Na+-Dependent Transport of Taurine by Membrane Vesicles of Neuroblastoma ± Glioma Hybrid Cells

The transport of taurine into membrane vesicles prepared from neuroblastoma x glioma hybrid cells 108CC5 was studied. A great part of the taurine uptake by the membrane preparation is due to the transport into an osmotically sensitive space of membrane vesicles. Taurine uptake by membrane vesicles is an active transport driven by the concentration gradient of Na+ across the membrane (outside concentration greater than inside). The Km value of 36 microM for Na+-dependent taurine uptake indicates a high-affinity transport system. The rate of taurine transport by the membrane vesicles is enhanced by the K+ gradient (inside concentration greater than outside) and the K+ ionophore valinomycin. Taurine transport is inhibited by several structural analogs of taurine: hypotaurine, beta-alanine, and taurocyamine. All these results indicate that the taurine transport system of the membrane vesicles displays properties almost identical to those of intact neuroblastoma X glioma hybrid cells.     

Identification of a System N-Like Na+-Dependent Glutamine Transport Activity in Rat Brain Neurons

Abstract: Glutamine is a primary precursor for the biosynthesis of the neurotransmitters glutamate and γ-aminobutyric acid. It is proposed that glutamine, synthesized and released by astrocytes, is transported into the neuron for subsequent conversion to neurotransmitters. To provide a more complete characterization of this process, we have delineated the transport systems for glutamine uptake in primary cultures of brain neuronal cells from 1-day-old rats. The Na⁺-dependent glutamine entry is mediated by system A, system ASC, and a third, previously unidentified, activity that has been tentatively designated as system N^b. System N^b activity can be monitored by assaying Na⁺-dependent [³H]glutamine uptake in the presence of 2 m M concentrations of both 2-(methylamino)isobutyric acid and threonine to block uptake by systems A and ASC, respectively. The newly identified transport activity exhibits an apparent substrate specificity that is unique compared with the hepatic system N, because it is inhibited by glutamine and asparagine, but not by histidine. Also, the affinity of system N^b for glutamine, as estimated from K _m values, is significantly greater than that observed for the hepatic and muscle Na⁺-dependent glutamine transporters, systems N and N^m. In sharp contrast to the hepatic system N transporter, system N^b exhibits a relative insensitivity to pH and does not permit Li⁺ substitution for Na⁺ as the cosubstrate. The substrate specificity, kinetic analysis, pH sensitivity, and cation dependence of this transport activity indicate that it represents a glutamine transport system not previously identified.     

Modulation of Human Glutamate Transporter Activity by Phorbol Ester

Abstract: Termination of synaptic glutamate transmission depends on rapid removal of glutamate by neuronal and glial high-affinity transporters. Molecular biological and pharmacological studies have demonstrated that at least five subtypes of Na⁺-dependent mammalian glutamate transporters exist. Our study demonstrates that Y-79 human retinoblastoma cells express a single Na⁺-dependent glutamate uptake system with a K _m of 1.7 ± 0.42 µ M that is inhibited by dihydrokainate and dl - threo -β-hydroxyaspartate (IC₅₀ = 0.29 ± 0.17 µ M and 2.0 ± 0.43 µ M , respectively). The protein kinase C activator phorbol 12-myristate 13-acetate caused a concentration-dependent inhibition of glutamate uptake (IC₅₀ = 0.56 ± 0.05 n M ), but did not affect Na⁺-dependent glycine uptake significantly. This inhibition of glutamate uptake resulted from a fivefold decrease in the transporter's affinity for glutamate, without significantly altering the V _max. 4α-Phorbol 12,13-didecanoate, a phorbol ester that does not activate protein kinase C, did not alter glutamate uptake significantly. The phorbol 12-myristate 13-acetate-induced inhibition of glutamate uptake was reversed by preincubation with staurosporine. The biophysical and pharmacological profile of the human glutamate transporter expressed by the Y-79 cell line indicates that it belongs to the dihydrokainate-sensitive EAAT2/GLT-1 subtype. This conclusion was confirmed by western blot analysis. Protein kinase C modulation of glutamate transporter activity may represent a mechanism to modulate extracellular glutamate and shape postsynaptic responses.     

