Dopamine Release In PC12 Cells Research Articles

Pharmacokinetic and Permeation Studies in Rat Brain of Natural Compounds Led to Investigate Eugenol as Direct Activator of Dopamine Release in PC12 Cells.

Eugenol, cinnamaldehyde and D-limonene, the main components of natural essential oils, are endowed with antioxidant and anti-inflammatory properties which allow them to induce beneficial effects on intestinal, cardiac and neuronal levels. In order to characterize their pharmacokinetic profiles and aptitude to permeate in the central nervous system after intravenous and oral administration to rats, new analytical procedures, easily achievable with HPLC-UV techniques, were developed. The terminal half-lives of these compounds range from 12.4 ± 0.9 (D-limonene) and 23.1 ± 1.6 min (cinnamaldehyde); their oral bioavailability appears relatively poor, ranging from 4.25 ± 0.11% (eugenol) to 7.33 ± 0.37% (cinnamaldehyde). Eugenol evidences a marked aptitude to permeate in the cerebrospinal fluid (CSF) of rats following both intravenous and oral administrations, whereas cinnamaldehyde appears able to reach the CSF only after intravenous administration; limonene is totally unable to permeate in the CSF. Eugenol was therefore recruited for in vitro studies of viability and time-/dose-dependent dopamine release in neuronal differentiated PC12 cells (a recognized cellular model mimicking dopaminergic neurons), evidencing its ability to increase cell viability and to induce dopamine release according to a U-shaped time-course curve. Moreover, concentration-response data suggest that eugenol may induce beneficial effects against Parkinson's disease after oral administration.

Ultrasonic stimulation of the brain to enhance the release of dopamine - A potential novel treatment for Parkinson's disease.

Parkinson's disease (PD) is characterized by the decrease of dopamine (DA) production and release in the substantia nigra and striatum regions of the brain. Transcranial ultrasound has been exploited recently for neuromodulation of the brain in a number of fields. We have stimulated DA release in PC12 cells using low-intensity continuous ultrasound (0.1W/cm2-0.3W/cm2, 1MHz), 12h after exposure at 0.2W/cm2, 40s, the amount of DA content eventually increased 78.5% (p=0.004). After 10-day ultrasonic treatment (0.3W/cm2, 5min/d), the DA content in the striatum of PD mice model restored to 81.07% of the control (vs 43.42% in the untreated PD mice model). In addition to this the locomotion activity was restored to the normal level after treatment. We suggest that the low intensity ultrasound-induced DA release can be attributed to a combination of neuron regeneration and improved membrane permeability produced by the mechanical force of ultrasound. Our study indicates that the application of transcranial ultrasound applied below FDA limits, could provide a candidate for relatively safe and noninvasive PD therapy through an amplification of DA levels and the stimulation of dopaminergic neuron regeneration without contrast agents.

Gintonin, a novel ginseng-derived lysophosphatidic acid receptor ligand, stimulates neurotransmitter release

Gintonin is a novel ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand. Gintonin elicits an intracellular calcium concentration [Ca2+]i transient via activation of LPA receptors and regulates calcium-dependent ion channels and receptors. [Ca2+]i elevation by neurotransmitters or depolarization is usually coupled to neurotransmitter release in neuronal cells. Little is known about whether gintonin-mediated [Ca2+]i transients are also coupled to neurotransmitter release. The PC12 cell line is derived from a pheochromocytoma of the rat adrenal medulla and is widely used as a model for catecholamine release. In the present study, we examined the effects of gintonin on dopamine release in PC12 cells. Application of gintonin to PC12 cells induced [Ca2+]i transients in concentration-dependent and reversible manners. However, ginsenoside Rg3, another active ingredient of ginseng, induced a lagged and irreversible [Ca2+]i increase. The induction of gintonin-mediated [Ca2+]i transients was attenuated or blocked by the LPA1/3 receptor antagonist Ki16425, a phospholipase C inhibitor, an inositol 1,4,5-triphosphate receptor antagonist, and an intracellular Ca2+ chelator. Repeated treatment with gintonin induced homologous desensitization of [Ca2+]i transients. Gintonin treatment in PC12 cells increased the release of dopamine in a concentration-dependent manner. Intraperitoneal administration of gintonin to mice also increased serum dopamine concentrations. The present study shows that gintonin-mediated [Ca2+]i transients are coupled to dopamine release via LPA receptor activation. Finally, gintonin-mediated [Ca2+]i transients and dopamine release via LPA receptor activation might explain one mechanism of gintonin-mediated inter-neuronal modulation in the nervous system.

