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Voltage-dependent anion channel-1 (VDAC-1) contributes to ATP release and cell volume regulation in murine cells.

Okada SF, O'Neal WK, Huang P, Nicholas RA, Ostrowski LE, Craigen WJ, Lazarowski ER, Boucher RC - J. Gen. Physiol. (2004)

Bottom Line: However, the mechanisms mediating ATP release onto airway surfaces remain unknown.Taken together, these studies suggest that VDAC-1, directly or indirectly, contributes to ATP release from murine cells.However, the observation that VDAC-1 knockout cells released a significant amount of ATP suggests that other molecules also play a role in this function.

View Article: PubMed Central - PubMed

Affiliation: Cystic Fibrosis/Pulmonary Research and Treatment Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. seiko_okada@med.unc.edu

ABSTRACT
Extracellular ATP regulates several elements of the mucus clearance process important for pulmonary host defense. However, the mechanisms mediating ATP release onto airway surfaces remain unknown. Mitochondrial voltage-dependent anion channels (mt-VDACs) translocate a variety of metabolites, including ATP and ADP, across the mitochondrial outer membrane, and a plasmalemmal splice variant (pl-VDAC-1) has been proposed to mediate ATP translocation across the plasma membrane. We tested the involvement of VDAC-1 in ATP release in a series of studies in murine cells. First, the full-length coding sequence was cloned from a mouse airway epithelial cell line (MTE7b-) and transfected into NIH 3T3 cells, and pl-VDAC-1-transfected cells exhibited higher rates of ATP release in response to medium change compared with mock-transfected cells. Second, ATP release was compared in cells isolated from VDAC-1 knockout [VDAC-1 (-/-)] and wild-type (WT) mice. Fibroblasts from VDAC-1 (-/-) mice released less ATP than WT mice in response to a medium change. Well-differentiated cultures from nasal and tracheal epithelia of VDAC-1 (-/-) mice exhibited less ATP release in response to luminal hypotonic challenge than WT mice. Confocal microscopy studies revealed that cell volume acutely increased in airway epithelia from both VDAC-1 (-/-) and WT mice after luminal hypotonic challenge, but VDAC-1 (-/-) cells exhibited a slower regulatory volume decrease (RVD) than WT cells. Addition of ATP or apyrase to the luminal surface of VDAC-1 (-/-) or WT cultures with hypotonic challenge produced similar initial cell height responses and RVD kinetics in both cell types, suggesting that involvement of VDAC-1 in RVD is through ATP release. Taken together, these studies suggest that VDAC-1, directly or indirectly, contributes to ATP release from murine cells. However, the observation that VDAC-1 knockout cells released a significant amount of ATP suggests that other molecules also play a role in this function.

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Hypotonicity-induced swelling and regulatory volume decrease (RVD) of tracheal cultures from FOXJ1/EGFP transgenic mice as measured by confocal microscopy. The cytosol of ciliated cells is defined by green fluorescence due to the ciliated cell–specific FOXJ1-driven expression of EGFP. Bar, 10 μm. (A) In the top panels, at t = 0, H2O was added gently onto the apical surface to generate 200 mOsm of hypotonicity. In the middle panels, cultures were preincubated with apyrase (10 U/ml) for 5 min, and H2O including apyrase (10 U/ml) was gently added to generate luminal hypotonicity at time 0. In the bottom panels, ATP (100 μM) was gently added together with luminal H2O challenge at t = 0. (B) Summary data for protocols illustrated in A. Points indicate mean relative tracheal cell height compared with t = 0, of cultures from two separate litters with n = 4 transwells/genotype/litter. * indicates significant difference (P < 0.05) of points for hypotonic challenge (H2O alone) group compared with t = 0. † indicates significant difference between hypotonic challenge alone (white) and apyrase pretreatment (blue) P < 0.05). ‡ indicates significant difference between hypotonic challenge alone and exogenous 100 μM ATP addition (red) (P < 0.05).
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fig6: Hypotonicity-induced swelling and regulatory volume decrease (RVD) of tracheal cultures from FOXJ1/EGFP transgenic mice as measured by confocal microscopy. The cytosol of ciliated cells is defined by green fluorescence due to the ciliated cell–specific FOXJ1-driven expression of EGFP. Bar, 10 μm. (A) In the top panels, at t = 0, H2O was added gently onto the apical surface to generate 200 mOsm of hypotonicity. In the middle panels, cultures were preincubated with apyrase (10 U/ml) for 5 min, and H2O including apyrase (10 U/ml) was gently added to generate luminal hypotonicity at time 0. In the bottom panels, ATP (100 μM) was gently added together with luminal H2O challenge at t = 0. (B) Summary data for protocols illustrated in A. Points indicate mean relative tracheal cell height compared with t = 0, of cultures from two separate litters with n = 4 transwells/genotype/litter. * indicates significant difference (P < 0.05) of points for hypotonic challenge (H2O alone) group compared with t = 0. † indicates significant difference between hypotonic challenge alone (white) and apyrase pretreatment (blue) P < 0.05). ‡ indicates significant difference between hypotonic challenge alone and exogenous 100 μM ATP addition (red) (P < 0.05).

