Lysophosphatidylcholine Induces Taurine Release from HeLa Cells

The putative role of lysophospholipids in activation and regulation of the volume-sensitive taurine efflux was investigated in HeLa cells using tracer technique. Lysophosphatidylcholine (LPC, 10 μm) with oleic acid increased taurine efflux during hypotonic and isotonic conditions. Substituting palmitic or stearic acid for oleic acid enhanced taurine release during isotonic conditions, whereas ethanolamine, serine or inositol containing lysophospholipids were ineffective. High concentrations of LPC (25 μm) induced Ca²⁺ influx, loss of adenosine nucleotides, taurine and the Ca²⁺-sensitive probe Fura-2, and thus reflected a general breakdown of the membrane permeability barrier. Low concentrations of LPC (5–10 μm) solely induced taurine efflux. The LPC-induced taurine release was unaffected by anion channel blockers (DIDS, MK196) and the 5-lipoxygenase inhibitor ETH 615-139, which all blocked the volume sensitive taurine efflux. Furthermore, LPC-induced taurine release was reduced by antioxidants (NDGA, vitamin E) and the protein tyrosine kinase inhibitor genistein. The swelling-induced taurine efflux was in the absence of LPC unaffected by vitamin E, blocked by genistein, and increased by H₂O₂ and the protein tyrosine phosphatase inhibitor vanadate. It is suggested that low concentrations of LPC permeabilizes the plasma membrane in a Ca²⁺-independent process that involves generation of reactive oxygen species and tyrosine phosphorylation, and that LPC is not a second messenger in activation of the volume sensitive taurine efflux in HeLa cells. Received: 17 December 1999/Revised: 13 April 2000     

Swelling-induced taurine transport: relationship with chloride channels, anion-exchangers and other swelling-activated transport pathways.

Cells have to regulate their volume in order to survive. Moreover, it is now evident that cell volume per se and the membrane transport processes which regulate it, comprise an important signalling unit. For example, macromolecular synthesis, apoptosis, cell growth and hormone secretion are all influenced by the cellular hydration state. Therefore, a thorough understanding of volume-activated transport processes could lead to new strategies being developed to control the function and growth of both normal and cancerous cells. Cell swelling stimulates the release of ions such as K(+) and Cl(-) together with organic osmolytes, especially the beta-amino acid taurine. Despite being the subject of intense research interest, the nature of the volume-activated taurine efflux pathway is still a matter of controversy. On the one hand it has been suggested that osmosensitive taurine efflux utilizes volume-sensitive anion channels whereas on the other it has been proposed that the band 3 anion-exchanger is a swelling-induced taurine efflux pathway. This article reviews the evidence for and against a role of anion channels and exchangers in osmosensitive taurine transport. Furthermore, the distinct possibility that neither pathway is involved in taurine transport is highlighted. The putative relationship between swelling-induced taurine transport and volume-activated anionic amino acid, alpha-neutral amino acid and K(+) transport is also examined.     

Volume-activated trimethylamine oxide efflux in red blood cells of spiny dogfish (Squalus acanthias)

The aims of this study were to determine the pathway of swelling-activated trimethylamine oxide (TMAO) efflux and its regulation in spiny dogfish (Squalus acanthias) red blood cells and compare the characteristics of this efflux pathway with the volume-activated osmolyte (taurine) channel present in erythrocytes of fishes. The characteristics of the TMAO efflux pathway were similar to those of the taurine efflux pathway. The swelling-activated effluxes of both TMAO and taurine were significantly inhibited by known anion transport inhibitors (DIDS and niflumic acid) and by the general channel inhibitor quinine. Volume expansion by hypotonicity, ethylene glycol, and diethyl urea activated both TMAO and taurine effluxes similarly. Volume expansion by hypotonicity, ethylene glycol, and diethyl urea also stimulated the activity of tyrosine kinases p72syk and p56lyn, although the stimulations by the latter two treatments were less than by hypotonicity. The volume activations of both TMAO and taurine effluxes were inhibited by tyrosine kinase inhibitors, suggesting that activation of tyrosine kinases may play a role in activating the osmolyte effluxes. These results indicate that the volume-activated TMAO efflux occurs via the organic osmolyte (taurine) channel and may be regulated by the volume activation of tyrosine kinases.     

