Involvement of GABA systems in acetylcholine release induced by 5-HT3 receptor blockade in slices from rat entorhinal cortex

The aim of the present study was to examine the role of 5-HT₃ receptors in spontaneous and K⁺-evoked acetylcholine (ACh) release from rat entorhinal cortex and striatal slices. The 5-HT3 receptor antagonists ondansetron and granisetron (0.01–10 μM) produced a concentration-dependent increase in both spontaneous and K⁺-evoked [³H]ACh release in the two brain regions studied. The release of ACh was Ca²⁺-dependent and tetrodotoxin-sensitive. 5-HT₃ receptor agonists, such as 2-methyl-5-HT and 1-phenylbiguanide, at concentrations up to 1 μM, did not show any intrinsic effect on [³H]ACh release in both rat brain regions. However, 2-methyl-5-HT, 1 μM, fully blocked the ondansetron-induced enhancement in both basal and K⁺-evoked ACh release, suggesting that 5-HT₃ through 5-HT₃ receptor activation, tonically inhibits ACh release. The possible implication of interposed inhibitory systems in ACh release after 5-HT₃ receptor blockade was subsequently analyzed. While the effect of ondansetron was not modified by haloperidol or naloxone, the GABA_A receptor antagonist bicuculline produced a marked potentiation of ACh release in the entorhinal cortex but not in the striatum. The results suggest that in this cortical area 5-HT activates 5-HT₃ receptors located on GABAergic neurons which in turn inhibit cholinergic function.     

KATP channel blockers selectively interact with A1-adenosine receptor mediated modulation of acetylcholine release in the rat hippocampus

In this study the role of ATP-sensitive K⁺ channels (K_ATP channels) in the A₁ receptor mediated presynaptic inhibitory modulation of acetylcholine release was investigated in the rat hippocampus. N⁶-Cyclohexyladenosine (CHA), the selective A₁-adenosine receptor agonist, reduced concentration-dependently the stimulation-evoked (2 Hz, 1 ms, 240 shocks) [³H]acetylcholine ([³H]ACh) release, from in vitro superfused hippocampal slices preloaded with [³H]choline, an effect prevented by the selective A₁ receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). By themselves, neither K_ATP channel openers, i.e. diazoxide, pinacidil and cromakalim, nor glibenclamide and glipizide, the inhibitors of K_ATP channels, exerted a significant effect on the resting and evoked release of [³H]ACh. Glibenclamide and glipizide (10–100 μM) completely prevented the inhibitory effect of 0.1 μM CHA and shifted the concentration response curve of CHA to the right. 4-Aminopyridine (10–100 μM), the non-selective potassium channel blocker, increased the evoked release of [³H]ACh, but in the presence of 4-aminopyridine, the inhibitory effect of CHA (0.1 μM) still persisted. Oxotremorine, the M₂ muscarinic receptor agonist, decreased the stimulation-evoked release of [³H]ACh, but its effect was not reversed by glibenclamide. 1,3-Diethyl-8-phenylxanthine (DPX), the selective A₁-antagonist, effectively displaced [³H]DPCPX in binding experiments, while in the case of glibenclamide and glipizide, only slight displacement was observed. In summary, our results suggest that K_ATP channels are functionally coupled to A₁ receptors present on cholinergic terminals of the hippocampus, and glibenclamide and glipizide, by interacting with K_ATP channels, relieve this inhibitory neuromodulation.     

Evidence for 5-HT4 receptor involvement in the enhancement of acetylcholine release by p-chloroamphetamine in rat frontal cortex

The roles of endogenous serotonin (5-HT) and 5-HT receptor subtypes in regulation of acetylcholine (ACh) release in frontal cortex of conscious rats were examined using a microdialysis technique. Systemic administration (1 and 3 mg/kg, i.p.) of the 5-HT-releasing agent p-chloroamphetamine (PCA) elevated ACh output in a dose-dependent manner. Depletion of endogenous 5-HT by p-chlorophenylalanine significantly attenuated the facilitatory effect of PCA on ACh release. The PCA (3 mg/kg)-induced increase in ACh release was significantly inhibited by local application of the 5-HT₄ receptor antagonists RS23597 (50 μM) and GR113803 (1 μM), while the 5-HT_1A antagonist WAY-100135 (10 mg/kg, i.p.; 100 μM), 5-HT_1A/1B/β-adrenoceptor antagonists (−)-pindolol (8 mg/kg, i.p.) and (−)-propranolol (150 μM), 5-HT_2A/2C antagonist ritanserin (1 mg/kg, i.p.; 10 μM) and 5-HT₃ antagonist ondansetron (1 mg/kg, i.p.; 10 μM) failed to significantly modify the effect of PCA. These results suggest that PCA-induced enhancement of 5-HT transmission facilitates ACh release from rat frontal cortex at least in part through 5-HT₄ receptors.     

