Fumonisin B1-induced apoptosis is associated with delayed inhibition of protein kinase C, nuclear factor-kappaB and tumor necrosis factor alpha in LLC-PK1 cells

Fumonisin B1 (FB1), the most potent of the fumonisin mycotoxins, is a carcinogen and causes a wide range of species-specific toxicoses. FB1 modulates the activity of protein kinase C (PKC), a family of phospholipid-dependent serine/threonine kinases that play important role in modulating a variety of biologic responses ranging from regulation of cell growth to cell death. Although it has been demonstrated that FB1 induces apoptosis in many cell lines, the precise mechanism of apoptosis is not fully understood. In this study, we investigated the membrane localization of various PKC isoforms, PKC enzyme activity, and its downstream targets, namely nuclear factor-kappa B (NF-kappaB), tumor necrosis factor alpha (TNFalpha), and caspase 3, in porcine renal epithelial (LLC-PK1) cells. FB1 repressed cytosol to membrane translocation of PKC-alpha, -delta, -epsilon, and -zeta isoforms over 24-72 h. The FB1-induced membrane PKC repression was corroborated by a concentration-dependent decrease in total PKC activity. Exposure of cells to phorbol 12-myristate 13-acetate (PMA) for this duration also resulted in repressed PKC membrane localization and activity comparable to FB1. Exposure of cells to FB1 (10 microM) was associated with inhibition of cytosol to nuclear translocation of NF-kappaB and NF-kappaB-DNA binding at 72 h. The expression of TNFalpha was significantly inhibited at 24 and 48 h in response to 1 and 10 microM FB1. Increased caspase 3 activity was observed in LLC-PK1 cells exposed to > or =1 microM FB1 at 48 h. PMA also increased the caspase 3 activity at 24 and 48 h. Results suggest that FB1-induced apoptosis involves the activation of caspase 3, which is associated with the repression of PKC and possibly its down-stream effectors, NF-kappaB and TNFalpha.     

Sphingosine kinase activity confers resistance to apoptosis by fumonisin B1 in human embryonic kidney (HEK-293) cells

Fumonisin B1 induces cytotoxicity in sensitive cells by inhibiting ceramide synthase due to its structural similarity to the long-chain backbones of sphingolipids. The resulting accumulation of sphingoid bases has been established as a mechanism for fumonisin B1 cytotoxicity. We found that despite the accumulation of sphinganine, human embryonic kidney (HEK-293) cells are resistant to fumonisin B1 toxicity; 25 microM fumonisin B1 exposure for 48 h did not increase apoptosis in these cells, while it did so in sensitive porcine kidney epithelial (LLC-PK1) cells. In this study, DL-threo-dihydrosphingosine, the sphingosine kinase inhibitor (SKI), considerably increased the sensitivity of HEK-293 cells to fumonisin B1. Treatment of these cells with 25 microM fumonisin B1 and 2.5 microM SKI increased apoptosis. Sphingoid bases, sphinganine or sphingosine, added to cell cultures induced apoptosis by themselves and their effects were potentiated by SKI or fumonisin B1. Addition of physiological amounts of sphingosine-1-phosphate prevented the toxic effects induced by SKI inhibition and fumonisin B1. Results indicated that HEK-293 cells are resistant to fumonisin B1 due to rapid formation of sphingosine-1-phosphate that imparts survival properties. Taken together, these findings suggest that sphingoid base metabolism by sphingosine kinase may be a critical event in rendering the HEK-293 cells relatively resistant to fumonisin B1-induced apoptosis.     

Increased susceptibility of renal epithelial cells to TNFalpha-induced apoptosis following treatment with fumonisin B1