Specificity of the volume-activated amino acid efflux pathway in cultured human breast cancer cells

It has been shown that cell swelling stimulates the efflux of taurine from MCF-7 and MDA-MB-231 cells via a pathway which has channel-like properties. The purpose of this study was to examine the specificity of the volume-activated taurine efflux pathway in both cell lines. A hyposmotic shock increased the efflux of glycine, L-alanine, AIB (α-aminoisobutyric acid), D-aspartate but not L-leucine from MDA-MB-231 and MCF-7 cells. It was evident that the time course of activation/inactivation of those amino acids whose efflux was affected by cell swelling was similar to that of volume-activated taurine efflux. The effect of exogenous ATP on swelling-induced glycine, AIB and D-aspartate efflux from MDA-MB-231 cells was similar to that found on taurine efflux. In addition, volume-activated AIB efflux from MDA-MB-231 cells, like that of swelling-induced taurine efflux, was inhibited by diiodosalicylate. Tamoxifen inhibited volume-activated taurine release from both MDA-MB-231 and MCF-7 cells. The results suggest that neutral and anionic α-amino acids are able to utilize the volume-activated taurine efflux pathway in both cell lines. The effect of tamoxifen on breast cancer growth may, in part, be related to perturbations in cell volume regulation.     

Cell swelling activates separate taurine and chloride channels in Ehrlich mouse ascites tumor cells

The taurine efflux from Ehrlich ascites tumor cells is stimulated by hypotonic cell swelling. The swelling-activated taurine efflux is unaffected by substitution of gluconate for extracellular Cl^– but inhibited by addition of MK196 (anion channel blocker) and 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS; anion channel and anion exchange blocker) and by depolarization of the cell membrane. This is taken to indicate that taurine does not leave the osmotically swollen Ehrlich cells in exchange for extracellular Cl^–, i.e., via the anion exchanger but via a MK196- and DIDS-sensitive channel that is potential dependent. An additional stimulation of the swelling-activated taurine efflux is seen after addition of arachidonic acid and oleic acid. Cell swelling also activates a Mini Cl^– channel. The Cl^– efflux via this Cl^– channel, in contrast to the swelling-activated taurine efflux, is unaffected by DIDS and inhibited by arachidonic acid and oleic acid. It is suggested that the swelling-activated Mini Cl^– channel and the swelling-activated taurine channel in the Ehrlich cell represent two distinct types of channels.This work was supported by the Danish Natural Research Council and by the NOVO foundation. Dr. Birte Kramhøft is acknowledged for critical reading of this paper.     

Characterization of regulatory volume decrease in the THP-1 and HL-60 human myelocytic cell linesn

Exposure to hypotonic stress produces a transient increase in cell volume followed by a regulatory volume decrease (RVD) in both THP-1 and HL-60 cells. In contrast, cells exposed to hypotonic stress in a high K/low Na Hanks' solution not only failed to volume regulate, but displayed a secondary swelling. Thus, while an outward K gradient was required ful KVD, the secondary swelling indicated that hypotonic stress increased permeability in the absence of a negative membrane potential. The K channel blocker quinine (1–4 mM) blocked RVD in both cell types. Gramicidin's ability to overcome the quinine block of RVD indicated that RVD is mediated by a quinine-sensitive cation transport mechanism that is independent of the swelling-induced anion transport mechanism. Barium (1–4 mM), another K channel blocker, slowed the rate of RVD, while 4-aminopyridine, charybdotoxin, tetraethylammonium chloride, tetrabutylammonium chloride, and gadolinium had no effect on RVD. Furthermore, RVD was not mediated by calcium-activated conductances, since it occurred normally in Ca-free medium, in medium containing cadmium, and in BAPTA-loaded cells. Gramicidin produced little or no volume change in isotonic medium, suggesting that basal C1 permeability of both THP-1 and HL-60 cells is low. However, swelling induced an anion efflux pathway that is permeable to both chloride and bromide, but is impermeable to methanesulfonate and glutamate. The anion channel blocker 3,5-diiodosalicylic acid (DISA) antagonized RVD in both cell types. In conclusion, RVD in THP-1 and HL-60 cells is mediated by independent anion and cation transport mechanisms that involve both a DISA-sensitive anion pathway and a quinine-inhibitable K efflux pathway, neither of which requires increases in intra-cellular calcium to be activated. © 1994 wiley-Liss, Inc.     

