NMDA and Non-NMDA Ionotropic Glutamate Receptors Modulate Striatal Acetylcholine Release via Pre- and Postsynaptic Mechanisms

Abstract: The effects of NMDA and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) on endogenous acetylcholine release from rat striatal slices and synaptosomes were investigated. Both agonists (1–300 µ M ) facilitated acetylcholine release from slices in a dose-dependent manner. NMDA (100–300 µ M ) and AMPA (30–300 µ M ), however, subsequently inhibited acetylcholine release. NMDA (100 µ M )-induced facilitation was antagonized by 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) and dizocilpine (both 1–10 µ M ), whereas the 10 µ M AMPA effect was antagonized by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 1–30 µ M ). NMDA (100 µ M )-induced inhibition was counteracted by CPP, but not dizocilpine, and by the nitric oxide synthase inhibitor l -nitroarginine (1–100 µ M ). Tetrodotoxin (0.5 µ M ) prevented the facilitatory effect of 3 µ M NMDA and AMPA, but left unchanged that of 30 µ M NMDA and 100 µ M AMPA. Acetylcholine release from synaptosomes was stimulated by KCI (7.5–100 m M ) in a dose-dependent manner. NMDA and AMPA maximally potentiated the 20 m M KCl effect at 1 µ M and 0.01 µ M , but were ineffective at 100 µ M and 10 µ M , respectively. Inhibition of acetylcholine release was never found in synaptosomes. The effects of 1 µ M NMDA and 0.01 µ M AMPA were antagonized by CPP (0.0001–1 µ M ) or dizocilpine (0.0001–10 µ M ) and by CNQX (0.001–1 µ M ), respectively. These data suggest that glutamatergic control of striatal acetylcholine release is mediated via both pre- and post-synaptic NMDA and non-NMDA ionotropic receptors.     

NMDA Receptor Subtype Selectivity: Eliprodil, Polyamine Spider Toxins, Dextromethorphan, and Desipramine Selectively Block NMDA-Evoked Striatal Acetylcholine but Not Spermidine Release

Abstract: NMDA receptor stimulation concomitantly increases the release of [¹⁴C]acetylcholine and [³H]spermidine from rat striatal slices in vitro. The NMDA-induced release of both acetylcholine and spermidine was blocked with equal potency by the NMDA channel blocker phencyclidine (0.1–10 µ M ). However, certain other channel blockers, including dextromethorphan (1–100 µ M ), which antagonized NMDA-evoked acetylcholine release without affecting NMDA-evoked spermidine release, and dextrorphan (1–100 µ M ) and memantine (1–100 µ M ), which block NMDA-evoked acetylcholine release more potently than NMDA-evoked spermidine release, showed greater selectivity of action. As previously shown for ifenprodil, eliprodil (SL82.0715; 1–100 µ M ) blocked NMDA-evoked acetylcholine but not spermidine release. This selectivity is also observed for other agents interacting with the polyamine site(s) on the NMDA receptor, including arcaine (1–1,000 µ M ), philanthotoxin₃₄₃, and argiotoxin₆₃₆ (10 µ M ) and was also noted for desipramine (1–100 µ M ). The NMDA-induced release of acetylcholine and spermidine is likely to be mediated by different native NMDA receptor subtypes, and several NMDA antagonists may be candidates for a selective action at a particular NMDA receptor subtype.     

Amphetamine and D1 Dopamine Receptor Agonists Produce Biphasic Effects on Glutamate Efflux in Rat Ventral Tegmental Area: Modification by Repeated Amphetamine Administration

Abstract: Amphetamine or selective D1 and D2 dopamine receptor agonists and antagonists were administered to the ventral tegmental area (VTA) through a microdialysis probe to determine their effects on glutamate and aspartate efflux in rats pretreated for 5 days with vehicle or 5 mg/kg (+)-amphetamine sulfate. In vehicle rats, glutamate efflux declined during 2 h of perfusion with the D1 receptor agonist SKF-82958 (10 and 100 µ M ). After SKF-82958 perfusion ended, glutamate efflux rebounded to basal levels and continued to increase gradually over the next 2 h. A similar biphasic pattern was observed with intra-VTA amphetamine (10 and 100 µ M ) and with another D1 agonist (100 µ M SKF-38393). The biphasic effects of SKF-82958 were prevented by coperfusion with a D1 antagonist (SCH-23390; 30 µ M ). Glutamate efflux was unaffected by a D2 agonist (100 µ M quinpirole) and by D1 or D2 antagonists administered alone (SCH 23390 and eticlopride; 30 µ M ). In amphetamine-pretreated rats tested 2 days after the last injection, both the decrease during SKF-82958 perfusion and the delayed increase in glutamate efflux were attenuated. In rats tested 12–14 days after the last amphetamine injection, only the decrease during SKF-82958 perfusion was attenuated. None of these drug treatments produced consistent effects on aspartate efflux. We showed previously that systemic amphetamine (5 mg/kg, i.p.) has no immediate effect on VTA glutamate efflux but produces a delayed increase in glutamate efflux that reaches statistical significance 2–3 h after injection. Because behavioral sensitization can be elicited either by repeated systemic or repeated intra-VTA administration, neurochemical effects common to both routes (such as the delayed increase in glutamate efflux) are most likely to contribute to its induction.     