Previous studies have shown that tumor necrosis factor alpha (TNFalpha) is involved in the pathogenic events following exposure to fumonisin B(1) (FB(1)), a potent inhibitor of ceramide synthase and sphingolipid biosynthesis. The intimate role of sphingolipid mediators in TNFalpha signaling and cellular death suggests that FB(1) may alter the sensitivity of cells to TNFalpha-induced apoptosis. We tested the hypothesis that FB(1) treatment will increase the sensitivity of porcine renal epithelial cells to TNFalpha. Porcine renal epithelial cells (LLC-PK(1)) were treated with FB(1) for 48 h prior to treatment with TNFalpha. A dose-dependent increase in TNFalpha-induced apoptosis was observed in cells pretreated with FB(1). Cells treated with FB(1) showed increased DNA fragmentation and terminal uridine nucleotide end labeling in response to TNFalpha treatment. FB(1) increased DNA synthesis and resulted in cell cycle arrest in the G(2)/M phase of the cell cycle. Flow cytometric analysis of the cell cycle indicated that TNFalpha predominantly killed cells in the G(2)/M phase. The activation of JNK, a mitogen-activated protein kinase (MAPK), was increased following 48 h exposure to FB(1). Phosphorylation of p38 and ERK remained unchanged following treatment with FB(1). FB(1) also increased free sphingoid base levels under identical treatment conditions. Results suggest that FB(1) increased free sphingoid base levels and the population of cells in the G(2)/M phase. This population was shown to be most susceptible to TNFalpha-induced apoptosis. Phosphorylation of pro-apoptotic JNK may play an important role in these effects.     

Long-Term Activation of Protein Kinase C by Nicotine in Bovine Adrenal Chromaffin Cells

Previous results from our laboratory suggest that long-term treatment of primary cultured bovine adrenal medullary (BAM) chromaffin cells with nicotine or phorbol 12-myristate 13-acetate, either of which directly activates protein kinase C (PKC), increases the mRNA levels encoding catecholamine-synthesizing enzymes and proenkephalin. In the present study, we have examined the effects of nicotine on BAM cell PKC activity with special emphasis on long-term effects. Nicotine increased particulate PKC activity in a concentration-dependent manner when measured using in vitro enzyme assay with histone as the substrate. This effect is mediated through nicotinic cholinergic receptors, because 1,1-dimethylphenylpiperazinium, a nicotinic agonist, had a similar effect. In addition, chlorisondamine, a specific nicotine-receptor blocking drug, antagonized the effect of nicotine. Nicotine also increased specific [3H]phorbol 12,13-dibutyrate ([3H]PdBu) binding within 1 min, the effect of which was maximal between 3 and 12 min. This effect was reversed by chlorisondamine similarly after 12 min and after 18 h of nicotine treatment, indicating that continual nicotinic-receptor occupancy is required for persistent PKC activation. Compared to PKC activation, the onset of nicotine-stimulated diacylglycerol production was slow, and it was observed after 12 min of incubation with nicotine. The diacylglycerol levels, specific [3H]PdBu binding, and PKC activity remained significantly elevated for at least 18 h with continuous nicotine incubation. Furthermore, nicotine increased the PKC immunoreactivity of a particulate protein with a molecular mass of 82 kDa in the western blot. These results suggest that nicotinic-receptor activation increases PKC activity and immunoreactivity in BAM cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Occurrence of fumonisin B1 in maize and poultry feeds in Haryana,India

A total of 100 maize and 50 poultry feed samples collected in 1998 at random from nine and eight districts of Haryana, respectively, were analysed for fumonisin B₁. The samples were collected from poultry farms, feed manufacturers and markets. Ninety one (91%) maize samples and forty two (84%) poultry feed samples were found to contain fumonisin B₁. Fumonisin B₁ contamination in the maize samples ranged from 0.1–87.0 ppm. Whereas the poultry feed samples contained fumonisin B₁ in the range of 0.02–28.0 ppm. It indicated widespread prevalence of fumonisin B₁ in maize and poultry feeds in different areas of Haryana. This revised version was published online in June 2006 with corrections to the Cover Date.     

Novel phosphorylation site markers of protein kinase C delta activation

Protein kinase C delta (PKCdelta) is a Ser/Thr kinase which regulates numerous cellular processes, including proliferation, differentiation, migration and apoptosis. Here, we demonstrate that PKCdelta undergoes in vitro autophosphorylation at three sites within its V3 region (S299, S302, S304), each of which is unique to this PKC isoform and evolutionarily conserved. We demonstrate that S299 and S304 can be phosphorylated in mammalian cells following phorbol ester stimulation and that S299-phosphorylated PKCdelta is localised to both the plasma and nuclear membranes. These data indicate that PKCdelta is phosphorylated upon activation and that phospho-S299 represents a useful marker of the activated enzyme.     

