Neurite Outgrowth and Protein Phosphorylation in Chick Embryonic Sensory Ganglia Induced by a Brief Exposure to 12-O-Tetradecanoylphorbol 13-Acetate

Abstract: An exposure to 12- O -tetradecanoylphorbol 13-acetate (TPA) at 20 n M for as short as 30 min was sufficient to elicit neurite outgrowth from explanted chick embryonic sensory ganglia. Attachment of the ganglia to the collagencoated substratum during exposure to TPA was essential for subsequent neurite outgrowth. Pulse-labeling with [³⁵S]-methionine indicated no significant difference in protein synthesis between control and TPA-treated ganglia. In vitro phosphorylation assay revealed a prominent protein kinase C substrate with an apparent molecular mass of 66,000 dalton (66 kDa) in chick embryo ganglia extracts. Treatment of intact ganglia with TPA for 30 min also specifically stimulated the phosphorylation of the same protein. When staurosporine, a potent inhibitor of protein kinase C, was present during TPA treatment, both neurite outgrowth and the phosphorylation of the 66-kDa protein were blocked. Biochemical analysis of the phosphorylated 66-kDa protein indicated that (1) phosphorylation was only in serine residue, (2) the pI value was 4.5, (3) after V8 protease digestion, two phosphorylated peptide fragments, 6.0 and 7.5 kDa in size, were produced, and (4) it cross-reacted with an antiserum raised against a 66-kDa neurofilament subunit from rat spinal cord. These results suggest that early activation of protein kinase C and the phosphorylation of the 66-kDa protein may be involved in neuritogenesis.     

Phorbol Esters Increase GTP-Dependent Adenylate Cyclase Activity in Rat Brain Striatal Membranes

Abstract: 4β-Phorbol 12-myristate 13-acetate (PMA), added to a lysed mitochondrial fraction of rat striatum, stimulates adenylate cyclase activity with an apparent time lag of ～30 s. Half-maximal and maximal enzyme stimulations are obtained with 8 and 200 nM PMA, respectively. The PMA stimulation is GTP dependent, reaching a maximum of ～60% at 50 μ.M GTP, and is associated with disappearance of the enzyme inhibition induced by micromolar concentrations of GTP. Enhancement of enzyme activity by cholera toxin and 3,4-dihydroxyphenylethylamine is amplified by PMA only at micromolar concentrations of GTP. PMA does not affect the enzyme stimulation by forskolin but reverses the inhibition of forskolin-stimulated enzyme by GTP. When guanyl-5′-yl-imidodiphosphate is substituted for GTP, PMA does not modify adenylate cyclase activity. Enzyme inhibition by acetylcholine, Leu-enkephalin, and R(-)N⁶-(2-phenylisopropyl)adenosine is magnified by PMA. Stimulation of adenylate cyclase by PMA is markedly reduced following EGTA treatment, is not observed when adenyl-5′-yl-imidodiphosphate is substituted for ATP as substrate for adenylate cyclase, and is enhanced by l-α-phosphatidyl-l-serine. Like PMA, 4β-phorbol 12,13-dibutyrate and 1-oleoyl-2-acetylglycerol stimulate striatal adenylate cyclase, whereas 4β-phorbol and 4β-phorbol 13-acetate are ineffective. The results indicate that phorbol esters increase striatal adenylate cyclase activity by reducing the GTP-induced inhibition of the enzyme, presumably as a result of protein kinase C activation.     

Stimulation of Phospholipase D Activity by Phorbol Esters in Cultured Astrocytes

The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) was found to stimulate phospholipase D activity in cultured primary astrocytes. Both the hydrolysis and the transphosphatidylation reaction catalyzed by phospholipase D were studied in cells labeled with [3H]glycerol. Phosphatidic acid (PA) synthesis was increased after addition of 100 nM TPA. When ethanol was present in the cell culture medium, phosphatidylethanol (Peth), a product of phospholipase D-catalyzed transphosphatidylation, was formed. The half-maximum effective concentrations (EC50) of TPA were 25 nM for PA increase as well as for Peth formation. The formation of Peth in ethanol-treated cells was accompanied by an inhibition of the TPA-induced increase in labeled PA. Increasing ethanol concentrations led to an increase in [3H]Peth and a decrease in [3H]PA. A protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), inhibited both the synthesis of PA and the formation of Peth observed after TPA addition to the astrocytes. Dioctanoyl-glycerol (100 microM) stimulated the formation of Peth in the presence of ethanol. In addition to the induction of Peth formation in astrocytes, TPA induced Peth formation in ethanol-treated neurons. The present results indicate that phospholipase D activity is stimulated by TPA in cultured primary brain cells. Modulation of phospholipase D activity by protein kinase C is a mechanism that may be important in signal transduction cascades.     