Adenosine A2a Receptor-Mediated Modulation of Striatal Acetylcholine Release In Vivo

Abstract: To determine the functions of striatal adenosine A_2a receptors in vivo, the effects of a selective agonist, 2-[4-(2-carboxyethyl)phenethylamino]-5'- N -ethylcarboxamidoadenosine hydrochloride (CGS 21680), and an antagonist, ( E )-8-(3,4-dimethoxystyryl)-1,3-dipropyl-7-methylxanthine (KF17837), on acetylcholine release were investigated in the striatum of awake freely moving rats using microdialysis. Intracerebroventricular injection of CGS 21680 (10 µg) increased acetylcholine release in striatum and KF17837 (30 mg/kg p.o.) antagonized the CGS 21680-induced acetylcholine elevation. To investigate the contribution of dopaminergic and GABAergic neurons on A_2a receptor-mediated acetylcholine release, the effects of CGS 21680 were studied by using dopamine-depleted rats in the presence or absence of GABA antagonists. In the dopamine-depleted striatum, the intrastriatal application of CGS 21680 (0.3–30 µ M ) increased extracellular acetylcholine, which was significantly greater than that in normal striatum. The CGS 21680-induced elevation of acetylcholine release was still observed in the presence of GABA antagonists bicuculline (30 µ M ) and 2-hydroxysaclofen (100 µ M ) and was similar in both normal and dopamine-depleted striatum. These results suggest that A_2a agonist stimulates acetylcholine release in vivo, and this effect of A_2a agonist is modulated by dopaminergic and GABAergic neurotransmission.     

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.     

Impact of Corticotropin-Releasing Hormone on Extracellular Norepinephrine in Prefrontal Cortex After Chronic Cold Stress

Abstract: We have previously demonstrated that exposing rats to cold (5°C) for 3–4 weeks potentiates the increase in extracellular norepinephrine (NE) in the medial prefrontal cortex produced by acute tail shock. In the present study, we used microdialysis to determine the duration of cold exposure required to produce this sensitization and explored the mechanism of the phenomenon. Tail shock elicited a twofold greater increase in extracellular NE in the medial prefrontal cortex of rats exposed to cold for 2 weeks than in naive control rats or in rats exposed to cold for 1 week and tested either immediately or after a 2-week delay. Local infusion of 10 µ M d -amphetamine or 30 m M K⁺ increased extracellular NE in the medial prefrontal cortex (∼350 and 190%, respectively) comparably in control rats and rats exposed to cold for 3 weeks. In contrast, intraventricular administration of 3.0 µg of corticotropin-releasing hormone increased extracellular NE in the medial prefrontal cortex by 65% in rats exposed to cold for 2 weeks, but only 35% in control rats. These results indicate that an enhanced responsiveness of noradrenergic neurons to acute tail shock (1) requires ∼2 weeks of cold exposure to develop and (2) may be mediated by a change at the level of the noradrenergic cell bodies rather than the nerve terminals.     

