Amal K. Dutta

Role of ATP‐conductive anion channel in ATP release from neonatal rat cardiomyocytes in ischaemic or hypoxic conditions

It is known that the level of ATP in the interstitial spaces within the heart during ischaemia or hypoxia is elevated due to its release from a number of cell types, including cardiomyocytes. However, the mechanism by which ATP is released from these myocytes is not known. In this study, we examined a possible involvement of the ATP‐conductive maxi‐anion channel in ATP release from neonatal rat cardiomyocytes in primary culture upon ischaemic, hypoxic or hypotonic stimulation. Using a luciferin–luciferase assay, it was found that ATP was released into the bulk solution when the cells were subjected to chemical ischaemia, hypoxia or hypotonic stress. The swelling‐induced ATP release was inhibited by the carboxylate‐ and stilbene‐derivative anion channel blockers, arachidonic acid and Gd3+, but not by glibenclamide. The local concentration of ATP released near the cell surface of a single cardiomyocyte, measured by a biosensor technique, was found to exceed the micromolar level. Patch‐clamp studies showed that ischaemia, hypoxia or hypotonic stimulation induced the activation of single‐channel events with a large unitary conductance (∼390 pS). The channel was selective to anions and showed significant permeability to ATP4‐ (PATP/PCl∼ 0.1) and MgATP2‐ (PATP/PCl∼ 0.16). The channel activity exhibited pharmacological properties essentially identical to those of ATP release. These results indicate that neonatal rat cardiomyocytes respond to ischaemia, hypoxia or hypotonic stimulation with ATP release via maxi‐anion channels.

Fluid flow induces mechanosensitive ATP release, calcium signalling and Cl− transport in biliary epithelial cells through a PKCζ-dependent pathway

ATP in bile is a potent secretogogue, stimulating cholangiocyte Cl− and fluid secretion via binding to membrane P2 receptors, though the physiological stimuli involved in biliary ATP release are unknown. The goal of the present studies was to determine the potential role of fluid flow in biliary ATP release and secretion. In both human Mz‐Cha‐1 biliary cells and normal rat cholangiocyte monolayers, exposure to flow increased relative ATP release which was proportional to the shear stress. In parallel studies, shear was associated with an increase in [Ca2+]i and membrane Cl− permeability, which were both dependent on extracellular ATP and P2 receptor stimulation. Flow‐stimulated ATP release was dependent on [Ca2+]i, exhibited desensitization with repetitive stimulation, and was regulated by PKCζ. In conclusion, both human and rat biliary cells exhibit flow‐stimulated, PKCζ‐dependent, ATP release, increases in [Ca2+]i and Cl− secretion. The finding that fluid flow can regulate membrane transport suggests that mechanosensitive ATP release may be a key regulator of biliary secretion and an important target to modulate bile flow in the treatment of cholestatic liver diseases.

Regulation of an ATP‐conductive large‐conductance anion channel and swelling‐induced ATP release by arachidonic acid

Mouse mammary C127 cells responded to hypotonic stimulation with activation of the volume‐dependent ATP‐conductive large conductance (VDACL) anion channel and massive release of ATP. Arachidonic acid downregulated both VDACL currents and swelling‐induced ATP release in the physiological concentration range with Kd of 4– 6 μm. The former effect observed in the whole‐cell or excised patch mode was more prominent than the latter effect observed in intact cells. The arachidonate effects were direct and not mediated by downstream metabolic products, as evidenced by their insensitivity to inhibitors of arachidonate‐metabolizing oxygenases, and by the observation that they were mimicked by cis‐unsaturated fatty acids, which are not substrates for oxygenases. A membrane‐impermeable analogue, arachidonyl coenzyme A was effective only from the cytosolic side of membrane patches suggesting that the binding site is localized intracellularly. Non‐charged arachidonate analogues as well as trans‐unsaturated and saturated fatty acids had no effect on VDACL currents and ATP release, indicating the importance of arachidonates negative charge and specific hydrocarbon chain conformation in the inhibitory effect. VDACL anion channels were inhibited by arachidonic acid in two different ways: channel shutdown (Kd of 4– 5 μm) and reduced unitary conductance (Kd of 13–14 μm) without affecting voltage dependence of open probability. ATP4‐‐conducting inward currents measured in the presence of 100 mm ATP in the bath were reversibly inhibited by arachidonic acid. Thus, we conclude that swelling‐induced ATP release and its putative pathway, the VDACL anion channel, are under a negative control by intracellular arachidonic acid signalling in mammary C127 cells.