Protein Kinase C in Primary Astrocyte Cultures: Cytoplasmic Localization and Translocation by a Phorbol Ester

The distribution of calcium-activated, phospholipid-dependent protein kinase (protein kinase C) in supernatant and particulate fractions of primary cultures of rat astrocytes and its translocation by a phorbol ester were studied. We observed that 91% of protein kinase C activity in astrocytes was in the supernatant fraction, as measured by lysine-rich histone phosphorylation assay. Attempts to uncover latent activity in the particulate fraction were unsuccessful. Approximately 75% of the supernatant protein kinase C activity could be translocated to the particulate fraction by prior treatment (30-60 min) of the cultures with 100 nM 12-O-tetradecanoyl-phorbol 13-acetate (TPA), but not with 4 alpha-phorbol, an inactive phorbol ester. Investigation of endogenous substrates for protein kinase C showed that TPA treatment brought about an increase in phosphorylation in membrane proteins and a decrease in phosphorylation of supernatant proteins. These findings indicate that the distribution of protein kinase C in astrocytes differs substantially from that in whole brain tissue, where approximately two-thirds of the protein kinase C activity is associated with the particulate fraction. Because protein kinase C is concentrated in the cytosol of astrocytes and most of this activity can be translocated to membranes, astrocytes may be particularly well-suited to respond to signals that activate phosphoinositide-linked receptors in brain.     

Phorbol Esters Enhance Neurotransmitter-Stimulated Cyclic AMP Production in Rat Brain Slices

The effect of phorbol esters on cyclic AMP production in rat CNS tissue was examined. Using a prelabeling technique for measuring cyclic AMP accumulation in brain slices, it was found that phorbol 12-myristate, 13-acetate (PMA) enhanced the cyclic AMP response to forskolin and a variety of neurotransmitter receptor stimulants while having no effect on second messenger accumulation itself. A short (15-min) preincubation period with PMA was required to obtain maximal enhancement, whereas the augmentation was lessened by prolonged exposure (3 h) to the phorbol. The response to PMA was concentration dependent (EC50 = 1 microM) and regionally selective, being most apparent in forebrain, and was not influenced by removal of extracellular calcium or by inhibition of phosphodiesterase or phospholipase A2. Only those phorbols known to stimulate protein kinase C augmented the accumulation of cyclic AMP. Moreover, the membrane substrates phosphorylated by endogenous C kinase and by a partially purified preparation of this enzyme were similar. The results suggest that phorbol esters, by activating protein kinase C, modify the cyclic AMP response to brain neurotransmitter receptor stimulation in brain by influencing a component of the adenylate cyclase system beyond the transmitter recognition site.     

Excess K+ and Phorbol Ester Activate Protein Kinase C and Support the Survival of Chick Sympathetic Neurons in Culture

The effects of phorbol esters were investigated on the survival of chick sympathetic neurons in a serum-free culture medium. The protein kinase C activator phorbol 12,13-dibutyrate (PDB) supported about 40% of the plated sympathetic neurons. This number was comparable to that supported by nerve growth factor (NGF). A combination of phorbol ester and NGF did not significantly increase the number of surviving neurons. Phorbol ester-supported sympathetic neurons possessed desipramine-sensitive [3H]-norepinephrine uptake mechanism, and therefore were noradrenegic in character. Two days after the start of cultures, if NGF was replaced by phorbol ester, or phorbol ester was replaced by NGF, the number of surviving sympathetic neurons was essentially the same in both groups, and the uptake of [3H]norepinephrine was also comparable when examined 2 days after the switchover. Interchangeability between phorbol ester and NGF in the survival of sympathetic neurons suggests that both agents act on the same subpopulation of neurons of the chick sympathetic ganglia. The protein kinase C activity of cytosol and particulate fractions of NGF-supported neurons was 0.14 and 0.09 pmol/min/mg protein, respectively. In phorbol ester-supported neurons the activity in the particulate fraction increased by about fivefold. Removal of the phorbol ester after 2 days resulted in restoration of the enzyme activity in less than 1 h, and readdition of the phorbol ester again increased the activity by fivefold. When NGF was added to these neurons (1 microgram for 15 min), there was no change in the enzyme activity. Phorbol 13-acetate was ineffective in supporting sympathetic neurons in culture, as well as in enhancing protein kinase C activity. We also compared the protein kinase C activity of sympathetic neurons supported in culture by NGF and excess potassium (35 mM K+) Neurons supported in culture by 35 mM K+ for 2 days had almost eightfold more protein kinase C activity in their particulate fraction than in cytosol fraction. In NGF-supported neurons were acutely treated with excess K+, the protein kinase C activity was increased in the particulate fraction by about sevenfold in a concentration- and time-dependent manner. Excess K+ plus phorbol ester did not produce an additive effect on protein kinase C activity. PDB and excess K+ had no effect on cyclic AMP content of sympathetic neurons. In summary, the present data suggest that the neurotrophic action of PDB and excess K+ is probably mediated through protein kinase C.     

