Role of ω-Conotoxin GVIA-Sensitive Ca2+ Entry in Angiotensin II-Stimulated [3H]Phorbol 12, 13-Dibutyrate Binding in Bovine Adrenal Medullary Cells

Abstract: The relative contributions of Ca²⁺ influx and intracellular Ca²⁺ mobilization were examined for angiotensin II-stimulated [³H]phorbol 12, 13-dibutyrate binding, which reflects the level of activated protein kinase C in bovine chromaffin cells. Angiotensin II receptors activate phospholipase C in chromaffin cells, leading to a shortlived mobilization of intracellular Ca²⁺. Angiotensin II-stimulated [³H]phorbol 12, 13-dibutyrate binding was largely blocked in Ca²⁺-free buffer and by pretreatment with the Ca²⁺-channel blocker ω-conotoxin GVIA. The [³H]phorbol 12, 13-dibutyrate binding response to [Sar¹]angiotensin II also appeared to be voltage sensitive, as no additivity was observed with the response to the depolarizing agent 4-aminopyridine (3 m M ). Threshold sensitivities of the extra-and intracellular Ca²⁺-mobilizing pathways to angiotensin II were similar, and all examined effects of angiotensin II in these cells were apparently mediated by losartan-sensitive (AT₁-Iike) receptors. The dependence of angiotensin II-stimulated [³H]phorbol 12, 13-dibutyrate binding on extracellular Ca²⁺ entry, in contrast to stimulation by other phospholipase C-linked receptor agonists (bradykinin and methacholine), suggests that angiotensin II preferentially stimulates protein kinase C translocation to the plasma membrane, rather than to internal membranes, in bovine adrenal medullary cells.     

Depolarization of Cerebellar Granule Cells Increases Phosphorylation of Rabphilin-3A

Abstract: Studies performed over the past several years have provided evidence that phosphorylation of proteins is important in the regulation of neurotransmitter release. In this study, it is shown that rabphilin-3A is present in cerebellar granule cells as a phosphoprotein, by using ³²P-labeling of cerebellar granule cells, immunoprecipitation, phosphoamino acid analysis, and phosphopeptide mapping. The level of phosphorylation was increased (224 ± 13%) (mean ± SEM) on depolarization of the cells with K⁺ (56 m M ) in the presence of external Ca²⁺ (1 m M ). Stimulation of protein kinase C with a phorbol ester (phorbol 12,13-dibutyrate) also enhanced the phosphorylation of rabphilin-3A (217 ± 21%). Inhibitors of Ca²⁺/calmodulin-stimulated protein kinases or protein kinase C reduced the depolarization-enhanced phosphorylation of rabphilin-3A, indicating that rabphilin-3A is one of the targets for Ca²⁺-activated protein kinases in the nerve terminal. Costimulation of cells with phorbol 12,13-dibutyrate and K⁺ depolarization produced an increased level of phosphorylation of rabphilin-3A compared with either stimulus alone (287 ± 61%). Phosphoamino acid analysis showed that serine was the main phosphorylated residue. A slight increase in the threonine phosphorylation could also be detected, whereas tyrosine phosphorylation could not be detected at all. These results suggest that rabphilin-3A is phosphorylated in vivo and undergoes synaptic activity-dependent phosphorylation during Ca²⁺-activated K⁺ depolarization.     

Agents that Promote Protein Phosphorylation Inhibit the Activity of the Na+/Ca2+ Exchanger and Prolong Ca2+ Transients in Bovine Chromaffin Cells

Abstract: The Na⁺/Ca²⁺ exchanger is an important element in the maintenance of calcium homeostasis in bovine chromaffin cells. The Na⁺/Ca²⁺ exchanger from other cell types has been extensively studied, but little is known about its regulation in the cell. We have investigated the role of reversible protein phosphorylation in the activity of the Na⁺/Ca²⁺ exchanger of these cells. Cells treated with 1 m M dibutyryl cyclic AMP (dbcAMP), 1 µ M phorbol 12,13-dibutyrate, 1 µ M okadaic acid, or 100 n M calyculin A showed lowered Na⁺/Ca²⁺ exchange activity and prolonged cytosolic Ca²⁺ transients caused by depolarization. A combination of 10 n M okadaic acid and 1 µ M dbcAMP synergistically inhibited Na⁺/Ca²⁺ exchange activity. Conversely, 50 µ M 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a protein kinase inhibitor, enhanced Na⁺/Ca²⁺ exchange activity. Moreover, we used cyclic AMP-dependent protein kinase and calcium phospholipid-dependent protein kinase catalytic subunits to phosphorylate isolated membrane vesicles and found that the Na⁺/Ca²⁺ exchange activity was inhibited by this treatment. These results indicate that reversible protein phosphorylation modulates the activity of the Na⁺/Ca²⁺ exchanger and suggest that modulation of the exchanger may play a role in the regulation of secretion.     