Activation of Amiloride-Sensitive Sodium Transport in C6 Glioma Cells

We have characterized, in C6 cells, an amiloride-sensitive Na+ entry pathway that can exchange for H+. In this report we demonstrate that this cation-exchange system can be induced within 24-36 h by either serum removal or by dibutyryl cyclic AMP; however, these modes of induction are not additive and are manifest only after activation by serum. In these glioma cells we found that activation by serum can be mimicked in part by specific serum factors, i.e., epidermal growth factor and bradykinin. We attempted to characterize this activation process further using several cell biologic probes. We had previously shown that that activation process involves a calcium-dependent step with full activation obtained in the presence of the calcium ionophore A23187. The activation by serum was inhibited by preincubation with colchicine but not with dihydrocytochalasin B, suggesting a cytoskeletal involvement in the activation process. Activation by epidermal growth factor and bradykinin was found to be unaffected by colchicine, suggesting that other factors must be present in serum that confer sensitivity to colchicine. Incubation of the cells with phorbol myristoyl acetate results in the activation of amiloride-sensitive transport, suggesting that stimulation of protein kinase C may be integral to the activation process. Unlike the effects of serum, activation by phorbol myristoyl acetate is not inhibited by colchicine, indicating that this drug works in a way that bypasses the cytoskeletal-dependent step. Since diacylglycerol is the presumed endogenous activator of protein kinase C, we studied the effects of dioleylglycerol. This intermediate of phospholipid turnover was found to increase specifically the amiloride-sensitive sodium pathway.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of Na+, K+ -ATPase Isoforms in Rat Neostriatum by Dopamine and Protein Kinase C

Our previous studies showed that dopamine inhibits Na+,K+-ATPase activity in acutely dissociated neurons from striatum. In the present study, we have found that in this preparation, dopamine inhibited significantly (by approximately 25%) the activity of the alpha3 and/or alpha2 isoforms, but not the alpha1 isoform, of Na+,K+-ATPase. Dopamine, via D1 receptors, activates cyclic AMP-dependent protein kinase (PKA) in striatal neurons. Dopamine is also known to activate the calcium- and phospholipid-dependent protein kinase (PKC) in a number of different cell types. The PKC activator phorbol 12,13-dibutyrate reduced the activity of Na+,K+-ATPase alpha3 and/or alpha2 isoforms (by approximately 30%) as well as the alpha1 isoform (by approximately 15%). However, dopamine-mediated inhibition of Na+,K+-ATPase activity was unaffected by calphostin C, a PKC inhibitor. Dopamine did not affect the phosphorylation of Na+,K+-ATPase isoforms at the PKA-dependent phosphorylation site. Phorbol ester treatment did not alter the phosphorylation of alpha2 or alpha3 isoforms of Na+,K+-ATPase in neostriatal neurons but did increase the phosphorylation of the alpha1 isoform. Thus, in rat neostriatal neurons, treatment with either dopamine or PKC activators results in inhibition of the activity of specific (alpha3 and/or alpha2) isoforms of Na+,K+-ATPase, but this is not apparently mediated through direct phosphorylation of the enzyme. In addition, PKC is unlikely to mediate inhibition of rat Na+,K+-ATPase activity by dopamine in neostriatal neurons.     

Stimulation of Phosphoinositide Hydrolysis by Serotonin in C6 Glioma Cells

5-Hydroxytryptamine (serotonin or 5-HT) stimulated the incorporation of 32Pi into phosphatidylinositol (PI) but not into polyphosphoinositides in C6 glioma cells with an EC50 of 1.2 X 10(-7) M. The phosphoinositide response was blocked by the 5-HT2 antagonists ketanserin and spiperone but inhibited only partly by methysergide and mianserin. Atropine, prazosin, and yohimbine did not block the response, whereas fluphenazine and haloperidol did so partially but also inhibited basal incorporation by approximately 30%. The 5-HT1A agonist 8-hydroxy-2(di-n-propylamino)tetralin did not cause stimulation. Incubation with 5-HT (1 microM) for 1 h increased the incorporation of [2-3H]myoinositol into all phosphoinositides but not into inositol phosphates (IPs). Li+ alone at 10 mM increased labeling in inositol bisphosphate (IP2) and trisphosphate (IP3), whereas labeling in IP and phosphoinositides remained unaltered. Addition of 5-HT had no effect on this increase. Mn2+ at 1 mM enhanced labeling in PI, PI-4-phosphate, lyso-PI, glycerophosphoinositol, and IP, but the presence of 5-HT again did not cause further stimulation. 5-HT also stimulated the release of IPs in cells prelabeled with [2-3H]myo-inositol, incubated with LiCl (10 mM) and inositol (10 mM), and then exposed to 5-HT (1 microM). Radioactivity in IP2 and IP3 was very low, was stimulated approximately 50% as early as 30 s, and remained elevated for at least 20 min. Radioactivity in IP was at least 10 times as high as in IP3 but was increased only from 3 min on with a peak at 20 min, when the elevation was approximately 40 times that in IP3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Na+-Dependent Uptake and Release of Taurine by Neuroblastoma × Glioma Hybrid Cells