Phorbol Esters Increase Dopamine Transporter Phosphorylation and Decrease Transport Vmax

Abstract: Sodium- and chloride-coupled transport of dopamine from synapses into presynaptic terminals plays a key role in terminating dopaminergic neurotransmission. Regulation of the function of the dopamine transporter, the molecule responsible for this translocation, is thus of interest. The primary sequence of the dopamine transporter contains multiple potential phosphorylation sites, suggesting that the function of the transporter could be regulated by phosphorylation. Previous work from this laboratory has documented that phorbol ester activation of protein kinase C (PKC) decreases dopamine transport V _max in transiently expressing COS cells. In the present report, we document in vivo phosphorylation of the rat dopamine transporter stably expressed in LLC-PK₁ cells and show that phosphorylation is increased threefold by phorbol esters. Dopamine uptake is also regulated by phorbol esters in these cells; phorbol 12-myristate 13-acetate (PMA) reduces transport V _max by 35%. Parallels between the time course, concentration dependency, and staurosporine sensitivity of alterations in transporter phosphorylation and transporter V _max suggest that dopamine transporter phosphorylation involving PKC could contribute to this decreased transporter function. Phosphorylation of the dopamine transporter by PKC or by a PKC-activated kinase could be involved in rapid neuroadaptive processes in dopaminergic neurons.     

The Differential Role of Protein Kinase C Isozyme in the Rapid Induction of Neurofilament Phosphorylation by Nerve Growth Factor and Phorbol Esters in PC12 Cells

We examined the short-term regulation of the phosphorylation of the mid-sized neurofilament subunit (NF-M) by kinases which were activated in rat pheochromocytoma (PC12) cells by nerve growth factor (NGF) and/or 12-O-tetradecanoylphorbol 13-acetate (TPA). We found that NGF and TPA, alone or in combination, increased (a) the incorporation of [32P]Pi into NF-M and (b) the rate of conversion of NF-M from a poorly phosphorylated to a more highly phosphorylated form. This was not due to increased synthesis of NF-M, because NGF alone did not increase NF-M synthesis and TPA alone or TPA and NGF together inhibited the synthesis of NF-M. Further, an increase in calcium/phospholipid-dependent kinase (PKC) activity resulting from the treatment of PC12 cells with NGF and TPA was observed concomitant with the increased phosphorylation of NF-M. This PKC activity was determined to be derived from the PKC alpha and PKC beta isozymes. Finally, when PC12 cells were rendered PKC-deficient by treatment with 1 muM TPA for 24 h, NGF maintained the ability to induce an increase in NF-M phosphorylation, though not to the level attained in cells which were not PKC-deficient. These data suggest that NGF with or without TPA stimulates NF-M phosphorylation as a result of a complex series of events which include PKC-independent and PKC-dependent pathways.     

