Ravshan Z. Sabirov

Macula densa cell signaling involves ATP release through a maxi anion channel

Macula densa cells are unique renal biosensor cells that detect changes in luminal NaCl concentration ([NaCl]L) and transmit signals to the mesangial cell/afferent arteriolar complex. They are the critical link between renal salt and water excretion and glomerular hemodynamics, thus playing a key role in regulation of body fluid volume. Since identification of these cells in the early 1900s, the nature of the signaling process from macula densa cells to the glomerular contractile elements has remained unknown. In patch–clamp studies of macula densa cells, we identified an [NaCl]L-sensitive ATP-permeable large-conductance (380 pS) anion channel. Also, we directly demonstrated the release of ATP (up to 10 μM) at the basolateral membrane of macula densa cells, in a manner dependent on [NaCl]L, by using an ATP bioassay technique. Furthermore, we found that glomerular mesangial cells respond with elevations in cytosolic Ca2+ concentration to extracellular application of ATP (EC50 0.8 μM). Importantly, we also found increases in cytosolic Ca2+ concentration with elevations in [NaCl]L, when fura-2-loaded mesangial cells were placed close to the basolateral membrane of macula densa cells. Thus, cell-to-cell communication between macula densa cells and mesangial cells, which express P2Y2 receptors, involves the release of ATP from macula densa cells via maxi anion channels at the basolateral membrane. This mechanism may represent a new paradigm in cell-to-cell signal transduction mediated by ATP.

Cells die with increased cytosolic ATP during apoptosis: a bioluminescence study with intracellular luciferase

Apoptosis is a distinct form of cell death, which requires energy. Here, we made real-time continuous measurements of the cytosolic ATP level throughout the apoptotic process in intact HeLa, PC12 and U937 cells transfected with the firefly luciferase gene. Apoptotic stimuli (staurosporine (STS), tumor necrosis factor α (TNFα), etoposide) induced significant elevation of the cytosolic ATP level. The cytosolic ATP level remained at a higher level than in the control for up to 6 h during which activation of caspase-3 and internucleosomal DNA fragmentation took place. When the STS-induced ATP response was abolished by glucose deprivation-induced inhibition of glycolysis, both caspase activation and DNA laddering were completely inhibited. Annexin V-binding induced by STS or TNFα was largely suppressed by glycolysis inhibition. Thus, it is suggested that the cells die with increased cytosolic ATP, and elevation of cytosolic ATP level is a requisite to the apoptotic cell death process.

ATP release via anion channels

ATP serves not only as an energy source for all cell types but as an ‘extracellular messenger-for autocrine and paracrine signalling. It is released from the cell via several different purinergic signal efflux pathways. ATP and its Mg2+ and/or H+ salts exist in anionic forms at physiological pH and may exit cells via some anion channel if the pore physically permits this. In this review we survey experimental data providing evidence for and against the release of ATP through anion channels. CFTR has long been considered a probable pathway for ATP release in airway epithelium and other types of cells expressing this protein, although non-CFTR ATP currents have also been observed. Volume-sensitive outwardly rectifying (VSOR) chloride channels are found in virtually all cell types and can physically accommodate or even permeate ATP4- in certain experimental conditions. However, pharmacological studies are controversial and argue against the actual involvement of the VSOR channel in significant release of ATP. A large-conductance anion channel whose open probability exhibits a bell-shaped voltage dependence is also ubiquitously expressed and represents a putative pathway for ATP release. This channel, called a maxi-anion channel, has a wide nanoscopic pore suitable for nucleotide transport and possesses an ATP-binding site in the middle of the pore lumen to facilitate the passage of the nucleotide. The maxi-anion channel conducts ATP and displays a pharmacological profile similar to that of ATP release in response to osmotic, ischemic, hypoxic and salt stresses. The relation of some other channels and transporters to the regulated release of ATP is also discussed.

Apoptotic and necrotic blebs in epithelial cells display similar neck diameters but different kinase dependency

AbstractApoptotic and necrotic blebs elicited by H2O2 were compared in terms of dynamics, structure and underlying biochemistry in HeLa cells and Clone 9 cells. Apoptotic blebs appeared in a few minutes and required micromolar peroxide concentrations. Necrotic blebs appeared much later, prior to cell permeabilization, and required millimolar peroxide concentrations. Strikingly, necrotic blebs grew at a constant rate, which was unaffected throughout successive cycles of budding and detachment. At 1 μm diameter, the necks of necrotic and apoptotic blebs were almost identical. ATP depletion was discarded as a major factor for both types of bleb. Inhibition of ROCK-I, MLCK and p38MAPK strongly decreased apoptotic blebbing but had no effect on necrotic blebbing. Taken together, these data suggest the existence of a novel structure of fixed dimensions at the neck of both types of plasma membrane blebs in epithelial cells. However, necrotic blebs can be distinguished from apoptotic blebs in their susceptibility to actomyosin kinase inhibition.

