The Effect of Colchicine and Cytochalasin B on the Release of Taurine from the Chick Retina

Abstract: The effect of colchicine (0.5 mM) and of cytochalasin B (10⁻⁴ M) on the release of [³⁵S]taurine from the isolated chick retina, upon stimulation by 68.5 mM-KCl, 10⁻⁵ M-veratridine and 10 mM-glutamate, was studied. Cytochalasin and colchicine effects on taurine release were compared with those on K⁺-stimulated release of [³H]dopamine and [³H]GABA. Colchicine caused a marked decrease of the [³⁵S]taurine release evoked by the three stimulatory agents; it also decreased [³H]dopamine release without affecting that of [³H]GABA. Cytochalasin B significantly decreased the efflux of [³⁵S]taurine stimulated by glutamate or veratridine without altering that evoked by 68.5 mM-KCl. Cytochalasin practically suppressed the [³H]dopamine-stimulated release and slightly decreased that of [³H]GABA. This drug produced a high increase in the spontaneous release of labeled GABA and taurine. These results suggest that the release of taurine and GABA from the chick retina probably occurs through different mechanisms. It is suggested that taurine release may be related to a process involving contractile proteins.     

TAURINE IN DEVELOPING RAT BRAIN: CHANGES IN BLOOD-BRAIN BARRIER

Abstract— The fate of [³⁵S]taurine injected intraperitoneally or intracranially has been compared in rats throughout neonatal development. The amount of [³⁵S]taurine present in the whole rat pup 24 h after intraperitoneal injection increases during development to a maximum 15 days after birth, and declines thereafter, whereas the amount of [³⁵S]taurine reaching the brain 24 h after intraperitoneal injection was greatest in the first 5 days after birth. The amount of [³⁵S]taurine remaining in the brain 24 h after intracranial injection does not vary throughout the period of neonatal development. These results suggest that the 'blood-brain' barrier is more accessible to taurine in the first few days after birth than later in neonatal development, and that factors other than simple exchange are involved.     

COMPARATIVE STUDIES ON THE DEGRADATION OF GABA and TAURINE IN THE BRAIN

Abstract— The degradation of taurine and GABA in mammalian brain was studied in vivo and in vitro. Small amounts of [³⁵S]isethionate (10–20 pmol/g brain wet weight) and [³⁵S]sulphate (about 2 pmol/g) were detected in mouse brain after intramuscular injection of [³⁵S]taurine. Taurine also produced isethionate in rat brain homogenates (about 20 nmol/h/g protein) and subcellular fractions (about 40 nmol/h/g protein in synaptosomes and about 300 nmol/h/g in mitochondria), but the reaction was not stimulated either by external electrical pulses or by the addition of various cofactors (NAD and NADP in both oxidized and reduced forms, riboflavin, glutathione. pyridoxal-5'-phosphate, ATP) to the incubation medium. [¹⁴C]GABA was readily metabolized to [¹⁴C]succinate both in vivo and in vitro. Isethionate formation activity was concentrated in the mitochondrial fraction, as was also GABA-T activity. Partially purified GABA-T from calf brain also slightly catalysed the formation of [³⁵S]isethionate (about 1.3 μmol/min/g protein) from [³⁵S]taurine. It appears that the slight formation of isethionate from taurine is coupled to GABA-T activity. The formation of isethionate from taurine is so small, that it apparently has no role in the control of the brain taurine pool.     

Formation and Utilization of the Active Sulfate Donor [35S]3'-Phosphoadenosine 5'-Phosphosulfate in Brain Slices: Effects of Depolarizing Agents

Abstract: The accumulation and utilization of [³⁵S]3'-phos-phoadenosine 5'-phosphosulfate (PAPS) were studied in slices from rat cerebral cortex incubated in the presence of inorganic [³⁵S]sulfate. [³⁵S]PAPS levels were directly evaluated after either isolation by ion-exchange chromatography or quantitative enzymatic transfer of its active [³⁵S]sulfate group to an acceptor phenol under the action of added phenolsulfotransferase activity. [³⁵S]PAPS formation was also indirectly followed by incubating slices in the presence of β-naphthol and measuring the levels of [³⁵S]β-naphthyl sulfate ([³⁵S]β-NS). Whereas [³⁵S]PAPS levels rapidly reached a plateau, [³⁵S]β-NS formation proceeded linearly with time for at least 1h, an observation indicating that the nucleotide was continuously synthesized and utilized for endogenous sulfation reactions. [³⁵S]PAPS formation in ices was completely and rather potently blocked by 2,6-dichloro-4-nitrophenol (IC₅₀= .10 μM), an inhibitor of the PAPS-synthesizing enzyme system in a cytosolic preparation. [³⁵S]PAPS accumulation and [³⁵S]β-NS'formation were strongly reduced by depolarizing agents such as potassium or veratridine. At millimolar concentrations, various excitatory amino acids (glutamate, aspartate, cysteate, quisqualate, and homocysteate) also elicited similar effects, whereas kainate and N -methyl-D-aspartate were inactive. This suggests that PAPS synthesis is turned off when cerebral cells are strongly depolarized.     

