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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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Effects of active linaclotide and lubiprostone on IFN-γ- induced changes of T84 epithelial cell barrier function and homeostasis: (A) ΔTER; (B) ΔFITC-LPS flux; (C) Δoccludin/actin ratio and (D) ΔΔmitochondrial membrane potential; and (E) Effect of active linaclotide and lubiprostone on TNF-α-induced changes of T84 epithelial cell barrier function measured as ΔTER and effect of methadone. 200 nM active linaclotide, 100 nM lubiprostone, 100 ng/ml IFN-γ and 50 ng/ml TNF-α were used. Data is plotted as mean ± S.E. (A) ΔTER: n = 3, #p < 0.02 wrt control, *p < 0.001 wrt DMSO, p < 0.0005 for IFN-γ/lin vs IFN-γ/lubi. Basal TERs = 1.56 ± 0.01 kΩ/cm2 and 1.26 ± 0.02 for control and DMSO respectively. Δlin vs Δlubi, p < 0.0005. (B) ΔFITC-LPS flux: n = 3, #p < 0.05 wrt control, *p < 0.005 wrt DMSO, p < 0.0005 for IFN-γ/lin vs IFN-γ/lubi. Basal LPS fluxes = 361.1 ± 3.4 and 378.1 ± 3.6 em units @ 530 nm for control and DMSO respectively. Δlin vs Δlubi, p < 0.01. (C) Δoccludin/actin ratio: n = 3, #p < 0.02 wrt control, *p < 0.0005 wrt DMSO, p < 0.0005 for IFN-γ/lin vs IFN-γ/lubi. Basal ratios = 0.98 ± 0.01 and 0.98 ± 0.01 for control and DMSO respectively. Δlin vs Δlubi, p < 0.0005. (D) ΔΔmitochondrial membrane potential: n = 8, #p < 0.001 wrt control, *p < 0.0005 wrt DMSO, p < 0.0005 for IFN-γ/lin vs IFN-γ/lubi. Basal Δ mito pds = 224.0 ± 0.2 and 211.1 ± 8.8 mV for control and DMSO respectively. Δlin vs Δlubi, p < 0.0005. (E) ΔTER: 1 μM methadone was used. NS, not significant wrt TNF-α/control, *p < 0.01 wrt TNF-α/DMSO, # p < 0.005 wrt TNF-α/lubi and p < 0.02 for TNF-α/lin vs TNF-α/lubi. Basal TERs = 1.32 ± 0.06 and 1.19 ± 0.01 kΩ/cm2 for control and DMSO respectively. Δlin vs Δlubi, p < 0.05.
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Figure 2: Effects of active linaclotide and lubiprostone on IFN-γ- induced changes of T84 epithelial cell barrier function and homeostasis: (A) ΔTER; (B) ΔFITC-LPS flux; (C) Δoccludin/actin ratio and (D) ΔΔmitochondrial membrane potential; and (E) Effect of active linaclotide and lubiprostone on TNF-α-induced changes of T84 epithelial cell barrier function measured as ΔTER and effect of methadone. 200 nM active linaclotide, 100 nM lubiprostone, 100 ng/ml IFN-γ and 50 ng/ml TNF-α were used. Data is plotted as mean ± S.E. (A) ΔTER: n = 3, #p < 0.02 wrt control, *p < 0.001 wrt DMSO, p < 0.0005 for IFN-γ/lin vs IFN-γ/lubi. Basal TERs = 1.56 ± 0.01 kΩ/cm2 and 1.26 ± 0.02 for control and DMSO respectively. Δlin vs Δlubi, p < 0.0005. (B) ΔFITC-LPS flux: n = 3, #p < 0.05 wrt control, *p < 0.005 wrt DMSO, p < 0.0005 for IFN-γ/lin vs IFN-γ/lubi. Basal LPS fluxes = 361.1 ± 3.4 and 378.1 ± 3.6 em units @ 530 nm for control and DMSO respectively. Δlin vs Δlubi, p < 0.01. (C) Δoccludin/actin ratio: n = 3, #p < 0.02 wrt control, *p < 0.0005 wrt DMSO, p < 0.0005 for IFN-γ/lin vs IFN-γ/lubi. Basal ratios = 0.98 ± 0.01 and 0.98 ± 0.01 for control and DMSO respectively. Δlin vs Δlubi, p < 0.0005. (D) ΔΔmitochondrial membrane potential: n = 8, #p < 0.001 wrt control, *p < 0.0005 wrt DMSO, p < 0.0005 for IFN-γ/lin vs IFN-γ/lubi. Basal Δ mito pds = 224.0 ± 0.2 and 211.1 ± 8.8 mV for control and DMSO respectively. Δlin vs Δlubi, p < 0.0005. (E) ΔTER: 1 μM methadone was used. NS, not significant wrt TNF-α/control, *p < 0.01 wrt TNF-α/DMSO, # p < 0.005 wrt TNF-α/lubi and p < 0.02 for TNF-α/lin vs TNF-α/lubi. Basal TERs = 1.32 ± 0.06 and 1.19 ± 0.01 kΩ/cm2 for control and DMSO respectively. Δlin vs Δlubi, p < 0.05.