The role of Pak-interacting exchange factor-β phosphorylation at serines 340 and 583 by PKCγ in dopamine release.

Protein kinase C (PKC) has been implicated in the control of neurotransmitter release. The AS/AGU rat, which has a nonsense mutation in PKCγ, shows symptoms of parkinsonian syndrome, including dopamine release impairments in the striatum. Here, we found that the AS/AGU rat is PKCγ-knock-out (KO) and that PKCγ-KO mice showed parkinsonian syndrome. However, the PKCγ substrates responsible for the regulated exocytosis of dopamine in vivo have not yet been elucidated. To identify the PKCγ substrates involved in dopamine release, we used PKCγ-KO mice and a phosphoproteome analysis. We found 10 candidate phosphoproteins that had decreased phosphorylation levels in the striatum of PKCγ-KO mice. We focused on Pak-interacting exchange factor-β (βPIX), a Cdc42/Rac1 guanine nucleotide exchange factor, and found that PKCγ directly phosphorylates βPIX at Ser583 and indirectly at Ser340 in cells. Furthermore, we found that PKC phosphorylated βPIX in vivo. Classical PKC inhibitors and βPIX knock-down (KD) significantly suppressed Ca(2+)-evoked dopamine release in PC12 cells. Wild-type βPIX, and not the βPIX mutants Ser340 Ala or Ser583 Ala, fully rescued the decreased dopamine release by βPIX KD. Double KD of Cdc42 and Rac1 decreased dopamine release from PC12 cells. These findings indicate that the phosphorylation of βPIX at Ser340 and Ser583 has pivotal roles in Ca(2+)-evoked dopamine release in the striatum. Therefore, we propose that PKCγ positively modulates dopamine release through β2PIX phosphorylation. The PKCγ-βPIX-Cdc42/Rac1 phosphorylation axis may provide a new therapeutic target for the treatment of parkinsonian syndrome.

Dopamine Release Suppression Dependent on an Increase of Intracellular Ca<sup>2+</sup> Contributed to Rotenone-induced Neurotoxicity in PC12 Cells

Rotenone is an inhibitor of mitochondrial complex I that produces a model of Parkinson’s disease (PD), in which neurons undergo dopamine release dysfunction and other features. In neurons, exocytosis is one of the processes associated with dopamine release and is dependent on Ca2+ dynamic changes of the cell. In the present study, we have investigated the exocytosis of dopamine and the involvement of Ca2+ in dopamine release in PC12 cells administrated with rotenone. Results demonstrated that rotenone led to an elevation of intracellular Ca2+ through Ca2+ influx by opening of the voltage-gated Ca2+ channel and influenced the soluble N-ethylmaleimide attachment protein receptor (SNARE) proteins expression (including syntaxin, vesicle-associated membrane protein 2 (VAMP2) and synaptosome-associated protein 25 (SNAP-25)); pretreatment with a blocker of L-type voltage-activated Ca2+ channels (nifedipine) decreased the intracellular dopamine levels and ROS formation, increased the cell viability and enhanced the neurite outgrowth and exocytosis of synaptic vesicles. These results indicated that the involvement of intracellular Ca2+ was one of the factors resulting in suppression of dopamine release suppression in PC12 cells intoxicated with rotenone, which was associated with the rotenone-induced dopamine neurotoxicity.