NEUROTRANSMITTER UPTAKE INTO SLICES OF RAT CEREBRAL CORTEX IN VITRO: EFFECT OF SLICE SIZE

Abstract— The uptake of [¹⁴C]GABA, [¹⁴C]taurine, [³H] β -alanine and [¹⁴C]dopamine was compared in slices of rat cerebral cortex of three different sizes (0.1 × 0.1 × 2 mm, 0.2 × 0.2 × 2 mm and 0.4 × 0.4 × 2 mm prepared with a mechanical tissue chopper). [¹⁴C]Taurine and [³H] β -alanine uptake increased whereas [¹⁴C]GABA uptake decreased with increasing slice size. [¹⁴C]Dopamine uptake was optimal in 0.2 × 0.2 × 2 mm slices. Increasing slice size was shown to decrease inhibition of [³H] β -alanine and [¹⁴C]GABA uptake by l -2,4-diaminobutyric acid. Lactate dehydrogenase activity increased with increasing slice size indicating decreased tissue damage or increased cellular integrity. The possibility that varying slice size can be used to distinguish between neuronal and glial uptake is discussed. It is suggested that taurine uptake in the cerebral cortex is predominantly glial.     

Modulatory Action of Taurine on the Release of GABA in Cerebellar Slices of the Guinea Pig

Abstract: For the purpose of demonstrating the action of taurine as a neuromodulator in addition to its suggested neurotransmitter function, the effects of taurine and muscimol on the depolarization-induced Ca-dependent release of [³H]γ-aminobutyric acid (pH]GABA) and l -[³H]glutamate in cerebellar slices from guinea pigs were investigated. The release of [³H]GABA was found to be greatly decreased by a GABA agonist, muscimol, and by taurine, but not by glycine. The release of l -[³H]glutamate was little affected by taurine. The release of [³H]GABA was enhanced by bicuculline and strychnine, but not by picrotoxin, and the suppressive action of muscimol on the GABA release was antagonized by bicuculline, picrotoxin, and strychnine, suggesting the possible existence of presynaptic autoreceptors for GABA in the cerebellum. The suppressive action of taurine on the release of [³H]GABA, on the other hand, was blocked only by bicuculline. These results suggest that taurine reduced the release of [³H]GABA from cerebellar slices by acting on the GABA autoreceptors or, more likely, on other types of receptors that are sensitive to bicuculline. As a possible mechanism for this modulatory action of taurine, the blockade by this amino acid of the influx of Ca²⁺ into cerebellar tissues was tentatively suggested.     

Cysteine Sulfinic Acid in the Central Nervous System: Specific Binding of [35S]Cysteic Acid to Cortical Synaptic Membranes-An Investigation of Possible Binding Sites for Cysteine Sulfinic Acid

Abstract: Specific binding sites for cysteine sulfinic acid, an excitatory amino acid, in crude synaptic membrane fractions of rat cerebral cortex were examined, using L-[³⁵S]cysteic acid as a ligand. Two specific binding systems of [³⁵S] cystec acid were found, one Na⁺-dependent and the other Na⁺-independent. The Na⁺-independent specific binding of [³⁵S]Cysteic acid was saturable, with a K_d of 474 n M and B_max of 3.29 pmol/mg protein. The binding was optimal at pH 7.4 and at 37°C. Treatment of the membranes with proteases, concanavalin A, or Triton X-100 markedly reduced the binding. Of various compounds related to cysteic acid, L-cysteine sulfinic acid was the most effective competitor of this binding. These results indicate the existence of an Na⁺-independent specific binding site for cysteic acid in the synaptic membrane of rat cerebral cortex, which may be different from that for glutamate. Possible involvement of cysteine sulfinic acid as an endogenous ligand for this binding site is discussed.     