Enigma of HIV-1 latent infection in astrocytes: an in-vitro study using protein kinase C agonist as a latency reversing agent

Purging HIV-1 to cure the infection in patients undergoing suppressive antiretroviral therapy requires targeting all possible viral reservoirs. Other than the memory CD4⁺ T cells, several other HIV-1 reservoirs have been identified. HIV-1 infection in the brain as a reservoir is well documented, but not fully characterized. There, microglia, perivascular macrophages, and astrocytes can be infected by HIV-1. HIV-1 infection in astrocytes has been described as a nonproductive and primarily a latent infection. Using primary human astrocytes, we investigated latent HIV-1 infection and tested phorbol 12-myristate 13-acetate (PMA), a protein kinase C agonist, as an HIV-1-latency- reversing agent in infected astrocytes. Chloroquine (CQ) was used to facilitate initial HIV-1 escape from endosomes in astrocytes. CQ significantly increased HIV-1 infection. But treatment with PMA or viral Tat protein was similar to untreated HIV-1-infected astrocytes. Long-term follow-up of VSV-envelope-pseudotyped HIV-1 infected astrocytes showed persistent infection for 110 days, indicating the active state of the virus.     

Functional role of extracellular signal-regulated kinase activation and c-Jun induction in phorbol ester-induced promoter activation of human 12(S)-lipoxygenase gene

The functional role of mitogen-activated protein kinase (MAPK) signaling and c-Jun induction in phorbol 12-myristate 13-acetate (PMA)-induced human 12(S)-lipoxygenase gene expression was studied in human epidermoid carcinoma A431 cells. Among the family of MAPK, PMA only increased the activity of extracellular signal-regulated kinase (ERK). Treatment of cells with PD98059, which is an inhibitor of mitogen-activated protein kinase kinase (MEK), decreased the PMA-induced expression of 12(S)-lipoxygenase. Transfection of cells with Ras, Raf and ERK2 dominant negative mutants inhibited the PMA-induced promoter activation of the 12(S)-lipoxygenase gene in all cases. PMA-induced expression of c-Jun was inhibited by pretreatment with PD98059. Following treatment with PMA, the interaction between c-Jun and simian virus 40 promoter factor 1 (Sp1) in cells increased with time. Enhancement of binding between the c-Jun-Sp1 complex and the Sp1 oligonucleotide was observed in cells treated with PMA, suggesting the possible interaction of c-Jun-Sp1 with GC-rich binding sites in the gene promoter. These results indicate that PMA treatment induced ERK activation mainly through the Raf-MEK-ERK signaling pathway following induction of c-Jun expression, and the formation of the c-Jun-Sp1 complex. Finally, PMA activated the promoter activity of the 12(S)-lipoxygenase gene in cells overexpressing protein kinase C (PKC)delta but not PKCalpha, indicating that PKCdelta played the functional role in mediating the gene activation of 12(S)-lipoxygenase induced by PMA.     

Effects of fumonisin B1 alone and combined with deoxynivalenol or zearalenone on porcine granulosa cell proliferation and steroid production

Fumonisin B₁ (FB₁) is a Fusarium mycotoxin frequently occurring in corn in combination with deoxynivalenol (DON) and zearalenone. The aim of this study was to determine if FB₁, alone and combined with DON or α-zearalenol (ZEA), zearalenone major active metabolite, can affect granulosa cell proliferation, steroid production, and gene expression in swine. Porcine granulosa cells were cultured for 2 days in serum-containing medium followed by 1 or 2 days in serum-free medium with or without added treatments. Fumonisin B₁ had inhibitory effects on granulosa cell proliferation. Deoxynivalenol strongly inhibited cell growth, and no significant difference was detected in combination with FB₁. α-Zearalenol showed a stimulatory effect on granulosa cell numbers even in combination with FB₁. Regarding steroid production, FB₁ increased progesterone production, and FB₁ had no effect on estradiol production. Deoxynivalenol strongly inhibited progesterone and estradiol production, and FB₁ had no significant effect on this response. α-Zearalenol increased progesterone production, and its combination with FB₁ produced additive effects. α-Zearalenol had no effect on estradiol production, whereas it decreased estradiol production when co-treated with FB₁. Fumonisin B₁ was found to decrease CYP11A1 messenger RNA abundance, and the stimulatory effect of FB₁ on progesterone production was found to be not dependent on 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity suggesting that FB₁ increases progesterone production through a different mechanism. The results show that these Fusarium mycotoxins can influence porcine granulosa cell proliferation and steroid production, thereby demonstrating their potential reproductive effects on swine.     