Mentions: Airway epithelial cultures exhibit a complex phenotype with differentiated surface cells, including ciliated cells and goblet cells, covering a single to several layers of basal cells. To investigate the role of ATP release in epithelial cell volume regulatory responses, we initiated studies with a model system that allowed accurate measurements of the volume responses of lumen-facing cells to luminal hypotonicity. Well-differentiated tracheal cultures from FOXJ1/EGFP transgenic mice that selectively express EGFP in ciliated cells (Ostrowski et al., 2003) were used. The height of each fluorescent ciliated cell was measured by confocal microscopy before (t = 0) and at designated times after dilution of the apical liquid to form a hypotonic (200 mOsm) solution. Tracheal epithelial cells exhibited a rapid increase in height after hypotonic challenge (Fig. 6 A). This initial increase (∼25%) was less than the expected level of volume increase (50%) if these cells acted as perfect osmometers in response to the dilution of apical liquid (300 to 200 mOsm) (Fig. 6 B). In comparison to previous reports describing the absence of regulatory volume increase (RVI) in human airway epithelia after hypertonic challenge (Willumsen et al., 1994; Matsui et al., 2000), mouse airway cells exhibited an RVD after hypotonic challenge.


Voltage-dependent anion channel-1 (VDAC-1) contributes to ATP release and cell volume regulation in murine cells.

Okada SF, O'Neal WK, Huang P, Nicholas RA, Ostrowski LE, Craigen WJ, Lazarowski ER, Boucher RC - J. Gen. Physiol. (2004)

Hypotonicity-induced swelling and regulatory volume decrease (RVD) of tracheal cultures from FOXJ1/EGFP transgenic mice as measured by confocal microscopy. The cytosol of ciliated cells is defined by green fluorescence due to the ciliated cell–specific FOXJ1-driven expression of EGFP. Bar, 10 μm. (A) In the top panels, at t = 0, H2O was added gently onto the apical surface to generate 200 mOsm of hypotonicity. In the middle panels, cultures were preincubated with apyrase (10 U/ml) for 5 min, and H2O including apyrase (10 U/ml) was gently added to generate luminal hypotonicity at time 0. In the bottom panels, ATP (100 μM) was gently added together with luminal H2O challenge at t = 0. (B) Summary data for protocols illustrated in A. Points indicate mean relative tracheal cell height compared with t = 0, of cultures from two separate litters with n = 4 transwells/genotype/litter. * indicates significant difference (P < 0.05) of points for hypotonic challenge (H2O alone) group compared with t = 0. † indicates significant difference between hypotonic challenge alone (white) and apyrase pretreatment (blue) P < 0.05). ‡ indicates significant difference between hypotonic challenge alone and exogenous 100 μM ATP addition (red) (P < 0.05).
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Related In: Results  -  Collection