Mechanism of thiyl radical-catalyzed isomerization of unsaturated fatty acid residues in homogeneous solution and in liposomes

Taurine is an important osmolyte involved in cell volume regulation. During regulatory volume decrease it is released via a volume-sensitive organic osmolyte/anion channel. Several molecules have been suggested as candidates for osmolyte release. In this study, we chose three of these, namely ClC-2, ClC-3 and ICln, because of their expression in rat astrocytes, a cell type which is known to release taurine under hypotonic stress, and their activation by hypotonic shock. As all three candidates were also suggested to be chloride channels, we investigated their permeability for both chloride and taurine under isotonic and hypotonic conditions using the Xenopus laevis oocyte expression system. We found a volume-sensitive increase of chloride permeability in ClC-2-expressing oocytes only. Yet, the taurine permeability was significantly increased under hypotonic conditions in oocytes expressing any of the tested candidates. Further experiments confirmed that the detected taurine efflux does not represent unspecific leakage. These results suggest that ClC-2, ClC-3 and ICln either participate in taurine transport themselves or upregulate an endogenous oocyte osmolyte channel. In either case, the taurine efflux of oocytes not being accompanied by an increased chloride flux suggests that taurine and chloride can be released via two separate pathways.     

Hypotonic-induced transport pathways in Xenopus laevis erythrocytes: taurine fluxes

Taurine fluxes in Xenopus laevis red cells were studied in vitro in media of different tonicities. Both influx and efflux increased 3-10 times reversibly when dilution of the medium exceeded 30%. The absolute values of uptake ranged between 5 and 30 micromol/l cells.h at extracellular taurine concentration of 1 mmol/l, but is poorly selective as almost the same uptake was measured for choline and sucrose. Q(10) of 2.77 and an activation energy of 71.90+/-7.37 kJ/mol were calculated for the uptake process. Taurine uptake was reduced 50% in the absence of Cl(-), whereas the alkali cations (Na(+), K(+), Li(+) and Rb(+)) supported it similarly. Taurine uptake was greatly increased in Ca(2+)-free solution, and was inhibited by alkaline pH. The inhibitor of anion exchange protein, 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (IC(50)=25 microM) and the Cl(-) channel blockers 5-nitro-2-(3-phenylpropylamino) benzoic acid and [(dihydro-indenyl) oxy] alkanoic acid (IC(50)<20 microM) inhibited taurine uptake effectively. Isoproterenol did not affect taurine uptake in isotonic, nor in hypotonic solution. The uptake was reduced slowly to near the original, control level within 15-30 min in hypotonic solutions, indicating deactivation of the hypotonic-induced taurine pathway.     

Hypotonic shock activated Cl- and K+ pathways in human fibroblasts.

The exposure of human fibroblasts to hypotonic medium (200 mosmolal) evoked the activation of both 36Cl- influx and efflux, which were insensitive to inhibitors of the anion exchanger and of the anion/cation cotransport, and conversely were inhibited by the Cl(-)-channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). 36Cl- efflux was linked to a parallel efflux of 86Rb+; thus conductive K+ and Cl- pathways are activated during volume regulation in human fibroblasts. This conclusion is supported by evidence that, in hypotonic medium, 36Cl- influx and 86Rb+ efflux were both enhanced by depolarization of the plasma membrane. Depletion of the intracellular K+ content, obtained by preincubation with the ionophore gramicidin in Na(+)-free medium, had no effect on Cl- efflux in hypotonic medium. This result has been interpreted as evidence for independent activation of K+ and Cl- pathways. It is also concluded that the anion permeability is the rate-limiting factor in the response of human fibroblasts to hypotonic stress.     

Effects of calcium, calmodulin, protein kinase C and protein tyrosine kinases on volume-activated taurine efflux in human erythroleukemia cells.

The effects of calcium, calmodulin, protein kinase C (PKC) and protein tyrosine kinase (PTK) modulators were examined on the volume-activated taurine efflux in the erythroleukemia cell line K562. Exposure to hypoosmotic solution significantly increased taurine efflux and intracellular calcium concentration ([Ca2+]i). The Ca2+ channel blockers La3+ (1 mM), verapamil (200 microM) and nifedipine (100 microM) inhibited the hypoosmotically-induced [Ca2+]i increase by more than 90%, while the volume-activated taurine efflux was inhibited by 61.3 +/- 9.5, 74.1 +/- 9.3 and 38.0 +/- 1.5%, respectively. Furthermore, the calmodulin inhibitors W7 (50 microM) and trifluoperazine (10 microM) and the Ca2+/calmodulin-dependent protein kinase II inhibitor KN-62 (2 microM) significantly blocked the volume-activated taurine efflux by 93.4 +/- 2.7, 77.9 +/- 3.5 and 61.3 +/- 15.8%, respectively. In contrast, the PKC inhibitor staurosporine (200 nM) or the PKC activator phorbol 12-myristate 13-acetate (100 nM) did not have significant effects on the volume-activated taurine efflux. However, pretreatment with PTK inhibitors genistein, tyrphostin A25, and tyrphostin A47 blocked the volume-activated taurine efflux. These results suggest that the volume-activated taurine efflux in K562 cells may not directly involve Ca2+, but may require the presence of calmodulin and/or PTK.     

