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Volume-regulated anion channels serve as an auto/paracrine nucleotide release pathway in aortic endothelial cells.

Hisadome K, Koyama T, Kimura C, Droogmans G, Ito Y, Oike M - J. Gen. Physiol. (2002)

Bottom Line: They did not, however, affect Ca(2+) oscillations and NO production induced by exogenously applied ATP.However, inhibition by ADP was less voltage sensitive, whereas adenosine did not affect VRAC currents, suggesting that the negative charges of the nucleotides are essential for their inhibitory action.We conclude from these observations that VRAC is a nucleotide-permeable channel, which may serve as a pathway for HTS-induced ATP release in BAEC.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan.

ABSTRACT
Mechanical stress induces auto/paracrine ATP release from various cell types, but the mechanisms underlying this release are not well understood. Here we show that the release of ATP induced by hypotonic stress (HTS) in bovine aortic endothelial cells (BAECs) occurs through volume-regulated anion channels (VRAC). Various VRAC inhibitors, such as glibenclamide, verapamil, tamoxifen, and fluoxetine, suppressed the HTS-induced release of ATP, as well as the concomitant Ca(2+) oscillations and NO production. They did not, however, affect Ca(2+) oscillations and NO production induced by exogenously applied ATP. Extracellular ATP inhibited VRAC currents in a voltage-dependent manner: block was absent at negative potentials and was manifest at positive potentials, but decreased at highly depolarized potentials. This phenomenon could be described with a "permeating blocker model," in which ATP binds with an affinity of 1.0 +/- 0.5 mM at 0 mV to a site at an electrical distance of 0.41 inside the channel. Bound ATP occludes the channel at moderate positive potentials, but permeates into the cytosol at more depolarized potentials. The triphosphate nucleotides UTP, GTP, and CTP, and the adenine nucleotide ADP, exerted a similar voltage-dependent inhibition of VRAC currents at submillimolar concentrations, which could also be described with this model. However, inhibition by ADP was less voltage sensitive, whereas adenosine did not affect VRAC currents, suggesting that the negative charges of the nucleotides are essential for their inhibitory action. The observation that high concentrations of extracellular ADP enhanced the outward component of the VRAC current in low Cl(-) hypotonic solution and shifted its reversal potential to negative potentials provides more direct evidence for the nucleotide permeability of VRAC. We conclude from these observations that VRAC is a nucleotide-permeable channel, which may serve as a pathway for HTS-induced ATP release in BAEC.

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Representative data showing the effects of adenine nucleotides and triphosphate nucleotides other than ATP on VRAC currents. Total concentration of each nucleotide is 1 mM, the calculated free concentration is given in the figure. (A) Current-voltage relationships in isotonic (i), and in hypotonic solution in the absence (ii) or presence (iii) of the nucleotide. Except for adenosine, all nucleotides affect the outward current but have no virtual effect on inward current. (B) Percent inhibition of VRAC by the various nucleotides. It is obvious that the AMP-block is hardly voltage-dependent. Data of all other nucleotides except for AMP could be fitted to Eq. 2 (solid lines). Values of Kd(0), δ and r for these fits were 0.81 mM, 0.40, and 0.26 for ADP; 4.1 mM, 0.44, and 0.057 for CTP; 0.41 mM, 0.44, and 0.24 for GTP; and 1.1 mM, 0.42, and 0.13 for UTP.
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fig5: Representative data showing the effects of adenine nucleotides and triphosphate nucleotides other than ATP on VRAC currents. Total concentration of each nucleotide is 1 mM, the calculated free concentration is given in the figure. (A) Current-voltage relationships in isotonic (i), and in hypotonic solution in the absence (ii) or presence (iii) of the nucleotide. Except for adenosine, all nucleotides affect the outward current but have no virtual effect on inward current. (B) Percent inhibition of VRAC by the various nucleotides. It is obvious that the AMP-block is hardly voltage-dependent. Data of all other nucleotides except for AMP could be fitted to Eq. 2 (solid lines). Values of Kd(0), δ and r for these fits were 0.81 mM, 0.40, and 0.26 for ADP; 4.1 mM, 0.44, and 0.057 for CTP; 0.41 mM, 0.44, and 0.24 for GTP; and 1.1 mM, 0.42, and 0.13 for UTP.