Dopamine release from rat pheochromocytoma (PC12) cells and rat brain striata induced by a series of straight carbon chain aldehydes with variations in carbon chain length and functional groups

Green odor compounds, a group of 6-carbon (C6) aldehydes and alcohols, are able to enhance dopamine release from pheochromocytoma (PC12) cells, rat brain striatal slices, and brain striata in living rats. In this study, we examined the effects of aldehydes and alcohols with varying carbon chain lengths (2–9 carbons) and functional groups on dopamine release in PC12 cells, brain slices, and living rat brain. In PC12 cells, n-aldehydes and n-alcohols promoted dopamine release, and this effect was stronger as the carbon chain length increased. In rat brain slices, however, the maximum dopamine release was detected when stimulated by n-hexanal, while n-nonanal promoted the lowest level of release. In addition, C6 compounds with a hydroxyl, aldehyde, or carboxyl group enhanced the dopamine release from PC12 cells and striatal slices. In the microdialysis study, n-hexanal and n-hexanol enhanced dopamine release, while n-nonanal promoted lower activity than n-hexanol. The relationship of the concentration of the odor-related compounds and the amount of dopamine released differed between PC12 cells and brain slices. Dopamine release in the living rat brains was similar to that in brain slices. These data suggested that the length of the carbon chain correlated with the strength of dopamine release, and the functional groups further modified it. The distinction between PC12 cells and rat striata might be due to the differences in the cell structure or the target molecules within the cells.

Identification of coffee components that stimulate dopamine release from pheochromocytoma cells (PC-12)

Coffee and caffeine are known to affect the limbic system, but data on the influence of coffee and coffee constituents on neurotransmitter release is limited. We investigated dopamine release and Ca2+-mobilization in pheochromocytoma cells (PC-12 cells) after stimulation with two lyophilized coffee beverages prepared from either Coffea arabica (AR) or Coffea canephora var. robusta (RB) beans and constituents thereof. Both coffee lyophilizates showed effects in dilutions between 1:100 and 1:10,000. To identify the active coffee compound, coffee constituents were tested in beverage and plasma representative concentrations. Caffeine, trigonelline, N-methylpyridinium, chlorogenic acid, catechol, pyrogallol and 5-hydroxytryptamides increased calcium signaling and dopamine release, although with different efficacies. While N-methylpyridinium stimulated the Ca2+-mobilization most potently (EC200: 0.14±0.29μM), treatment of the cells with pyrogallol (EC200: 48±14nM) or 5-hydroxytryptamides (EC200: 10±3nM) lead to the most pronounced effect on dopamine release. In contrast, no effect was seen for the reconstituted biomimetic mixture.We therefore conclude that each of the coffee constituents tested stimulated the dopamine release in PC-12 cells. Since no effect was found for their biomimetic mixture, we hypothesize other coffee constituents being responsible for the dopamine release demonstrated for AR and RB coffee brews.

High concentrations of MDMA (‘ecstasy’) and its metabolite MDA inhibit calcium influx and depolarization-evoked vesicular dopamine release in PC12 cells

The popular synthetic drug of abuse 3,4-methylenedioxymethampetamine (MDMA) and its metabolite 3,4-methylenedioxyamphetamine (MDA) act mainly on the serotonergic system, though they also increase the amount of extracellular dopamine (DA) in the brain, presumably via reversal of the membrane dopamine transporter (DAT). As the involvement of exocytotic DA release is debated, we investigated if these drugs alter the intracellular calcium concentration ([Ca 2+] i) and subsequent DA exocytosis in single PC12 cells using respectively Fura-2 imaging and amperometry. MDMA and MDA did not change basal [Ca 2+] i or exocytosis, but inhibited depolarization-evoked increases in [Ca 2+] i and exocytosis following 15 min exposure to high concentrations of drugs (1 mM). Surprisingly, MDA was more potent in inhibiting exocytosis than MDMA and already inhibited exocytosis at concentrations that did not inhibit depolarization-evoked Ca 2+ influx (10–100 μM). Without 15 min pre-exposure, both drugs failed to inhibit depolarization-evoked Ca 2+ influx. These results indicate that at high concentrations both MDMA and MDA inhibit exocytosis via indirect inhibition of Ca 2+ influx, whereas at lower concentrations MDA may also reduce vesicle cycling. Our data suggest that the DAT-independent increase in extracellular DA in vivo is not due to direct stimulation of exocytosis, but rather to effects of these drugs on other neurotransmitter systems that innervate the dopaminergic system.

