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Letter to the Editor
Center for Neuropharmacology and Neuroscience Albany Medical College; Department of Brain and Heart Cell Rescue Ordway Research Institute Albany, New York 12208
To the Editor: We read with great interest the paper by Darby et al. in the April 2003 issue of the Journal of Neurophysiology (Darby et al. 2003
). The authors demonstrated that, in cultured astrocytes, hyposmotic cell swelling causes release of ATP via a pathway with pharmacological profile similar to the multidrug resistance transporter. This ATP then stimulates P2Y1-like receptors and activates, via appropriate intracellular signaling mechanisms, volume-regulated anion channels (VRACs), measured as whole cell Cl– currents. They also show that in nonswollen astrocytes, exogenous ATP activates Cl– currents resembling currents carried by VRACs. These data fit the previously proposed model of autocrine ATP release that is obligatory for the activation of VRACs in hepatoma cells (Wang et al. 1996). It is important to note that the significance of such findings in astrocytes extends beyond the "academic" question of how volume-sensitive chloride channels are activated in response to cell volume changes. Astrocytic VRACs, as well as their counterparts in other cells, are permeable toward a variety of small organic anions and uncharged molecules, including the excitatory amino acids glutamate and aspartate (Kimelberg et al. 1990). Therefore in the brain, ATP released during synaptic transmission or astrocyteto-astrocyte communication may cause VRAC-mediated glutamate release from astrocytes. Such astrocytic glutamate release is now considered a key feedback signal in neuron-astrocyte communication (Haydon 2001), with ATP likely acting as the main neuron-to-astrocyte and astrocyte-to-astrocyte intercellular messenger (Fields and Stevens 2000).
We have also studied the role of ATP in activation of astrocytic VRACs, measured as release of preloaded [3H]labeled excitatory amino acid D-aspartate. We found that, in cultured astrocytes, exogenous 10 µM ATP activates a transient excitatory amino acid release in nonswollen cells and strongly potentiates amino acid release in hyposmotically swollen cells (Mongin and Kimelberg 2002; Fig. 1). This ATP-induced D-[3H]aspartate efflux was potently inhibited by the VRAC blockers 5-nitro-2-(3-phenylpropylamino) benzoic acid, DIDS, and phloretin. It was also suppressed by a 10% increase in medium osmolarity (Mongin and Kimelberg 2002). On the basis of these data, we propose that ATP is incapable of direct VRAC activation, but instead, positively modulates a small fraction of VRACs that are active in nonswollen cells. The ATP-induced D-[3H]aspartate release in our experiments involving nonswollen cells did not exceed one-tenth of the release induced by a 100 mOsm reduction in medium osmolarity. In contrast, Darby and colleagues report a substantially higher activation of the Cl– currents by ATP in nonswollen astrocytes, reaching one-half of the value of the hyposmotic Cl– currents (Darby et al. 2003). If translated to ATP-induced VRAC-mediated glutamate release, such effect should have a large impact on normal brain physiology.
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The effect of ATP seen by Darby et al. was concentration-dependent, with the maximum values at 1 and 5 mM. However, millimolar concentrations of extracellular ATP block VRAC-mediated excitatory amino acid release in astrocytes (Haskew et al. 2002; Mongin and Kimelberg 2002), as well as VRAC-mediated Cl– currents in several cell lines, with the IC50 varying between 0.2 and 5 mM ATP (Okada 1997). As seen in Fig. 1, in cultured astrocytes, 10 µM, 100 µM, and 1 mM ATP activated the transient D-[3H]aspartate release with equal potency. However, at 5 and 10 mM ATP, the first transient phase of D-[3H]aspartate release is not present and is presumably inhibited by high concentration of ATP. Instead we observed a delayed and sustained amino acid efflux. This latter release phase is unlikely to be due to VRAC activation. It may be due to extracellular Mg2+/Ca2+ chelation by ATP, promoting glutamate release through astrocytic connexin hemi-channels or P2X7 receptor-channels, as reported recently (Duan et al. 2003; Ye et al. 2003).
Based on the foregoing, we propose that ATP release is not obligatory or sufficient for VRAC activation in astroglial cells. Instead ATP likely modulates active VRACs in both swollen and nonswollen cells. A similar conclusion has been reached in a few recent studies in intestinal epithelial and hepatoma cells employing both an electrophysiological approach and ion flux measurements (Hazama et al. 1999; Junankar et al. 2002). The ATP-induced Cl– currents in nonswollen astrocytes may result from activation of multiple Cl– channels. At high ATP concentrations (such as 5–10 mM), the contribution of non-VRAC channels likely predominates. Distinction between permeability pathways is very important since VRAC activation may be a source of physiological astrocytic glutamate release. On the other hand, it has been shown that cell swelling activates more than one type of Cl– channel (Zhang and Jacob 1997). Therefore the differences between the ATP effects on Cl– currents and organic osmolyte efflux may also be explained by contribution of several permeability pathways to what is currently collectively known as a VRAC. We believe that further studies looking at the mechanisms of VRAC activation in nonswollen cells may have important implications for our understanding of astrocyte functions in the brain.