Dual regulation of phospholipase D1 by protein kinase C alpha in vivo

The regulation of phospholipase D1 (PLD1), which has been shown to be activated by protein kinase C (PKC) alpha, was investigated in the human melanoma cell lines. In G361 cell line, which lacks PKCalpha, 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced PLD activation was potentiated by introducing PKCalpha by the adenovirus vector. The kinase-negative PKCalpha elevated TPA-induced PLD activity less significantly than the wild type. A PKC specific inhibitor GF109203X lowered PLD activation in the cells expressing PKCalpha, but did not prevent PLD potentiation induced by the kinase-negative PKCalpha. Expression of PKCbetaII and the kinase-negative PKCbetaII enhanced TPA-stimulated PLD activity moderately in MeWo cell line, in which PKCbetaII is absent. Furthermore, the TPA treatment increased the association of PKCalpha, PKCbetaII, and their kinase-negative mutants with PLD1 in melanoma cells. These results indicate that PLD1 is dually regulated through phosphorylation as well as through the protein-protein interaction by PKCalpha, and probably by PKCbetaII, in vivo.     

Ganglioside GM(1) biphasically regulates the activity of protein kinase C by the effects on the structure of the lipid bilayer

Addition of a small amount of ganglioside GM(1) to phosphatidylserine (PS) liposomes, a gradual increase of protein kinase C (PKC) activity was recorded up to about 2 mol% GM(1) where the maximal enzyme activity was obtained. Then the activity of PKC began to decline and even turned to be inhibited with the further increase of GM(1) content. It was also indicated that GM(1)/PS binary liposomes had the highest membrane fluidity and very low spatial density of lipid headgroups which was demonstrated in the MC-540 studies due to the interposition of GM(1) when the liposomes contained about 2 mol% GM(1). Besides, the liposomes containing about 2 mol% GM(1) provided a more hydrophobic environment for PKC than the liposomes containing less or more GM(1) which was indicated in the Acrylodan experiments. These factors commonly induced PKC to be stimulated maximally. Whether at the lower or higher GM(1) content, the membrane structure was not the most suitable to support the activity of PKC, which declined as a consequence.     

Oleic acid induces endothelin-1 expression through activation of protein kinase C and NF-kappa B

This study investigated the effect of oleic acid on the expression levels of endothelin-1 (ET-1) and on the signaling pathways mediating it in human aortic endothelial cells (HAECs). ET-1 mRNA expression was significantly increased by oleic acid in a dose- and time-dependent manner. Elevation of ET-1 expression in response to oleic acid was inhibited by the protein kinase C (PKC) inhibitor, GF109203X, or the NF-kappa B inhibitor, pyrrolidine dithiocarbamate. In addition, both PKC and NF-kappa B activities were significantly increased by oleic acid. Immunoblot analysis revealed that conventional PKCs (PKC-alpha and -beta II isoforms) were significantly increased in the membranous fractions of HAECs treated with oleic acid. PKC inhibitor completely abolished oleic acid-induced NF-kappa B activation, suggesting that PKC activation is upstream of NF-kappa B activation in oleic acid-induced ET-1 expression. These data suggest that elevated plasma oleic acid levels observed in obese, insulin-resistant subjects result in endothelial dysfunction, at least in part, through an increase in ET-1 expression.     

Potentiation of large-conductance calcium-activated potassium (BK(Ca)) channels by a specific isoform of protein kinase C

The phosphorylation state of large-conductance calcium-activated potassium (BK_Ca) channels regulates their activity and is dynamically regulated by protein phosphatases and kinases, including protein kinase C (PKC). In this study, we showed that PKC activators up-regulate the activity of the BK_Ca channel alpha (α)-subunit, Slo1, in cell-attached patches of transfected COS7 cells. In an immune complex kinase assay, BK_Ca channels isolated from rat brain were phosphorylated in the presence of PKC activators, without the addition of exogenous PKC, which suggests that PKC and BK_Ca channels functionally interact in vivo. Four different PKC isozymes, including PKCδ, phosphorylated the C-terminus of Slo1 and the addition of purified PKCδ-activated BK_Ca channels in excised patches of transfected HEK293 cells. Our results demonstrate that PKC up-regulates BK_Ca channels and that PKCδ may functionally interact with BK_Ca channel complexes in vivo.     