THE SELECTIVE NEURONAL UPTAKE AND RELEASE OF [3H]DL-2,4-DIAMINOBUTYRIC ACID BY RAT CEREBRAL CORTEX

Abstract— At 25°C the accumulation of [³H] dl -2,4-diaminobutyric acid (DABA) into small rat cortical slices was linear with time and a tissue: medium ratio of 35:1 was attained after 60 min. At 37°C the uptake was no longer linear and the tissue: medium ratio at 60 min was 66:1. Uptake was unaffected by the addition of 10 μ m -AOAA and dependent on the presence of Na⁺ in the incubation media. The uptake was shown to have a high affinity component with a K _m of 20.7 μ m and a V _max of 28.6 nmol/g/min. IC50's for the inhibition of [³H]DABA uptake by dl -DABA, l -DABA and GABA were 80, 40 and 17 μ m respectively. Two m m β -alanine, however, caused less than 13% inhibition of [³H]DABA uptake. Electron microscopic autoradiographs showed the [³H]DABA to be accumulated by 22% of the identifiable nerve terminals and, after 14 days exposure, the density of silver grains over nerve terminals was 36–38 times higher than that over the rest of the electron micrograph. On the other hand, [³H]DABA was not taken up into rat sensory ganglia and light level autoradiography showed the small amount of [³H]DABA accumulated by the ganglia to be evenly distributed throughout the tissue. Both electrical stimulation for 30 s and exposure of the tissue to a medium containing 47 m m -K⁺ for 2 min caused a marked increase in the efflux of [³H]DABA from the tissue. Both these effects were abolished by a reduction in Ca²⁺ concentration and an increase in the Mg²⁺ concentration of the superfusing medium. These results suggest that l -DABA acts as a 'false transmitter' for the neuronal uptake, storage and release of GABA.     

STUDIES ON THE POLYSACCHARIDE-SULFATING SYSTEM IN SEA URCHIN EMBRYOS

The sulfating system in sea urchin embryos was examined, using the labeled precursor inorganic [³⁵S]sulfate in vivo and [³⁵S]3'-phosphoadenosine 5'-phosphosulfate ([³⁵S]PAPS) in a cell-free system. In vivo incorporation of [³⁵pS]sulfate into the trichloroacetic acid (TCA)-insolubte fraction increased gradually during sea urchin development, whereas radioactivity of [³⁵S]sulfate contained in the TCA-soluble fraction showed a conspicuous peak at the late gastrula stage.
In a cell-free system, the particulate fraction showed marked incorporation of [³⁵pS]JPAPS. This sulfating activity was highest at pH 6.4 to 7.2 and at 27°C, and it was strongly inhibited by Hg ²⁺and p-chloromercuribenzoic acid.
The sulfating activity was quite low in fertilized eggs, but then increased rapidly up to the swimming blastula stage. The activity in the particulate fraction precipitated at 10,000 xg increased gradually and that in the particulate fraction precipitated at 100,000 xg was almost constant from the swimming blastula stage to the pluteus stage.     

EFFECTS OF AMINO-OXYACETIC ACID ON [3H]GABA UPTAKE BY RAT BRAIN SLICES

Abstract— The effect of amino-oxyacetic acid on the uptake of [³H]GABA by rat brain slices was studied. When added simultaneously with [³H]GABA, amino-oxyacetic acid had no significant effect on [³H]GABA uptake. However, preincubation of brain slices with amino-oxyacetic acid prior to addition of [³H]GABA produced inhibition of uptake, which increased with longer duration of preincubation. The inhibitory effect of amino-oxyacetic acid was maximal at 2 mM concentration and concentrations sufficient to inhibit significantly GABA:glutamate transaminase (10^--⁶ M) had no effect on [³H]GABA uptake. D-Cycloserine and β-hydrazino-propionic acid also inhibited [³H]GABA uptake, but the amounts required were considerably in excess of those needed to inhibit GABA:glutamate transaminase. 4-Deoxypyridoxine inhibited [³H]GABA uptake, whether given in vivo or in vitro , and the inhibitory effect of amino-oxyacetic acid was reversed with pyridoxine. GABA transport appears to be dependent on pyridoxal phosphate and interference with this function of the vitamin is suggested as the basis for the inhibitory effect of amino-oxyacetic acid on [³H]GABA uptake.     