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.

Bradykinin-induced astrocyte–neuron signalling: glutamate release is mediated by ROS-activated volume-sensitive outwardly rectifying anion channels

Glial cells release gliotransmitters which signal to adjacent neurons and glial cells. Previous studies showed that in response to stimulation with bradykinin, glutamate is released from rat astrocytes and causes NMDA receptor‐mediated elevation of intracellular Ca2+ in adjacent neurons. Here, we investigate how bradykinin‐induced glutamate release from mouse astrocytes signals to neighbouring neurons in co‐cultures. Astrocyte‐to‐neuron signalling and bradykinin‐induced glutamate release from mouse astrocytes were both inhibited by the anion channel blocker 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid (DIDS) and phloretin. Glutamate release was also sensitive to 4‐(2‐Butyl‐6,7‐dichlor‐2‐cyclopentylindan‐1‐on‐5‐yl) oxybutyric acid (DCPIB), a specific blocker of the volume‐sensitive outwardly rectifying anion channel (VSOR). Astrocytes, but not neurons, responded to bradykinin with activation of whole‐cell Cl− currents. Although astrocytes stimulated with bradykinin did not undergo cell swelling, the bradykinin‐activated current exhibited properties typical of VSOR: outward rectification, inhibition by osmotic shrinkage, sensitivity to DIDS, phloretin and DCPIB, dependence on intracellular ATP, and permeability to glutamate. Bradykinin increased intracellular reactive oxygen species (ROS) in mouse astrocytes. Pretreatment of mouse astrocytes with either a ROS scavenger or an NAD(P)H oxidase inhibitor blocked bradykinin‐induced activation of VSOR, glutamate release and astrocyte‐to‐neuron signalling. By contrast, pretreatment with BAPTA‐AM or tetanus neurotoxin A failed to suppress bradykinin‐induced glutamate release. Thus, VSOR activated by ROS in mouse astrocytes in response to stimulation with bradykinin, serves as the pathway for glutamate release to mediate astrocyte‐to‐neuron signalling. Since bradykinin is an initial mediator of inflammation, VSOR might play a role in glia–neuron communication in the brain during inflammation.

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.

Genetic Demonstration That the Plasma Membrane Maxianion Channel and Voltage-dependent Anion Channels Are Unrelated Proteins

The maxianion channel is widely expressed in many cell types, where it fulfills a general physiological function as an ATP-conductive gate for cell-to-cell purinergic signaling. Establishing the molecular identity of this channel is crucial to understanding the mechanisms of regulated ATP release. A mitochondrial porin (voltage-dependent anion channel (VDAC)) located in the plasma membrane has long been considered as the molecule underlying the maxianion channel activity, based upon similarities in the biophysical properties of these two channels and the purported presence of VDAC protein in the plasma membrane. We have deleted each of the three genes encoding the VDAC isoforms individually and collectively and demonstrate that maxianion channel (∼400 picosiemens) activity in VDAC-deficient mouse fibroblasts is unaltered. The channel activity is similar in VDAC1/VDAC3-double-deficient cells and in double-deficient cells with the VDAC2 protein depleted by RNA interference. VDAC deletion slightly down-regulated, but never abolished, the swelling-induced ATP release. The lack of correlation between VDAC protein expression and maxianion channel activity strongly argues against the long held hypothesis of plasmalemmal VDAC being the maxianion channel.

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.

Hypertonic activation of a non‐selective cation conductance in HeLa cells and its contribution to cell volume regulation

In whole‐cell recordings on single HeLa cells, the hypertonic activation of a cation conductance with a selectivity ratio PNa:PLi:PK:PCs:PNMDG:PCa:PCl of 1.00:0.86:0.84:0.56:0.10:0.07:0.15 was observed. This (non‐selective) cation conductance was reduced to 59 and 30% of maximal stimulation by Gd3+ and flufenamate, respectively, but it was insensitive to amiloride (with each compound applied at 100 μmol/l). As was determined by the Coulter counter technique, the cation conductance was the main mechanism of regulatory volume increase (RVI) in HeLa cells. Whereas a significant contribution of Na+/H+ antiport was also detectable, Na+‐K+‐2Cl− symport most likely did not contribute to RVI.