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

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

Protein Kinase C-Mediated Down-Regulation of Voltage-Dependent Sodium Channels in Adrenal Chromaffin Cells

Abstract: Treatment of cultured bovine adrenal chromaffin cells with 12- O -tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C (PKC), decreased [³H]saxitoxin ([³H]STX) binding in a concentration (IC₅₀ = 19 n M )- and time ( t _1/2 = 4.5 h)-dependent manner. TPA (100 n M for 15 h) lowered the B _max of [³H]STX binding by 53% without altering the K _D value. Phorbol 12,13-dibutyrate (PDBu) also reduced [³H]STX binding, whereas 4α-TPA, an inactive analogue, had no effect. The inhibitory effect of TPA was abolished when H-7 (an inhibitor of PKC), but not H-89 (an inhibitor of cyclic AMP-dependent protein kinase), was included in the culture medium for 1 h before and during TPA treatment. Simultaneous treatment with TPA in combination with either actinomycin D or cycloheximide, an inhibitor of protein synthesis, nullified the effect of TPA. TPA treatment also attenuated veratridine-induced ²²Na⁺ influx but did not alter the affinity of veratridine for Na channels as well as an allosteric potentiation of veratridine-induced ²²Na⁺ influx by brevetoxin. These results suggest that an activation of PKC down-regulates the density of Na channels without altering their pharmacological features; this down-regulation is mediated via the de novo synthesis of an as yet unidentified protein(s), rather than an immediate effect of Na channel phosphorylation.     

Accumulation of N-Arachidonoylethanolamine (Anandamide) into Cerebellar Granule Cells Occurs via Facilitated Diffusion

Abstract: N -Arachidonoylethanolamine (anandamide, AEA) is a putative endogenous ligand of the cannabinoid receptor. Intact cerebellar granule neurons in primary culture rapidly accumulate AEA. [³H]AEA accumulation by cerebellar granule cells is dependent on incubation time ( t _1/2 of 2.6 ± 0.8 min at 37°C) and temperature. The accumulation of AEA is saturable and has an apparent K _m of 41 ± 15 µ M and a V _max of 0.61 ± 0.04 nmol/min/10⁶ cells. [³H]AEA accumulation by cerebellar granule cells is significantly reduced by 200 µ M phloretin (57.4 ± 4% of control) in a noncompetitive manner. [³H]AEA accumulation is not inhibited by either ouabain or removal of extracellular sodium. [³H]AEA accumulation is fairly selective for AEA among other naturally occurring N -acylethanolamines; only N -oleoylethanolamine significantly inhibited [³H]AEA accumulation at a concentration of 10 µ M . The ethanolamides of palmitic acid and linolenic acid were inactive at 10 µ M . N -Arachidonoylbenzylamine and N -arachidonoylpropylamine, but not arachidonic acid, 15-hydroxy-AEA, or 12-hydroxy-AEA, compete for AEA accumulation. When cells are preloaded with [³H]AEA, temperature-dependent efflux occurs with a half-life of 1.9 ± 1.0 min. Phloretin does not inhibit [³H]AEA efflux from cells. These results suggest that AEA is accumulated by cerebellar granule cells by a protein-mediated transport process that has the characteristics of facilitated diffusion.     

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.     

Stimulatory Effects of Protein Kinase C and Calmodulin Kinase II on N-Methyl-d-Aspartate Receptor/Channels in the Postsynaptic Density of Rat Brain

