多巴胺对PC12细胞的双重影响

本实验运用PC12细胞,研究不同浓度多巴胺（dopamine,DA)对细胞的影响,同时利用兼具促进多巴胺释放和抑制多巴胺摄取双重作用的安排它命（amphetamine,AMP)观察胞内外多巴胺对细胞的不同作用。结果显示;胞外高浓度DA能引起细胞抗氧化能力下降,胞内游离Ca^2 浓度上升。细胞存活率大幅度降低,部分细胞出现凋亡;低浓度DA对细胞存活率无明显影响,而使细胞抗氧化能力有一定提高,长时间安非它命单独作用也可引起细胞存活率下降,并伴随胞内GSH水平降低;安非它命与多巴胺共同作用在一定程度上可导致细胞内抗氧化物质水平低于多巴胺单独作用,表明细胞内一定浓度DA可以维持或提高细胞抗氧化物质水平。结果提示,脑内同样存在的多巴胺神经元对DA重摄取功能下降,胞外氧化应激增强,可能是引起脑内多巴胺神经元退行性病变的重要原因之一。     

Bisperoxovanadium complex promotes dopamine exocytosis in PC12 cells.

The effects of the peroxovanadium complex potassium bisperoxo(1,10-phenanthroline)-oxovanadate (bpV[phen]) have been studied on dopamine (DA) exocytosis in PC12 cells. Bisperoxo(1,10-phenanthroline)-oxovanadate does not elicit dopamine secretion in PC12 cells. However, treatment of PC12 cells with 30 microM bpV[phen] for 20 min significantly enhances the secretion induced by the Ca(2+)-ionophore A23187. The effects appear to be irreversible, and strikingly different from the transient and suppressing effects of orthovanadate, which, like bpV[phen], is also a protein tyrosine phosphatase inhibitor. Contrastingly, the short-lived peroxovanadates, formed in situ by the addition of hydrogen peroxide and orthovanadate, are relatively ineffective. The Ca(2+) chelating agent EGTA abolishes bpV[phen]-enhanced dopamine release. The extracellular-regulated protein kinases (ERK) and synaptophysin, proteins implicated in exocytosis, are both tyrosine-phosphorylated by bpV[phen] in a dose- and time-dependent manner, with a maximal effect at 30 microM. Pre-treatment of cells with PD98059 significantly reduced dopamine release (P<0.05). These results suggest that this peroxovanadium complex enhances dopamine exocytosis, at least in part, by ERK-mediated signaling pathway and synaptophysin-associated phosphatase(s).     

Non-genomic modulation of dopamine release by bisphenol-A in PC12 cells

An endocrine disruptor chemical, bisphenol-A (BPA), is reported to have several short-term actions in various tissues and/or cells; however, the mechanisms of these actions have not been fully elucidated. We investigated short-term actions evoked by BPA in pheochromocytoma PC12 cells. BPA elicited dopamine release in PC12 cells in a dose-dependent manner. A selective N-type calcium channel antagonist (omega-conotoxin GVIA) and a ryanodine receptor blocker (ryanodine) inhibited the BPA-induced dopamine release. The expression of ryanodine receptor mRNA was detected by RT-PCR in PC12 cells. Subsequently, in order to prove whether membrane receptors participate in BPA-evoked dopamine release, a guanine nucleotide-binding protein inhibitor [guanosine 5'-(beta-thio) diphosphate], cyclic AMP antagonist (Rp-cAMPS) or protein kinase A inhibitor (H7 or H89) was added to PC12 cells prior to BPA-treatment. All of these agents suppressed BPA-evoked dopamine release, indicating that multiple signaling pathways may be involved in BPA-evoked dopamine release in PC12 cells. In conclusion, we demonstrated that BPA induced dopamine release in a non-genomic manner through guanine nucleotide-binding protein and N-type calcium channels. These findings illustrate a novel function of BPA and suggest that exposure to BPA influences the function of dopaminergic neurons.     