Detecting ATP Release by a Biosensor Method

Cells release adenosine 5′-triphosphate (ATP) into the extracellular space in response to various stimuli. This released ATP plays an important physiological role in cell-to-cell signal transduction. The bulk ATP concentration can be detected using a conventional luciferin-luciferase assay. However, the ATP concentration in the vicinity of the cell surface is often different from the bulk concentration because of its rapid degradation by ecto-ATPases and because of delayed diffusion due to unstirred layer effects. Here, we describe a simple biosensor method to measure the local ATP concentration on the cell surface in real time. The method is based on the ATP-dependent opening of ligand-gated cation channels of purinergic P2X receptors expressed in undifferentiated pheochromocytoma (PC12) cells or in human embryonic kidney 293 (HEK293) cells stably transfected with recombinant P2X2 purinergic receptors. Under the whole-cell configuration of patch-clamp, a sensor PC12 cell or HEK293 is positioned within the proximity of a target cell, and the P2X-mediated currents induced by ATP released from a given site on the target cell surface is measured. The ATP release is quantified by a calibration procedure utilizing local puff applications of ATP at preset concentrations.

The Puzzles of Volume-Activated Anion Channels

This chapter deals with the anion channels and their multiple functions. Anion channels (ACs) are present both in the plasma membrane and in the membranes of intracellular organelles. Animal cells express a large variety of anion channels in their plasma membrane. ACs are involved in a wide range of functions, such as inhibitory synaptic transmission through plasma membrane hyperpolarization, epithelial Clˉ transport, as well as transport of other organic anions such as glutamate and anionic forms of ATP. In contrast to cation channels, ACs are not directly involved in the initiation and termination of action potentials in nerves and muscles. In neurons and other cell types, membrane potential is determined by the relative electromotive forces and the conductances of each ion permeation pathway.

Bile acids stimulate cholangiocyte fluid secretion by activation of transmembrane member 16A Cl- channels

Bile acids stimulate a bicarbonate‐rich choleresis, in part, through effects on cholangiocytes. Because Cl− channels in the apical membrane of cholangiocytes provide the driving force for secretion and transmembrane member 16A (TMEM16A) has been identified as the Ca2+‐activated Cl− channel in the apical membrane of cholangiocytes, the aim of the present study was to determine whether TMEM16A is the target of bile‐acid–stimulated Cl− secretion and to identify the regulatory pathway involved. In these studies of mouse, rat, and human biliary epithelium exposure to ursodeoxycholic acid (UDCA) or tauroursodeoxycholic acid (TUDCA) rapidly increased the rate of exocytosis, ATP release, [Ca2+]i, membrane Cl− permeability, and transepithelial secretion. Bile‐acid–stimulated Cl− currents demonstrated biophysical properties consistent with TMEM16A and were inhibited by pharmacological or molecular (small‐interfering RNA; siRNA) inhibition of TMEM16A. Bile acid–stimulated Cl− currents were not observed in the presence of apyrase, suramin, or 2‐aminoethoxydiphenyl borate (2‐APB), demonstrating that current activation requires extracellular ATP, P2Y, and inositol 1,4,5‐trisphosphate (IP3) receptors. TUDCA did not activate Cl− currents during pharmacologic inhibition of the apical Na+‐dependent bile acid transporter (ASBT), but direct intracellular delivery of TUDCA rapidly activated Cl− currents. Conclusion: Bile acids stimulate Cl− secretion in mouse and human biliary cells through activation of membrane TMEM16A channels in a process regulated by extracellular ATP and [Ca2+]i. These studies suggest that TMEM16A channels may be targets to increase bile flow during cholestasis. (Hepatology 2018;68:187‐199).