Abstract: To clarify the regulatory mechanism of the N -methyl- d -aspartate (NMDA) receptor/channel by several protein kinases, we examined the effects of purified type II of protein kinase C (PKC-II), endogenous Ca²⁺/calmodulin-dependent protein kinase II (CaMK-II), and purified cyclic AMP-dependent protein kinase on NMDA receptor/ channel activity in the postsynaptic density (PSD) of rat brain. Purified PKC-II and endogenous CaMK-II catalyzed the phosphorylation of 80–200-kDa proteins in the PSD and l -glutamate-(or NMDA)-induced increase of (+)-5-[³H]methyl-10, 11-dihydro-5 H -dibenzo[a, d]cyclohepten-5, 10-imine maleate ([³H]MK-801; open channel blocker for NMDA receptor/channel) binding activity was significantly enhanced. However, the pretreatment of PKC-II-and CaMK-II-catalyzed phosphorylation did not change the binding activity of l -[³H]glutamate, cis -4-[³H](phospho-nomethyl)piperidine-2-carboxylate ([³H]CGS-19755; competitive NMDA receptor antagonist), [³H]glycine, α-[³H]-amino-3-hydroxy-5-methyl-isoxazole-4-propionate, or [³H]-kainate in the PSD. Pretreatment with PKC-II-and CaMK-II-catalyzed phosphorylation enhanced l -glutamate-induced increase of [³H]MK-801 binding additionally, although purified cyclic AMP-dependent protein kinase did not change l -glutamate-induced [³H]MK-801 binding. From these results, it is suggested that PKC-II and/or CaMK-II appears to induce the phosphorylation of the channel domain of the NMDA receptor/channel in the PSD and then cause an enhancement of Ca²⁺ influx through the channel.     

Novel Pharmacological Sensitivity of the Presynaptic Calcium Channels Controlling Acetylcholine Release in Skate Electric Organ

Abstract: The presynaptic terminals of skate ( Raja montagui ) electric organ were tested for their sensitivity to calcium channel antagonists. Acetylcholine (ACh) release and the elevation of intraterminal Ca²⁺ concentrations triggered by K⁺ depolarisation were studied. ACh release was measured as ³H efflux from slices of organ prelabelled with [³H]choline. Depolarisation caused a marked, Ca²⁺-dependent increase in ³H efflux that was completely blocked by 100 µ M Cd²⁺ and by 300 n M ω-conotoxin-MVIIC (MVIIC). Inhibition by MVIIC was concentration dependent (IC₅₀ of ∼20 n M ) and reversible. No inhibition was seen with nifedipine (5 µ M ) or the two other peptide antagonists studied: ω-conotoxin-GVIA (GVIA) at 5 µ M and ω-agatoxin-IVA (Aga-IVA) at 1 µ M . In a "nerve plate" preparation (a presynaptic plexus of nerve fibres, Schwann cells, and nerve terminals) changes in intraterminal Ca²⁺ concentrations were measured by microfluorimetry using fluo-3. An increase in fluorescence, indicating a rise in the free [Ca²⁺], rapidly followed K⁺ depolarisation, and this change was restricted to the nerve terminals. This response was insensitive to nifedipine (5 µ M ), GVIA (5 µ M ), and Aga-IVA (300 n M ) but almost completely abolished by MVIIC (1 µ M ). MVIIC inhibition was concentration dependent and partially reversible. These results show that the nerve terminals in skate electric organ have calcium channels with a pharmacological sensitivity that is markedly different from the established L, N, and P types in other systems but shares some, but not all, of the features of the recently described Q type.     

Lithium Modulation of Phosphoinositide Signaling System in Rat Cortex: Selective Effect on Phorbol Ester Binding

Abstract— Recent work indicates that the therapeutic action of lithium may be mediated through perturbation of postreceptor second messenger systems. To elucidate further the postreceptor cellular sites of action(s) of lithium, the effect of chronic lithium treatment on various components of the receptor-activated phosphoinositide pathway was investigated. We found that chronic administration of lithium (0.2% LiCI, 21 days) to adult male rats did not significantly affect phosphoinositide hydrolysis in cerebral cortical slices induced by carbachol (1 m M ) or NaF (10 m M ). Nor did the same treatment alter the carbachol (1 m M ) potentiation of guanosine 5'-(γ-thio)triphosphate (30 μ M ) stimulation of phosphoinositide hydrolysis (an index of receptor/G protein coupling) in cortical membranes. Immunoblotting studies revealed no changes in the levels of Gα_q/11 immunoreactivity in the cortex after chronic lithium treatment. The levels of protein kinase C, as revealed by specific binding of [³H]phorbol dibutyrate ([³H]PDBu), were significantly reduced in the cytosolic fraction and increased in the particulate fraction of rat cortex after chronic lithium, whereas the K _D of [³H]PDBu binding remained relatively constant. A small and insignificant decrease in the density of [³H]inositol 1,4,5-trisphosphate binding was also found in the cortex. The above data suggest that chronic lithium treatment affects neither the muscarinic cholinergic-linked phosphoinositide turnover nor the putative G protein α subunit (Gα_q/11) responsible for phospholipase C activation. However, a possible translocation and activation of protein kinase C activity may be significant in the therapeutic effect of this mood-stabilizing agent.     