Modulation of T-type Ca2+ channels by corticotropin-releasing factor through protein kinase C pathway in MN9D dopaminergic cells

Corticotrophin-releasing factor (CRF) is the main regulator of the body's stress axis and its signal is translated through G-protein-coupled CRF receptors (CRF-R1, CRF-R2). Even though CRF receptors are present in the midbrain dopamine neurons, the cellular mechanism of CRF action is not clear yet. Since voltage-dependent Ca(2+) channels are highly expressed and important in dopamine neuronal functions, we tested the effect of CRF on voltage-dependent Ca(2+) channels in MN9D cells, a model of dopamine neurons. The application of CRF-related peptide, urocortin 1, reversibly inhibited T-type Ca(2+) currents, which was a major Ca(2+) channel in the cells. The effect of urocortin was abolished by specific CRF-R1 antagonist and was mimicked by protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate. PKC inhibitors abolished the effect of urocortin. These results suggest that urocortin modulates T-type Ca(2+) channel by interacting with CRF-R1 via the activation of PKC signal pathway in MN9D cells.     

Phosphatidylcholine could be the source of 1,2-DAG which activates protein kinase C in EGF-stimulated colon carcinoma cells (HT29)

In our previous study (A. Balogh et al, Cell. Signalling 5 (6), 795-802, 1993.), we have shown that epidermal growth factor (EGF) increased protein kinase C (PKC) activities in colon carcinoma cell line (HT29), possibly through the increased 1,2-diacylglycerol (1,2-DAG) production via phosphatidylcholine (PC). Here we investigate the effect of the well-known PKC activator 12-O-tetradecanoyl-2 phorbol-13-acetate (TPA), on the levels of ³²P incorporation into EGF induced phosphatidylinositols (PI, PI4P, PI4, 5P₂) and different phospholipids (PC, PA, PS) as well as on induced tyrosine kinase activity. TPA significantly decreased the effects of EGF and it had the biggest inhibitory effect on EGF induced PC level. These data support our contention that PC plays an important role in the activation of PKC via 1,2-DAG production in the EGF stimulated pathway.     

Modulation of the expression of GABAA receptors in rat cerebellar granule cells by protein tyrosine kinases and protein kinase C

The expression of GABA_A receptors in rat cerebellar granules in culture has been studied by β_2/3 subunit immunocytochemistry and fluorescence confocal microscopy. These cells show labeling all over the cell bodies' plasma membrane and dendrites. Treatment with the protein tyrosine kinase (PTK) inhibitor genistein results in a decrease of the labeling associated with the β_2/3 subunit in both cell bodies and dendrites. No effect was found with an inactive genistein analogue, daidzein. A similar effect was found with a protein kinase C (PKC) activator, phorbol myristate acetate (PMA). The effects of genistein and PMA are additive.The interpretation of the results is that PTK inhibition blocks exocytotic deposit of newly synthesized GABA_A receptors onto the neuronal plasma membrane. On the other hand, PKC activation speeds up endocytotic removal of GABA_A receptors.     

Toxicity of the mycotoxins fumonisins B1 and B2 and Alternaria alternata f. sp. lycopersici toxin (AAL) in cultured mammalian cells

Fumonisins B₁ and B₂ and AAL toxin are a series of structurally related mycotoxins. Fumonisins B₁ and B₂, produced by Fusarium moniliforme Sheldon induce toxic hepatitis and hepatomas in rats and leukoencephalomalacia in horses. The cancer-promotion assay which has been used to guide their purification is slow and consumes large amounts of sample. We have examined a series of cultured mammalian cell lines in order to develop a more rapid and sensitive bioassay system, which may be useful for examining structure-activity relationships and the mechanism(s) of action of these toxins. Of 9 rat hepatoma cell lines tested, all except the two most de-differentiated lines were sensitive to the three toxins, with a toxic response visible by 48 h. Approximate IC₅₀ values for the most sensitive hepatoma line, H4TG, were 4, 2 and 10 g/ml for fumonisins B₁, B₂ and AAL toxin, respectively in 100 l cultures. Among 15 cell lines from other sources, only MDCK dog kidney epithelial cells were sensitive (IC₅₀ = 2.5, 2 and 5 g/ml, respectively). Studies in co-cultures of sensitive and insensitive cell lines and in cultures of a sensitive cell line over a range of cell densities indicated that cytotoxicity of fumonisins B₁ and B₂ does not involve metabolite activation to a derivative stable enough to diffuse to adjacent cells.Abbreviations AAL toxin Alternaria alternata f. sp. lycopersici toxin - IC₅₀ concentration giving 50% inhibition of cell proliferation     