Mentions: IFN-γ, a pro-inflammatory cytokine, reduces TER in part through reduction of occludin levels and is thought to be involved in the pathology of celiac sprue enteropathy and inflammatory bowel disease (ulcerative colitis and Crohn’s disease) [33-37]. Similar to IFN-γ, TNF-α also causes loss of barrier function [34,36,37]. Lubiprostone has been shown to promote repair of intestinal barrier properties after ischemia in the pig intestine model [22-24] whereas nothing is known of the effect of linaclotide. Therefore the effects of active linaclotide and lubiprostone on IFN-γ- and TNF-α-induced loss/disruption of T84 epithelial cell barrier and cell function were next examined. TER, mucosal to serosal FITC-LPS flux, occludin/actin ratio and mitochondrial membrane potential were measured. The results shown in Figure 2 are plotted as changes compared to vehicle controls (basal levels are given in the legend). IFN-γ (100 ng/ml) significantly reduced TER (Figure 2A, p < 0.0005), increased mucosal to serosal FITC-LPS flux (Figure 2B, p < 0.0005), decreased the occludin/actin ratio (Figure 2C, p < 0.0005) and decreased the mitochondrial membrane potential (Figure 2D, p < 0.0005). Active linaclotide had small IFN-γ-counteracting effects (ca. 5.7%, 20%, 23% and 30%, Figures 2A, B,C and D respectively), while lubiprostone had greater effects (ca. 27.3%, 89%, 43.8% and 77.8%, Figure 2A, B, C and D respectively), partially or totally preventing/repairing the effects of IFN-γ. The significance of the Δlinaclotide versus Δlubiprostone in the presence of IFN-γ for Figure 2A2B2C &2D were p < 0.0005, p < 0.0005, p < 0.02 and p < 0.0005 respectively. Guanylin (200 nM), STa (50 nM) and 8Br-cGMP (1 mM) had effects similar to active linaclotide (data not shown). Active linaclotide was relatively ineffective, while lubiprostone was significantly effective in protecting from or repairing the detrimental effects of IFN-γ on T84 epithelial cell barrier function and cell homeostasis. Thus, after IFN-γ lubiprostone, but not linaclotide, protected or repaired barrier and cell function.


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)

Effects of active linaclotide and lubiprostone on IFN-γ- induced changes of T84 epithelial cell barrier function and homeostasis: (A) ΔTER; (B) ΔFITC-LPS flux; (C) Δoccludin/actin ratio and (D) ΔΔmitochondrial membrane potential; and (E) Effect of active linaclotide and lubiprostone on TNF-α-induced changes of T84 epithelial cell barrier function measured as ΔTER and effect of methadone. 200 nM active linaclotide, 100 nM lubiprostone, 100 ng/ml IFN-γ and 50 ng/ml TNF-α were used. Data is plotted as mean ± S.E. (A) ΔTER: n = 3, #p < 0.02 wrt control, *p < 0.001 wrt DMSO, p < 0.0005 for IFN-γ/lin vs IFN-γ/lubi. Basal TERs = 1.56 ± 0.01 kΩ/cm2 and 1.26 ± 0.02 for control and DMSO respectively. Δlin vs Δlubi, p < 0.0005. (B) ΔFITC-LPS flux: n = 3, #p < 0.05 wrt control, *p < 0.005 wrt DMSO, p < 0.0005 for IFN-γ/lin vs IFN-γ/lubi. Basal LPS fluxes = 361.1 ± 3.4 and 378.1 ± 3.6 em units @ 530 nm for control and DMSO respectively. Δlin vs Δlubi, p < 0.01. (C) Δoccludin/actin ratio: n = 3, #p < 0.02 wrt control, *p < 0.0005 wrt DMSO, p < 0.0005 for IFN-γ/lin vs IFN-γ/lubi. Basal ratios = 0.98 ± 0.01 and 0.98 ± 0.01 for control and DMSO respectively. Δlin vs Δlubi, p < 0.0005. (D) ΔΔmitochondrial membrane potential: n = 8, #p < 0.001 wrt control, *p < 0.0005 wrt DMSO, p < 0.0005 for IFN-γ/lin vs IFN-γ/lubi. Basal Δ mito pds = 224.0 ± 0.2 and 211.1 ± 8.8 mV for control and DMSO respectively. Δlin vs Δlubi, p < 0.0005. (E) ΔTER: 1 μM methadone was used. NS, not significant wrt TNF-α/control, *p < 0.01 wrt TNF-α/DMSO, # p < 0.005 wrt TNF-α/lubi and p < 0.02 for TNF-α/lin vs TNF-α/lubi. Basal TERs = 1.32 ± 0.06 and 1.19 ± 0.01 kΩ/cm2 for control and DMSO respectively. Δlin vs Δlubi, p < 0.05.