Searching for mechanisms of N-methyl-D-aspartate-induced glutathione efflux in organotypic hippocampal cultures

N-Methyl-d-aspartate (NMDA)-receptor stimulation evoked a selective and partly delayed elevated efflux of glutathione, phosphoethanolamine, and taurine from organotypic rat hippocampus slice cultures. The protein kinase inhibitors H9 and staurosporine had no effect on the efflux. The phospholipase A₂ inhibitors quinacrine and 4-bromophenacyl bromide, as well as arachidonic acid, a product of phospholipase A₂ activity, did not affect the stimulated efflux. Polymyxin B, an antimicrobal agent that inhibits protein kinase C, and quinacrine in high concentration (500 µM), blocked efflux completely. The stimulated efflux after but not during NMDA incubation was attenuated by a calmodulin antagonist (W7) and an anion transport inhibitor (DNDS). Omission of calcium increased the spontaneous efflux with no or small additional effects by NMDA. In conclusion, NMDA receptor stimulation cause an increased selective efflux of glutathione, phosphoethanolamine and taurine in organotypic cultures of rat hippocampus. The efflux may partly be regulated by calmodulin and DNDS sensitive channels.     

In Vivo Microdialysis Evidence for Transient Dopamine Release by Benzazepines in Rat Striatum

Abstract: The effects of benzazepine derivatives on extracellular levels of dopamine (DA) and l -3,4-dihydroxyphenylacetic acid (DOPAC) in the dorsal striatum of freely moving rats were studied using in vivo microdialysis. Direct injection of SKF-38393 (0.5 or 1.5 µg/0.5 µl), a selective D₁ receptor agonist, into the striatum through a cannula secured alongside a microdialysis probe produced a rapid dose-dependent transient increase in striatal DA efflux and a more gradual reduction in efflux of DOPAC. The rapid increase in DA efflux was not affected by infusion of tetrodotoxin (TTX; 2 µ M ) or Ca²⁺-free Ringer's solution and occurred after either enantiomer of SKF-38393. A TTX-insensitive increase in DA level similar to that induced by SKF-38393 was also seen after other benzazepines acting as agonists (SKF-75670 and SKF-82958, each 1.5 µg in 0.5 µl) and antagonists (SCH-23390, 1.5 µg in 0.5 µl) at the D₁ receptor and after (+)-amphetamine. These effects were inhibited by infusion of nomifensine (100 µ M ). It is concluded that the transient increases in striatal DA efflux seen after intrastriatal injection of SKF-38393 and other benzazepines are not mediated by presynaptic D₁ receptors but by an amphetamine-like action on the dopamine transporter.     

Pharmacological Characterization of Tachykinin Receptors Controlling Acetylcholine Release from Rat Striatum: An In Vivo Microdialysis Study

Abstract: The regulation of striatal cholinergic function by tachykinins was examined in urethane-anesthetized rats by using microdialysis. Substance P (0.01–1 µ M ), [Sar⁹,Met(O₂)¹¹]substance P (1–10 µ M ), septide (0.1–3 µ M ), neurokinin (NK) A (0.1–10 µ M ), and senktide (0.1–10 µ M ) produced concentration-dependent increases in striatal acetylcholine (ACh) release. Septide was the most potent agonist for inducing release of ACh, whereas the stimulating effect of senktide was less pronounced and more progressive in onset. The response to septide was prevented by intraperitoneal administration of the nonpeptide NK₁ antagonist SR 140333 (1–3 mg/kg) but not by the nonpeptide NK₂ receptor antagonist SR 48968, indicating that the effect was mediated specifically by NK₁ receptors. ACh release caused by NKA was reduced by SR 48968 (1–3 mg/kg) and slightly affected by SR 140333, indicating a principal role for NK₂ receptors in the peptide response. The similar efficacy of SR 140333 and SR 48968 in blocking substance P-induced ACh release suggested that the effect of this peptide involves the stimulation of both NK₁ and NK₂ receptors. Finally, our results indicate that the increase in striatal ACh release induced by the D₁ agonist (+)-SKF-38393 (3 µ M ) may be mediated indirectly through local release of NKA or substance P acting at NK₂ receptors.     

Stress-Induced Sensitization of Dopamine and Norepinephrine Efflux in Medial Prefrontal Cortex of the Rat

