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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 arachidonate's 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.
Extracellular ATP is a well-recognized autocrine and paracrine regulator of a multitude of physiological functions at cellular as well as at organ level (Bodin & Burnstock, 2001). Mechanical stress and osmotic swelling are among the most effective physiological stimuli for ATP release (Burnstock, 1999; Bodin & Burnstock, 2001). Although purinergic receptor proteins located on the plasma membrane are well characterized, the molecular nature of the ATP-releasing pathway remains poorly understood at present. As most ATP molecules exist in anionic forms at physiological pH, it is plausible that some anion channels can conduct ATP, thereby serving as a pathway for ATP release. Indeed, ATP-conducting currents associated with the expression of CFTR (Reisin et al. 1994; Schwiebert et al. 1995; Cantiello et al. 1997,1998; Pasyk & Foskett, 1997; Lader et al. 2000) or MDR1 (Abraham et al. 1993; Bosch et al. 1996; Roman et al. 1999), or independent of CFTR and MDR1 expression (Grygorczyk & Hanrahan, 1997; Sugita et al. 1998; Bodas et al. 2000) have so far been observed. We recently demonstrated that in mouse mammary C127 cells (Hazama et al. 2000b) and human Intestine 407 cells (Hazama et al. 1999) neither CFTR nor volume-sensitive outwardly rectifying (VSOR) Cl− channels were responsible for swelling-induced release of ATP. On the other hand, we demonstrated (Sabirov et al. 2001) that cell swelling activated not only conventional VSOR channels but also another type of anion channel which exhibits a large unitary conductance (≈400 pS), bell-shaped voltage dependence and ATP permeability. This volume- (and voltage-) dependent ATP-conductive large conductance (VDACL) anion channel had a pharmacological profile distinct from that of the VSOR channel, but strikingly similar to that of swelling-induced ATP release, and thus the VDACL channel was proposed to be an ATP-releasing conductive pathway in mammary C127 cells (Sabirov et al. 2001).
At present, little information is available on physiological regulators of swelling-induced ATP release machinery in general and the VDACL channel as a putative pathway of ATP release in particular. Arachidonic acid is an unsaturated fatty acid liberated from membrane phospholipids by phospholipases (Irvine, 1982; Holtzman, 1991; Meves, 1994; Brash, 2001) upon stimulation by a variety of stimuli, including hypotonic stress (Thoroed et al. 1997; Tinel et al. 1997; Basavappa et al. 1998; Pedersen et al. 2000; Hoffmann, 2000). Thus, we tested the hypothesis that the arachidonic acid signalling pathway is involved in the regulation of swelling-induced ATP release mediated by the VDACL channel. Here we report that arachidonic acid, at physiologically relevant concentrations, downregulates both VDACL channel activity and swelling-induced ATP release in C127 cells, a fact providing new evidence that the VDACL channel serves as a pathway for swelling-induced ATP release in C127 cells. Also, we provide evidence that anionic arachidonate exerts a downregulatory effect from the intracellular side of the VDACL anion channel, not only by reducing the number of active channels but also by reducing the unitary conductance of open channels. A negative charge and the cis-conformation of the hydrophobic chain constitute the structural determinants essential for the inhibitory activity of arachidonic acid.
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Translocation of ATP from the intracellular compartment to the extracellular fluid is a fundamental process that provides the substrate for purinergic autocrine and paracrine cell signalling. Although the physiological importance of this process is well recognized, the cellular mechanisms are poorly understood. Existence of a conductive pathway for ATP release has been reported in a number of studies, and CFTR was suggested to be a determinant of this process (Reisin et al. 1994; Schwiebert et al. 1995; Cantiello et al. 1997, 1998; Pasyk & Foskett, 1997; Lader et al. 2000). In human epithelial Intestine 407 and mouse mammary C127 cell lines, the pathway of swelling-induced ATP release was shown to be distinct from both CFTR and VSOR chloride channels (Hazama et al. 1998, 1999, 2000a,b). In our previous study (Sabirov et al. 2001), we demonstrated that C127 cells express a large-conductance anion channel, which was silent under normal conditions but could be activated under hypotonic conditions. Based on pharmacological analysis and ATP4- current measurements, we concluded that this VDACL anion channel serves as a conductive pathway for swelling-induced ATP release from C127 cells. We found a similar channel in a previous study of kidney macula densa cells, where the channel was regulated by luminal NaCl and was suggested to mediate the ATP release-dependent tubulo-glomerular feedback mechanism (Bell et al. 2000).
