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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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Extracellular ATP concentrations in the apical surface liquid of tracheal cultures from WT and VDAC-1 (−/−) mice after apical hypotonic challenge. (A) Apical hypotonic challenge: H2O was added gently to the apical surface at t = 0 to generate a 200 mOsm solution, and samples were collected from the apical solution at each indicated time and analyzed by luminometry. (B) Apical isosmotic challenge: a 300 mOsm mannitol solution was added to the lumen at t = 0 and samples obtained and processed as in A. Points represent mean, ±SEM, of experiments conducted on three separate litters with n = 8 transwells/genotype/litter. * indicates significant difference between WT (⋄) and VDAC-1 (−/−) (•) mice (P < 0.05). (C and D) Profile of adenyl purines in the apical surface liquid covering (D) basal (t = 0) and (C) hypotonicity-stimulated (2 min after challenge) tracheal epithelia. Samples at each point were analyzed by etheno-derivertization. Bars represent mean, ±SEM, of experiments conducted on three separate litters with n = 12 transwells/genotype/litter. * indicates significant difference between WT (□) and VDAC-1 (−/−) (▪) mice (P < 0.05). (E) Intracellular ATP levels determined after extraction by TCA. Bars represent mean, ±SEM, of three litters with n = 4 transwells/genotype /litter.
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fig5: Extracellular ATP concentrations in the apical surface liquid of tracheal cultures from WT and VDAC-1 (−/−) mice after apical hypotonic challenge. (A) Apical hypotonic challenge: H2O was added gently to the apical surface at t = 0 to generate a 200 mOsm solution, and samples were collected from the apical solution at each indicated time and analyzed by luminometry. (B) Apical isosmotic challenge: a 300 mOsm mannitol solution was added to the lumen at t = 0 and samples obtained and processed as in A. Points represent mean, ±SEM, of experiments conducted on three separate litters with n = 8 transwells/genotype/litter. * indicates significant difference between WT (⋄) and VDAC-1 (−/−) (•) mice (P < 0.05). (C and D) Profile of adenyl purines in the apical surface liquid covering (D) basal (t = 0) and (C) hypotonicity-stimulated (2 min after challenge) tracheal epithelia. Samples at each point were analyzed by etheno-derivertization. Bars represent mean, ±SEM, of experiments conducted on three separate litters with n = 12 transwells/genotype/litter. * indicates significant difference between WT (□) and VDAC-1 (−/−) (▪) mice (P < 0.05). (E) Intracellular ATP levels determined after extraction by TCA. Bars represent mean, ±SEM, of three litters with n = 4 transwells/genotype /litter.

Mentions: With luminometry, no differences in ATP concentrations were observed between VDAC-1 (−/−) and WT tracheal cells in the basal (resting) state in either the apical (Fig. 5 A) or basolateral (0.1 ± 0.03 nM in VDAC (−/−) and 0.11 ± 0.02 nM in WT, three experiments on three separate litters with n = 4 transwells/genotype/litter) liquids. 2 min after 10 μl H2O was gently added to the apical surface to generate a 66% hypotonic challenge, a robust increase in ATP in apical surface liquid was observed in WT cells, whereas VDAC-1 (−/−) mouse cultures exhibited a smaller increase in levels of released ATP (Fig. 5 A). Basolateral bath ATP concentrations showed no change in response to apical hypotonic challenge for either genotype (ΔATP at 2 min = 0.02 ± 0.005 nM in VDAC-1 (−/−) and 0.03 ± 0.01 nM in WT, three experiments on three separate litters with n = 4 transwells/genotype/litter).


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)

Extracellular ATP concentrations in the apical surface liquid of tracheal cultures from WT and VDAC-1 (−/−) mice after apical hypotonic challenge. (A) Apical hypotonic challenge: H2O was added gently to the apical surface at t = 0 to generate a 200 mOsm solution, and samples were collected from the apical solution at each indicated time and analyzed by luminometry. (B) Apical isosmotic challenge: a 300 mOsm mannitol solution was added to the lumen at t = 0 and samples obtained and processed as in A. Points represent mean, ±SEM, of experiments conducted on three separate litters with n = 8 transwells/genotype/litter. * indicates significant difference between WT (⋄) and VDAC-1 (−/−) (•) mice (P < 0.05). (C and D) Profile of adenyl purines in the apical surface liquid covering (D) basal (t = 0) and (C) hypotonicity-stimulated (2 min after challenge) tracheal epithelia. Samples at each point were analyzed by etheno-derivertization. Bars represent mean, ±SEM, of experiments conducted on three separate litters with n = 12 transwells/genotype/litter. * indicates significant difference between WT (□) and VDAC-1 (−/−) (▪) mice (P < 0.05). (E) Intracellular ATP levels determined after extraction by TCA. Bars represent mean, ±SEM, of three litters with n = 4 transwells/genotype /litter.
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fig5: Extracellular ATP concentrations in the apical surface liquid of tracheal cultures from WT and VDAC-1 (−/−) mice after apical hypotonic challenge. (A) Apical hypotonic challenge: H2O was added gently to the apical surface at t = 0 to generate a 200 mOsm solution, and samples were collected from the apical solution at each indicated time and analyzed by luminometry. (B) Apical isosmotic challenge: a 300 mOsm mannitol solution was added to the lumen at t = 0 and samples obtained and processed as in A. Points represent mean, ±SEM, of experiments conducted on three separate litters with n = 8 transwells/genotype/litter. * indicates significant difference between WT (⋄) and VDAC-1 (−/−) (•) mice (P < 0.05). (C and D) Profile of adenyl purines in the apical surface liquid covering (D) basal (t = 0) and (C) hypotonicity-stimulated (2 min after challenge) tracheal epithelia. Samples at each point were analyzed by etheno-derivertization. Bars represent mean, ±SEM, of experiments conducted on three separate litters with n = 12 transwells/genotype/litter. * indicates significant difference between WT (□) and VDAC-1 (−/−) (▪) mice (P < 0.05). (E) Intracellular ATP levels determined after extraction by TCA. Bars represent mean, ±SEM, of three litters with n = 4 transwells/genotype /litter.
Mentions: With luminometry, no differences in ATP concentrations were observed between VDAC-1 (−/−) and WT tracheal cells in the basal (resting) state in either the apical (Fig. 5 A) or basolateral (0.1 ± 0.03 nM in VDAC (−/−) and 0.11 ± 0.02 nM in WT, three experiments on three separate litters with n = 4 transwells/genotype/litter) liquids. 2 min after 10 μl H2O was gently added to the apical surface to generate a 66% hypotonic challenge, a robust increase in ATP in apical surface liquid was observed in WT cells, whereas VDAC-1 (−/−) mouse cultures exhibited a smaller increase in levels of released ATP (Fig. 5 A). Basolateral bath ATP concentrations showed no change in response to apical hypotonic challenge for either genotype (ΔATP at 2 min = 0.02 ± 0.005 nM in VDAC-1 (−/−) and 0.03 ± 0.01 nM in WT, three experiments on three separate litters with n = 4 transwells/genotype/litter).

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