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Figure 2: Effects of active linaclotide and lubiprostone on IFN-γ- induced changes of T84 epithelial cell barrier function and homeostasis: (A) ΔTER; (B) ΔFITC-LPS flux; (C) Δoccludin/actin ratio and (D) ΔΔmitochondrial membrane potential; and (E) Effect of active linaclotide and lubiprostone on TNF-α-induced changes of T84 epithelial cell barrier function measured as ΔTER and effect of methadone. 200 nM active linaclotide, 100 nM lubiprostone, 100 ng/ml IFN-γ and 50 ng/ml TNF-α were used. Data is plotted as mean ± S.E. (A) ΔTER: n = 3, #p < 0.02 wrt control, *p < 0.001 wrt DMSO, p < 0.0005 for IFN-γ/lin vs IFN-γ/lubi. Basal TERs = 1.56 ± 0.01 kΩ/cm2 and 1.26 ± 0.02 for control and DMSO respectively. Δlin vs Δlubi, p < 0.0005. (B) ΔFITC-LPS flux: n = 3, #p < 0.05 wrt control, *p < 0.005 wrt DMSO, p < 0.0005 for IFN-γ/lin vs IFN-γ/lubi. Basal LPS fluxes = 361.1 ± 3.4 and 378.1 ± 3.6 em units @ 530 nm for control and DMSO respectively. Δlin vs Δlubi, p < 0.01. (C) Δoccludin/actin ratio: n = 3, #p < 0.02 wrt control, *p < 0.0005 wrt DMSO, p < 0.0005 for IFN-γ/lin vs IFN-γ/lubi. Basal ratios = 0.98 ± 0.01 and 0.98 ± 0.01 for control and DMSO respectively. Δlin vs Δlubi, p < 0.0005. (D) ΔΔmitochondrial membrane potential: n = 8, #p < 0.001 wrt control, *p < 0.0005 wrt DMSO, p < 0.0005 for IFN-γ/lin vs IFN-γ/lubi. Basal Δ mito pds = 224.0 ± 0.2 and 211.1 ± 8.8 mV for control and DMSO respectively. Δlin vs Δlubi, p < 0.0005. (E) ΔTER: 1 μM methadone was used. NS, not significant wrt TNF-α/control, *p < 0.01 wrt TNF-α/DMSO, # p < 0.005 wrt TNF-α/lubi and p < 0.02 for TNF-α/lin vs TNF-α/lubi. Basal TERs = 1.32 ± 0.06 and 1.19 ± 0.01 kΩ/cm2 for control and DMSO respectively. Δlin vs Δlubi, p < 0.05.
Mentions: IFN-γ, a pro-inflammatory cytokine, reduces TER in part through reduction of occludin levels and is thought to be involved in the pathology of celiac sprue enteropathy and inflammatory bowel disease (ulcerative colitis and Crohn’s disease) [33-37]. Similar to IFN-γ, TNF-α also causes loss of barrier function [34,36,37]. Lubiprostone has been shown to promote repair of intestinal barrier properties after ischemia in the pig intestine model [22-24] whereas nothing is known of the effect of linaclotide. Therefore the effects of active linaclotide and lubiprostone on IFN-γ- and TNF-α-induced loss/disruption of T84 epithelial cell barrier and cell function were next examined. TER, mucosal to serosal FITC-LPS flux, occludin/actin ratio and mitochondrial membrane potential were measured. The results shown in Figure 2 are plotted as changes compared to vehicle controls (basal levels are given in the legend). IFN-γ (100 ng/ml) significantly reduced TER (Figure 2A, p < 0.0005), increased mucosal to serosal FITC-LPS flux (Figure 2B, p < 0.0005), decreased the occludin/actin ratio (Figure 2C, p < 0.0005) and decreased the mitochondrial membrane potential (Figure 2D, p < 0.0005). Active linaclotide had small IFN-γ-counteracting effects (ca. 5.7%, 20%, 23% and 30%, Figures 2A, B,C and D respectively), while lubiprostone had greater effects (ca. 27.3%, 89%, 43.8% and 77.8%, Figure 2A, B, C and D respectively), partially or totally preventing/repairing the effects of IFN-γ. The significance of the Δlinaclotide versus Δlubiprostone in the presence of IFN-γ for Figure 2A2B2C &2D were p < 0.0005, p < 0.0005, p < 0.02 and p < 0.0005 respectively. Guanylin (200 nM), STa (50 nM) and 8Br-cGMP (1 mM) had effects similar to active linaclotide (data not shown). Active linaclotide was relatively ineffective, while lubiprostone was significantly effective in protecting from or repairing the detrimental effects of IFN-γ on T84 epithelial cell barrier function and cell homeostasis. Thus, after IFN-γ lubiprostone, but not linaclotide, protected or repaired barrier and cell function.

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