REFERENCES
Darby M, Kuzmiski JB, Panenka W, Feighan D, and MacVicar BA. ATP released from astrocytes during swelling activates chloride channels. J Neurophysiol 89: 1870–1877, 2003; 10.1152/jn.00510.2002.
Duan S, Anderson CM, Keung EC, Chen Y, Chen Y, and Swanson RA. P2X7 receptor-mediated release of excitatory amino acids from astrocytes. J Neurosci 23: 1320–1328, 2003.
Fields RD and Stevens B. ATP: an extracellular signaling molecule between neurons and glia. Trends Neurosci 23: 625–633, 2000.[ISI][Medline]
Haskew RE, Mongin AA, and Kimelberg HK. Peroxynitrite enhances astrocytic volume-sensitive excitatory amino acid release via a src tyrosine kinase-dependent mechanism. J Neurochem 82: 903–912, 2002.[ISI][Medline]
Haydon PG. Glia: listening and talking to the synapse. Nat Rev Neurosci 2: 185–193, 2001.[ISI][Medline]
Hazama A, Shimizu T, Ando-Akatsuka Y, Hayashi S, Tanaka S, Maeno E, and Okada Y. Swelling-induced, CFTR-independent ATP release from a human epithelial cell line: lack of correlation with volume-sensitive Cl- channels. J Gen Physiol 114: 525–533, 1999.
Junankar PR, Karjalainen A, and Kirk K. The role of P2Y1 purinergic receptors and cytosolic Ca2+ in hypotonically activated osmolyte efflux from a rat hepatoma cell line. J Biol Chem 277: 40324–40334, 2002.
Kimelberg HK, Goderie SK, Higman S, Pang S, and Waniewski RA. Swelling-induced release of glutamate, aspartate, and taurine from astrocyte cultures. J Neurosci 10: 1583–1591, 1990.[Abstract]
Mongin AA and Kimelberg HK. ATP potently modulates anion channel-mediated excitatory amino acid release from cultured astrocytes. Am J Physiol Cell Physiol 283: C569–C578, 2002. Published online April 18, 2002; 10.1152/ajpcell.00438.2001.
Okada Y. Volume expansion-sensing outward-rectifier Cl- channel: fresh start to the molecular identity and volume sensor. Am J Physiol Cell Physiol 273: C755–C789, 1997.
Wang Y, Roman R, Lidofsky SD, and Fitz JG. Autocrine signaling through ATP release represents a novel mechanism for cell volume regulation. Proc Natl Acad Sci USA 93: 12020–12025, 1996.
Ye ZC, Wyeth MS, Baltan-Tekkok S, and Ransom BR. Functional hemichannels in astrocytes: a novel mechanism of glutamate release. J Neurosci 23: 3588–3596, 2003.
Zhang JJ and Jacob TJC. Three different Cl– channels in the bovine ciliary epithelium activated by hypotonic stress. J Physiol 499: 379–389, 1997.[ISI][Medline]
; Seki et al. 1999). We have evidence that glutamate release from these channels contributes to spreading depression propagation (Basarsky et al. 1999). However, we would like to clarify some issues that are raised by this letter. Certainly, the final point of the letter by Mongin and Kimelberg should be kept foremost in mind in any discussion on this issue. As they state there are several species of Cl– channels that are activated by swelling (Zhang and Jacob 1997). In addition, there are several mechanisms by which glutamate and aspartate efflux can occur from astrocytes and other cell types. Mongin and Kimelberg should also be cautious in interpreting their measurements of radiolabeled aspartate efflux as solely due to efflux from anion channels. Their measurements are quite distant from Cl– channel activation, which is turning out to be a complex phenomenon in astrocytes (Crepel et al. 1998).