Increase in Chloride-Dependent l-Glutamate Transport Activity in Synaptic Membrane After In Vitro Ischemic Treatment

Abstract: The effect of energy failure on Cl⁻-dependent l -glutamate ( l -Glu) transport was examined with an in vitro preparation. Rat brain slices were incubated in low oxygen and glucose-deprived medium (in vitro ischemia), and a synaptic membrane fraction was prepared from the slices. Cl⁻-dependent l -[³H]Glu uptake into vesicles increased about twofold after 20 min of in vitro ischemia. The increased l -[³H]Glu uptake was inhibited by l -Glu, dl -2-amino-4-phosphonobutyrate, l -homocysteic acid, l -cystine, 4,4'-diisothiocyano-2,2'-disulfonic stilbene, and removal of Cl⁻. Uptakes of Na⁺-dependent l -[³H]-Glu, [³H]GABA, and [³H]taurine were not changed by the in vitro ischemia. In vitro ischemia increased the V _max value without affecting the K _m value. The increased l -[³H]Glu uptake by in vitro ischemia was reduced by subsequent incubation in a normoxic glucose-containing solution. ATP content in brain slices decreased to <10% of control values by in vitro ischemia for 10 min. The decrease in ATP content was restored by subsequent incubation in normoxic glucose-containing solution. Treatment with veratrine, 2,4-dinitrophenol, carbonyl cyanide m -chlorophenylhydrazone, and NaCN in normoxic conditions increased l -[³H]Glu uptake with a concomitant decrease in ATP content in slices. These results suggest that Cl⁻-dependent l -Glu transport activity in synaptic membranes increases in ischemia- or hypoxia-induced brain energy failures.     

Characterization, Distribution, and Protein Kinase C-Mediated Regulation of [35S]Glutathione Binding Sites in Mouse and Human Spinal Cord

Abstract: We have characterized a high-affinity [³⁵S]-glutathione ([³⁵S]GSH) binding site in mouse and human spinal cord. [³⁵S]GSH binding sites in mouse and human spinal cord were observed largely within the gray matter in both the dorsal and ventral horns of spinal cord at cervical, thoracic, and lumbosacral segments. High-affinity [³⁵S]GSH binding was saturable, showing a B _max of 72 fmol/mg of protein and a K _D of 3.0 n M for mouse spinal cord and a B _max of 52 fmol/mg of protein and a K _D of 1.6 n M for human spinal cord. [³⁵S]GSH binding was displaceable by GSH, l -cysteine, and S -hexyl-GSH, but not by glutamate, glycine, or NMDA. These [³⁵S]GSH binding sites exhibited kinetic and saturation characteristics similar to GSH binding sites in rat brain astrocytes. To determine whether [³⁵S]GSH binding sites could be regulated by protein kinase C, we exposed human spinal cord sections to phorbol 12,13-diacetate for 1 h before ligand binding. Phorbol ester treatment increased [³⁵S]GSH binding by ∼60%, an effect that could be blocked by exposure of spinal cord sections to 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a general protein kinase inhibitor. [³⁵S]GSH binding sites in the spinal cord of both species exhibited many of the characteristics of a receptor including saturable binding, high affinity, ligand specificity, and modulation by kinase activity. These data suggest that GSH is a neurotransmitter in the CNS.     

Potassium-Stimulated Release of Radiolabelled Taurine and Glycine from the Isolated Rat Retina

Abstract: The release of preloaded [³H]glycine and [³H]taurine in response to a depolarising stimulus (12.5-50 m M KCl) has been studied in the superfused rat retina. High external potassium concentration immediately increased the spontaneous efflux of [3H]glycine, the effect of 50 m M K⁺ apparently being abolished by omitting calcium from the superfusing medium. In contrast, although high potassium concentrations increased the spontaneous emux of [³H]taurine from the superfused rat retina, this release was not evident until the depolarising stimulus was removed from the superfusing medium. The magnitude of this "late" release of [³H]taurine was dependent on external K⁺ concentrations, and appeared immediately after cessation of the stimulus irrespective of whether it was applied for 4, 8, or 12 min. Potassium (50 m M )-induced release of taurine appeared partially calcium-dependent, being significantly reduced (p < 0.01) but not abolished by replacing calcium with 1 mM EDTA in the superfusate. High-affinity uptake systems for both [³H]glycine and [³H]taurine were demonstrated in the rat retina in vitro ( K _m values, 1.67 μ M and 2.97 μ M ; V_max values, 19.3 and 23.1 nmol/g wet weight tissue/h, respectively). The results are discussed with respect to the possible neuro-transmitter roles of both amino acids in the rat retina.     