Abstract In neuroblastoma × glioma hybrid cells, a cell line of neuronal character, a saturable uptake system for taurine is found which displays high affinity and high capacity ( K _m= 38 μ m , V = 1.25 nmol mg⁻¹ min⁻¹)- Only the closely related structural analogues hypotaurine and β-alanine are able to inhibit the transport of radioactively labeled taurine. Imipramine or haloperidol at 100 μ m effectively blocks taurine uptake. High-affinity taurine uptake shows an absolute and highly specific requirement for Na⁺. The hybrid cells internalize taurine very slowly and, with 1 m m extracellular taurine, attain a plateau only after more than 20 h, at which time approximately 34 m m labeled taurine has accumulated in the cytosol. Generally there is hardly any spontaneous release of accumulated taurine. Efflux can, however, be induced by increasing the intracellular Na⁺ content and is then accelerated by lowering the extracellular Na⁺ concentration. The hypothesis is forwarded that taurine may exert its function by driving the extrusion of Na⁺ in emergency situations.     

Subtypes of Sodium-Dependent High-Affinity L-[3H]Glutamate Transport Activity: Pharmacologic Specificity and Regulation by Sodium and Potassium

Abstract: Some data suggest that the sodium-dependent, high-affinity L-glutamate (Glu) transport sites in forebrain are different from those in cerebellum. In the present study, sodium-dependent transport of L-[³H]Glu was characterized in cerebellum and cortex. In both cerebellar and cortical tissue, activity was enriched in synaptosomes. Approximately 100 excitatory amino acid analogues were tested as potential inhibitors of transport activity. Many of the compounds tested inhibited transport activity by <65% at 1 mM and were not studied further. One group of compounds exhibited inhibition conforming to theoretical curves with Hill coefficients of 1 and were <10-fold selective as inhibitors of transport activity. These included three of the putative endogenous substrates for transport: L-Glu, L-aspartate, and L-cysteate. Four of the compounds exhibited inhibition conforming to theoretical curves with Hill coefficients of 1 and were > 10-fold selective as inhibitors. These included β-N-oxalyl-L-α,β-diaminopropionate, α-methyl-DL-glutamate, (2S, 1′S,2′S)-2-(carboxycyclopropyl)glycine, and (2S, 1′S,2′S,3′S)-2-(2-carboxy-3-methoxymethylcyclopropyl)glycine. Data obtained with a few of the inhibitors were consistent with two sites in one or both of the brain regions. (2S, 1′R,2′R)-2-(Carboxycyclopropyl)glycine (L-CCG-II) was identified as the most potent (IC₅₀= 5.5 μM) and selective (60–100-fold) inhibitor of transport activity in cerebellum. One of the potential endogenous substrates, L-homocysteate, was also a selective inhibitor of cerebellar transport activity. The data for inhibition of transport activity in cortex by both L-CCG-II and L-homocysteate were best fit to two sites. Kainate was equipotent as an inhibitor of transport activity, and in both brain regions the data for inhibition were best fit to two sites. The possibility that there are four subtypes of excitatory amino acid transport is discussed. Altering sodium and potassium levels affects cerebellar and cortical transport activity differently, suggesting that the differences extend to other recognition sites on these transporters.     