Characterization of cellular uptake and distribution of coenzyme Q10 and vitamin E in PC12 cells

Coenzyme Q (CoQ) is a well-known electron transporter in the mitochondrial respiratory chain. Furthermore, ubiquinol (UQH(2))--a reduced form of ubiquinone (UQ)--has been shown to act as a radical-scavenging antioxidant. Some studies have reported the beneficial effect of CoQ addition to cultured cells; however, the cellular uptake and distribution of CoQ have not been elucidated. In the present study, we used rat pheochromocytoma PC12 cells to investigate and compare the cellular uptake and distribution of CoQ(10) and alpha-tocopherol (alphaT). UQ(10) or UQ(10)H(2) treatment resulted in an increase in the cellular content of both CoQ(10) in a time- and concentration-dependent manner. A subcellular fractionation study revealed that the added UQ(10) as well as UQ(10)H(2) mainly localized in the mitochondrial fraction, which is similar to the localization of endogenous CoQ but different from that of alphaT. The cellular distribution of alphaT directly corresponded to the lipid distribution, while the CoQ distribution did not show any relationship with the lipid distribution, particularly in the mitochondrial and microsomal fractions. These results indicate that the cellular distribution of CoQ is completely different from that of alphaT; moreover, a certain system which accumulates CoQ preferentially in mitochondria may be suggested.     

Regulation of nicotine-evoked dopamine release from PC12 cells.

The nature of second messengers involved in the nicotine-evoked release of dopamine from PC12 cells was examined. Calmidazolium, a calmodulin inhibitor, abolished the nicotine-evoked release. A23187, a Ca2+ ionophore, enhanced dopamine release, and this was inhibited by calmidazolium. Further, 2', 5'-dideoxyadenosine abolished both the nicotine- and A23187-evoked release. Forskolin, dibutyryl-cyclic AMP, and rolipram (a cyclic AMP phosphodiesterase inhibitor) all enhanced dopamine release. 1, 9-Dideoxyforskolin, a forskolin analog which does not activate adenylate cyclase, did not alter dopamine release. These results suggest an obligatory role for Ca2+ and calmodulin-sensitive adenylate cyclase in the nicotine-evoked release process.     

Uncoupling protein 2 influences dopamine secretion in PC12h cells

Uncoupling protein 2 (UCP2) belongs to the UCP family, and is distributed in many organs including the brain. Although UCP2 is known to be related to many functions such as the regulation of insulin secretion or the scavenging of the radicals, the role of UCP2 in the central nervous system remains unclear. In this report, rat UCP2 (rUCP2) and its mutants were overexpressed in the PC12h cells to determine the physiological roles played by UCP2 in neural cells and to elucidate the mechanisms that regulate these functions. It was found that rUCP2 was activated by the stimulation of the cAMP-protein kinase A (PKA) cascade. Moreover, the activation of rUCP2 suppressed intracellular ATP levels and inhibited the cAMP-dependent increase of dopamine secretion. Thus, UCP2 appears to be regulated by the excitatory stimulus via the cAMP-PKA cascade and serves to negatively control the synaptic output by reducing intracellular ATP levels.     

Characterization of tumor-associated fucogangliosides from PC 12 pheochromocytoma cells

PC 12h pheochromocytoma cells were subcutaneously transplanted into rat. We found the transplanted tumors accumulated some fucogangliosides associated with PC 12 cells. These gangliosides were isolated and purified by DEAE-Sephadex A-25 and Iatrobeads column chromatographies. Their structures were determined by fast atom bombardment mass spectrometry, proton nuclear magnetic resonance spectrometry, permethylation study, and sequential degradation using various exoglycosidases and mild acid hydrolysis. Two tumor-associated fucogangliosides were found to possess the blood group B determinant as follows: G6: IV2Fuc alpha, IV3Gal alpha, II3NeuAc, GgOse4Cer; G11: IV2Fuc alpha, IV3Gal alpha, II3 (NeuAc)2, GgOse4Cer. A ganglioside with the similar structure as ganglioside G6 was isolated from rat hepatoma cells (Holmes, E.H., and Hakomori, S-I. (1982) J. Biol. Chem. 257, 7698-7703). However, ganglioside G11 has not previously been reported in the literature. These fucogangliosides reacted with the monoclonal antibody prepared by immunizing mice with PC 12h cells. Other fucogangliosides were also found to accumulate in the transplanted tumor tissues. They were identified as fucosyl-GM1 and fucosyl-GDlb. These fucogangliosides did not react with the monoclonal antibody against PC 12h cells.     