Effects of scoparone on dopamine release in PC12 cells

The effects of scoparone on dopamine release in PC12 cells were investigated. Scoparone at 50–200 µM increased dopamine release into the culture medium. However, the released levels of dopamine by scoparone were not altered in the absence of extracellular Ca 2+ and by adenylyl cyclase inhibitor MDL-12,330A. Scoparone increased phosphorylation of PKA, CaMK II and synapsin I. Scoparone also enhanced K +-induced levels of dopamine release by CaMK II phosphorylation. These results suggest that scoparone increases dopamine release by synapsin I phosphorylation via activation of PKA and CaMK II, which are mediated by cyclic AMP levels and Ca 2+ influx.

Evaluation of anti-Fas ligand-induced apoptosis and neural differentiation of PC12 cells treated with nerve growth factor using small interfering RNA method and sampling by microdialysis

The small interfering RNA (siRNA) method is an effective technique for silencing gene expression and is a useful tool for screening the gene functions in drug discovery. Our study found that nerve growth factor (NGF) can increase the cell viability of PC12 cells and that NGF induction up-regulates the expression of Bcl-2 detected by real-time reverse transcription–polymerase chain reaction (RT–PCR). To further investigate the role of Bcl-2 expression in NGF-treated PC12 cells, the plasmid of Bcl-2 siRNA was then transfected into PC12 cells. Moreover, to investigate and continuously monitor the real-time dynamic neurotransmitter release, and to compare with the time course of Bcl-2 expression, a liquid chromatography coupled with electrochemical detection (LC–ED) and with a microdialysis device was used. After 6 h of NGF being added to the PC12 cell culture medium, the dopamine (DA) concentrations were significantly increased ( P < 0.05). This result is simultaneously compatible with the up-regulated messenger RNA (mRNA) expressions of tyrosine hydroxylase (TH), aromatic acid decarboxylase (AADC), and Bcl-2 by RT–PCR. Using the Bcl-2 siRNA method, our data revealed that NGF can inhibit Fas ligand (FasL)-induced apoptosis in PC12 cells through the activation of Bcl-2. The in vitro observation further demonstrated that NGF can stimulate the neurite development in PC12 cells through the activation of Bcl-2. Moreover, the DA concentrations of NGF induction were decreased specifically by Bcl-2 siRNA ( P < 0.05). In sum, our data support that NGF prevents Fas-induced apoptosis, facilitates neural differentiation, promotes dendritic formation, and increases DA release in PC12 cells through activation of Bcl-2.

Salviae miltiorrhizae radix increases dopamine release of rat and pheochromocytoma PC12 cells

The Radix of Salvia miltiorrhiza Bunge (Labiatae) (SMR), an eminent herb, is often included as an ingredient in various herbal remedies recommended for vascular circulation therapies. The present study investigated the effect of SMR on dopaminergic neurotransmission. Various extracts prepared from the stems of SMR were tested for cytotoxic activity on pheochromocytoma PC12 cells using the XTT assay method. The ethanol extract (IC50 > 100 microg/mL), water extract (IC50 > 100 microg/mL) and chloroform (IC50 = 90 microg/mL) fraction exhibited weak cytotoxic activity. However, the butanol (IC50 = 80 microg/mL) and ethyl acetate (EtOAc; IC50 = 70 microg/mL) fractions exhibited strong cytotoxic activity. Also, the extracts and fractions were investigated for dopamine release effects. The EtOAc fraction showed a stronger stimulatory effect on dopamine release activity than the other fractions. The effect of the crude EtOAc fraction (50 microg/mL) of SMR on K+ (20 mm)-stimulated dopamine (DA) release from rat striatal slices was compared with amphetamine (10(-4) m) using high-performance liquid chromatography with electrochemical detection to measure endogenous DA. The EtOAc fraction significantly increased K+ -stimulated DA release (p < 0.001) from rat striatal slices when compared with K+ -stimulated alone. The EtOAc fraction potentiated the effect of amphetamine on K+ -stimulated DA release (p < 0.001) when compared with amphetamine alone. To examine whether in vitro the EtOAc fraction treatment induces DA release in PC12 cells, the role of protein kinases was investigated in the induction of the EtOAc fraction-mediated events by using inhibitors of protein kinase C (PKC), mitogen activated protein kinase (MAP kinase) or protein kinase A (PKA). The PKC inhibitors chelerythrine (50 nm and 100 nm) and Ro31-8220 (100 nm) and the MAP kinase kinase inhibitor, PD98059 (20 microm), inhibited the ability of the EtOAc fraction of SMR to elicit the EtOAc fraction-stimulated DA release. The PKC activator, 12-O-tetradecanoyl phorbol 13-acetate (TPA, 100 nm) mimicked the ability of the EtOAc fraction of SMR to elicit DA release. In contrast, a selective PKA inhibitor, 50 microm Rp-8-Br-cAMP, blocked the development of EtOAc fraction-stimulated DA release. It was demonstrated that the EtOAc fraction of SMR stimulated DA release. Therefore the mechanism by which the EtOAc fraction of SMR induced the enhancement in EtOAc fraction-stimulated DA release is apparent.

