Yushi Ito

Volume-regulated Anion Channels Serve as an Auto/Paracrine Nucleotide Release Pathway in Aortic Endothelial Cells

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 Ca2+ oscillations and NO production. They did not, however, affect Ca2+ 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.

Sequential activation of RhoA and FAK/paxillin leads to ATP release and actin reorganization in human endothelium

We have investigated the cellular mechanisms of mechanical stress‐induced immediate responses in human umbilical vein endothelial cells (HUVECs). Hypotonic stress (HTS) induced ATP release, which evoked a Ca2+ transient, followed by actin reorganization within a few minutes, in HUVECs. Disruption of the actin cytoskeleton did not suppress HTS‐induced ATP release, and inhibition of the ATP‐mediated Ca2+ response did not affect actin reorganization, thereby indicating that these two responses are not interrelated. ATP release and actin reorganization were also induced by lysophosphatidic acid (LPA). HTS and LPA induced membrane translocation of RhoA, which occurs when RhoA is activated, and tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin. Tyrosine kinase inhibitors (herbimycin A or tyrphostin 46) inhibited both HTS‐ and LPA‐induced ATP release and actin reorganization, but did not affect RhoA activation. In contrast, Rho‐kinase inhibitor (Y27632) inhibited all of the HTS‐ and LPA‐induced responses. These results indicate that the activation of the RhoA/Rho‐kinase pathway followed by tyrosine phosphorylation of FAK and paxillin leads to ATP release and actin reorganization in HUVECs. Furthermore, the fact that HTS and LPA evoke exactly the same intracellular signals and responses suggests that even these immediate mechanosensitive responses are in fact not mechanical stress‐specific.

Constitutive nitric oxide production in bovine aortic and brain microvascular endothelial cells: a comparative study.

Vascular endothelium constitutively generates nitric oxide (NO) in large vessels and induces a relaxation of smooth muscle cells. However, little is known about the production of NO in microvessels, where smooth muscle layers are thin or absent. In this study, we have compared the constitutive production of NO in bovine brain microvascular endothelial cells (BBECs) with that in bovine aortic endothelial cells (BAECs). ATP, acetylcholine (ACh) and A23187 induced Ca2+ transients both in BBECs and BAECs. In contrast, although ATP and A23187 evoked a similar degree of [Ca2+]i increase in both types of cell, they failed to induce NO production in BBECs, as measured with an NO‐sensitive fluorescent dye DAF‐2, whereas in BAECs there was an increase in DAF‐2 fluorescence. Hypotonic stress induced ATP release and subsequent NO production in BAECs, but not in BBECs. We have developed an in vitro model vessel system that consists of aortic smooth muscle cells embedded in a collagen gel lattice and overlaid with endothelial cells. Precontracted gels showed relaxation in response to ACh, when BAECs were overlaid. However, ACh‐induced relaxation was not observed in BBEC‐overlaid gels. Expression of eNOS protein as well as cellular uptake of l‐[3H]arginine were significantly lower in BBECs than in BAECs. These results indicate that Ca2+‐dependent NO production is at an undetectable level in BBEC, for which at least two factors, i.e. low levels of eNOS expression and l‐arginine uptake, are responsible.

Theophylline and cAMP inhibit lysophosphatidic acid‐induced hyperresponsiveness of bovine tracheal smooth muscle cells

We have established an in vitro model of airway hyperresponsiveness, using a bovine tracheal smooth muscle cell (BTSMC)‐embedded collagen gel lattice. When the gel was pretreated with lysophosphatidic acid (LPA), which activates the small G protein RhoA, ATP‐ and high K+ solution‐induced gel contraction was significantly augmented. This was not due to the modulation of Ca2+ mobilizing properties, since ATP‐ and high K+‐induced Ca2+ transients were not significantly different between control and LPA‐treated BTSMC. Y‐27632, an inhibitor of Rho‐kinase, suppressed the LPA‐induced augmentation of gel contraction, whereas it did not inhibit the contraction of control gels. Theophylline (> 1 μm) reversed the LPA‐induced augmentation of gel contraction, whereas it inhibited control gel contraction only with a very high concentration (100 μm). We confirmed that theophylline increased the intracellular concentration of cAMP ([cAMP]i) in BTSMC. Elevation of [cAMP]i with dibutyryl cAMP or forskolin also reversed the LPA‐induced augmentation of gel contraction. Furthermore, theophylline, as well as dibutyryl cAMP and forskolin, suppressed the LPA‐induced membrane translocation of RhoA, indicating that they prevented airway hyperresponsiveness by inhibiting RhoA. We conclude from these results that theophylline inhibits LPA‐induced, RhoA/Rho‐kinase‐mediated hyperresponsiveness of tracheal smooth muscle cells due to the accumulation of cAMP.