Stimulation of [3H]GABA and β-[3H]Alanine Release from Rat Brain Slices by cis-4-Aminocrotonic Acid

Abstract: cis -4-Aminocrotonic acid (CACA; 100 µ M ), an analogue of GABA in a folded conformation, stimulated the passive release of [³H]GABA from slices of rat cerebellum, cerebral cortex, retina, and spinal cord and of β-[³H]alanine from slices of cerebellum and spinal cord without influencing potassium-evoked release. In contrast, CACA (100 µ M ) did not stimulate the passive release of [³H]taurine from slices of cerebellum and spinal cord or of d -[³H]aspartate from slices of cerebellum and did not influence potassium-evoked release of [³H]taurine from the cerebellum and spinal cord and d -[³H]aspartate from the cerebellum. These results suggest that the effects of CACA on GABA and β-alanine release are due to CACA acting as a substrate for a β-alanine-sensitive GABA transport system, consistent with CACA inhibiting the uptake of β-[³H]alanine into slices of rat cerebellum and cerebral cortex. The observed K _i for CACA against β-[³H]alanine uptake in the cerebellum was 750 ± 60 µ M . CACA appears to be 10-fold weaker as a substrate for the transporter system than as an agonist for the GABA_c receptor. The effects of CACA on GABA and β-alanine release provide indirect evidence for a GABA transporter in cerebellum, cerebral cortex, retina, and spinal cord that transports GABA, β-alanine, CACA, and nipecotic acid that has a similar pharmacological profile to that of the GABA transporter, GAT-3, cloned from rat CNS. The structural similarities of GABA, β-alanine, CACA, and nipecotic acid are demonstrated by computer-aided molecular modeling, providing information on the possible conformations of these substances being transported by a common carrier protein.     

Ethylenediamine as a Specific Releasing Agent of γ-Aminobutyric Acid in Rat Striatal Slices

Abstract: Following incubation with [¹⁴C]y-aminobutyric acid (GABA) or [³H]dopamine, slices of rat striatum were superfused with media containing 36 mM K⁺ or ethylenediamine (EDA), 1 or 5 mM. Both K⁺ and EDA induced a release of [¹⁴C]GABA, the K⁺-induced release being largely Ca²⁺-dependent, while the EDA-induced release was not. Whereas K⁺ also evoked a Ca²⁺-dependent release of [³H]dopamine, EDA evoked no release of dopamine. EDA may therefore have potential as a specific GABA releasing agent.     

Cysteine Sulfinic Acid in the Central Nervous System: Uptake and Release of Cysteine Sulfinic Acid by a Rat Brain Preparation

Abstract: Uptake and release of cysteine sulfinic acid by synaptosomal fractions (P₂) and slices of rat cerebral cortex were investigated. The P₂ fraction had a Na⁺-dependent high-affinity uptake system for cysteine sulfinic acid (K_m, 12μM), which was restricted to the synaptosomes. High-affinity uptake of cysteine sulfinic acid was competitively inhibited by glutamate, aspartate, and cysteic acid. None of the various centrally acting drugs tested specifically inhibited this transport system. Release of [¹⁴C]cysteine sulfinic acid from preloaded cortical slices or P₂ fractions was examined by a superfusion method, which avoided reuptake of released [¹⁴C]cysteine sulfinic acid. High K⁺ (56 m M ) and veratridine (10μM) stimulated the release of cysteine sulfinic acid from slices and the P₂ fraction in a partly Ca²⁺-dependent manner. Diazepam at concentrations of 10 and 100 μM markedly inhibited the stimulated release, but not the spontaneous release, by cortical slices. On the contrary, it had no effect on the stimulated release of cysteine sulfinic acid from the P₂ fraction.     