Autocrine regulation of volume-sensitive anion channels in airway epithelial cells by adenosine

The activity of volume-sensitive Cl- channels was studied in human tracheal epithelial cells (9HTEo-) by taurine efflux experiments. The efflux elicited by a hypotonic shock was partially inhibited by adenosine receptor antagonists, by alpha,beta-methyleneadenosine 5'-diphosphate (alphabetaMeADP), an inhibitor of the 5'-ectonucleotidase, and by adenosine deaminase. On the other hand, dipyridamole, a nucleoside transporter inhibitor, increased the swelling-induced taurine efflux. Extracellular ATP and adenosine increased taurine efflux by potentiating the effect of hypotonic shock. alphabetaMeADP strongly inhibited the effect of extracellular ATP but not that of adenosine. These results suggest that anion channel activation involves the release of intracellular ATP, which is then degraded to adenosine by specific ectoenzymes. Adenosine then binds to purinergic receptors, causing the activation of the channels. To directly demonstrate ATP efflux, cells were loaded with [3H]AMP, and the release of radiolabeled molecules was analyzed by high performance liquid chromatography. During hypotonic shock, cell supernatants showed the presence of ATP, ADP, and adenosine. alphabetaMeADP inhibited adenosine formation and caused the appearance of AMP. Under hypotonic conditions, elevation of intracellular Ca2+ by ionomycin caused an increase of ATP and adenosine in the extracellular solution. Our results demonstrate that volume-sensitive anion channels are regulated with an autocrine mechanism involving swelling-induced ATP release and then hydrolysis to adenosine.     

Regulation of taurine transport in Ehrlich ascites tumor cells

Summary Taurine influx is inhibited and taurine efflux accelerated when the cell membrane of Ehrlich ascites tumor cells is depolarized. Taurine influx is inhibited at acid pH partly due to the concomitant depolarization of the cell membrane partly due to a reduced availability of negatively charged free carrier. These results are in agreement with a 2Na, 1Cl, 1taurine cotransport system which is sensitive to the membrane potential due to a negatively charged empty carrier. Taurine efflux from Ehrlich cells is stimulated by addition of LTD4 and by swelling in hypotonic medium. Cell swelling in hypotonic medium is known to result in stimulation of the leukotriene synthesis and depolarization of the cell membrane. The taurine efflux, activated by cell swelling, is dramatically reduced when the phospholipase A₂ is inhibited indirectly by addition of the anti-calmodulin drug pimozide, or directly by addition of RO 31-4639. The inhibition is in both cases lifted by addition of LTD₄. The swelling-induced taurine efflux is also inhibited by addition of the 5-lipoxygenase inhibitors ETH 615-139 and NDGA. It is concluded that the swelling-induced activation of the taurine leak pathway involves a release of arachidonic acid from the membrane phospholipids and an increased oxidation of arachidonic acid into leukotrienes via the 5-lipoxygenase pathway. LTD₄ seems to act as a second messenger for the swelling induced activation of the taurine leak pathway either directly or indirectly via its activation of the Cl^– channels, i.e., via a depolarization of the cell membrane.     

Transport of organic substrates via a volume-activated channel.

We have investigated the volume-activated transport of organic solutes in flounder erythrocytes. Osmotic swelling of cells suspended in a Na(+)-free medium led to increased membrane transport of taurine, glucose, and uridine. For each compound there was a significant lag period (1-2 min at 10 degrees C) between cell swelling and activation of the flux. The volume-activated fluxes of each of the substrates increased in parallel with increasing cell volume, and those of taurine and uridine increased linearly with concentration (up to 19 mM). The volume-activated fluxes of each of the three compounds showed similar sensitivities to a number of anion-selective channel blockers (5-nitro-2-(3-phenylpropylamino)benzoic acid > 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid approximately MK-196 > niflumic acid > furosemide); the IC50 for the inhibition of the volume-activated fluxes by NPPB was around 12 microM. The results are consistent with the hypothesis that the volume-activated transport of organic osmolytes is via a pathway with the characteristics of a volume-activated "chloride channel." This raises the question of whether the transport of organic substrates might represent a physiological role for such channels in other cell types.     