Activation of Protein Kinase C by Phorbol Esters and Arachidonic Acid Required for the Optimal Potentiation of Glutamate Exocytosis

Abstract: The effects of arachidonic acid and phorbol esters in the Ca²⁺-dependent release of glutamate evoked by 4-aminopyridine (4-AP) in rat cerebrocortical synaptosomes were studied. In the absence of arachidonic acid, high concentrations (500 n M ) of 4β-phorbol dibutyrate (4β-PDBu) were required to enhance the release of glutamate. However, in the presence of arachidonic acid, low concentrations of 4β-PDBu (1–50 n M ) were effective in potentiating glutamate exocytosis. This potentiation of glutamate release by phorbol esters was not observed with the methyl ester of arachidonic acid, which does not activate protein kinase C. Moreover, pretreatment of synaptosomes with the protein kinase inhibitor staurosporine also prevented the stimulatory effect by arachidonic acid and phorbol esters. These results suggest that the activation of protein kinase C by both arachidonic acid and phorbol esters may play a role in the potentiation of glutamate exocytosis.     

Phorbol Ester-Induced Upregulation of Angiotensin II Receptors in Neuronal Cultures Is Potentiated by a Calcium Ionophore

Previous studies have suggested that protein kinase C is important in the regulation of angiotensin II receptors in neuronal cultures, because the C-kinase agonists, phorbol esters, are able to increase the number of these receptors. In the present study, we have further investigated the role of protein kinase C in angiotensin II receptor regulation. This enzyme is calcium dependent, and so we investigated the effects of A23187, a calcium ionophore, on phorbol ester-stimulated and basal angiotensin II receptor regulation. A23187, at concentrations that increased 45Ca2+ influx, caused a dose-dependent potentiation of phorbol-12-myristate-13-acetate (TPA)-stimulated upregulation of angiotensin II receptors. This potentiation by A23187 was a further increase in angiotensin II receptor number and was abolished in calcium-free medium. In the absence of TPA, A23187 caused a decrease in angiotensin II receptor number, an effect not observed in calcium-free medium. The results suggest at least two pathways for angiotensin II receptor regulation in neuronal cells: (a) by calcium-dependent protein kinase C and (b) via an influx of calcium into the cell.     

Effects of Phorbol Esters and Forskolin on Basal and Histamine-induced Accumulation of Inositol Phosphates in Cultured Bovine Adrenal Chromaffin Cells

The effect of phorbol esters and forskolin pretreatment on basal and histamine-induced accumulation of inositol phosphates and catecholamine release was examined in cultures of bovine adrenal chromaffin cells. Histamine caused a dose-dependent, Ca2+-dependent accumulation of total inositol phosphates with an EC50 at approximately 1 microM and an eight- to 10-fold increase at 100 microM within 30 min of incubation. Histamine (10 microM) also caused the release of cellular catecholamines amounting to some 2.8% of cellular stores released over a 20-min period. Both the inositol phosphate and catecholamine responses were completely blocked by the H1-antagonist mepyramine and were insensitive to the H2-antagonist cimetidine. Examination of the time course of accumulation of the individual inositol phosphates stimulated by histamine revealed an early and sustained rise in inositol 1,4-bisphosphate content but not inositol 1,4,5-trisphosphate content at 1 min and the overall largest accumulation of inositol monophosphate after 30 min of stimulation. Pretreatment with the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA) resulted in a dose-dependent, time-dependent inhibition of histamine-induced inositol phosphate formation and catecholamine secretion. In this inhibitory action, PMA exhibited high potency (IC50 of approximately 0.5 nM), an effect not shared by the inactive phorbol ester 4-alpha-phorbol 12,13-didecanoate. Pretreatment with forskolin, on the other hand, only marginally inhibited the histamine-induced inositol phospholipid metabolism and catecholamine secretion. These data suggest that protein kinase C activation in chromaffin cells may mediate a negative feedback control on inositol phospholipid metabolism.     

Activation of Protein Kinase C Augments Peptide Release from Rat Sensory Neurons

Abstract: To determine whether protein kinase C (PKC) mediates release of peptides from sensory neurons, we examined the effects of altering PKC activity on resting and evoked release of substance P (SP) and calcitonin gene-related peptide (CGRP). Exposing rat sensory neurons in culture to 10 or 50 n M phorbol 12,13-dibutyrate (PDBu) significantly increased SP and CGRP release at least 10-fold above resting levels, whereas the inactive 4α-PDBu analogue at 100 n M had no effect on release. Furthermore, 100 n M bradykinin increased peptide release approximately fivefold. Down-regulation of PKC significantly attenuated the release of peptides evoked by either PDBu or bradykinin. PDBu at 1 n M or 1-oleoyl-2-acetyl- sn -glycerol at 50 µ M did not alter resting release of peptides, but augmented potassium- and capsaicin-stimulated release of both SP and CGRP approximately twofold. This sensitizing action of PKC activators on peptide release was significantly reduced by PKC down-regulation or by pretreating cultures with 10 n M staurosporine. These results establish that activation of PKC is important in the regulation of peptide release from sensory neurons. The PKC-induced enhancement of peptide release may be a mechanism underlying the neuronal sensitization that produces hyperalgesia.     