Abstract: We examined whether prior exposure to chronic cold (17–28 days, 5°C) alters basal or stress-evoked (30-min tail shock) catecholamine release in medial prefrontal cortex, nucleus accumbens, and striatum, using in vivo microdialysis. Basal norepinephrine (NE) concentrations in medial prefrontal cortex did not differ between chronically cold-exposed rats and naive control rats (2.7 ± 0.3 vs. 2.5 ± 0.2 pg/20 µl, respectively). Basal dopamine (DA) efflux in any of the brain regions was not significantly different between chronically cold-exposed rats and naive rats. However, a trend for lower basal DA efflux in the cold-exposed relative to naive rats was observed in medial prefrontal cortex (1.5 ± 0.2 vs. 2.2 ± 0.3 pg/20 µl, respectively), nucleus accumbens (3.7 ± 0.8 vs. 5.4 ± 0.9 pg/20 µl, respectively), and striatum (4.4 ± 0.5 vs. 7.2 ± 1.5 pg/20 µl, respectively). In medial prefrontal cortex of rats previously exposed to cold, tail shock elicited a greater increase from baseline in both DA and NE efflux relative to that measured in naive rats (DA, 2.3 ± 0.3 vs. 1.2 ± 0.1 pg, respectively; NE, 3.8 ± 0.4 vs. 1.4 ± 0.2 pg, respectively). However, in nucleus accumbens or striatum of rats previously exposed to cold, the stress-induced increase in DA efflux was not significantly different from that of naive rats (nucleus accumbens, 1.8 ± 0.7 vs. 1.5 ± 0.3 pg, respectively; striatum, 1.9 ± 0.4 vs. 2.6 ± 0.7 pg, respectively). Thus, both cortical NE projections and cortically projecting DA neurons sensitize after chronic exposure to cold. In contrast, subcortical DA projections do not sensitize under these conditions.     

Kainate-Evoked Release of Adenosine from the Hippocampus of the Anaesthetised Rat: Possible Involvement of Free Radicals

Abstract: Using microdialysis in the hippocampus of anaesthetised rats, the concentration of extracellular adenosine was estimated to be 0.8 µ M . Kainic acid (0.1–25 m M ) in the perfusate evoked a concentration-dependent release of adenosine with an EC₅₀ of 940 µ M . Two 5-min pulses of 1 m M kainic acid in the perfusate increased the dialysate levels with an S2/S1 ratio of 0.52 ± 0.03. Kainate-evoked release of adenosine was reduced significantly by 10 µ M tetrodotoxin and by a κ-receptor agonist, U50,488H (100 µ M ). The S2/S1 ratio was reduced by 4.5 µ M 6-cyano-7-nitroquinoxaline-2,3-dione, a non-NMDA receptor antagonist, but not by the NMDA receptor blockers (+)-MK-801 (dizocilpine; 100 µ M ) or (±)-2-amino-5-phosphonopentanoic acid (1 m M ), indicating a non-NMDA receptor-mediated process. The S2/S1 ratio was also reduced significantly by 10 m M ascorbic acid, 10 m M glutathione (a scavenger of hydroperoxides), and 1 m M oxypurinol (a xanthine oxidase inhibitor), indicating the possible involvement of free radicals. Neither the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (100 µ M ) nor the A1 adenosine receptor agonist R (−)- N ⁶-(2-phenylisopropyl)adenosine (100 µ M ) affected release. Adenosine release evoked by kainic acid is therefore mediated by activation of non-NMDA receptors and may involve the propagation of action potentials and the production of free radicals.     

In Vivo Evidence that GABAB Receptors Are Negatively Coupled to Adenylate Cyclase in Rat Striatum

Abstract: The characteristics of the cerebral GABA_B receptor/cyclic AMP (cAMP)-generating system were investigated using the in vivo microdialysis technique in freely moving rats. Addition of forskolin, an activator of adenylate cyclase, to perfusate for 20 min resulted in a dose-dependent increase of cAMP efflux from the striatum. Pre- and coinfusions of baclofen for 80 min had no effect on the basal efflux of cAMP from the striatum but induced a significant decrease of forskolin (10 µ M )-stimulated cAMP efflux from the striatum in a dose-dependent manner. SKF 97541 (100 µ M ), a GABA_B receptor agonist, and GABA (50 µ M ) also decreased forskolin-induced cAMP efflux from the striatum. Coinfusion of CGP 54626A (100 µ M ), a GABA_B receptor antagonist, counteracted the effect of baclofen on the forskolin-stimulated cAMP efflux. In contrast, the isoproterenol (5 m M )-induced increase of cAMP efflux from the striatum was significantly enhanced by pre- and coinfusions with baclofen. These results suggest that this test system using in vivo microdialysis may be useful for examining the effect of drugs on the GABA_B receptor-linked cAMP-generating system in vivo.     