Regulation of Dopamine D1 and D2 Receptors on Striatal Acetylcholine Release in Rats

The effects of dopamine (DA) D₁ and D₂ receptors on striatal acetylcholine (ACh) releases were investigated by in vivo microdialysis. All drugs were applied via dialysis membrane directly to the striatum. The levels of ACh release were increased by 10⁻⁴ M SKF38393, a D₁ receptor agonist. Although 10⁻⁴ M SCH23390, a D₁ receptor antagonist, exhibited an increase in the levels of ACh release, the agonist (10⁻⁴ M) induced-increase in the levels of ACh release was suppressed by coperfusion of the antagonist (10⁻⁴ M). In contrast, the levels of ACh release were decreased by the D₂ receptor agonist, N-434, in a dose-dependent manner (10⁻⁵ M to 10⁻⁷ M) and increased by the D₂ receptor antagonist, sulpiride, in a dose-dependent manner (10⁻⁵ M to 10⁻⁷ M). The agonist (10⁻⁵ M) induced-decrease in the levels of ACh release was suppressed by coperfusion of the antagonist (10⁻⁶ M). Coperfusion of D₁ (10⁻⁴ M) and D₂ (10⁻⁵ M) agonists blocked both effects of respective drug alone. In order to clarify the effect of endogenous DA, two drugs with different mechanisms for enhancing DA concentration in the synaptic cleft, the DA release-inducer methamphetamine, and the DA uptake inhibitor nomifensine were perfused separately. Both (10⁻⁴ M to 10⁻⁶ M) produced a dose- and a time-dependent decrease in the levels of ACh release. Significant higher levels of ACh release were observed in the striatum of the 6-hydroxydopamine (8

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)-treated rats with significant depletion of striatal DA content. These results suggest that in striatal DA-ACh interaction ACh release, as cholinergic interneuron's activity, is tonically inhibited via the D₂ receptor, mainly by dopaminergic input, and the D₁ receptor probably modifies the effect of the D₂ receptor indirectly.     

Biphasic effect of tacrine on acetylcholine release in rat brain via M1 and M2 receptors

Rat cortical synaptosomes preloaded with [³H]choline were superfused and stimulated with K⁺ in order to investigate the effect of the cholinesterase inhibitor tacrine on the in vitro release of acetylcholine (ACh). Tacrine was found to biphasically both increase (10⁻⁶ and 5 × 10⁻⁶ M) and decrease (10⁻⁵−10-⁻⁴ M) the release of ACh in a concentration-dependent manner. The facilitatory effect of tacrine was prevented by atropine and the M₁ antagonist pirenzepine, whereas the inhibitory effect induced by tacrine was blocked by atropine and the M₂ antagonist AF-DX 116. These results indicate that tacrine causes a biphasic effect on K⁺ stimulated ACh release in the brain via M₁ and M₂ muscarinic receptors. The tacrine induced enhancement of the ACh release occurs at clinical relevant tacrine concentrations and might therefore be of importance for the treatment outcome of Alzheimer's disease.     

5-HT2 receptor regulation of acetylcholine release induced by dopaminergic stimulation in rat striatal slices