Gastrin''s trophic effect in the colon: Identification of a signaling pathway mediated by protein kinase C

In previous studies we have reported that gastrin exerts a trophic effect on rat colonic epithelial cells in vitro. The effect of gastrin appeared to be mediated through a protein kinase C mechanism. In this study, we have characterized the role of protein kinase C in the gastrin-induced stimulation. Gastrin, in a time- and dose-dependent manner, increased protein kinase C translocation from the cytosol to the membrane, an index of enzyme activation. Maximum translocation occurred in 1 to 2 min following exposure to gastrin (10⁻⁸ M), before declining back to baseline level within 5 min. Gastrin did not change total protein kinase C activity in the colonic cells. Staurosporine, an inhibitor of protein kinase C, totally abolished the basal as well as the gastrin-stimulated activity of protein kinase C. The tumor promoter phorbol 12-myristate 13-acetate also stimulated colonic epithelial protein kinase C. However, prolonged treatment of cells with phorbol inhibited their subsequent response to gastrin stimulation. The response to gastrin was also prevented by the gastrin receptor antagonist proglumide. These observations suggest that protein kinase C mediates the stimulatory effect of gastrin on colonic epithelial cells, possibly through a receptor mechanism.     

Neurofilament Phosphorylation in Cultured Bovine Adrenal Chromaffin Cells Is Stimulated by Phorbol Ester

Primary cultures of bovine adrenal chromaffin cells contain neurofilament proteins that are hypophosphorylated. When the cells were grown in medium containing 32Pi and 0.1 microM 12-O-tetradecanoyl-phorbol 13-acetate (TPA), 32P-labelling of the three neurofilament subunits was increased 6- to 20-fold relative to controls, the highest level of stimulation occurring for the mid-sized subunit. Addition of the protease inhibitor leupeptin to the growth medium had no effect on TPA-stimulated phosphorylation. The increased 32P incorporation was accompanied by a marked reduction in the gel electrophoretic mobilities of the two largest subunits. The augmented phosphorylation was observed 10 min after addition of TPA to a concentration of 0.1 microM or after 1 h of incubation in the presence of 0.01 microM TPA. One-dimensional peptide mapping and phosphoamino acid analysis indicated that TPA stimulated the phosphorylation of seryl residues at new sites in the mid-sized subunit. All of the latter subunit contained in the cytoskeletal fraction of chromaffin cells was converted to a more highly phosphorylated state after the cells were grown in the presence of TPA for 1 h.     

Phorbol Esters Induce Neurotransmitter Release in Cholinergic Synaptosomes from Torpedo Electric Organ

The effect of phorbol esters and so the involvement of Ca2+/phospholipid-dependent protein kinase (protein kinase C;PKC) in the release of acetylcholine (ACh) was studied using Torpedo electric organ synaptosomes. 12-O-Tetradecanoylphorbol 13-acetate (TPA), a known activator of PKC, induced neurotransmitter release in a concentration-dependent manner and increased the potassium-evoked release of ACh. The effect of TPA was shown to be independent of the extrasynaptosomal calcium concentration. TPA-induced ACh release was reversed by H-7, an inhibitor of PKC activity. This drug showed no effect on potassium-evoked ACh release. Botulinum toxin, a strong blocker of potassium-induced ACh release in that synaptosomal preparation, showed no inhibitory effect on the TPA-induced ACh release. Our results suggest that activation of PKC potentiates the release of an ACh pool that is not releasable by potassium depolarization, independently of the extracellular calcium concentration.     