Salviae Miltiorrhizae BGE Radix Increases Rat Striatal K+-Stimulated Dopamine Release and Activates the Dopamine Release with Protection Against Hydrogen Peroxide-Induced Injury in Rat Pheochromocytoma PC12 Cells

The present study investigated the effect of the medicinal plant Salviae miltiorrhizae radix (SMR) on dopaminergic neurotransmission in comparison with amphetamine. The effect of SM (0.1 g/ml) on K(+) (20 mM)-stimulated dopamine (DA) release from rat striatal slices was compared with amphetamine (10(-4) M). Amphetamine and SMR significantly increased K(+)-stimulated DA release (P<0.001) from rat striatal slices when compared with K(+)-stimulated alone. On the other hand, to examine whether in vitro SMR treatment induces DA release in PC12 cells, the role of protein kinases has been investigated in the induction of the SMR-mediated events by using inhibitors of protein kinase C (PKC), mitogen activated protein kinase (MAP kinase) or protein kinase A (PKA). PKC inhibitors chelerythrine (50 and 100 nM), Ro31-8220 (100 nM) and the MAP kinase inhibitor, PD98059 (20 microM) inhibited the ability of SMR to elicit the SMR-stimulated DA release. The direct-acting PKC activator, 12-O-tetradecanoyl phorbol 13-acetate (TPA, 100 nM) mimicked the ability of SMR to elicit DA release. On the contrary, a selective PKA inhibitor, 50 microM Rp-8-Br-cAMP, blocked the development of SMR-stimulated DA release. The results demonstrated that SMR may stimulate DA release and that SMR-induced increases in MAP kinase and PKC are important for induction of the enhancement in transporter-mediated DA release and PKA was also required for the enhancement in SMR-stimulated DA release. SMR treatment (0.1-10 microg/ml) to the hydrogen peroxide (H(2)O(2))-treated PC12 cells activated the enzyme activities such as catalase, superoxide dismutase and glutathione peroxidase, and decreased the malondialdehyde level, indicating that SMR has also protective effects against free radical-induced cell toxicity. Therefore, the mechanism by which SMR induces the enhancement in SMR-stimulated DA release is apparent. It remains to be determined whether the effect of SMR on DA function is important in its therapeutic use in the treatment of drug addiction.

Dopamine release in PC12 cells is mediated by Ca2+‐dependent production of ceramide via sphingomyelin pathway

A presynaptic membrane disturbance is an essential process for the release of various neurotransmitters. Ceramide, which is a tumor suppressive lipid, has been shown to act as a channel-forming molecule and serve as a precursor of ceramide-1-phosphate, which can disturb the cellular membrane. This study found that while permeable ceramide increases the rate of dopamine release in the presence of a Ca(2+)-ionophore, A23187, permeable ceramide-1-phosphate provoked its release even without the ionophore. The treatment of PC12 cells with the ionophore at concentrations < 2 microM produced ceramide via the sphingomyelin (SM) pathway with a concomitant release of dopamine, and no cell damage was observed. The addition of a Ca(2+) chelator, EGTA, to the medium inhibited the increase in the release of both the ceramide and dopamine. This suggests that ceramide might be produced by Ca(2+) and is implicated in the membrane disturbance associated with the release of dopamine as a result of its conversion to ceramide-1-phosphate. Consistent with these results, this study detected a membrane-associated and neutral pH optimum sphingomyelinase (SMase) whose activity was increased by Ca(2+). Together, these results demonstrate that ceramide can be produced via the activation of a neutral form of SMase through Ca(2+), and is involved in the dopamine release in concert with Ca(2+).