The role of 5-hydroxytryptamine (5-HT) receptor subtypes in acetylcholine (ACh) release induced by dopamine or neurokinin receptor stimulation was studied in rat striatal slices. The dopamine D₁ receptor agonist SKF 38393 potentiated in a tetrodotoxin-sensitive manner the K⁺-evoked [³H]ACh release while SCH 23390, a dopamine D₁ receptor antagonist, had no effect. [³H]ACh release was decreased by the dopamine D₂ receptor agonist LY 171555 (quinpirole) and slightly potentiated by the dopamine D₂ receptor antagonist haloperidol. The selective neurokinin NK₁ receptor agonist [Sar⁹, met(O₂)¹¹]SP also potentiated K⁺-evoked release of [³H]ACh. GR 82334, a NK₁ receptor antagonist, blocked not only the effect of [Sar⁹, met(O₂)¹¹]SP but also the release of ACh induced by the D₁ receptor agonist SKF 38393. Among the 5-HT agents studied, only the 5-HT_2A receptor antagonists ketanserin and ritanserin were able to reduce the ACh release induced by dopamine D₁ receptor stimulation. Mesulergine, a more selective 5-HT_2C antagonist, showed an intrinsic releasing effect but did not affect K⁺-evoked ACh release induced by SKF 38393. Methysergide and methiothepin, mixed 5-HT_1/2 antagonists, as well as ondansetron, a 5-HT₃ receptor antagonist, showed an intrinsic effect on ACh release, their effects being additive to that of SKF 38393. 5-HT₂ receptor agonists were ineffective. However, the 5-HT₂ agonist DOI was able to prevent the antagonism by ketanserin of the increased [³H]ACh efflux elicited by SKF 38393, suggesting a permissive role of 5-HT_2A receptors. None of the above indicated 5-HT agents was able to reduce the ACh release induced by the selective NK₁ agonist. The results suggest that 5-HT₂ receptors, probably of the 5-HT_2A subtype, modulate the release of ACh observed in slices from the rat striatum after stimulation of dopamine D₁ receptors. It seems that this serotonergic control is exerted on the interposed collaterals of substance P-containing neurons which promote ACh efflux through activation of NK₁ receptors located on cholinergic interneurons.     

Partial characterization of K-induced increase in [Ca]cyt and GnRH release in GT1-7 neurons

Secretion of pituitary gonadotropins is regulated centrally by the hypothalamic decapeptide gonadotropin releasing hormone (GnRH). Using the immortalized hypothalamic GT1-7 neuron, we characterized pharmacologically the dynamics of cytosolic Ca²⁺ and GnRH release in response to K⁺-induced depolarization of GT1-7 neurons. Our results showed that K⁺ concentrations from 7.5 to 60 mM increased [Ca²⁺]_cyt in a concentration-dependent manner. Resting [Ca²⁺]_cyt in GT1-7 cells was determined to be 69.7 ± 4.0 nM (mean ± S.E.M.; N = 69). K⁺-induced increases in [Ca²⁺]_cyt ranged from 58.2 nM at 7.5 mM [K⁺] to 347 nM at 60 mM [K⁺]. K⁺-induced GnRH release ranged from about 10 pg/ml at 7.5 mM [K⁺] to about 60 pg/ml at 45 mM [K⁺]. K⁺-induced increases in [Ca²⁺]_cyt and GnRH release were enhanced by 1 μM BayK 8644, an L-type Ca²⁺ channel agonist. The BayK enhancement was completely inhibited by 1 μM nimodipine, an L-type Ca²⁺ channel antagonist. Nimodipine (1 μM) alone partially inhibited K⁺-induced increases in [Ca²⁺]_cyt and GnRH release. Conotoxin (1 μM) alone had no effect on K⁺-induced GnRH release or [Ca²⁺]_cyt, but the combination of conotoxin (1 μM) and nimodipine (1 μM) inhibited K⁺-induced increase in [Ca²⁺]_cyt significantly more (p < 0.02) than nimodipine alone, suggesting that N-type Ca²⁺ channels exist in GT1-7 neurons and may be part of the response to K⁺. The response of [Ca²⁺]_cyt to K⁺ was linear with increasing [K⁺] whereas the response of GnRH release to increasing [K⁺] appeared to be saturable. K⁺-induced increase in [Ca²⁺]_cyt and GnRH release required extracellular [Ca²⁺]. These experiments suggest that voltage dependent N- and L-type Ca²⁺ channels are present in immortalized GT1-7 neurons and that GnRH release is, at least in part, dependent on these channels for release of GnRH.     