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fig6: Hypotonicity-induced swelling and regulatory volume decrease (RVD) of tracheal cultures from FOXJ1/EGFP transgenic mice as measured by confocal microscopy. The cytosol of ciliated cells is defined by green fluorescence due to the ciliated cell–specific FOXJ1-driven expression of EGFP. Bar, 10 μm. (A) In the top panels, at t = 0, H2O was added gently onto the apical surface to generate 200 mOsm of hypotonicity. In the middle panels, cultures were preincubated with apyrase (10 U/ml) for 5 min, and H2O including apyrase (10 U/ml) was gently added to generate luminal hypotonicity at time 0. In the bottom panels, ATP (100 μM) was gently added together with luminal H2O challenge at t = 0. (B) Summary data for protocols illustrated in A. Points indicate mean relative tracheal cell height compared with t = 0, of cultures from two separate litters with n = 4 transwells/genotype/litter. * indicates significant difference (P < 0.05) of points for hypotonic challenge (H2O alone) group compared with t = 0. † indicates significant difference between hypotonic challenge alone (white) and apyrase pretreatment (blue) P < 0.05). ‡ indicates significant difference between hypotonic challenge alone and exogenous 100 μM ATP addition (red) (P < 0.05).
Mentions: Airway epithelial cultures exhibit a complex phenotype with differentiated surface cells, including ciliated cells and goblet cells, covering a single to several layers of basal cells. To investigate the role of ATP release in epithelial cell volume regulatory responses, we initiated studies with a model system that allowed accurate measurements of the volume responses of lumen-facing cells to luminal hypotonicity. Well-differentiated tracheal cultures from FOXJ1/EGFP transgenic mice that selectively express EGFP in ciliated cells (Ostrowski et al., 2003) were used. The height of each fluorescent ciliated cell was measured by confocal microscopy before (t = 0) and at designated times after dilution of the apical liquid to form a hypotonic (200 mOsm) solution. Tracheal epithelial cells exhibited a rapid increase in height after hypotonic challenge (Fig. 6 A). This initial increase (∼25%) was less than the expected level of volume increase (50%) if these cells acted as perfect osmometers in response to the dilution of apical liquid (300 to 200 mOsm) (Fig. 6 B). In comparison to previous reports describing the absence of regulatory volume increase (RVI) in human airway epithelia after hypertonic challenge (Willumsen et al., 1994; Matsui et al., 2000), mouse airway cells exhibited an RVD after hypotonic challenge.

Bottom Line: However, the mechanisms mediating ATP release onto airway surfaces remain unknown.Taken together, these studies suggest that VDAC-1, directly or indirectly, contributes to ATP release from murine cells.However, the observation that VDAC-1 knockout cells released a significant amount of ATP suggests that other molecules also play a role in this function.

View Article: PubMed Central - PubMed

Affiliation: Cystic Fibrosis/Pulmonary Research and Treatment Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. seiko_okada@med.unc.edu

ABSTRACT
Extracellular ATP regulates several elements of the mucus clearance process important for pulmonary host defense. However, the mechanisms mediating ATP release onto airway surfaces remain unknown. Mitochondrial voltage-dependent anion channels (mt-VDACs) translocate a variety of metabolites, including ATP and ADP, across the mitochondrial outer membrane, and a plasmalemmal splice variant (pl-VDAC-1) has been proposed to mediate ATP translocation across the plasma membrane. We tested the involvement of VDAC-1 in ATP release in a series of studies in murine cells. First, the full-length coding sequence was cloned from a mouse airway epithelial cell line (MTE7b-) and transfected into NIH 3T3 cells, and pl-VDAC-1-transfected cells exhibited higher rates of ATP release in response to medium change compared with mock-transfected cells. Second, ATP release was compared in cells isolated from VDAC-1 knockout [VDAC-1 (-/-)] and wild-type (WT) mice. Fibroblasts from VDAC-1 (-/-) mice released less ATP than WT mice in response to a medium change. Well-differentiated cultures from nasal and tracheal epithelia of VDAC-1 (-/-) mice exhibited less ATP release in response to luminal hypotonic challenge than WT mice. Confocal microscopy studies revealed that cell volume acutely increased in airway epithelia from both VDAC-1 (-/-) and WT mice after luminal hypotonic challenge, but VDAC-1 (-/-) cells exhibited a slower regulatory volume decrease (RVD) than WT cells. Addition of ATP or apyrase to the luminal surface of VDAC-1 (-/-) or WT cultures with hypotonic challenge produced similar initial cell height responses and RVD kinetics in both cell types, suggesting that involvement of VDAC-1 in RVD is through ATP release. Taken together, these studies suggest that VDAC-1, directly or indirectly, contributes to ATP release from murine cells. However, the observation that VDAC-1 knockout cells released a significant amount of ATP suggests that other molecules also play a role in this function.

Show MeSH
Related in: MedlinePlus