NaNP was the positive control. Basal [cGMP]is =1.7 ± 0.001, 3.1 ± 0.01 and 23.9 ± 0.002 pmol/105T84 cells for lin, lubi and PGE1 respectively. (B) Δ[Ca2+]i: n = 3, *p < 0.01, **p < 0.0005, #p < 0.02, ##p < 0.0025, xp < 0.001 all wrt 1 nM drug. Basal [Ca2+]is = 175.5 ± 7.1, 117.6 ± 6.4 and 80.8 ± 2.4 nM for lin, lubi and PGE1 respectively. (C) Δplasma membrane potential: n = 5, ##p < 0.001, *p < 0.02 all wrt 1 nM drug. PGE1 was used as positive control.
Mentions: Increases in [cGMP]i and [Ca2+i have been shown with linaclotide and guanylin and changes in occludin occur with breakdown of barrier function [8,33,35,36,40]. Therefore active linaclotide effects on [cGMP]i, [Ca2+i and plasma membrane potential were investigated and compared with effects of lubiprostone. The results are shown in Figure 3 as changes that occurred compared to vehicle controls (basal levels are given in the legend). Active linaclotide significantly increased [cGMP]i while lubiprostone significantly decreased [cGMP]i (Figure 3A, p < 0.0005). Active linaclotide and PGE1 significantly increased [Ca2+i, while lubiprostone significantly decreased [Ca2+i (Figure 3B, p values are given in the figure legend). Figure 3C shows that active linaclotide significantly depolarized the plasma membrane potential, while lubiprostone significantly hyperpolarized the plasma membrane potential (p values are given in the figure legend). Thus active linaclotide, but not lubiprostone, resulted in increased [cGMP]i as expected and increased [Ca2+i. Effects on the plasma membrane potential suggest that lubiprostone, but not linaclotide, leads to cell stabilization, that helps maintain cellular homeostasis.

Bottom Line: Linaclotide, but not lubiprostone increased [cGMP]i as expected and [Ca(2+)]i and linaclotide depolarized while lubiprostone hyperpolarized the T84 plasma membrane potential suggesting that lubiprostone may lead to greater cellular stability compared to linaclotide.In T84 cells, as found with linaclotide but not with lubiprostone, transepithelial resistance was slightly but significantly decreased by guanylin, STa and 8-bromo cGMP and fluorescent dextran fluxes were increased by guanylin.However the physiological implications of these small but statistically significant changes remain unclear.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular & Cellular Physiology, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0576, USA. John.Cuppoletti@uc.edu

ABSTRACT

Background: Linaclotide has been proposed as a treatment for the same gastrointestinal indications for which lubiprostone has been approved, chronic idiopathic constipation and irritable bowel syndrome with constipation. Stressors damage the epithelial cell barrier and cellular homeostasis leading to loss of these functions. Effects of active linaclotide on repair of barrier and cell function in pig jejunum after ischemia and in T84 cells after treatment with proinflammatory cytokines, interferon-γ and tumor necrosis factor-α were examined. Comparison with effects of lubiprostone, known to promote repair of barrier function was carried out.

Results: In ischemia-damaged pig jejunum, using measurements of transepithelial resistance, (3)H-mannitol fluxes, short-circuit current (Cl(-) secretion) and occludin localization, active linaclotide failed to effectively promote repair of the epithelial barrier or recovery of short-circuit current, whereas lubiprostone promoted barrier repair and increased short-circuit current. In control pig jejunum, 1 μM linaclotide and 1 μM lubiprostone both caused similar increases in short-circuit current (Cl(-) secretion). In T84 cells, using measurements of transepithelial resistance, fluxes of fluorescent macromolecules, occludin and mitochondrial membrane potential, active linaclotide was virtually ineffective against damage caused by interferon-γ and tumor necrosis factor-α, while lubiprostone protected or promoted repair of epithelial barrier and cell function. Barrier protection/repair by lubiprostone was inhibited by methadone, a ClC-2 inhibitor. Linaclotide, but not lubiprostone increased [cGMP]i as expected and [Ca(2+)]i and linaclotide depolarized while lubiprostone hyperpolarized the T84 plasma membrane potential suggesting that lubiprostone may lead to greater cellular stability compared to linaclotide. In T84 cells, as found with linaclotide but not with lubiprostone, transepithelial resistance was slightly but significantly decreased by guanylin, STa and 8-bromo cGMP and fluorescent dextran fluxes were increased by guanylin. However the physiological implications of these small but statistically significant changes remain unclear.

Conclusions: Considering the physiological importance of epithelial barrier function and cell integrity and the known impact of stressors, the finding that lubiprostone, but not active linaclotide, exhibits the additional distinct property of effective protection or repair of the epithelial barrier and cell function after stress suggests potential clinical importance for patients with impaired or compromised barrier function such as might occur in IBS.

Show MeSH
Related in: MedlinePlus