Effects of Pb and Cd on acetylcholine release and Ca movements in synaptosomes and subcellular fractions from rat brain and Torpedo electric organ

In this work we examined the effects of Pb²⁺ and Cd²⁺ on (a) [³H]ACh release and voltage-sensitive Ca²⁺ channels in rat brain synaptosomes, and (b)⁴⁵Ca²⁺ binding to isolated brain mitochondria and microsomes, and synaptic vesicles isolated from Torpedo electric organs. Pb²⁺ (K_i ≈ 1.1 μM) and Cd²⁺ (K_i ≈ 2.2) competitively block the K⁺-evoked influx of⁴⁵Ca²⁺ through the ‘fast’ calcium channels in synaptosomes. The K_is obtained with synaptosomes are in good agreement with the K_i values obtained from electrophysiological experiments at the frog neuromuscular junction (K_Pb:0.99 μM, K_Cd: 1.7 μM)⁷. The K_i for the inhibition of ACh release from synaptosomes by Cd²⁺ is 4.5 μM. Pb²⁺ is a less effective inhibitor of transmitter release (K_i ≈ 16 μM) because it secondarily augments spontaneous transmitter efflux. Cd²⁺ has no effect on spontaneous release at concentrations ≤ 100 μM. The enhancing effect of Pb²⁺ on spontaneous release is (a) not abolished by omission of Ca²⁺ from the bathing medium, (b) is delayed by 1–2 min after the beginning of Pb²⁺ exposure, (c) is reversed upon the removal of Pb²⁺. In the presence of physiological concentrations of ATP (1 mM), Mg²⁺ (1 mM) and P_i (2 mM), 1–10 μM Pb²⁺ inhibits calcium uptake but Pb²⁺ > 10μM causes a several-fold stimulation of passive binding of calcium to the organelles. This effect is associated with Pb²⁺-induced enhancement of P_i uptake. Cd²⁺ inhibits Ca²⁺ binding at all concentrations tested (1–50 μM) and reduces the Pb²⁺-induced Ca²⁺-binding to organelles. Neither Pb²⁺ nor Cd²⁺ have any discernible effects on spontaneous loss of calcium from mitochondria or microsomes preloaded with⁴⁵Ca. In summary, these data are consistent with the notion that Pb²⁺ and Cd²⁺ are potent blockers of presynaptic voltage-sensitive Ca²⁺ channels and the evoked release of transmitter which is contingent on Ca²⁺ influx through these channels. Our results are not consistent with the hypothesis that Pb²⁺ augments spontaneous release by interfering with intraterminal Ca²⁺-buffering by mitochondria, endoplasmic reticulum, or synaptic vesicles.     

Blockade of bepridil on IA and IK in acutely isolated hippocampal CA1 neurons

The effects of bepridil, an antianginal agent with antiarrhythmic action, on voltage-dependent K⁺ currents in the CA1 pyramidal neurons acutely isolated from rat hippocampus were studied by means of whole-cell patch clamp techniques. Current recordings were made in the presence of TTX to block Na⁺ current. Depolarizing test pulses activated two components of outward K⁺ currents: a rapidly activating and inactivating component, I_A; and a delayed component, I_K. Results showed that bepridil reduced the amplitude of I_A and I_K, and exerted its inhibitory action in time- and dose-dependent manner. Half-blocking concentrations (IC₅₀) of bepridil on I_A and I_K were 17.8 μM and 1.7 μM, respectively. 10 μM bepridil suppressed I_A and I_K by 46.7% and 77.1% at +30 mV of depolarization, respectively. When I_K was activated nearly uncontaminated with I_A by holding at −50 mV, 10 μM bepridil inhibited I_K by 71.6% at +30 mV of depolarization; 10 μM bepridil positively shifted the voltage-dependent of activation curves of I_A and I_K 12.1 mV and 28.7 mV, respectively. These results suggested that blockade on K⁺ currents by bepridil is preferential for I_K, and contributes to the protection brain against ischemic damage.     

Different pathways by which extracellular Ca promotes calcitonin gene-related peptide release from central terminals of capsaicin-sensitive afferents of guinea pigs: effect of capsaicin, high K and low pH media