Mentions: Whole cell membrane current was recorded in the conventional ruptured whole cell configuration (Hamill et al., 1981) with an EPC-9 amplifier (Heka Elekronik GmbH). The pipette solution for examining the effects of VRAC inhibitors and brefeldin A contained (in mM): KCl 40, K-aspartate 100, MgCl2 1, Na2ATP 5, HEPES 10, and EGTA 5 (pH adjusted to 7.3 with KOH). For the effects of 1 mM extracellular nucleotides (Figs. 4 and 5), the pipette solution contained (in mM): CsCl 45, Cs-aspartate 100, MgCl2 1, Na2ATP 5, HEPES 10, BAPTA 5, and CaCl2 1.436 (to give free [Ca2+]i of 30 nM, pH adjusted to 7.3 with CsOH). To examine the contribution of extracellular ADP to the VRAC current (see Fig. 6), we have used a pipette solution with reduced Cl− concentration containing (in mM): Cs-aspartate 145, MgCl2 1, Na2ATP 1, HEPES 10, BAPTA 5, and CaCl2 1.503 (to give free [Ca2+]i of 30 nM, pH adjusted to 7.3 with CsOH). The osmolarity of each solution was adjusted to 300 mOsm with a freezing point depression osmometer (OM-801; Vogel) by adding mannitol. In the experiments with extracellular nucleotides, we pretreated the cells with 1 μM thapsigargin for 30 min to deplete intracellular Ca2+ stores in order to avoid a possible contamination with Ca2+-activated chloride currents (Nilius et al., 1997b) that might be activated by nucleotide-induced Ca2+ release.


Volume-regulated anion channels serve as an auto/paracrine nucleotide release pathway in aortic endothelial cells.

Hisadome K, Koyama T, Kimura C, Droogmans G, Ito Y, Oike M - J. Gen. Physiol. (2002)

Representative data showing the effects of adenine nucleotides and triphosphate nucleotides other than ATP on VRAC currents. Total concentration of each nucleotide is 1 mM, the calculated free concentration is given in the figure. (A) Current-voltage relationships in isotonic (i), and in hypotonic solution in the absence (ii) or presence (iii) of the nucleotide. Except for adenosine, all nucleotides affect the outward current but have no virtual effect on inward current. (B) Percent inhibition of VRAC by the various nucleotides. It is obvious that the AMP-block is hardly voltage-dependent. Data of all other nucleotides except for AMP could be fitted to Eq. 2 (solid lines). Values of Kd(0), δ and r for these fits were 0.81 mM, 0.40, and 0.26 for ADP; 4.1 mM, 0.44, and 0.057 for CTP; 0.41 mM, 0.44, and 0.24 for GTP; and 1.1 mM, 0.42, and 0.13 for UTP.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2233868&req=5