Cell volume regulation in the perfused liver of a freshwater air-breathing catfishClarias batrachus under aniso-osmotic conditions: Roles of inorganic ions and taurine

The roles of various inorganic ions and taurine, an organic osmolyte, in cell volume regulation were investigated in the perfused liver of a freshwater air-breathing catfishClarias batrachus under aniso-osmotic conditions. There was a transient increase and decrease of liver cell volume following hypotonic (-80 mOsmol/l) and hypertonic (+80 mOsmol/l) exposures, respectively, which gradually decreased/increased near to the control level due to release/ uptake of water within a period of 25–30 min. Liver volume decrease was accompanied by enhanced efflux of K⁺ (9.45 ± 0.54 μmol/g liver) due to activation of Ba²⁺- and quinidine-sensitive K⁺ channel, and to a lesser extent due to enhanced efflux of Cl^- (4.35 ± 0.25 μmol/g liver) and Na⁺ (3.68 ± 0.37 μmol/g liver). Conversely, upon hypertonic exposure, there was amiloride- and ouabain-sensitive uptake of K⁺(9.78 ± 0.65 μmol/g liver), and also Cl^- (3.72 ± 0.25 μmol/g liver). The alkalization/acidification of the liver effluents under hypo-/hypertonicity was mainly due to movement of various ions during volume regulatory processes. Taurine, an important organic osmolyte, appears also to play a very important role in hepatocyte cell volume regulation in the walking catfish as evidenced by the fact that hypo- and hyper-osmolarity caused transient efflux (5.68 ± 0.38 μmol/g liver) and uptake (6.38 ± 0.45 μmol/g liver) of taurine, respectively. The taurine efflux was sensitive to 4,4′-di-isothiocyanatostilbene-2,2′-disulphonic acid (DIDS, an anion channel blocker), but the uptake was insensitive to DIDS, thus indicating that the release and uptake of taurine during volume regulatory processes are unidirectional. Although the liver of walking catfish possesses the RVD and RVI mechanisms, it is to be noted that liver cells remain partly swollen and shrunken during anisotonic exposures, thereby possibly causing various volume-sensitive metabolic changes in the liver as reported earlier.     

Muscarinic receptor regulation of osmosensitive taurine transport in human SH-SY5Y neuroblastoma cells

The ability of G protein‐coupled receptors to regulate osmosensitive uptake of the organic osmolyte, taurine, into human SH‐SY5Y neuroblastoma cells has been examined. When monitored under isotonic conditions and in the presence of physiologically relevant taurine concentrations (1–100 μM), taurine influx was mediated exclusively by a Na⁺‐dependent, high‐affinity (K_m = 2.5 μM) saturable transport mechanism (V_max = 0.087 nmol/mg protein/min). Reductions in osmolarity of > 20% (attained under conditions of a constant NaCl concentration) resulted in an inhibition of taurine influx (> 30%) that could be attributed to a reduction in V_max, whereas the K_m for uptake remained unchanged. Inclusion of the muscarinic cholinergic agonist, oxotremorine‐M (Oxo‐M), also resulted in an attenuation of taurine influx (EC₅₀～0.7 μM). Although Oxo‐M‐mediated inhibition of taurine uptake could be observed under isotonic conditions (～25–30%), the magnitude of inhibition was significantly enhanced by hypotonicity (～55–60%), a result that also reflected a reduction in the V_max, but not the K_m, for taurine transport. Oxo‐M‐mediated inhibition of taurine uptake was dependent upon the availability of extracellular Ca²⁺ but was independent of protein kinase C activity. In addition to Oxo‐M, inclusion of either thrombin or sphingosine 1‐phosphate also attenuated volume‐dependent taurine uptake. The ability of Oxo‐M to inhibit the influx of taurine was attenuated by 4‐[(2‐butyl‐6,7‐dichloro‐2‐cyclopentyl‐2,3‐dihydro‐1‐oxo‐1H‐inden‐5‐yl)oxy]butanoic acid, an inhibitor of the volume‐sensitive organic osmolyte and anion channel. 4‐[(2‐Butyl‐6,7‐dichloro‐2‐cyclopentyl‐2,3‐dihydro‐1‐oxo‐1H‐inden‐5‐yl)oxy]butanoic acid also prevented receptor‐mediated changes in the efflux and influx of K⁺ under hypoosmotic conditions. The results suggest that muscarinic receptor activation can regulate both the volume‐dependent efflux and uptake of taurine and that these events may be functionally coupled.     