Different modes by which Ca²⁺, entering the nerve terminal, promotes transmitter secretion as well as the ability of protons to release neuropeptides, have been shown in peripheral endings of capsaicin-sensitive afferents. We have studied these two aspects in the central endings of these neurons by measuring the release of calcitonin-gene related peptide-like immunoreactivity (CGRP-LI) from slices of the dorsal half of the guinea pig spinal cord. Altough capsaicin (1 μM) released both CGRP-LI and substance P-like immunoreactivity (SP-LI), CGRP-LI was chosen as the sole suitable marker of peptides released from central terminals of capsaicin-sensitive afferents, since after in vitro desensitization to capsaicin (1μM capsaicin for 20 min), high K⁺ (80 mM) failed to evoke CGRP-LI release, whereas SP-LI release was still observed. The capsaicin (1 μM)-evoked CGRP-LI release was entirely dependent on extracellular Ca²⁺. It was unaffected by 0.3 μM tetrodotoxin (TTX), slightly reduced by 0.1 μM ω-conotoxin (CTX) and blocked by 10 μM Ruthenium red (RR). The Ca²⁺-dependent K⁺ (80 mM)-evoked CGRP-LI release was unaffected by TTX, markedly reduced by CTX and only moderatedly inhibited by RR. Low pH (pH 5) produced a remarkable increase in CGRP-LI outflow that was abolished after exposure to capsaicin, reduced by about 50% in Ca²⁺-free medium and unaffected by TTX (0.3 μM). The Ca²⁺-dependent component of the proton-evoked CGRP-LI release was abolished in the presence of RR (10 μM) and slightly inhibited by CTX (0.1 μM). The mode by which capsaicin or high K⁺ promote Ca²⁺ entry into the central endings of capsaicin-sensitive afferents, and hence promote neuropeptide release may be distinguished on a pharmacological basis. Protons release CGRP in the spinal cord by a mechanism that shares a common pathway with that activated by capsaicin.     

Blockade of N- and Q-type Ca channels inhibit K-evoked [H]acetylcholine release in rat hippocampal slices

In the present study, we examined the contribution of specific Ca²⁺ channels to K⁺-evoked hippocampal acetylcholine (ACh) release using [³H]choline loaded hippocampal slices. [³H]ACh release was Ca²⁺-dependent, blocked by the nonspecific Ca²⁺ channel blocker verapamil, but not by blockade of L-type Ca²⁺ channels. The N-type Ca²⁺ channel blocker, ω-conotoxin GVIA (ω-CgTx GVIA; 250 nM) inhibited [³H]ACh release by 44% and the P/Q-type Ca²⁺ channel blocker ω-agatoxin IVA (ω-Aga IVA; 400 nM) inhibited [³H]ACh release by 27%, with the combination resulting in a nearly additive 79% inhibition. Four hundred or one thousand nM ω-Aga IVa was necessary to inhibit [³H]ACh release, ω-Conotoxin MVIIC (ω-CTx-MVIIC) was used after first blocking N-type Ca²⁺ channels with ω-CgTx GVIA (1 μM). Under these conditions, 500 nM ω-CTx-MVIIC led to a nearly maximal inhibition of the ω-CgTx GVIA-insensitive [³H]ACh release. Based on earlier reports about the relative sensitivity of cloned and native Ca²⁺ channels to these toxins, this study indicates that N- and Q-type Ca²⁺ channels primarily mediate K⁺-evoked hippocampal [³H]ACh release.     

Differential effects of the adenosine A1 receptor allosteric enhancer PD 81,723 on agonist binding to brain and adipocyte membranes

The benzoylthiophene analog, PD 81,723, has been shown to allosterically enhance agonist binding and functional activation of the mammalian adenosine (ADO) A₁ receptor subtype by putatively maintaining the receptor in a high affinity state. The present studies were conducted to evaluate the ability of PD 81,723 to enhance the binding of [³H]cyclohexyladenosine ([³H]CHA) to A₁ receptors of neural (cerebral cortex) and non-neural (adipocyte) origin in three different species; rat, guinea pig and dog. PD 81,723 (0.3–100 μM) produced a concentration-dependent enhancement of [³H]CHA binding to rat brain A₁ receptors. These effects were also species-dependent with larger enhancements (150–200% of control) observed in guinea pig and dog brain membranes as compared to the rat (120% of control). In contrast, PD 81,723 did not produce any enhancement of [³H]CHA binding to A₁ receptors in adipocyte membranes from any of the species examined. Additional binding studies were conducted using pharmacological manipulations that have previously been shown to enhance the allosteric effects of PD 81,723. In the presence of 1 mM GTP, the allosteric effects of PD 81,723 (15 μM) were increased in rat, guinea pig and dog brain membranes, however, in adipocyte membranes from each species, no significant alteration in agonist binding was observed. Similarly, the A₁ receptor selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (added to effectively reduce the intrinsic antagonist properties of PD 81,723) was found to enhance the allosteric effects of PD 81,723 (15 μM) in brain, but produce no alteration of agonist binding in adipocyte membranes from each species. Examination of the dissociation kinetics of [³H]CHA binding from rat brain and adipocyte membranes revealed that PD 81,723 (15 μM) differentially slowed agonist dissociation from brain, but not adipocyte, membranes. Taken together, the present data support the hypothesis that in tissues that are sensitive to PD 81,723, this benzyolthiophene functions to maintain the A₁ receptor in a high-affinity state and that the relative proportions of high-affinity A₁ receptors present in specific tissues may contribute, at least in part, to the apparent differential effects of PD 81,723 on agonist binding. The tissue specific modulation of A₁ receptor function by PD 81,723 also illustrates the possibility that the locus of allosteric modulation by PD 81,723 may be manifest via a specific, but indirect and tissue-dependent, interaction with the A₁ receptor.     