fig5: Representative data showing the effects of adenine nucleotides and triphosphate nucleotides other than ATP on VRAC currents. Total concentration of each nucleotide is 1 mM, the calculated free concentration is given in the figure. (A) Current-voltage relationships in isotonic (i), and in hypotonic solution in the absence (ii) or presence (iii) of the nucleotide. Except for adenosine, all nucleotides affect the outward current but have no virtual effect on inward current. (B) Percent inhibition of VRAC by the various nucleotides. It is obvious that the AMP-block is hardly voltage-dependent. Data of all other nucleotides except for AMP could be fitted to Eq. 2 (solid lines). Values of Kd(0), δ and r for these fits were 0.81 mM, 0.40, and 0.26 for ADP; 4.1 mM, 0.44, and 0.057 for CTP; 0.41 mM, 0.44, and 0.24 for GTP; and 1.1 mM, 0.42, and 0.13 for UTP.
Mentions: Whole cell membrane current was recorded in the conventional ruptured whole cell configuration (Hamill et al., 1981) with an EPC-9 amplifier (Heka Elekronik GmbH). The pipette solution for examining the effects of VRAC inhibitors and brefeldin A contained (in mM): KCl 40, K-aspartate 100, MgCl2 1, Na2ATP 5, HEPES 10, and EGTA 5 (pH adjusted to 7.3 with KOH). For the effects of 1 mM extracellular nucleotides (Figs. 4 and 5), the pipette solution contained (in mM): CsCl 45, Cs-aspartate 100, MgCl2 1, Na2ATP 5, HEPES 10, BAPTA 5, and CaCl2 1.436 (to give free [Ca2+]i of 30 nM, pH adjusted to 7.3 with CsOH). To examine the contribution of extracellular ADP to the VRAC current (see Fig. 6), we have used a pipette solution with reduced Cl− concentration containing (in mM): Cs-aspartate 145, MgCl2 1, Na2ATP 1, HEPES 10, BAPTA 5, and CaCl2 1.503 (to give free [Ca2+]i of 30 nM, pH adjusted to 7.3 with CsOH). The osmolarity of each solution was adjusted to 300 mOsm with a freezing point depression osmometer (OM-801; Vogel) by adding mannitol. In the experiments with extracellular nucleotides, we pretreated the cells with 1 μM thapsigargin for 30 min to deplete intracellular Ca2+ stores in order to avoid a possible contamination with Ca2+-activated chloride currents (Nilius et al., 1997b) that might be activated by nucleotide-induced Ca2+ release.

Bottom Line: They did not, however, affect Ca(2+) oscillations and NO production induced by exogenously applied ATP.However, inhibition by ADP was less voltage sensitive, whereas adenosine did not affect VRAC currents, suggesting that the negative charges of the nucleotides are essential for their inhibitory action.We conclude from these observations that VRAC is a nucleotide-permeable channel, which may serve as a pathway for HTS-induced ATP release in BAEC.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan.

ABSTRACT
Mechanical stress induces auto/paracrine ATP release from various cell types, but the mechanisms underlying this release are not well understood. Here we show that the release of ATP induced by hypotonic stress (HTS) in bovine aortic endothelial cells (BAECs) occurs through volume-regulated anion channels (VRAC). Various VRAC inhibitors, such as glibenclamide, verapamil, tamoxifen, and fluoxetine, suppressed the HTS-induced release of ATP, as well as the concomitant Ca(2+) oscillations and NO production. They did not, however, affect Ca(2+) oscillations and NO production induced by exogenously applied ATP. Extracellular ATP inhibited VRAC currents in a voltage-dependent manner: block was absent at negative potentials and was manifest at positive potentials, but decreased at highly depolarized potentials. This phenomenon could be described with a "permeating blocker model," in which ATP binds with an affinity of 1.0 +/- 0.5 mM at 0 mV to a site at an electrical distance of 0.41 inside the channel. Bound ATP occludes the channel at moderate positive potentials, but permeates into the cytosol at more depolarized potentials. The triphosphate nucleotides UTP, GTP, and CTP, and the adenine nucleotide ADP, exerted a similar voltage-dependent inhibition of VRAC currents at submillimolar concentrations, which could also be described with this model. However, inhibition by ADP was less voltage sensitive, whereas adenosine did not affect VRAC currents, suggesting that the negative charges of the nucleotides are essential for their inhibitory action. The observation that high concentrations of extracellular ADP enhanced the outward component of the VRAC current in low Cl(-) hypotonic solution and shifted its reversal potential to negative potentials provides more direct evidence for the nucleotide permeability of VRAC. We conclude from these observations that VRAC is a nucleotide-permeable channel, which may serve as a pathway for HTS-induced ATP release in BAEC.

Show MeSH
Related in: MedlinePlus