Mitochondrial damage by nitric oxide is potentiated by dopamine in PC12 cells

Mitochondrial damage in PC12 cells, a model for dopaminergic cells, was examined in terms of the contribution of oxidative stress, nitric oxide (*NO), and dopamine to impairment of mitochondrial respiratory control (RC). A kinetic analysis suggested that the oxidative deamination of dopamine catalyzed by monoamine oxidase (MAO) was not a significant source of hydrogen peroxide, because of constrains imposed by the low cytosolic level of dopamine. *NO induced irreversible damage of mitochondrial complex I in PC12 cells: this damage followed a sigmoid response on *NO concentration with a well-defined threshold level. Dopamine did not elicit damage of mitochondria in PC12 cells; however, the amine potentiated the effects of *NO at or near the threshold level, thus leading to irreversible impairment of mitochondrial respiration. This synergism between *NO and dopamine was not observed at *NO concentrations below the threshold level. Depletion of dopamine from the storage vesicles by reserpine protected mitochondria from *NO damage. Dopamine oxidation by *NO increased with pH, and occurred at modest levels at pH 5.5. In spite of this, calculations showed that the oxidation of dopamine in the storage vesicles (pH 5.5) was higher than that in the cytosol (pH 7.4), due to the higher dopamine concentration in the storage vesicles (millimolar range) compared to that in the cytosol (micromolar range). It is suggested that storage vesicles may be the cellular sites where the potential for dopamine oxidation by *NO is higher.These data provide further support to the hypothesis that dopamine renders dopaminergic cells more susceptible to the mitochondrial damaging effects of *NO. In the early stages of Parkinson's disease, *NO production increases until reaching a point near the threshold level that induces neuronal damage. Dopamine stored in dopaminergic cells may cause these cells to be more susceptible to the deleterious effects of *NO, which involve irreversible impairment of mitochondrial respiration.     

Nocardia asteroides culture filtrates cause dopamine depletion and cytotoxicity in PC12 cells

Experimental infection of BALB/c mice with the gram-positive bacterium Nocardia asteroides produces marked loss of nigrostriatal dopamine neurons, resulting in striatal dopamine depletion. To investigate the mechanism(s) responsible for this neuronal loss, we evaluated the influence of N. asteroides cell-free culture filtrates on rat pheochromocytoma PC12 cells, an in vitro model for dopamine neurons. Changes in cell viability and cell numbers were minimal after 24 h, but increased with longer incubation. In contrast, dopamine depletion occurred after 30 min incubation, and was greater with GUH-2 filtrate than with filtrate from the less virulent strain 10905. Incubation with the culture filtrate decreased viability in neuroblastoma and glioma cell lines, indicating that cytotoxic effects were not limited to dopaminergic cells. These findings suggest that the loss of nigrostriatal dopamine neurons and concomitant striatal dopamine depletion in Nocardia-infected mice may be due, at least in part, to the neurotoxicity of nocardial secretory products.     

Involvement of dopamine receptors and beta-arrestin in metamphetamine-induced inclusions formation in PC12 cells

Exposure of PC12 cells to metamphetamine (MA) induces the formation of multilamellar structures (whorls) resembling autophagic granules that subsequently develop as intracellular inclusions. These inclusions stain for a variety of antigens belonging to the ubiquitin proteasome pathway. Since MA-induced intracellular bodies require the presence of dopamine in the present study we analyzed the role of dopamine (DA) receptors in producing neuronal inclusions. Moreover, we investigated potential signaling pathways which could lead to ubiquitination in the presence of MA. Based on recent reports that ubiquitination of β-adrenergic receptors is promoted by β-arrestin which shuttles proteins from the plasma membrane to the ubiquitin proteasome system we investigated whether β-arrestin is involved in MA-induced inclusion formation. Our experiments document that (i) β-arrestin was associated with MA-induced intracellular bodies; (ii) MA induced a rapid and reversible ubiquitination of β-arrestin; (iii) dopamine antagonists reduced both MA-induced β-arrestin ubiquitination and intracellular whorls formation; (iv) the number of MA-induced intracellular bodies was reduced in cells transfected with the β-arrestin dominant negative mutant, βarrV53D and was increased by the persistently ubiquitinated β-arrestin-ubiquitin fusion protein. In conclusion, the present study demonstrates the involvement of β-arrestin in MA-induced intracellular bodies and the participation of dopamine receptors in this process.     