Modulation of noradrenaline release from hippocampal slices by hexachlorocyclohexane isomers. Effects of GABAergic compounds

The effects of hexachlorocyclohexane (HCH) isomers and some GABAergic compounds on [³H]noradrenaline (NA) release from rat hippocampal slices prelabelled with 80 nM [³H]NA were determined. The convulsant γ-HCH isomer facilitated (EC₅₀ = 21 μM) and the depressant δ-HCH isomer reduced (EC₅₀ = 48 μM) the Ca²⁺-dependent K⁺-evoked release of [³H]NA, whereas α- and β-HCH isomers did not show any effect. Moreover, α- and δ-HCH isomers antagonized the facilitation of evoked [³H]NA release induced by the γ-HCH isomer. The GABAergic convulsant drugs, bicuculline, picrotoxin and pentylenetetrazol, did not cause any modification of the evoked [³H]NA release even at high concentrations. Neither bicuculline nor picrotoxin blocked the effects of HCH isomers on K⁺-evoked release of [³H]NA. Exposure of slices to diazepam reduced the K⁺-evoked release of [³H]NA (EC₅₀ = 33 μM) in a manner similar to that of the δ-HCH isomer. In addition, diazepam (50 μM) blocked the γ-HCH effect and caused an additive inhibitory response with the δ-HCH isomer. On the other hand, diazepam and δ-HCH induced a time-dependent Ca²⁺-independent enhancement of basal [³H]NA release. The results suggest that modulation of [³H]NA release in the hippocampus by HCH isomers may be involved in the central actions of these compounds, and that sites other than the classic GABA_A receptor may underlie their presynaptic mechanisms of action.     

Effects of taurine on depolarization-evoked release of amino acids from rat cortical synaptosomes

Effects of taurine on endogenous aspartic acid (Asp), glutamic acid (Glu) and γ-aminobutyric acid (GABA) release has been investigated using synaptosomes prepared from rat cerebral cortex. Although basal release of these amino acids was not affected, taurine inhibited KCl (30 mM)-evoked overflow of Asp, Glu and GABA in a concentration-dependent manner with potencies (IC₅₀) of 1 μM, 0.8 μM and 5 nM, respectively. Taurine (10 μM) maximally inhibited K⁺-evoked Asp, Glu and GABA overflow by 28, 37 and 65%, respectively. Phaclofen (10 μM, a GABA_B receptor antagonist), but not bicuculline (10 μM, a GABA_A receptor antagonist), counteracted the inhibition of GABA overflow, although the inhibition of Asp and Glu overflow was not attenuated. These data suggest that taurine may inhibit GABA release through the activation of presynaptic GABA_B autoreceptors and, at high concentration, also act on Asp- and Glu-nerve terminals to regulate release of excitatory amino acids in rat cortex.     

Adenosine A1 receptors inhibit Ca channels coupled to the release of ACh, but not of GABA, in cultured retina cells

We investigated the effect of adenosine A₁ receptors on the release of acetylcholine (ACh) and GABA, and on the intracellular calcium concentration ([Ca²⁺]_i) response in cultured chick amacrine-like neurons, stimulated by KCl depolarization. The KCl-induced release of [³H]ACh, but not the release of [¹⁴C]GABA, was potentiated when adenosine A₁ receptor activation was prevented by perfusing the cells with adenosine deaminase (ADA) or with 1,3-dipropyl-8-cycloentylxanthine (DPCPX). The changes in the [Ca²⁺]_i induced by KCl depolarization, measured in neurite segments of single cultured cells, were also modulated by endogenous adenosine, acting on adenosine A₁ receptors. Our results show that adenosine A₁ receptors inhibit Ca²⁺ entry coupled to ACh release, but not to the release of GABA, suggesting that the synaptic vesicles containing each neurotransmitter are located in different zones of the neurites, containing different VSCC and/or different densities of adenosine A₁ receptors.     