Functional involvement of sulphonylurea receptor (SUR) type 1 and 2B in the activity of pig urethral ATP-sensitive K+ channels

We have investigated the possible roles of sulphonylurea receptor (SUR) type 1 and 2B in the activity of pig urethral ATP‐sensitive K+ channels (KATP channels) by use of patch‐clamp and reverse transcriptase–polymerase chain reaction (RT–PCR) techniques. In voltage‐clamp experiments, not only diazoxide, a SUR1 and weak SUR2B activator, but also pinacidil, a selective SUR2 activator, caused an inward current at a holding potential of −50 mV in symmetrical 140 mM K+ conditions. Gliclazide (1 μM), a selective SUR1 blocker, inhibited the 10 μM pinacidil‐induced currents (Ki=177 μM) and the 500 μM diazoxide‐induced currents (high‐affinity site, Ki1=5 nM; low‐affinity site, Ki2=108 μM) at −50 mV. Application of tolbutamide (100 μM) reversibly caused an inhibition of the 500 μM diazoxide‐induced current at –50 mV. MCC‐134, a SUR type‐specific KATP channel regulator (1–100 μM), produced a concentration‐dependent inward K+ current, which was suppressed by the application of glibenclamide at −50 mV. The amplitude of the MCC‐134 (100 μM)‐induced current was approximately 50% of that of the 100 μM pinacidil‐induced currents. Using cell‐attached configuration, MCC‐134 activated a glibenclamide‐sensitive KATP channel which was also activated by pinacidil. RT–PCR analysis demonstrated the presence of SUR1 and SUR2B transcripts in pig urethra. These results indicate that both SUR1 and SUR2B subunits play a functional role in regulating the activity of pig urethral KATP channels and that SUR1 contributes less than 25% to total KATP currents.

Propofol prevents endothelial dysfunction induced by glucose overload

Surgical operations often induce acute hyperglycemia, which is known to affect endothelial functions. In this study, we examined the effects of propofol, a commonly used general anaesthetic, on bovine aortic endothelial cell (BAEC) dysfunction induced by glucose overload. D‐glucose overload (23 mM) induced an accumulation of superoxide anion (O2−), assessed by MCLA chemiluminescence, to a similar extent as that generated by 233 μU ml−1 xanthine oxidase (XO) and 100 μM xanthine. Propofol inhibited this accumulation with an IC50 of 0.21 μM, whereas much higher concentrations of propofol were required to scavenge O2− generated by 250 μU ml−1 XO and 100 μM xanthine (IC50: 13.5 μM). D‐glucose overload attenuated ATP‐induced NO production which was detected using diaminofluorescence‐2 (DAF‐2). The inhibition was reversed by propofol with an EC50 of 0.60 μM. In contrast, inhibitions caused by xanthine/XO were not altered by propofol (1 μM). D‐glucose overload suppressed ATP‐induced Ca2+ oscillations and capacitative Ca2+ entry (CCE), which were both restored by superoxide dismutase, indicating that O2− was responsible. Propofol restored these attenuated Ca2+ oscillations and CCE with EC50 of 0.31 and 1.0 μM, respectively. D‐glucose overload (23 mM) increased the intracellular glucose concentration 4 fold, compared with cells exposed to 5.75 mM glucose, and 1 μM propofol reduced this increase to 2.8 fold. We conclude from these results that anaesthetic concentrations of propofol prevent the impairment of Ca2+‐dependent NO production in BAEC induced by glucose overload. This effect is mainly due to the reduction of O2− accumulation, and involves, at least in part, the inhibition of cellular glucose uptake.