Inhibition of Maitotoxin-Induced Ca2+ Influx in Rat Glioma C6 Cells by Brevetoxins and Synthetic Fragments of Maitotoxin

Abstract: ⁴⁵Ca²⁺ influx in rat glioma C6 cells induced by 0.3 n M maitotoxin (MTX) was markedly inhibited by brevetoxin A (PbTx1) and brevetoxin B (PbTx2), with EC₅₀ values of 16 and 13 µ M , respectively. This inhibition was observed immediately after addition of MTX when monitored with fura-2, which suggests that PbTx2 directly blocks the action of MTX. This blockade by PbTx2 was not affected by tetrodotoxin, which excludes the involvement of voltage-sensitive sodium channels. The depolarizing effects of these brevetoxins were also not a likely cause of this inhibition, because melittin, a channel-forming peptide, did not significantly block MTX-induced ⁴⁵Ca²⁺ influx. Instead, this inhibition was thought to be mediated by blockade of an MTX-binding site by the brevetoxins, based on the fact that these toxins, particularly PbTx2, closely mimic the partial structure of MTX. Synthetic fragments of MTX corresponding to the hydrophilic EF-GH rings (200 µ M ) and LM-NO rings (500 µ M ) of MTX significantly reduced MTX-elicited Ca²⁺ influx. The observation that the effects of MTX were inhibited by structural mimics of both its hydrophobic and hydrophilic portions implies that both portions of the MTX molecule recognize its target.     

Inhibition by K+ of Na+-Dependent D-Aspartate Uptake into Brain Membrane Saccules

Na+-dependent uptake of dicarboxylic amino acids in membrane saccules, due to exchange diffusion and independent of ion gradients, was highly sensitive to inhibition by K+. The IC50 was 1-2 mM under a variety of conditions (i.e., whole tissue or synaptic membranes, frozen/thawed or fresh, D-[3H]aspartate (10-1000 nM) or L-[3H]glutamate (100 nM), phosphate or Tris buffer, NaCl or Na acetate, presence or absence of Ca2+ and Mg2+). The degree of inhibition by K+ was also not affected on removal of ion gradients by ionophores, or by extensive washing with H2O and reloading of membrane saccules with glutamate and incubation medium in the presence or absence of K+ (3 mM, i.e., IC70). Rb+, NH4+, and, to a lesser degree Cs+, but not Li+, could substitute for K+. [K+] showed a competitive relationship to [Na+]2. Incubation with K+ before or after uptake suggested that the ion acts in part by allowing net efflux, thus reducing the internal pool of amino acid against which D-[3H]aspartate exchanges, and in part by inhibiting the interaction of Na+ and D-[3H]aspartate with the transporter. The current model of the Na+-dependent high-affinity acidic amino acid transport carrier allows the observations to be explained and reconciled with previous seemingly conflicting reports on stimulation of acidic amino acid uptake by low concentrations of K+. The findings correct the interpretation of recent reports on a K+-induced inhibition of Na+-dependent "binding" of glutamate and aspartate, and partly elucidate the mechanism of action.     

Phorbol Ester Pretreatment Desensitizes the Inhibition of Ca2+ Channels Induced by k-Opiate, α2-Adrenergic, and Muscarinic Receptor Agonists

Acute treatment of rat spinal cord-dorsal root ganglion cocultured neurons with 12-O-tetradecanoylphorbol 13-acetate (TPA), a known activator of protein kinase C, inhibited the dihydropyridine-sensitive voltage-dependent 45Ca2+ influx measured in these cells (IC50 of approximately 100 nM, 66% inhibition at 1 microM TPA). However, prolonged preincubation (24 h) of the cells with 100 nM TPA followed by extensive washing completely abolished, i.e., desensitized, the capacity of a second application of TPA to inhibit the activity of the voltage-dependent Ca2+ channels. Moreover, this treatment also abolished the inhibition of Ca2+ influx produced by kappa-opiate as well as by alpha 2-adrenergic and muscarinic receptor agonists. Substantial desensitization was already observed following a 1-h pretreatment with 100 nM TPA. In contrast to TPA, an inactive phorbol ester (4 beta-phorbol 13-acetate) did not affect the inhibition of the voltage-dependent Ca2+ influx by these receptor agonists. These results suggest that protein kinase C may have a role in the modulation of Ca2+ channels by kappa-opiate, alpha 2-adrenergic, and muscarinic receptor agonists.     