Arginine vasopressin controls p27 protein expression by PKC activation and irreversibly inhibits the proliferation of K-Ras-dependent mouse Y1 adrenocortical malignant cells

The neurohypophyseal hormone arginine vasopressin (AVP) is a classic mitogen in many cells. In K-Ras-dependent mouse Y1 adrenocortical malignant cells, AVP elicits antagonistic responses such as the activation of the PKC and the ERK1/2 mitogenic pathways to down-regulate cyclin D1 gene expression, which induces senescence-associated β-galactosidase (SA-βGal) and leads to cell cycle arrest. Here, we report that in the metabolic background of Y1 cells, PKC activation either by AVP or by PMA inhibits the PI3K/Akt pathway and stabilises the p27^Kip1 protein even in the presence of the mitogen fibroblast growth factor 2 (FGF2). These results suggest that p27^Kip1 is a critical signalling node in the mechanisms underlying the survival of the Y1 cells. In Y1 cells that transiently express wild-type p27^Kip1, AVP caused a severe reduction in cell survival, as shown by clonogenic assays. However, AVP promoted the survival of Y1 cells transiently expressing mutant p27-S10A or mutant p27-T187A, which cannot be phosphorylated at Ser10 and Thr187, respectively. In addition, PKC activation by PMA mimics the toxic effect caused by AVP in Y1 cells, and inhibition of PKC completely abolishes the effects caused by both PMA and AVP in clonogenic assays. The vulnerability of Y1 cells during PKC activation is a phenotype conditioned upon K-ras oncogene amplification because K-Ras down-regulation with an inducible form of the dominant-negative mutant H-RasN17 has resulted in Y1 cells that are resistant to AVP's deleterious effects. These data show that the survival destabilisation of K-Ras-dependent Y1 malignant cells by AVP requires large quantities of the p27^Kip1 protein as well as phosphorylation of the p27^Kip1 protein at both Ser10 and Thr187.     

The regulation of glycine transporter GLYT1 is mainly mediated by protein kinase Calpha in C6 glioma cells

Glycine has been shown to possess important functions as a bidirectional neurotransmitter. At synaptic clefts, the concentration of glycine is tightly regulated by the uptake of glycine released from nerve terminals into glial cells by the transporter GLYT1. It has been recently demonstrated that protein kinase C (PKC) mediates the downregulation of GLYT1 activity in several cell systems. However, it remains to be elucidated which subtypes of PKC might be important in the regulation of GLYT1 activity. In this study, we attempted to make clear the mechanism of the phorbol 12-myristate 13-acetate (PMA)-suppressed uptake of glycine in C6 glioma cells which have the native expression of GLYT1. In C6 cells, the expression of PKCα, PKCδ, and PKC of the PMA-activated subtypes was detected. The PMA-suppressed action was fully reversed by the removal of both extracellular and intracellular Ca²⁺. Furthermore, the inhibitory effects of PMA or thymeleatoxin (THX), which is a selective activator of conventional PKC (cPKC), were blocked by the downregulation of all PKCs expressed in C6 cells by long-term incubation with THX, or pretreatment with GF109203X or Gö6983, which are broad inhibitors of PKC, or Gö6976, a selective inhibitor of cPKC. On the other hand, treatment of C6 cells with ingenol, a selective activator of novel PKCs, especially PKCδ and PKC, did not affect the transport of glycine. Silencing of PKCδ expression by using RNA interference or pretreatment with the inhibitor peptide for PKC had no effect on the PMA-suppressed uptake of glycine. Together, these results suggest PKCα to be a crucial factor in the regulation of glycine transport in C6 cells.