Endomorphin-Stimulated [35S]GTPγS Binding in Rat Brain: Evidence for Partial Agonist Activity at μ-Opioid Receptors

Abstract: Endomorphin-1 is a peptide whose binding selectivity suggests a role as an endogenous ligand at μ-opioid receptors. In the present study, the effect of endomorphin-1 on μ receptor-coupled G proteins was compared with that of the μ agonist DAMGO by using agonist-stimulated [³⁵S]GTPγS binding in rat brain. [³⁵S]GTPγS autoradiography revealed a similar localization of endomorphin-1 and DAMGO-stimulated [³⁵S]GTPγS binding in areas including thalamus, caudate-putamen, amygdala, periaqueductal gray, parabrachial nucleus, and nucleus tractus solitarius. Naloxone blocked endomorphin-1-stimulated labeling in all regions examined. Although the distribution of endomorphin-1-stimulated [³⁵S]GTPγS binding resembled that of DAMGO, the magnitude of endomorphin-1-stimulated binding was significantly lower than that produced by DAMGO. Concentration-effect curves of endomorphin-1 and DAMGO in thalamic membranes confirmed that endomorphin-1 produced only 70% of DAMGO-stimulated [³⁵S]GTPγS binding. Differences in maximal stimulation of [³⁵S]GTPγS binding between DAMGO and endomorphin-1 were magnified by increasing GDP concentrations, and saturation analysis of net endomorphin-1-stimulated [³⁵S]GTPγS binding revealed a lower apparent B _max value than that obtained with DAMGO. Endomorphin-1 also partially antagonized DAMGO stimulation of [³⁵S]GTPγS binding. These results demonstrate that endomorphin-1 is a partial agonist for G protein activation at the μ-opioid receptor in brain.     

Involvement of Protein Kinase C in γ-Aminobutyric Acid Release from Xenopus Oocytes Injected with Rat Brain mRNA

Abstract: Involvement of protein kinase C (PKC) in the release of γ-aminobutyric acid (GABA) was examined in Xenopus laevis oocytes injected with mRNA from rat cerebellum, as compared with findings in slices of rat cerebellum. The mRNA-injected oocytes preloaded with [³H]GABA showed spontaneous release of [³H]GABA, ∼0.5% of GABA content per 1 min. Stimulation with either Ca²⁺ ionophore (A23187) or a high K⁺ concentration increased the release of [³H]GABA from slices of rat deep cerebellar nucleus and mRNA-injected oocytes but not from noninjected and water-injected oocytes. 12- O -Tetradecanoylphorbol 13-acetate (10–300 n M ) but not 4α-phorbol 12,13-didecanoate (300 n M ) potentiated the A23187-stimulated release of [³H]GABA from slices and from mRNA-injected oocytes, in a concentration-dependent manner. Thus, machinery associated with release processes of GABA can be expressed in oocytes by injecting rat cerebellar mRNA, and PKC participates in GABA release from the functionally expressed GABAergic nerve terminals.     

Ca2+-Dependent Enhancement of [3H]Dopamine Uptake in Rat Striatum: Possible Involvement of Calmodulin-Dependent Kinases

Abstract: We studied effects of Ca²⁺ in the incubation medium on [³H]dopamine ([³H]DA) uptake by rat striatal synaptosomes. Both the duration of the preincubation period with Ca²⁺ (0–30 min) and Ca²⁺ concentration (0–10 m M ) in Krebs-Ringer medium affected [³H]DA uptake by the synaptosomes. The increase was maximal at a concentration of 1 m M Ca²⁺ after a 10-min preincubation (2.4 times larger than the uptake measured without preincubation), which reflected an increase in V _max of the [³H]DA uptake process. On the other hand, [³H]DA uptake decreased rapidly after addition of ionomycin in the presence of 1 m M Ca²⁺. The Ca²⁺-dependent enhancement of the uptake was still maintained after washing synaptosomes with Ca²⁺-free medium following preincubation with 1 m M Ca²⁺. Protein kinase C inhibitors did not affect apparently Ca²⁺-dependent enhancement of the uptake, whereas 1-[ N,O -bis(1,5-isoquinolinesulfonyl)- N -methyl- l -tyrosyl]-4-phenylpiperazine (KN-62; a Ca²⁺/calmodulin-dependent kinase II inhibitor) and wortmannin (a myosin light chain kinase inhibitor) significantly reduced it. Inhibitory effects of KN-62 and wortmannin appeared to be additive. N -(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7; a calmodulin antagonist) also remarkably inhibited the enhancement. These results suggest that Ca²⁺-dependent enhancement of [³H]DA uptake is mediated by activation of calmodulin-dependent protein kinases.