Hydrogen peroxide preconditioning protects PC12 cells against apoptosis induced by dopamine

Dopamine (DA), one of the major sources of reactive oxygen species (ROS), is implicated in neuronal death associated with Parkinson's disease (PD). Preconditioning with oxidative stress has been shown to provide cytoprotection similar to ischemic preconditioning (IPC), against cell apoptosis. In this study, using the model neurosecretory cell line, PC12, we investigated whether hydrogen peroxide (H(2)O(2)) at low concentration (10 microM) can protect PC12 cells against apoptosis induced by DA. PC12 cells were preconditioned with 10 microM H(2)O(2) for 90 min, followed by 24-h recovery and subsequent exposures to different concentrations (20, 50, 100 and 200 microM) of DA for 24-h, respectively. DA induced apoptotic cell death with significant morphological nuclear changes and DNA fragmentation as well as the dysfunction of mitochondria. Preconditioning with H(2)O(2) at 10 microM significantly reduced the percentage of apoptotic cells and partly blocked the decreases in 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) reduction and mitochondrial membrane potential (MMP) induced by DA. These results suggest that preconditioning with low concentration of H(2)O(2) protected PC12 cells against DA-induced apoptosis, the part restoration of the damaged mitochondrial functions might be one of the underlying mechanisms of this cytoprotection.     

Transmitter uptake and release in PC12 cells overexpressing plasma membrane monoamine transporters

Transmitter uptake and exocytosis of secretory vesicles are two essential aspects of neurotransmission. Here we show that transient overexpression of plasma membrane monoamine transporters in rat pheochromocytoma PC12 cells induced an approximate 20-fold enhancement of cellular uptake of monoamines. Intravesicular amine concentration was greatly increased, as demonstrated directly by carbon fibre amperometry. However, the amount of stored monoamines diminished over a 5-h period, unless monoamine oxidase was inhibited, indicating that monoamines leak out from secretory vesicles. This efflux of monoamines accounts for the reported dependence of vesicular monoamine content (the quantal size) on the kinetics of vesicular monoamine uptake. Measuring radiolabelled monoamines release from the cell population provided accurate determination of the secretory activity of the subpopulation (10-20%) of cells transfected with monoamine transporters, since they contained about 95% of the radiolabel. Accordingly, significant modification of the secretory responses was observed, at the cell population level, upon transient expression of the serotonin transporter and of proteins known to interfere with exocytosis, such as botulinum neurotoxin C1, GTPase-deficient Rab3 proteins, truncated Rabphilin constructs or Rim. The co-transfection assay described here, based on transient expression of monoamine transporters, should prove useful in functional studies of the secretory machinery.     

Characterization of small-molecular-mass guanine-nucleotide-binding regulatory proteins in insulin-secreting cells and PC12 cells.

The distribution of ras-related small-molecular-mass guanine-nucleotide-binding regulatory proteins (SMG) of two insulin-secreting cell lines, RINm5F and HIT-T15, and of a catecholamine-secreting cell line, PC12, have been studied using different techniques. About ten such proteins were detected by [32P]GTP binding after two-dimensional gel electrophoresis and transfer to nitrocellulose membranes. In insulin-secreting cells, rho protein(s) that cannot be detected with the GTP-binding technique were identified by ADP ribosylation with Clostridium botulinum C3 exoenzyme. After subcellular fractionation, SMG displayed specific distributions. The insulin-secreting cell line RINm5F and the catecholamine-secreting cell line PC12 expressed a similar set of these proteins with analogous localization. [32P]GTP binding analysis revealed that at least seven SMG were associated with the secretory granule enriched fraction of RINm5F cells and with the fraction containing dense secretory granules from PC12 cells, proteins of 27 (pI 5.4), 23 (pI 6.8) and 25 kDa (pI 6.7) being the most abundant. These proteins were present in a highly purified granule fraction of a solid rat insulinoma. The 23 kDa (pI 6.8) and 25 kDa (pI 6.7) proteins, but not the protein migrating at 27 kDa (pI 5.4), were detected in the corresponding fraction from HIT-T15 cells. A monoclonal antibody directed against smg25A/rab3A recognized the SMG in secretory granules migrating at 25 kDa (pI 6.7) and 27 kDa (pI 5.4). This antibody also revealed the presence of such protein(s) in homogenates of rat pancreatic islets. During stimulation of insulin secretion of either intact or permeabilized cells, there was no detectable redistribution to the cytosol or to the plasma membrane of the major proteins located on secretory granules. In view of the invariable presence of at least two of the SMG in granules of secretory cells, these proteins are good candidates for regulation of hormone secretion.     