Muscarinic Modulation of Striatal Dopamine, Glutamate, and GABA Release, as Measured with In Vivo Microdialysis

Abstract: Intrastriatal microdialysis was used to administer muscarinic drugs in freely moving rats for 40 min at a flow rate of 2 µl/min. Administration of the nonselective agonist pilocarpine at 10 m M increased striatal dopamine release and decreased extracellular GABA and glutamate overflow. Perfusion with the muscarinic M₂ antagonist methoctramine at 75 µ M increased extracellular dopamine and glutamate concentrations but exerted no changes on extracellular GABA levels. Intrastriatal administration of the M₁ antagonist pirenzepine at 0.05 µ M decreased extracellular dopamine overflow. Application of pirenzepine (0.05 and 5 µ M ) exerted no effects on the measured GABA or glutamate levels. There are thus important differences in applied doses of muscarinic drugs needed to obtain modulatory effects. High doses of agonists are probably needed to superimpose on the background of tonic influences of striatal acetylcholine, whereas antagonists can block the receptors in small doses. We further suggest that M₁ receptors might tonically facilitate striatal dopamine release, that M₂ receptors might tonically inhibit striatal glutamate efflux, and that acetylcholine does not exert tonic effects on striatal GABA release. The link with the pilocarpine animal model for temporal lobe epilepsy will be discussed.     

Cholinomimetics Increase Glutamate Outflow via an Action on the Corticostriatal Pathway: Implications for Alzheimer's Disease

Abstract: Physostigmine, the acetylcholinesterase inhibitor (0.3 mg/kg, i.m.), increased extracellular glutamate but not aspartate concentrations in the striatum of anaesthetised rats, determined using microdialysis and HPLC. The rise was both tetrodotoxin and calcium dependent. In contrast, neither physostigmine (10 µ M ) added to the perfusion fluid nor vehicle (injected intramuscularly) affected amino acid concentrations. To obtain evidence that the action of acetylcholine was to modulate positively cortical pyramidal neurone activity via the M₁ receptor, the selective M₁ agonist PD 142505-0028 (10 µ M ) was topically applied to the frontal cortex. Like physostigmine, PD 142505-0028 rapidly increased glutamate but not aspartate concentrations in the striatum. Moreover, the effect of intramuscular physostigmine was blocked by a topically applied M₁ antagonist. These new data add to our hypothesis that cholinomimetics increase pyramidal neurone function.     

Modulation of α-Amino-3-Hydroxy-5-Methylisoxazole-4-Propionic Acid (AMPA) Binding Sites by Nitric Oxide

Abstract: Nitric oxide release is reported to be involved in physiological processes associated with altered sensitivity of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) class of glutamate receptor. A series of compounds liberating nitric oxide were therefore tested for their ability to modulate in vitro the characteristics of [³H]AMPA binding to sections of rat brain. Pretreatment of forebrain or cerebellar sections with sodium nitroprusside (1 m M ), S -nitroso- N -acetylpenicillamine (SNAP, 200 µ M ), glyceryl trinitrate (1 µ M ), or isosorbide dinitrate (0.5 m M ) all increased the binding of 3 n M [³H]AMPA by 15–30%. These actions were reproduced by 8-bromo-cyclic GMP (200 µ M ) in the cerebellum but not in the forebrain. In a similar manner, the effect of SNAP was attenuated by an inhibitor of cyclic GMP-dependent protein kinase in the cerebellum but not in the forebrain. The elevated [³H]AMPA binding observed after pretreatment with SNAP was caused by an increase in binding affinity, but the capacity of the sites was unchanged. Autoradiographic analysis showed that forebrain binding was enhanced in the cerebral cortex and hippocampus but not in the striatum. Nitric oxide therefore appears to be able to increase the affinity of AMPA binding sites via two distinct mechanisms in different brain areas. This action may contribute to synaptic plasticity associated with nitric oxide release.     

The Effect of Chronic Treatment with the GABA Transaminase Inhibitors γ-Vinyl-GABA and Ethanolamine-O-Sulphate on the In Vivo Release of GABA from Rat Hippocampus