Cerebellar CB1 receptor mediation of Δ-THC-induced motor incoordination and its potentiation by ethanol and modulation by the cerebellar adenosinergic A1 receptor in the mouse

The effect of intracerebellar microinfusion of antisense oligodeoxynucleotide to Δ⁹-tetrahydrocannabinol (Δ⁹-THC) and other naturally occurring cannabinoid receptor (CB₁) mRNA on Δ⁹-THC-induced motor impairment was investigated in mice. Δ⁹-THC (15–30 μg/μl; intracerebellar) resulted in a significant motor impairment in a dose-related manner. The intracerebellar pretreatment with antisense oligodeoxynucleotide (3.0 μg/100 nl/12 h; six administrations/mouse) virtually abolished Δ⁹-THC (15 and 25 μg/1 μl; intracerebellar)-induced motor impairment. However, intracerebellar pretreatment with the mismatched oligodeoxynucleotide in exactly the same manner as the antisense was completely ineffective in altering the Δ⁹-THC-induced motor impairment. These results strongly suggest the involvement of CB₁ receptor in the expression of Δ⁹-THC-induced motor impairment. The intracerebellar microinfusion of adenosine A₁-selective agonist, N⁶-cyclohexyladenosine (CHA) (4 ng/100 nl) significantly enhanced Δ⁹-THC-induced motor impairment, suggesting a cerebellar A₁ adenosinergic modulation of motor impairment. A pretreatment with the antisense and the mismatched oligodeoxynucleotide also markedly attenuated and did not alter, respectively, the cerebellar A₁ adenosinergic modulation (enhancement) of Δ⁹-THC-induced motor impairment. There was no change in the normal motor coordination due to intracerebellar pretreatment with antisense and its mismatch, in the presence as well as absence of intracerebellar CHA indicating the selectivity of interactions with Δ⁹-THC. The Δ⁹-THC-induced motor incoordination was also significantly enhanced dose-dependently by systemic (i.p.) ethanol administration suggesting behavioral synergism between the two psychoactive drugs. Pretreatment (intracerebellar) with pertussis toxin (PTX) markedly attenuated Δ⁹-THC- and Δ⁹-THC+CHA-induced motor incoordination suggesting coupling of CB₁ receptor to PTX-sensitive G-protein (G_i/G_o). These data suggested co-modulation by cerebellar cannabinoid and adenosine system of Δ⁹-THC-induced motor impairment. Conversely, the results in the present study also suggested co-modulation by cerebellar adenosine A₁ and CB₁ receptors of ethanol-induced motor impairment, thereby indicating a possible common signal transduction pathway in the expression of motor impairment produced by Δ⁹-THC as well as ethanol.     

Arachidonic acid and prostaglandin D2 cooperatively accelerate desensitization of nicotinic acetylcholine receptor channel in mouse skeletal muscles

To clarify the effects of arachidonic acid (AA) and its metabolites on desensitization of nicotinic acetylcholine (ACh) receptor channel in mouse skeletal muscle cells, we investigated the time-dependent decrease in the channel opening frequency of ACh (1 μM)-activated channel currents by the cell-attached patch clamp technique. AA (30–100 μM) applied to a patched membrane or to non-patched membrane accelerated the decrease in the channel opening frequency. A cyclooxygenase inhibitor, indomethacin (10 μM), prevented the acceleration elicited by 30 μM AA, but not by 100 μM AA. A lipoxygenase inhibitor, nordihydroguaiaretic acid (10 μM), and a cytochrome P-450 inhibitor, ketoconazole (3 μM), did not affect the acceleration by 30 μM AA. Prostaglandin (PG) D₂ at 10 μM alone and at 25 nM in combination with 10 μM AA accelerated the decrease in the channel opening frequency. No acceleration was observed with PGE₂ at 10 μM alone and at 25 nM in combination with 10 μM AA. Pretreatment with a protein kinase (PK) C inhibitor, staurosporine (10 nM), but not with a PKA inhibitor, H-89 (3 μM), prevented the acceleration elicited by AA+PGD₂. These results suggest that AA, and PGD₂ of its metabolites, cooperatively accelerate desensitization of nicotinic ACh receptor channel. The activation of PKC by AA and PGD₂ may be involved in the mechanism of the cooperative acceleration of desensitization.     