Synergistic Stimulation of Interleukin 6 Release and Gene Expression by Phorbol Esters and Interleukin 1β in Rat Cortical Astrocytes: Role of Protein Kinase C Activation and Blockade

Abstract: The involvement of protein kinase C and its interaction with interleukin 1β in the control of interleukin 6 release by cortical astrocytes was studied. The blockade of protein kinase C catalytic domain, by staurosporine, as well as the desensitization of protein kinase C by short-term phorbol 12-myristate 13-acetate pretreatment, increased the basal release of interleukin 6 by rat cortical astrocytes, whereas calphostin C, an antagonist of phorbol ester binding on protein kinase C regulatory domain, did not affect the basal release of the cytokine. The activation of protein kinase C by phorbol 12-myristate 13-acetate enhanced concentration- and time-dependently interleukin 6 release. This stimulatory action of phorbol 12-myristate 13-acetate was significantly reduced by staurosporine, by calphostin C, and by the desensitization of protein kinase C. Interleukin 1β increased interleukin 6 release in a concentration-related manner. Protein kinase C inhibition, by staurosporine or desensitization, potentiated severalfold, whereas calphostin C reduced interleukin 1β stimulation of interleukin 6 release. The treatment of cortical astrocytes with both interleukin 1β (3 ng/ml) and phorbol 12-myristate 13-acetate (10 nM) caused a synergistic stimulation of interleukin 6 release and its gene expression, an effect that was not relieved by either 20 nM staurosporine or by calphostin C but was slightly affected by protein kinase C desensitization. In conclusion, our data show that in rat cortical astrocytes the basal release of interleukin 6 is under a tonic inhibition exerted by a protein kinase C isoform or isoforms sensitive to blockade by staurosporine and desensitization but insensitive to calphostin C. Interleukin 1β stimulated interleukin 6 secretion via a mechanism that is also negatively modulated by a protein kinase C isoform or isoforms sensitive to staurosporine and desensitization. Finally, we showed that interleukin 1β and phorbol 12-myristate 13-acetate synergistically stimulated interleukin 6 release and its gene expression, operating in a manner insensitive to protein kinase C blockers and slightly reduced by protein kinase C desensitization.     

Phorbol Myristate Acetate Inhibition of Phospholipase C Activation by Carbachol in Slices of Rat Brain Cortex Is a Delayed and Indirect Effect

We examined the effect of phorbol esters on phospholipase C activation in rat brain cortical slices and membranes. There was little effect of concurrent addition of phorbol 12-myristate 13-acetate (PMA) with carbachol on phosphoinositide breakdown due to carbachol over a 1-h incubation of brain slices. However, if slices were preincubated for 3 h with 1 microM PMA or 200 microM sphingosine before addition of carbachol, there was a 35-50% inhibition of phosphoinositide breakdown. There was also a marked loss of protein kinase C (PKC) activity from both cytosol and membranes after a 3-h exposure to PMA. The loss in responsiveness to the muscarinic agonists in slices was not reflected in carbachol-stimulated phospholipase C activation using isolated membranes. However, the decrease in carbachol-induced phosphoinositide breakdown seen in slices after a 3-h exposure to PMA was abolished if the extracellular K+ concentration was elevated from 5.9 to 55mM. Because elevation of the K+ level induces depolarization and increases Ca2+ entry, we examined the effect of ionomycin, a Ca2+ ionophore. Ionomycin potentiated the effects of carbachol on phosphoinositide breakdown but was unable to reverse the effects of a 3-h incubation with PMA. Because apamin, an inhibitor of Ca2(+)-dependent K+ channels, mimicked the effects of exposure to PMA for 3 h, it is possible that these channels are involved in muscarinic cholinergic regulation of phosphoinositide breakdown in rat brain slices. These results support the hypothesis that prolonged PMA treatment in rat brain cortex has no direct effect on phospholipase C activation by muscarinic cholinergic stimulation.     

Activation of High-Affinity Uptake of Glutamate by Phorbol Esters in Primary Glial Cell Cultures

The effects of 12-O-tetradecanoylphorbol 13-acetate (TPA), a potent activator of protein kinase C, on high-affinity Na(+)-dependent glutamate transport were investigated in primary cultures of neurons and glial cells from rat brain cortex. Incubation of glial cells with TPA led to concentration- and time-dependent increases in the glutamate transport that could be completely suppressed by the addition of the protein kinase C (PKC) inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine. The TPA effects could be mimicked by oleoylacetylglycerol and by the diacylglycerol kinase inhibitor R59022. The effects of TPA were potentiated by the Ca2+ ionophore A23187. Under the chosen experimental conditions TPA had no effect on glutamate transport in neurons. We conclude that PKC activates the sodium-dependent high-affinity glutamate transport in glial cells and that it has dissimilar effects on neurons and glial cells.