Na+-Dependent and Phlorizin-Inhibitable Transport of Glucose and Cycasin in Brain Endothelial Cells

Abstract: Although cycasin (methylazoxymethanol β- d -glucoside) is proposed to be a significant etiological factor for the prototypical neurodegenerative disorder Western Pacific amyotrophic lateral sclerosis and parkinsonism-dementia complex, the mechanism underlying transport of cycasin across the blood-brain barrier (BBB) is unknown. We examined cycasin transport in cultured bovine brain endothelial cells, a major element of the BBB. Cycasin was taken up into endothelial cells in a dose-dependent manner with maximal uptake observed at a concentration of 10 µ M . Cycasin uptake was significantly inhibited by α-methyl- d -glucoside, a specific analogue for the Na⁺-dependent glucose transporter (SGLT), by the SGLT inhibitor phlorizin, by replacement of extracellular NaCl with LiCl, and by dinitrophenol (DNP), an inhibitor of energy metabolism. In addition, cycasin produced inward currents in a whole-cell voltage clamp configuration. Peak currents were observed at 10 µ M with a trend toward reduction at higher concentrations, and currents were clearly blocked by α-methyl- d -glucoside, phlorizin, and DNP. In addition, cycasin never evoked currents in Na⁺-free extracellular solution. These results suggest that cycasin is selectively transported across brain endothelial cells, possibly across the BBB by a Na⁺/energy-dependent glucose transporter.     

Pharmacology of Sodium-Dependent High-Affinity l-[3H]Glutamate Transport in Glial Cultures

Abstract: Pharmacological and molecular biological studies provide evidence for subtypes of sodium-dependent high-affinity glutamate (Glu) transport in the mammalian CNS. At least some of these transporters appear to be selectively expressed in different brain regions or by different cell types. In the present study, the properties of l -[³H]Glu transport were characterized using astrocyte-enriched cultures prepared from cerebellum and cortex. In both brain regions, the kinetic data for sodium-dependent transport were consistent with a single site with K_m values of 91 ± 17 µM in cortical glial cells and 66 ± 23 µM in cerebellar glial cells. The capacities were 6.1 ± 1.6 nmol/mg of protein/min in cortical glial cells and 8.4 ± 0.9 nmol/mg of protein/min in cerebellar glial cells. The potencies of ～40 excitatory amino acid analogues for inhibition of sodium-dependent transport into glial cells prepared from cortex and cerebellum were examined, including compounds that are selective inhibitors of transport in synaptosomes prepared from either cerebellum or cortex. Of the analogues tested, 14 inhibited transport activity by >50% at 1 mM concentrations. Unlike l -[³H]Glu transport in synaptosomes prepared from cerebellum or cortex, there were no large differences between the potencies of compounds for inhibition of transport measured in glial cells prepared from these two brain regions. With the exception of (2S,1′R,2′R)-2-(carboxycyclopropyl)glycine and l -α-aminoadipate, all of the compounds examined were ～10–200-fold less potent as inhibitors of l -[³H]Glu transport measured in glial cells than as inhibitors of transport measured in synaptosomes prepared from their respective brain regions. The pharmacology of transport measured in these glial cells differs from the reported pharmacology of the cloned Glu transporters, suggesting the existence of additional uncloned Glu transporters or Glu transporter subunits.     

Effect of Desipramine on a Glycoprotein Sialyltransferase Activity in C6 Cultured Glioma Cells

The tricyclic antidepressant desipramine, when added to culture medium, gave rise in C6 rat glioma cells to a decrease of the activity of the enzyme asialofetuin sialyltransferase. The inhibition was dose and time dependent and was observed in both multiplying cells and cells blocked with 2 mM thymidine or depletion of amino acids. This inhibition was rather specific to the sialyltransferase, as under the conditions where this enzyme was inhibited up to 70%, other enzymes such as dolichol phosphate mannose synthetase, glutamine synthetase, and glycerol phosphate dehydrogenase remained unaffected. This inhibition was not reversed after removal of desipramine from the medium and was not observed by direct addition of desipramine to the sialyltransferase incubation assay. Under the same conditions, W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], which is known to be a potent calmodulin antagonist and an inhibitor of calmodulin-dependent kinases, gave the same concentration-dependent inhibition profile of sialyltransferase as desipramine, whereas H-7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine], which is an inhibitor of protein kinase C and cyclic nucleotide-dependent kinases, had no effect. So, it is suggested that desipramine inhibits the sialyltransferase activity in C6 glioma cells through a calmodulin-dependent system.     