5-HT1A and 5-HT2 receptors differentially regulate the excitability of 5-HT-containing neurones of the guinea pig dorsal raphe nucleus in vitro

We have used intracellular recording techniques to examine the effects of 5-hydroxytryptamine (5-HT, serotonin) on 5-HT-containing neurones of the guinea pig dorsal raphe nucleus in vitro. Bath-applied 5-HT (30–300 μM) had two opposing effects on the membrane excitability of these cells, reflecting the activation of distinct 5-HT receptor subtypes. As demonstrated previously in the rat, 5-HT evoked a hyperpolarization and inhibition of 5-HT neurones, which appeared to involve the activation of an inwardly rectifying K⁺ conductance. This hyperpolarizing response was blocked by the 5-HT_1A receptor-selective antagonist WAY-100635 (30–100 nM). In the presence of WAY-100635, 5-HT induced a previously unreported depolarizing, excitatory response of these cells, which was often associated with an increase in the apparent input resistance of the neurone, likely due to the suppression of a K⁺ conductance. Like the hyperpolarizing response to 5-HT, this depolarization could be recorded in the presence of the Na⁺ channel blocker tetrodotoxin. In addition, the response was not significantly attenuated by the α₁-adrenoceptor antagonist prazosin (500 nM), indicating that it is not due to the release of noradrenaline, or to the direct activation of α₁-adrenoceptors by 5-HT. The 5-HT₃ receptor antagonist granisetron (1 μM) and the 5-HT₄ receptor antagonist SB 204070 (100 nM) failed to reduce the depolarizing response to 5-HT; however, ketanserin (100 nM), mesulergine (100 nM) and lysergic acid diethylamide (1 μM) significantly reduced or abolished the depolarization, indicating that this effect of 5-HT is mediated by 5-HT₂ receptors.     

Inhibition of glutamate release: A potential mechanism of action for the anticonvulsant U-54494A in the guinea pig hippocampus

U-54494A, a 1,2-diamine, is a potent inhibitor of glutamate release in a synaptosomal preparation that is highly enriched with hippocampal mossy fiber (MF) nerve endings. At a concentration of 100 μM, U-54494A significantly reduced the availability of cytosolic free calcium (Ca²⁺) in depolarized MF-enriched synaptosomes by 30% and inhibited the K⁺-evoked release of endogenous glutamate by 85%. The extent to which glutamate release was inhibited allows us to conclude that U-54494A acts directly on the MF subpopulation of glutamatergic nerve endings in the guinea pig hippocampus. In addition, this anticonvulsant effectively countered the presynaptic facilitation of K⁺-evoked glutamate release that is induced by kainic acid (KA). Thus, while KA (1 mM) by itself nearly doubled the rate of K⁺-evoked glutamate release, there was no net increase in the presence of both KA and U-54494A (100 μM). However, the opposed effects of these two compounds on glutamate release do not appear to be due to a direct interaction. In the presence of U-54494A (100 μM), KA (1 mM) significantly enhanced the K⁺-evoked release of glutamate. Finally, it is demonstrated that the KA-induced enhancement of glutamate release does not require the depolarization-induced entry of extracellular Ca²⁺.     

Plasmalogens, phospholipases A2 and signal transduction

Several lines of evidence indicate that the breakdown of plasmalogens in neural membranes during neurodegenerative diseases is a receptor-mediated process catalyzed by a plasmalogen-selective phospholipase A₂. This enzyme has recently been purified from bovine brain. It does not require Ca²⁺ and is localized in cytosol. It has a molecular mass of 39 kDa and is strongly inhibited by glycosaminoglycans, with the pattern of inhibition being heparan sulfate0 > hyaluronic acid > chondroitin sulfate > heparin. This plasmalogen-selective phospholipase A₂ is also inhibited by gangliosides and sialoglycoproteins. Substrate specificity and the effects of metal ions, detergents and inhibitors suggest that this phospholipase A₂ is different from the well-known 85 kDa Ca²⁺-dependent cytosolic phospholipase A₂ that has recently been cloned and is not plasmalogen-selective. The plasmalogen-selective phospholipase A₂ may be regulated by glycosaminoglycans and sialoglycoconjugates and may be involved in the regulation of K⁺ channels. This enzyme, which plays a major role in the release of fatty acids during ischemic injury and reperfusion, shows promise as a major target for drug therapy.