The major issue mentioned by Mongin and Kimelberg is our use of high concentrations of ATP (1 to 5 mM). This experiment should be understood in the context of the study. Our argument that ATP release is involved in activating volume-activated Cl– currents (ICl,swell) is based on: 1) ATP receptor (P2Y1) antagonists depressed the activation of ICl,swell; 2) apyrase, an enzyme that degrades ATP, reduced ICl,swell; and 3) application of ATP and other P2Y agonists activated a Cl– current that appeared similar to ICl,swell. We observed that the degree of block of ICl,swell by P2Y1 antagonists and activation by ATP was only a fraction (
50%) of the total current. We therefore applied quite high concentrations of ATP (1 to 5 mM) to ensure that we were at saturating concentrations. We did not observe ATP-mediated depression under these conditions. Another reason that we used high ATP concentrations was to examine the potential role for P2X7 receptors. Previously we showed that P2X7 receptor activation triggered Erk1/2 and p38 MAP kinase activation in astrocytes, thereby leading to increased MCP-1 synthesis (Panenka et al. 2001). P2X7 receptors are activated by high ATP concentrations and by BzATP. Therefore, we tested the possibility of P2X7 receptor activation in our paper with high ATP concentrations and with BzATP. Our data did not support a P2X7 receptor involvement because the P2X7 agonist BzATP was not more effective than ATP at 100 µM and there was no substantial further activation at the high ATP concentrations. Finally, it should be noted that ATP concentrations of 100 µM were used in our experiments to examine the effectiveness of ATP vs. other P2 agonists (see Fig. 3 in our article, Darby et al. 2003). ADP was more efficacious than ATP at activating ICl,swell, which is consistent with a P2Y1 receptor involvement.
In their previous studies and in this letter, Mongin and Kimelberg have provided interesting data on factors that modify the release of aspartate from astrocytes in cell culture. However, it is unreasonable to dismiss actual recordings of Cl– channel activation based on measurements of radiolabeled release of aspartate. The cellular response to swelling is complex, and it is conceivable that cellular swelling and the attendant activation of MAP kinases could be an important modifier of numerous cellular processes, some of which could modify aspartate release or Cl– channel activation. The recent elegant description of glutamate release through hemichannels stresses the multiplicity of glutamate release mechanisms (Ye et al. 2003). In light of these findings, it will be important for future experiments to delineate unequivocally the roles for hemichannels vs. anion channels in the release of glutamate.
Finally, our results indicate that ATP release only contributes to 50% of the activation of ICl,swell. This suggests that either another signaling factor is released or that the residual current represents a different population of Cl– channels (Zhang and Jacob 1997). The analysis of these currents and the relationship of these currents to the release of aspartate that Mongin and Kimelberg have studied is an important next step.
REFERENCES
Basarsky TA, Feighan D, and MacVicar BA. Glutamate release through volume-activated channels during spreading depression. J Neurosci 19: 6439–6445, 1999.
Crepel V, Panenka W, Kelly ME, and MacVicar BA. Mitogen-activated protein and tyrosine kinases in the activation of astrocyte volume-activated chloride current. J Neurosci 18: 1196–1206, 1998.
Darby M, Kuzmiski JB, Panenka W, Feighan D, and MacVicar BA. ATP released from astrocytes during swelling activates chloride channels. J Neurophysiol 89: 1870–1877, 2003; 10.1152/jn.00510.2002.
Mongin AA and Kimelberg HK. ATP potently modulates anion channel-mediated excitatory amino acid release from cultured astrocytes. Am J Physiol Cell Physiol 283: C569–C578, 2002. Published online April 18, 2002; 10.1152/ajpcell.00438.2001.
Panenka W, Jijon H, Herx LM, Armstrong JN, Feighan D, Wei T, Yong VW, Ransohoff RM, and MacVicar BA. P2X7-like receptor activation in astrocytes increases chemokine monocyte chemoattractant protein-1 expression via mitogen-activated protein kinase. J Neurosci 21: 7135–7142, 2001.
Seki Y, Feustel PJ, Keller RW Jr, Tranmer BI, and Kimelberg HK. Inhibition of ischemia-induced glutamate release in rat striatum by dihydrokinate and an anion channel blocker. Stroke 30: 433–440, 1999.
Ye ZC, Wyeth MS, Baltan-Tekkok S, and Ransom BR. Functional hemichannels in astrocytes: a novel mechanism of glutamate release. J Neurosci 23: 3588–3596, 2003.
Zhang JJ and Jacob TJ. Three different Cl– channels in the bovine ciliary epithelium activated by hypotonic stress. J Physiol 499: 379–389, 1997.
Brain Research Center University of British Columbia Vancouver, British Columbia
This article has been cited by other articles:
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  A. A. Mongin and H. K. Kimelberg ATP regulates anion channel-mediated organic osmolyte release from cultured rat astrocytes via multiple Ca2+-sensitive mechanisms Am J Physiol Cell Physiol, January 1, 2005; 288(1): C204 - C213. [Abstract] [Full Text] [PDF]
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