Amphetamine reduces vesicular dopamine content in dexamethasone-differentiated PC12 cells only following l-DOPA exposure

Amphetamine (AMPH) increases brain dopamine (DA) levels via reversal of the membrane DA transporter. Additional mechanisms have been suggested, including inhibition of vesicular monoamine transporters and vesicular leakage of DA and Ca²⁺. According to the widely-accepted weak base theory, AMPH disrupts the proton gradient required for filling vesicles with DA. As a result, DA and Ca²⁺ will leak from vesicles, giving rise to exocytosis of less-filled vesicles. As several contradictions have been described, the aim of the present study was to re-examine this theory using amperometry and Fura-2 imaging to measure AMPH-induced changes in exocytosis and intracellular Ca²⁺ levels, respectively, in PC12 and chromaffin cells. Unexpectedly, 15 min exposure to AMPH (20–200 μM) does not affect the amount of DA released per vesicle, the frequency of exocytosis or intracellular Ca²⁺ levels in PC12 cells or chromaffin cells. Comparable results were found following prolonged exposure to AMPH (45 min) or at 37°C. When cells were pre-treated with the DA precursor l -DOPA, vesicle content increased to ∼150%. When these pre-treated cells are exposed to AMPH, vesicle content is strongly reduced. These results indicate that in dexamethasone-differentiated PC12 cells AMPH-induced vesicle leakage occurs only under specific conditions, therefore arguing for re-evaluation of the theory of AMPH-induced vesicular DA leakage.     

On the Ca2+ dependence of non-transferrin-bound iron uptake in PC12 cells

Non-transferrin-bound iron (NTBI) uptake has been reported to follow two pathways, Ca(2+)-dependent and Ca(2+)-independent (Wright, T. L., Brissot, P., Ma, W. L., and Weisiger, R. A. (1986) J. Biol. Chem. 261, 10909-10914; Sturrock, A., Alexander, J., Lamb, J., Craven, C. M., and Kaplan, J. (1990) J. Biol. Chem. 265, 3139-3145). Studies reporting the two pathways have ignored the weak interactions of Ca(2+) with the chelator nitrilotriacetate (NTA) and the reducing agent ascorbate. These studies used a constant ratio of total Fe(2+) to NTA with and without Ca(2+). We observed Ca(2+) activation of NTBI uptake in PC12 cells with the characteristics reported for other cells upon using 1 mm ascorbate and a constant ratio of total Fe(2+) to NTA with or without Ca(2+). However, Ca(2+) did not affect NTBI uptake in solutions without NTA. We then determined conditional stability constants for NTA binding to Ca(2+) and Fe(2+) by potentiometry under conditions of NTBI uptake experiments (pH, ionic strength, temperature, ascorbate, total Fe(2+), and total Ca(2+) concentrations). In solutions based on these constants and taking Ca(2+) chelation into account, Ca(2+) did not affect NTBI uptake over a range of free Fe(2+) concentrations. Thus, the Ca(2+) activation of NTBI uptake observed using the constant total Fe(2+) to NTA ratio was because of Ca(2+)-NTA chelation rather than an activation of the NTBI transporter itself. It is suggested that the previously reported Ca(2+) dependence of NTBI uptake be re-evaluated.     

L-dopa and dopamine enhance the formation of aggregates under proteasome inhibition in PC12 cells

The formation of inclusion bodies in dopaminergic neurons is associated with the pathogenesis of Parkinson's disease. In order to clarify the role of dopamine/L-dopa in the formation of protein aggregates, we investigated dopamine/L-dopa-related aggregation using an experimental inclusion model. The inhibition of tyrosine hydroxylase (TH) by alpha-methyltyrosine dramatically decreased MG132-induced aggregate formation. In addition, the inhibition of TH caused the upregulation of proteasomes in cultured cells and the dopamine/L-dopa induced non-enzymatic polymerization of ubiquitin. This inhibition did not affect cell viability. These results suggest that dopamine/L-dopa might enhance aggregate formation, and that intracellular aggregates may not be toxic to cells.     

Characterization of ATP receptor which mediates norepinephrine release in PC12 cells.