Kinetics, Pharmacology, and Autoradiographic Distribution of l-[3H]Nitroarginine Binding Sites in Rat Cerebellum

Abstract: The kinetics and pharmacology of N ^G-nitro- l -[2,3,4,5-³H]arginine ( l -[³H]NOARG) binding to rat cerebellum were investigated using in vitro radioligand binding. Specific l -[³H]NOARG binding in cerebellum was of nanomolar affinity, reversible, saturable, and best fit to a single-site model. Specific binding was Ca²⁺ dependent and sensitive to pH (with an optimum of 5.5–7.0). Added calmodulin (1.5–40 µg/ml) had no influence on specific l -[³H]NOARG binding. However, the calmodulin antagonists W-5, W-13, and calmidazolium inhibited l -[³H]NOARG binding with IC₅₀ values in the micromolar range, and calmodulin (10 µg/ml) competitively reversed this inhibition. Nitric oxide synthase (NOS) inhibitors ( N ^G-nitro- l -arginine methyl ester and N ^G-monomethyl- l -arginine acetate) and l -arginine displaced l -[³H]NOARG binding with IC₅₀ values in the nanomolar range, whereas d -arginine and basic amino acids ( l -lysine and l -histidine) displaced l -[³H]NOARG binding with IC₅₀ values in the millimolar range. A comparison of the NOS functional assay with l -[³H]NOARG binding in rat cerebellum showed similar profiles of Ca²⁺ dependency and inhibitory kinetics. Quantitative autoradiographic distribution of l -[³H]NOARG binding sites was significantly higher in the molecular layer than in the granular layer of cerebellum. These studies confirm the potential use of l -[³H]NOARG binding to study the regional distribution and functional properties of NOS.     

Modulation of Human Glutamate Transporter Activity by Phorbol Ester

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

Tyrosine Hydroxylase Phosphorylation in Digitonin-Permeabilized Bovine Adrenal Chromaffin Cells: The Effect of Protein Kinase and Phosphatase Inhibitors on Ser19 and Ser40 Phosphorylation

Abstract: The protein kinases and protein phosphatases that act on tyrosine hydroxylase in vivo have not been established. Bovine adrenal chromaffin cells were permeabilized with digitonin and incubated with [γ-³²P]ATP, in the presence or absence of 10 µ M Ca²⁺, 1 µ M cyclic AMP, 1 µ M phorbol dibutyrate, or various kinase or phosphatase inhibitors. Ca²⁺ increased the phosphorylation of Ser¹⁹ and Ser⁴⁰. Cyclic AMP, and phorbol dibutyrate in the presence of Ca²⁺, increased the phosphorylation of only Ser⁴⁰. Ser³¹ and Ser⁸ were not phosphorylated. The Ca²⁺-stimulated phosphorylation of Ser¹⁹ was incompletely reduced by inhibitors of calcium/calmodulin-stimulated protein kinase II (46% with KN93 and 68% with CaM-PKII 273–302), suggesting that another protein kinase(s) was contributing to the phosphorylation of this site. The Ca²⁺-stimulated phosphorylation of Ser⁴⁰ was reduced by specific inhibitors of protein kinase A (56% with H89 and 38% with PKAi 5–22 amide) and protein kinase C (70% with Ro 31-8220 and 54% with PKCi 19–31), suggesting that protein kinases A and C contributed to most of the phosphorylation of this site. Results with okadaic acid and microcystin suggested that Ser¹⁹ and Ser⁴⁰ were dephosphorylated by PP2A.     

