Maxi-anion channel as a candidate pathway for osmosensitive ATP release from mouse astrocytes in primary culture

Abstract

In the present study, we aimed to evaluate the pathways contributing to ATP release from mouse astrocytes during hypoosmotic stress. We first examined the expression of mRNAs for proteins constituting possible ATPreleasing pathways that have been suggested over the past several years. In RT-PCR analysis using both control and osmotically swollen astrocytes, amplification of cDNA fragments of expected size was seen for connexins (Cx32, Cx37, Cx43), pannexin 1 (Px1), the P2X7 receptor, MRP1 and MDR1, but not CFTR. Inhibitors of exocytotic vesicular release, gap junction hemi-channels, CFTR, MRP1, MDR1, the P2X7 receptor, and volume-sensitive outwardly rectifying chloride channels had no significant effects on the massive ATP release from astrocytes. In contrast, the hypotonicity-induced ATP release from astrocytes was most effectively inhibited by gadolinium (50 µM), an inhibitor of the maxi-anion channel, which has recently been shown to serve as a pathway for ATP release from several other cell types. Thus, we propose that the maxi-anion channel constitutes a major pathway for swelling-induced ATP release from cultured mouse astrocytes as well.

The maxi-anion channel: a classical channel playing novel roles through an unidentified molecular entity

“…Modified from Sabirov et al [8] 2 day old rats [18] and adult humans [47] as well as in freshly dissected rat spinal root Schwann cells [48]. These channels were similar to those observed in cultured cortical astrocytes from rats [4,49,50] and mice [5][6][7]51] as well as in a rat astrocytic RGCN cell line [52]. In epithelia, the urinary bladder [53], gastric [54], pancreatic [55][56][57], colonic [58][59][60], airway [61][62][63], choroid plexus [64], bile duct [65,66], ciliary [67][68][69], renal [9,19,[70][71][72][73][74][75][76][77][78], vestibular [79], placental [80][81][82][83][84][85][86], ruminal …”

“…The maxi-anion channel is a very likely candidate for this role due to the following five reasons: (1) The stimuli, which effectively activated the maxi-anion channel, also produced a massive release of ATP. This has been confirmed in mammary C127 cells [8], cardiomyocytes [2,12] and astrocytes [6,7] for osmotic stress; in kidney macula densa cells for the salt stress [70]; and in cultured neonatal [2] and acutely isolated adult cardiomyocytes [12] as well as in primary cultured astrocytes [6,7] for the ischemic and hypoxic stresses. (2) In all these studies, the inhibitors of the maxi-anion channel, such as SITS, NPPB and Gd 3? , effectively suppressed the stimulated ATP release, whereas blockers of VSOR, phloretin and glibenclamide, had no notable effect on the stimulated release of ATP from the cells tested in these studies.…”

“…Astrocytes may communicate with neurons by releasing extracellular signaling molecules called gliotransmitters such as ATP and glutamate [143,144]. Thus, it is suggested that purinergic cell-to-cell signaling in the brain occurs via activation of maxi-anion channels [5][6][7].…”

“…Consistent with the phenotype of the whole-cell VSOR current, this channel current exhibits outward rectification and voltage-dependent inactivation at large positive potentials ([?50 to ?100 mV). However, many authors have also reported the single-channel event with a much larger unitary conductance (300-400 pS) observed in the cell-attached mode after cell swelling [2][3][4][5][6][7][8][9]. A representative example of the maxianion channel currents recorded in the cell-attached patch on an osmotically swollen mammary C127 cells is shown in Fig.…”

“…Since then, we have carried out extensive studies on the maxianion channel along the following four lines: (1) its biophysical properties including the pore size [11], (2) its role in ATP release from astrocytes [6,7] and cardiomyocytes [2,12] under ischemic or osmotic stress, (3) its role in glutamate release from astrocytes under ischemic or osmotic stress [5], and (4) its molecular identification [13,14]. The present review article, thus, gives an updated account of the biophysical properties, the roles in release of ATP and glutamate under pathophysiological conditions, and the molecular identification of the maxianion channel.…”