Protein Kinase C of Sympathetic Neuronal Membrane Is Activated by Phorbol Ester-Correlation Between Transmitter Release, 45Ca2+ Uptake, and the Enzyme Activity

The effects of phorbol esters [phorbol 12,13-dibutyrate (PDB), 12-O-tetradecanoylphorbol 13-acetate (TPA), and phorbol 13-acetate] were investigated on the release of [3H]norepinephrine, 45Ca2+ accumulation, and protein kinase C activity in cultured sympathetic neurons of the chick embryo. Sympathetic neurons derived from 10-day-old chick embryo were cultured in serum-free medium supplemented with insulin, transferrin, and nerve growth factor. After 3 days, neurons were loaded with [3H]-norepinephrine and the release of [3H]norepinephrine was determined before and after electrical stimulation. Stimulation at 1 Hz for 15 s increased the release of [3H]-norepinephrine over the nonstimulation period. Stimulation-evoked release gradually declined with time during subsequent stimulation periods. Incubation of neurons in Ca2+-free Krebs solution containing 1 mM EGTA completely blocked stimulation-evoked release of [3H]-norepinephrine. Stimulation-evoked release of [3H]-norepinephrine was markedly facilitated by 3 and 10 nM PDB or TPA. The spontaneous release was also enhanced by PDB and TPA. The net accumulation of 45Ca2+ during stimulation of sympathetic neurons was increased by two- to fourfold in the presence of PDB or TPA. PDB at 1-100 nM produced a concentration-dependent increase in the activation of protein kinase C. PDB at 30 nM increased the activity of protein kinase C of the particulate fraction from 0.09 to 0.58 pmol/min/mg protein. There was no significant change in protein kinase C activity of the cytosolic fraction (0.14 pmol/min/mg versus 0.13 pmol/min/mg protein). The ratio of the particulate to cytosolic protein kinase C increased from a control value of 0.62 to 4.39 after treatment with 30 nM PDB. TPA (10 and 30 nM) also increased protein kinase C activity of the particulate fraction by six- to eightfold. Phorbol 13-acetate had no effect on protein kinase C activity, [3H]norepinephrine release, and 45Ca2+ accumulation. These results provide direct evidence that activation of protein kinase C enhances Ca2+ accumulation, which in turn leads to the facilitation of transmitter release in sympathetic neurons.     

Stimulation of Na+-Dependent, Veratridine-Sensitive Synaptosomal Amino Acid Uptakes by Serum Albumin

Abstract: Bovine serum albumin (BSA) is shown to stimulate selectively the synaptosomal uptakes of those amino acids that are dependent on external Na⁺ and that are inhibited by veratridine. Thus, the stimulation can be seen in the case of aspartic acid, glutamic acid, glycine, proline, and γ-aminobutyric acid, but not with serine and threonine. Further, studies on the interaction of veratridine, valinomycin, and BSA on the uptake of proline suggest that the primary action of the albumin is to increase the influx of proline. Such an action could result as a consequence of stabilization of the Na⁺ gradient by increased endogenous levels of ATP. Intrasynaptosomal ATP was increased in the presence of BSA but significantly decreased by veratridine.     

Chronic Imipramine Administration Amplifies the Serotonin2A Receptor-Induced Intracellular Ca2+ Mobilization in C6 Glioma Cells Through a Calmodulin-Dependent Pathway

Abstract: In the present study, we examined whether chronic exposure of C6BU-1 cells to 100 n M of several different types of antidepressants directly influences serotonin_2A (5-HT_2A) receptor-stimulated intracellular Ca²⁺ mobilization. Imipramine, desipramine, clomipramine, and maprotiline amplified the 5-HT response at 48, but not at 2, h. Imipramine increased the maximum response to 5-HT without altering the EC₅₀ of the dose-response curve. This effect was time dependent and cycloheximide blocked the maximal induction, suggesting an essential role for protein synthesis in this process. Previous exposure of the cells to thrombin or isoproterenol did not influence 5-HT_2A receptor function and pretreatment with imipramine did not alter the thrombin- or bradykinin-induced Ca²⁺ mobilization, which indicates that the effects of imipramine appear to be specific to the 5-HT_2A receptor. The effect of imipramine was potently suppressed by a calmodulin antagonist, W-13, in a dose-dependent manner. Furthermore, this amplified 5-HT response was blocked by KN-93, but not by H-7. Taken together, these results suggest that imipramine has a modulatory effect on the 5-HT_2A receptor-coupled intracellular Ca²⁺ in C6 cells through a calmodulin-dependent pathway, possibly involving Ca²⁺/calmodulin kinase activation.     