Administration of Dexamethasone Up-Regulates Protein Kinase C Activity and the Expression of γ and ε Protein Kinase C Isozymes in the Rat Brain

Abstract : Altered hypothalamic-pituitary-adrenal (HPA) function (increased plasma cortisol level) has been shown to be associated with mood and behavior. Protein kinase C (PKC), an important component of the phosphatidyl-inositol signal transduction system, plays a major role in mediating various physiological functions. The present study investigates the effects of acute (single) and repeated (10-day) administrations of 0.5 or 1.0 mg/kg doses of dexamethasone (DEX), a synthetic glucocorticoid, on B _max and K _D of [³H]phorbol 12,13-dibutyrate ([³H]PDBu) binding, PKC activity, and protein expression of PKC isozymes, α, β, γ, δ, and ε in the membrane and the cytosolic fractions of rat cortex and hippocampus. It was observed that repeated administration of 1.0 mg/kg DEX for 10 days caused a significant increase in B _max of [³H]PDBu binding to PKC, in PKC activity, and in expressed protein levels of the γ and ε isozymes in both the cytosolic and the membrane fractions of the cortex and the hippocampus, whereas a lower dose of DEX (0.5 mg/kg for 10 days) caused these changes only in the hippocampus. On the other hand, a single administration of DEX (0.5 or 1.0 mg/kg) had no significant effect on PKC in the cortex or in the hippocampus. These results suggest that alterations in HPA function from repeated administration of glucocorticoids may modulate PKC-mediated functions.     

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.     

Cytotoxic Effect of β-Amyloid on a Human Differentiated Neuron Is Not Mediated by Cytoplasmic Ca2+ Accumulation

Abstract: The effects of synthetic β-amyloid (Aβ1–42) on cell viability and cellular Ca²⁺ homeostasis have been studied in the human neuron-like NT2N cell, which differentiates from a teratocarcinoma cell line, NTera2/C1.D1, by retinoic acid treatment. NT2N viability was measured using morphological criteria and fluorescent live/dead staining and quantified using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide metabolism. Aβ1–42 dose-dependently caused NT2N cell death when it was present in the cell culture for 14 days but had no effect on viability when it was present for 4 days. The lowest effective concentration was 4 µ M , and the strongest effect was produced by 40 µ M . Control NT2N cells produced spontaneous cytosolic Ca²⁺ oscillations under basal conditions. These oscillations were inhibited dose-dependently (0.4–40 µ M ) by Aβ1–42 that was present in the cell culture for 1 or 4 days. Ca²⁺ wave frequency was decreased from 0.21 ± 0.02 to 0.05 ± 0.02/min, amplitude from 88 ± 8 to 13 ± 4 n M , and average Ca²⁺ level from 130 ± 8 to 58 ± 3 n M . The Ca²⁺ responses to 30 m M K⁺ and 100 µ M glutamate were not different between control and Aβ-treated cells. Thus, the results do not support the hypothesis that cytosolic early Ca²⁺ accumulation mediates Aβ-induced NT2N cell death.     

Fenfluramine Releases Serotonin from Human Brain Nerve Endings by a Dual Mechanism

Abstract: Fenfluramine is the most widely used anorexigenic drug in humans. In animal experiments d -fenfluramine has been shown to act as a potent releaser of brain serotonin [5-hydroxytryptamine (5-HT)]. Here we have investigated the effects of d -fenfluramine on the release of [³H]5-HT from isolated nerve endings of human neocortex. The drug elicited release of unmetabolized [³H]5-HT, and this effect was concentration dependent. However, the mechanism of release seems to differ profoundly depending on the concentrations of d -fenfluramine used. At 5 µ M , the release of [³H]5-HT was blocked by the 5-HT transporter inhibitor fluoxetine and was Ca²⁺ independent and insensitive to the human autoreceptor 5-HT_1D agonist sumatriptan. The release of [³H]5-HT elicited by 0.5 µ M d -fenfluramine was similarly blocked by fluoxetine, but it was strongly Ca²⁺ dependent and sensitive to sumatriptan. It is suggested that, at relatively high concentrations, d -fenfluramine largely diffuses into serotonergic terminals and causes release of 5-HT through the 5-HT carrier working in the inside-outside direction; at relatively low concentrations d -fenfluramine enters the terminals through the 5-HT transporter but elicits release of 5-HT by an exocytotic-like mechanism.     