Abstract: The effects of chronic treatment with the specific, mechanism-based, irreversible inhibitors of 4-aminobutyrate aminotransferase (EC 2.6.1.19; GABA transaminase), ethanolamine O -sulphate (EOS), and 4-aminohexenoate [vigabatrin; γ-vinyl-GABA (GVG)] on the extracellular concentrations of GABA in the hippocampus have been studied using in vivo microdialysis in conscious animals. Oral dosing [3 mg/ml of drinking water, giving doses of GVG of 194 ± 38 mg/kg/day and of EOS of 303 ± 42 mg/kg/day (mean ± SD)] was followed by microdialysis at 2, 8, and 21 days. The basal outflow of GABA (in the range of ∼1–2 pmol/30 µl/30-min sample) after 2 and 8 days of treatment was not significantly different from that in control animals, but the 21-day treatment gave significant rises in the extracellular GABA concentration (up to ∼6–8 pmol/30 µl/30-min sample). Both inhibitors gave similar results. Depolarisation with 100 m M K⁺ gave large increases in GABA release in control (∼20–60 pmol/30 µl/30-min sample) and treated animals. The 8- and 21-day-treated animals showed significant increases in the stimulated release compared with control animals (∼80–100 pmol/30 µl/30-min sample). Excluding Ca²⁺ had no significant effect on either basal or stimulated release. The significant increases in K⁺-evoked release of GABA show that the increased intracellular pool of GABA is available for release, and this may be related to the anticonvulsant action of these compounds.     

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.     

Activation of Protein Kinase C by Trimethyltin: Relevance to Neurotoxicity

Abstract: The differentiated PC12 cell neuronal model was used to determine the effect of trimethyltin (TMT) on protein kinase C (PKC). Cells treated with 5–20 µ M TMT showed a partial and sustained PKC translocation within 30 min and persisted over a 24-h period. TMT treatment was accompanied by a low level of PKC down-regulation over 24 h, which was small compared with that produced by phorbol esters. Confocal imaging of differentiated PC12 cells showed that PKC translocates to the plasma membrane and the translocation is blocked by the PKC inhibitor chelerythrine (1 µ M ). Phorbol myristate-induced PKC down-regulation or inhibition with chelerythrine provided protection against TMT-induced cytotoxicity. It was concluded that TMT-induced PKC translocation and activation contribute to the cytotoxicity of TMT in differentiated PC12 cells.     

Different GABAB-Mediated Effects on Protein Kinase C Activity and Immunoreactivity in Neonatal and Adult Rat Hippocampal Slices

Abstract: The effects of GABA on protein kinase C (PKC) were investigated in rat hippocampal slices at various postnatal ages [postnatal day (P) 1-P60]. At P4, GABA (300 µ M ) induced a rapid (in 1–2 min) 40–50% increase of PKC activity in the membrane fraction and a decrease in the cytosol. These effects were mediated by GABA_B receptors because (a) they were neither blocked by 10 µ M bicuculline nor reproduced by 10 µ M isoguvacine and (b) they were mimicked by the GABA_B agonist baclofen (3–30 µ M ), an effect fully antagonized by the GABA_B antagonist 2-hydroxysaclofen (10 µ M ). A baclofen-induced increased PKC activity in the membrane fraction was only present during the early postnatal period (P1–P14); it was associated with a translocation from the cytosol to the membrane of the immunoreactivity of some PKC isoforms (α-, β-, and ε-PKCs). In contrast, after P21, PKC activity and α-, β-, ε-, and γ-PKC immunoreactivities were decreased by baclofen in the membrane fraction and increased in the cytosol. These results suggest that the stimulation of GABA_B receptors differentially modulates PKC activity via distinct second messenger pathways in developing and mature hippocampi.     

Muscarinic Receptor-Mediated Increase in Extracellular Inositol 1,4,5-Trisphosphate Levels in the Rat Hippocampus: An In Vivo Microdialysis Study

Abstract: The extracellular concentration of inositol 1,4,5-trisphosphate (IP₃) has been monitored in the ventral hippocampus of the anesthetized rat by using a microdialysis technique coupled to a radioreceptor assay. Three hours after the implantation of the cannula, basal extracellular concentration of IP₃ (corrected for a 9% recovery) was 71 n M (0.39 pmol/60-µl fraction) and remained stable for at least 5 h. Local infusion of carbachol for 60 min caused a significant concentration-related increase in extracellular IP₃ levels (0, 24, and 57% at 1, 50, and 100 µ M , respectively). Acetylcholine (100 µ M ) and muscarine (100 µ M ) increased IP₃ outflow by 40 and 42%, respectively. The effect of carbachol was fully prevented by coinfusion of 10 µ M pirenzepine and reduced by 1 µ M tetrodotoxin indicating that the carbachol response is mediated by neuronal muscarinic receptors. These data demonstrate the feasibility of using microdialysis and a radioreceptor assay to measure IP₃ in the extracellular space. This approach could prove useful for the study of the in vivo operation of muscarinic and, by extension, a number of receptors coupled to phosphoinositide turnover.