Arachidonic acid is an abundant constituent of the cell. The concentration of esterified arachidonate in resting platelets, for instance, has been estimated to be as high as 5 mm (Brash, 2001). Free arachidonic acid levels are low in the plasma and cytosol, but can rise upon stimulation to 10–100 μm (Brash, 2001). Although most biological activities of arachidonic acid occur through its conversion to prostaglandins, leukotrienes and other products by the cyclooxygenase, lipoxygenase and monooxygenase pathways (Irvine, 1982; Brash, 2001), arachidonic acid itself is also an important regulator of many cellular functions, including cell volume regulation (Lambert, 1987; Kubo & Okada, 1992; Margalit et al. 1993; Civan et al. 1994; Sanchez-Olea et al. 1995; Gosling et al. 1996; Mignen et al. 1999; Hoffmann, 2000). Arachidonic acid has been shown to upregulate ClC-2 Cl− channels (Tewari et al. 2000; Cupoletti et al. 2001) but to downregulate VSOR (Kubo & Okada, 1992; Nilius et al. 1994; Sakai et al. 1996; Gosling et al. 1996; Xu et al. 1997) and other types of Cl− channel (Anderson & Welsh, 1990; Hwang et al. 1990; Zachar & Hurnak, 1994; Riquelme & Parra, 1999; Linsdell, 2000).
In the present study, we demonstrate that arachidonic acid is an effective inhibitor of the VDACL anion channel in C127 cells. This result is consistent with previous observations for maxi-Cl− channels from L6 myoblasts (Zachar & Hurnak, 1994) and large anion channels from human term placenta reconstituted in giant liposomes (Riquelme & Parra, 1999). The Kd value of 4–5 μm obtained here is similar to the values observed for a variety of ion channels (Meves, 1994) and is within the physiological range for arachidonic acid concentrations detected in different cells (Brash, 2001). Km values of 5 μm for cyclooxygenase and 3.4-28 μm for lipoxygenases (Needleman et al. 1986) also indicate that the arachidonic acid concentration in the range of 5–10 μm is physiologically relevant. Therefore, we conclude that arachidonic acid-mediated regulation of VDACL anion channel function actually takes place under physiological conditions.
Arachidonic acid effectively inhibited whole-cell and outside-out macropatch VDACL currents from the extracellular side, and inside-out patch currents from the intracellular side. However, given the rapid, diffusional movement of fatty acids across phospholipid bilayers (Kamp & Hamilton, 1993), it is reasonable to assume that arachidonic acid added to the extracellular fluid can easily reach the intracellular moiety and exert its effect from the intracellular side unless the cytosol is perfused. Consistent with this notion, an impermeant analogue, arachidonyl CoA, exerted the inhibitory action only from the intracellular side of inside-out patches. In addition, we did not observe any inhibitory change in VDACL channel activity in inside-out patches, when 20 μm arachidonic acid was added into the pipette solution (data not shown, n/ 10). This observation is similar to that of Zachar & Hurnak (1994) for maxi-Cl− channels from L6 cells. Taken together with arachidonyl CoA data, it is concluded that the site of arachidonate action exists on the cytosolic side of the VDACL anion channel.