Phorbol Ester Binding Sites in the Fish Retina: Correlation with Stimulation of Endogenous Phosphorylation and Protein Kinase C Activation

Abstract: The injection of phorbol esters into the eyes of dark-adapted teleost fish can mimic light effects in the retina and induces corresponding synaptic plasticity of horizontal cells (HCs). It is therefore very likely that protein kinase C (PKC) mediates light-induced synaptic plasticity. In the present study, we investigated the distribution of PKC, the phorbol ester receptor, in isolated HCs and in the whole retina by using tritiated phorbol 12,13-dibutyrate ([³H]PDBu). The binding characteristics analyzed for HC homogenates and retinal homogenates revealed that [³H]PDBu binding is time dependent, specific, saturable, and reversible. Binding sites in HCs displayed a dissociation constant of 11.5 n M and a total number of 2.8 pmol/mg of protein. Autoradiography revealed that [³H]PDBu labeling is present in all retinal layers, including HCs, where it is associated with the somata. Furthermore, the treatment with PDBu strongly affected the endogenous phosphorylation of several membrane, cytosolic, and HC proteins and led to PKC activation as measured by H1 histone phosphorylation. In HCs, the treatment with PDBu in particular affected the amount of ³²P incorporated into a group of phosphoproteins (68, 56/58, 47, 28, and 15 kDa) that were recently shown to be affected by light adaptation. These proteins might therefore be considered as important components of the observed morphological and physiological synaptic plasticity of HCs in the course of light adaptation.     

Synthesis of Urokinase-Type Plasminogen Activator and of Type- 1 Plasminogen Activator Inhibitor in Neuronal Cultures of Human Fetal Brain: Stimulation by Phorbol Ester

Human neuronal brain cultures established from 12- and 14-week-old fetuses synthesize and secrete urokinase-type plasminogen activator (uPA) and limited amounts of tissue-type plasminogen activator (tPA). These cells also produce and secrete the endothelial cell-type PA inhibitor (PAI-1), which forms sodium dodecyl sulfate-stable tPA/PAI-1 complexes in the culture medium. Immunocytochemistry shows a predominant localization of uPA, tPA, and PAI-1 in neuronal cells, with only a very weak positivity detectable in the few glial cells present in these cultures. The protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulates the synthesis of both uPA and PAI-1, resulting in a final increase in the plasmin-generating capacity of neuronal cell cultures. No significant effect is observed, however, when cells are treated with the TPA analogue 4 alpha-phorbol 12,13-didecanoate, which is inactive as a PKC inducer, or with the neurotrophic polypeptide basic fibroblast growth factor. These data represent the first characterization of the plasmin-generating system in human fetal brain neurons and suggest a role for PKC in the modulation of uPA and PAI-1 synthesis.     

自噬在声动力疗法抑制C6胶质瘤细胞增殖中的作用

目的:探讨自噬在血卟啉单甲醚(Hematoporphyrin monomethyl ether,HMME)介导的声动力疗法(Sonodynamic therapy,SDT)抑制C6胶质瘤细胞增殖中的作用。方法:选取对数期生长的C6胶质瘤细胞并随机分为四组:对照组(未予处理)、超声组(单独超声照射)、HMME组(单独加入HMME)、SDT组(超声照射+HMME)。透射电镜观察SDT处理的C6胶质瘤细胞中自噬体数量的改变。应用qRT-PCR和免疫印迹分析SDT处理对C6胶质瘤细胞中的LC3、Beclin1、Bcl-2 m RNA及蛋白表达水平的影响。MTT检测C6胶质瘤细胞的活力变化。结果:透射电子显微镜显示SDT组自噬体数量较对照组明显增多。SDT组C6胶质瘤细胞中微管相关蛋白1轻链3 (Microtubule associated protein 1 light chain 3, LC3)、Beclin1 m RNA和蛋白水平高于对照组,B细胞淋巴瘤-2(B cell lymphoma-2, Bcl-2) m RNA和蛋白水平低于对照组。与对照组相比,SDT组C6胶质瘤细胞存活率从0 h至6 h逐渐下降,从12 h至72 h逐渐升高。3-甲基腺嘌呤(3-Methyladenine,3-MA)+SDT、氯喹(Chloroquine,CQ)+SDT处理后C6胶质瘤细胞存活率较SDT组明显降低。结论:SDT可能通过诱导自噬抑制C6胶质瘤细胞增殖。