PC12 cells, a rat pheochromocytoma cell line, has been reported to release norepinephrine in response to extracellular ATP in the presence of extracellular Ca2+. The potency order of ATP analogues was adenosine 5'-O-(3-thiotriphosphate) greater than ATP greater than adenosine 5'-O-(1-thiotriphosphate) = 2-methylthioadenosine 5'-triphosphate (MeSATP) greater than 2'- and 3'-O-(4-benzoyl-benzoyl)ATP (BzATP) greater than ADP greater than 5-adenylylimidodiphosphate. Adenosine 5'-O-(2-thiodiphosphate), beta, gamma-methyleneadenosine 5'-triphosphate, AMP and adenosine were inactive. The ATP action in the absence of extracellular Ca2+, suggests a small but appreciable contribution of intracellular Ca2+ mobilization, for norepinephrine release. However, for some ATP derivatives, like BzATP, almost no contribution of the phospholipase C-Ca2+ pathway is suggested, based on their low activity in inositol phosphates production. To identify the ATP-receptor protein, PC12 cell membranes were photoaffinity-labeled with [32P]BzATP. SDS-PAGE analysis showed that a 53-kDa protein labeling was inhibited by ATP and its derivatives, as well as by P2-antagonists, suramin and reactive blue 2, which inhibit the nucleotide-induced norepinephrine release. The inhibitory activity of the nucleotides was, in parallel with their potency, to induce norepinephrine release. Despite their inability to release norepinephrine, GTP and GTP gamma S inhibited the BzATP labeling, suggesting the participation of a putative G protein in the ATP-receptor-mediated actions. We suggest that the 53-kDa protein on the PC12 cell surface is an ATP receptor, which mediates the norepinephrine release, depending, mainly, on extracellular Ca2+ gating.     

Mechanism of biosynthesis of soluble and membrane-bound forms of dopamine beta-hydroxylase in PC12 pheochromocytoma cells

Dopamine beta-hydroxylase was present as 2 subunit forms (apparent Mr = 77,000 and 73,000) in the PC12 pheochromocytoma cell line as detected by immunoprecipitation from [35S]methionine-labeled cultures, and analyzed by sodium dodecyl sulfate gel electrophoresis and fluorography. The Mr = 77,000 form was present in a crude membrane fraction, while the Mr = 73,000 form was soluble. Both forms appeared to be present in approximately equal amounts, and both were glycosylated. Treatment of PC12 cells with tunicamycin, a potent inhibitor of core glycosylation in the endoplasmic reticulum, completely inhibited the appearance of the Mr = 77,000 and Mr = 73,000 forms, and 2 new immunoreactive polypeptides were obtained (apparent Mr = 67,000 and 63,000). Pulse-chase experiments suggested that the Mr = 77,000 form is initially synthesized (by 5 min) and a portion is converted in 15-90 min to the Mr = 73,000 form. Thereafter, the ratio between forms remains relatively constant, at least for several hours. Translation of mRNA from bovine and rat adrenals, and immunoprecipitation, indicated that dopamine beta-hydroxylase is initially synthesized as a single polypeptide (apparent Mr = 67,000). The subcellular site of biosynthesis of dopamine beta-hydroxylase was determined by isolation of mRNA from free and membrane-bound polysomes from bovine adrenal medulla. Translation in a cell free system and immunoprecipitation localized the synthesis of dopamine beta-hydroxylase on membrane-bound polysomes. These experiments suggest that both soluble and membrane-bound forms of dopamine beta-hydroxylase are synthesized and core glycosylated in the endoplasmic reticulum, and that there probably is a precursor-product relationship between the Mr = 77,000 and the Mr = 73,000 subunit forms of dopamine beta-hydroxylase.     

Nicardipine-sensitive enhancement of high K+-evoked dopamine release in PC12 cells pretreated with 12-O-tetradecanoylphorbol 13-acetate

The effects of the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) on stimulus-evoked dopamine release were studied in PC12 cells. Pretreatment of the cells with TPA resulted in an enhancement of dopamine release which could be further stimulated by high concentrations of K⁺, A23187, but not with carbamylcholine. TPA-dependent, high-K⁺-evoked enhancement of dopamine release was studied in detail: a maximum release was observed (169% of control) in response to 50 mM KCl upon treatment with 10⁻⁷ M TPA for 5 min at 37°C. This enhancement of dopamine release was associated with the concomitant reduction of the concentration rise of intracellular Ca²⁺ ([Ca²⁺]_i) induced by a high concentration of K⁺ monitored by a fluorescent indicator, fura2. Thus, these data provide an example for alteration in the efficiency of stimulus-secretion coupling as pointed out in our previous paper. Moreover, we have shown that nicardipine, CdCl₂, and CoCl₂ inhibit high-K⁺-evoked dopamine release more effectively in TPA treated cells than that of untreated cells, and that the TPA-dependent, high-K⁺-evoked dopamine release observed in TPA treated cells is completely abolished by the presence of nicardipine, Cd²⁺ or Co²⁺, but is only partially inhibited in the presence of verapamil. These relevant findings suggest the possible involvement of protein kinase C in regulating the efficiency of a high-K⁺-evoked dopamine release through the modification of nicardipine-sensitive Ca²⁺ channels.