In our experiments, arachidonic acid inhibited VDACL channels in two different ways: (i) channel shutdown (decrease in number of active channels) due to a high-affinity binding with a Kd value of 4–5 μm and (ii) reduced unitary conductance due to low-affinity binding with a Kd value of 13–14 μm. The effects of arachidonic acid were direct and not mediated by downstream metabolic products as evidenced by (i) lack of an effect of inhibitors of arachidonate-metabolizing oxygenases and (ii) similar VDACL inhibition by cis-unsaturated fatty acids that could not be substrates for oxygenases. Neither trans-unsaturated nor saturated fatty acids affected VDACL currents, indicating the importance of the specific conformation of arachidonate's hydrocarbon chain in its inhibitory effect on the VDACL channel. Removing the negative charge of the carboxyl group either by substitution with hydroxyl (arachidonyl alcohol) or by esterification (arachidonyl methyl ester) completely abolished the inhibitory action of arachidonic acid, suggesting the importance of this charge. The necessity for negative charges is in apparent contradiction to voltage independence of both unitary conductance reduction and channel shutdown. We propose that the inhibitory arachidonate-binding site is located close to the internal entrance to the VDACL channel pore, where little or no voltage drop occurs. Arachidonate binding to the blocking site situated outside the electric field may lead to voltage-independent reduction of single-channel conductance due to either fast open-channel block or a conformational change of the channel pore.
According to the proposed physiological role for the VDACL channel as a conductive pathway for ATP release (Sabirov et al. 2001), arachidonic acid must block not only ATP currents but also mass release of ATP induced by cell swelling. Indeed, small inward currents carried by ATP4- were reversibly inhibited by application of arachidonic acid (Fig. 9). Swelling-induced ATP release was also suppressed by arachidonate (Fig. 10). Since arachidonic acid is constantly consumed by endogenous oxygenases, only partial inhibition by arachidonate was observed when these metabolic pathways were still active in intact non-patched cells. In whole-cell configuration, constant perfusion of the intracellular space with the pipette solution may have allowed more profound suppression of VDACL currents by arachidonate due to an increase in effective concentration of arachidonate by washout or decreased specific activity of oxygenases. Inhibition of arachidonate degradation by a cocktail of specific inhibitors for the oxygenases led to ≈40 % reduction in ATP release, presumably due to accumulation of arachidonic acid generated endogenously in response to the hypotonic stress (Thoroed et al. 1997; Tinel et al. 1997; Basavappa et al. 1998; Hoffmann, 2000; Pedersen et al. 2000). In these conditions, exogenously added arachidonate further suppressed ATP release, though still not completely. The Kd values of 4–6 μm were very close to those observed in patch-clamp experiments, providing strong evidence for the involvement of the VDACL channel in swelling-induced ATP release. This idea was further supported by the observation that only cis-unsaturated fatty acids (oleic and linoleic), but not trans-unsaturated (elaidic) or saturated (palmitic) fatty acids, could noticeably affect both swelling-induced ATP release and VDACL channel activity.
Arachidonic acid failed to completely suppress the mass ATP release from intact cells even in the presence of an inhibitory cocktail, whereas arachidonic acid nearly completely eliminated VDACL currents recorded in the whole-cell or excised patch mode. This discrepancy may be related to a complex regulation of ATP-releasing pathways in intact cells compared to excised patches or cells in the whole-cell mode. Swelling-induced activation of phospholipase A2 was reported to lead to an arachidonate-mediated increase in intracellular Ca2+ (Oike et al. 1994). On the other hand, a Ca2+-mobilizing mitogen, bombesin, and a Ca-ionophore, A23187, were shown to activate the maxi-Cl− channel (Kawahara & Takuwa, 1991). Thus, we may suppose that, in intact cells, a direct inhibiting effect of arachidonic acid on VDACL channel might be attenuated or even counteracted by the Ca2+-mediated enhancing effect on the VDACL activation system. Another possible explanation for the above discrepancy would be that arachidonate at high concentrations can form hydrophobic micelles (see Meves, 1994). A residual whole-cell current apparent in the presence of arachidonate (Fig. 1) and an arachidonate-insensitive component of macropatch current in Fig. 2B might represent a non-specific leak induced by the arachidonate micelles. We may suppose that a similar leak can be induced by arachidonate in cell membranes in ATP release experiments and might be responsible for the arachidonate-insensitive component of ATP release. On the other hand, we cannot exclude an alternative possibility that the VDACL channel is not the sole ATP-releasing pathway in C127 cells.
In summary, arachidonic acid at micromolar concentrations was found to directly downregulate both the VDACL anion channel and swelling-induced ATP release before being metabolized by oxygenases, in C127 cells. This fact provides new evidence for our previous conclusion that the VDACL anion channel serves as a pathway for swelling-induced ATP release, both being under a negative control by intracellular arachidonic acid signalling.