Melatonin inhibition of nicotine-stimulated dopamine release in PC12 cells

Melatonin, a pineal hormone, modifies numerous physiologic processes including circadian rhythms and sleep. In specific tissues, melatonin appears to have an inverse relationship with dopamine. To examine this relationship, a pheochromocytoma cell line (PC12) was used to determine the extent of melatonin’s ability to inhibit nicotine-stimulated dopamine release. Multiple experiments were conducted that examined: (1) the dose response of acute melatonin (5 min); (2) the effects of chronic melatonin (16 h pre-exposure); (3) the effects of prior nicotine or melatonin exposure (5 min) on melatonin’s ability to alter dopamine release from a second 5-min nicotine exposure; and (4) the role of melatonin receptors (by pertussis toxin inhibition) on nicotine-stimulated dopamine release. In the dose response studies, melatonin inhibited nicotine-stimulated dopamine release with an ED50 of 8.6 μM. Chronic exposure to melatonin had no effect on melatonin’s acute inhibition of nicotine-stimulated dopamine release. Prior nicotine or melatonin exposure had little effect on subsequent melatonin or nicotine exposure, except that the cells exposed to nicotine were not responsive to a second exposure to nicotine. Blockade of melatonin receptor function by pre-exposure to pertussis toxin (16 h) did not prevent melatonin’s inhibition of nicotine-stimulated dopamine release. However, the toxin-treated cells were less inhibited by melatonin when compared to control cells suggesting a partial role for melatonin receptors. These results indicate that melatonin can acutely inhibit nicotine-stimulated dopamine release in PC12 cells. This model system allows detailed examination of melatonin’s cellular actions as well as supporting a role for melatonin on neuronal dopamine release.

GAP-43 mRNA suppression by the ribozyme in PC12 cells and inhibition of evoked dopamine release.

We constructed a ribozyme designed to cleave the GAP-43 mRNA and which contained a bacteriophage T7 transcription terminator at its 3' site to maintain stability. The ribozyme was overexpressed in PC12 cells by pEF-BOS, a powerful mammalian expression vector. Consequently, PC12 cells overexpressing the GAP-43 ribozyme revealed a drastic decrease in the levels of GAP-43 mRNA expression, and the evoked dopamine release was significantly suppressed in these cell lines. These results support the previous observations that GAP-43 is associated with Ca-dependent dopamine release in PC12 cells, and the ribozyme expression system used in the present study was demonstrated to be useful for suppression of the functions of specific mRNAs and exploration of specific gene products.

Identification of the alternative spliced form of the α2/δ subunit of voltage sensitive Ca 2+ channels expressed in PC12 cells

The α2/δ subunit of voltage sensitive Ca 2+ channels expressed in PC12 has been cloned and partially sequenced. The message observed in Northern blot analysis displays a 7.5 kb transcript, identical in size to mRNA of rabbit skeletal muscle and rat brain. the nucleotide sequence of the cloned α2 subunit of the PC12 specific cDNA is >99% identical to rat brain sequence and 85% to skeletal muscle. Reverse-transcriptase-polymerase chain reaction (RT-PCR) of the alternative splicing region identifies two deleted regions of 57 bp and 21 bp in PC12 expressed α2/δ transcript. The alternative variant α 2e of α2/δ subunit which is expressed in PC12 cells was previously identified in human embryonic kidney (HEK293) cells. RT-PCR analysis show two different sized alternative PCR fragments in rat lung and none in rat spleen, kidney and intestine. Antibodies prepared against a 19 amino acid peptide within the alternative spliced region effectively inhibits [ 3H]dopamine release in PC12 cells. This implies that the alternatively spliced region is positioned extracellularly and is involved in regulation of the L-type Ca 2+ channel-mediated transmitter release.

P-155 - Modulation by serotonin of ATP-evoked dopamine release in PC12 cells.

P-155 - Modulation by serotonin of ATP-evoked dopamine release in PC12 cells.

α-Latrotoxin-induced CA2+-independent dopamine release in PC12 cells

α-Latrotoxin-induced CA2+-independent dopamine release in PC12 cells

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 Ca 2+ ([Ca 2+] 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, CdCl 2, and CoCl 2 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, Cd 2+ or Co 2+, 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 Ca 2+ channels.