Cell Specificity of Molecular Changes During Memory Storage

Abstract: The aeolid nudibranch, Hermissenda crassicornis , exhibits Pavlovian conditioning to paired light and rotational stimuli and it has been suggested that protein kinase C (PKC) may play a critical role in the cellular mechanism for this conditioned behavioral response in the B-cell photoreceptor. The present study was designed to further examine learning-specific PKC involvement in identified cellular areas, particularly those in the visual-vestibular network, of the Hermissenda nervous system after Pavlovian conditioning. As used in previous vertebrate studies, the highly specific PKC radioligand, [³H]phorbol-12,13-dibutyrate ([³H]-PDBU), was used to determine the binding characteristics of the molluscan protein receptor considered to be PKC. The binding was specific, saturable, and could be displaced by a soluble diacylglycerol analogue. The binding activity was distributed evenly between the cytosol and the membrane. All of these analyses suggest that [³H]PDBU binds primarily to PKC in Hermissenda as it does in many other systems. Computerized grain image analysis was then used to determine the cellular localization of PKC as a function of Pavlovian conditioning. The medial and intermediate B photoreceptor and the optic ganglion showed significantly increased [³H]PDBU binding in conditioned animals. The present results provide the first report of an associative learning change of a key signal transduction component in identified neurons.     

Effects of Protein Kinase Inhibitors on Morphology and Function of Cultured Bovine Adrenal Chromaffin Cells: KN-62 Inhibits Secretory Function by Blocking Stimulated Ca2+ Entry

Abstract: In cultured bovine adrenal chromaffin cells, a nonselective protein kinase inhibitor, staurosporine, inhibits secretory function and induces neurite outgrowth. In the present study, effects of other nonselective protein kinase inhibitors (K-252a, H-7, and H-8) and reportedly selective protein kinase inhibitors (KN-62 and chelerythrine chloride) were examined on bovine adrenal chromaffin cell morphology, secretory function, and ⁴⁵Ca²⁺ uptake. Treatment of chromaffin cells with 10 µ M K-252a, 50 µ M H-7, or 50 µ M H-8 induced changes in cell morphology within 3 h; these compounds also induced a time-dependent inhibition of stimulated catecholamine release. Chelerythrine chloride, a selective inhibitor of Ca²⁺/phospholipid-dependent protein kinase, did not induce outgrowth or inhibit secretory function under our treatment conditions. KN-62, a selective inhibitor of Ca²⁺/calmodulin-dependent protein kinase II (CaMK II), significantly inhibited stimulated catecholamine release (IC₅₀ value of 0.32 µ M ), but had no effect on cell morphology. The reduction of secretory function induced by 1 µ M KN-62 was significant within 5 min and rapidly reversible. Unlike H-7, H-8, and staurosporine, KN-62 significantly inhibited stimulated ⁴⁵Ca²⁺ uptake. KN-04, a structural analogue of KN-62 that does not inhibit CaMK II, inhibited stimulated ⁴⁵Ca²⁺ uptake and catecholamine release like KN-62. These studies indicate that KN-62 inhibits secretory function via the direct blockade of activated Ca²⁺ influx. The nonselective inhibitors, K-252a, H-7, H-8, and staurosporine, inhibit secretory function by another mechanism, perhaps one involving alterations in the cytoskeleton.     

[3H]Dizocilpine Association Kinetics Distinguish Stimulatory and Inhibitory Polyamine Sites of N-Methyl-d-Aspartate Receptors

Abstract: Spermine and other polyamines both stimulate and inhibit N -methyl- d -aspartate receptor function, probably by interacting with two separate sites. To characterize these two actions, the effect of spermine on the binding kinetics of the channel blocker [³H]dizocilpine was studied in the presence of glutamate and glycine. Low concentrations (10 µ M ) of spermine increased the association and dissociation rates without modifying equilibrium binding, indicating that spermine increases the accessibility of [³H]dizocilpine to the channel by interacting with a high-affinity, stimulatory site. At higher concentrations (1 m M ), spermine markedly decreased equilibrium [³H]-dizocilpine binding by decreasing both affinity and B _max, indicating that spermine allosterically inhibits binding by interacting with a second, low-affinity site. The presumed polyamine antagonists arcaine, diethylenetriamine, and 1,10-diaminodecane completely inhibited equilibrium [³H]dizocilpine binding, probably by interacting with the inhibitory polyamine site or other sites, but not with the stimulatory polyamine site. Low concentrations (10 µ M ) of ifenprodil completely reversed the increase in association rate produced by spermine, whereas higher concentrations (IC₅₀ = 123 µ M ) inhibited equilibrium binding, indicating that ifenprodil is both a potent antagonist of the stimulatory site and a low-affinity ligand of the inhibitory site. The polyamine agonists spermine, spermidine, and neomycin interacted with the inhibitory site, but produced only partial inhibition of equilibrium [³H]dizocilpine binding.