Volume-sensitive taurine efflux from mammary tissue is not obliged to utilize volume-activated anion channels

Cell-swelling, induced by a hyposmotic shock, activates the release of taurine from lactating rat mammary tissue expiants. The degree of stimulation of taurine efflux was dependent upon the extent of cell-swelling. Volume-sensitive taurine release was attenuated by the anion transport inhibitors NPPB, DIOA, DIDS, niflumate, flufenamate, mefenamate and diiodosalicylate but not by salicylate. Cell-swelling, following a hyposmotic challenge, did not increase the unidirectional efflux of radiolabelled I^– or D-asparate from mammary tissue expiants. The results suggest that although mammary tissue expresses a volume-sensitive amino acid transport system which is inhibited by anion transport blockers the pathway has no identity with volume-activated anion channels.     

Osmoregulation of taurine efflux from cultured human breast cancer cells: comparison with volume activated Cl- efflux and regulation by extracellular ATP.

The properties and regulation of volume-activated taurine efflux from MDA-MB-231 and MCF-7 cells have been investigated. Volume-activated taurine release from both cell lines was almost completely inhibited by diidosalicylate. DIDS , was more effective at inhibiting swelling-induced taurine release from MCF-7 than from MDA-MB-231 cells. On the basis of comparing taurine, Cl(-) and I(-) efflux time courses, it appears that volume-activated taurine efflux does not utilize volume-sensitive anion channels in MDA-MB- 231 and MCF-7 cells. Extracellular ATP stimulated volume-activated taurine release from MDA-MB-231 cells but not from MCF-7 cells. The effect of ATP was mimicked by UTP and was dependent upon external calcium and inhibited by suramin. However, suramin inhibited volume-activated taurine efflux from both MDA-MB-231 and MCF-7 cells even in the absence of exogenously added ATP suggesting that it acts directly on the taurine efflux pathway and/or is inhibiting the effect of ATP released from the cells. Volume-activated taurine efflux from MDA-MB-231 cells was stimulated by ionomycin. In contrast, ionomycin had no effect on taurine release from MCF-7 cells. Adenosine also stimulated volume-activated taurine efflux from MDA-MB-231 cells. The results suggest that purines regulate taurine transport in MDA-MB- 231 cells via more than one type of receptor.     

Chloride transport in human fibroblasts is activated by hypotonic shock

Incubation of human skin fibroblasts in hypotonic media induced the activation of 36Cl- efflux which was roughly proportional to the decrease in the osmolality of the media. The efflux of 36Cl- was insensitive to DIDS plus furosemide and inhibited by addition of a Cl- channel blocker such as 5-nitro-2-(3-phenyl propylamino) benzoic acid (NPPB). We propose that a conductive pathway for Cl- transport, almost silent in isotonic conditions, is activated by exposing human fibroblasts to hypotonic shock, this conclusion being supported by evidence that also 36Cl- influx was enhanced by hypotonic medium.     

Apoptotic inducers activate the release of d-aspartate through a hypotonic stimulus-triggered mechanism in PC12 cells

We have characterized release of d-aspartate (d-Asp), a regulator of hormone synthesis and secretion, via a volume-sensitive organic anion channel (VSOC) in PC12 cells by studying its response to apoptotic stimuli. PC12 cells have been demonstrated to endogenously synthesize d-Asp. Apoptotic inducers, including staurosporin (STS), tumor necrosis factor (TNF)-α, H₂O₂, and C2-ceramide, activate the release of d-Asp through a hypotonic stimulus-triggered mechanism. Putative blockers of the anion channel, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and 4,4′-diisothiocyanostilbene-2,2′-sulphonic acid (DIDS), significantly inhibited stress-induced d-Asp release under hypotonic conditions following the application of apoptotic inducers. Hypotonic conditions are essential for activation by apoptotic inducers. Phorbol 12-mirystate 13-acetate and the Ca²⁺ ionophore A23187 increased d-Asp efflux via the VSOC, implying the involvement of intracellular Ca²⁺ in the activation of the d-Asp efflux. However, hypotonic stress and STS had no effect on the concentration of intracellular Ca²⁺ in PC12 cells. Furthermore, an unknown EGTA-sensitive factor(s), other than Ca²⁺, and peroxynitrite may play pivotal roles in STS-enhanced d-Asp release.