Amperometric measurement of glutamate release modulation by gabapentin and pregabalin in rat neocortical slices: role of voltage-sensitive Ca2+ α2δ-1 subunit

Gabapentin (GBP; Neurontin) and pregabalin (PGB; Lyrica, S-(+)-3-isobutylgaba) are used clinically to treat several disorders associated with excessive or inappropriate excitability, including epilepsy; pain from diabetic neuropathy, postherpetic neuralgia, and fibromyalgia; and generalized anxiety disorder. The molecular basis for these drugs' therapeutic effects are believed to involve the interaction with the auxiliary α(2)δ subunit of voltage-sensitive Ca(2+) channel (VSCC) translating into a modulation of pathological neurotransmitter release. Glutamate as the primary excitatory neurotransmitter in the mammalian central nervous system contributes, under conditions of excessive glutamate release, to neurological and psychiatric disorders. This study used enzyme-based microelectrode arrays to directly measure extracellular glutamate release in rat neocortical slices and determine the modulation of this release by GBP and PGB. Both drugs attenuated K(+)-evoked glutamate release without affecting basal glutamate levels. PGB (0.1-100 μM) exhibited concentration-dependent inhibition of K(+)-evoked glutamate release with an IC(50) value of 5.3 μM. R-(-)-3-Isobutylgaba, the enantiomer of PGB, did not significantly reduce K(+)-evoked glutamate release. The decrease of K(+)-evoked glutamate release by PGB was blocked by the l-amino acid l-isoleucine, a potential endogenous ligand of the α(2)δ subunit. In neocortical slices from transgenic mice having a point mutation (i.e., R217A) of the α(2)δ-1 (subtype) subunit of VSCC, PGB did not affect K(+)-evoked glutamate release yet inhibited this release in wild-type mice. The results show that GBP and PGB attenuated stimulus-evoked glutamate release in rodent neocortical slices and that the α(2)δ-1 subunit of VSCC appears to mediate this effect.     

Gabapentin inhibits the substance P-facilitated K(+)-evoked release of [(3)H]glutamate from rat caudial trigeminal nucleus slices.

The effect of gabapentin on the release of the spinal sensory neurotransmitter glutamate has been investigated in an in vitro model using a perfused thin slice preparation from the rat brainstem containing the spinal trigeminal caudal subnucleus (Sp5C) and pre-incubated with [(3)H]glutamate. Addition of excess K(+) to the perfusing solution increased the content of tritium in the perfusate. The prior addition of substance P increased this index of glutamate release in a concentration-dependent manner, with the mean maximum of around 50% increase obtained at 1-3 microM. The action of substance P to increase the evoked release of glutamate was blocked by the antagonist CP-99994, suggesting a specific involvement of the NK(1) receptor in mediating the facilitatory effect. On its own, gabapentin at up to 100 microM did not modify the baseline level of K(+)-evoked release of glutamate; however, gabapentin caused a concentration-dependent decrease of the facilitatory effect of substance P (EC(50)=6.49 microM). The R-(-)- and S-(+)-isomers of 3-isobutylgaba were then tested against the increase in K(+)-evoked release of glutamate by substance P. S-(+)-3-isobutylgaba (pregabalin) at 30 microM acted like gabapentin to reduce the substance P-mediated increase of release almost to the baseline level of K(+)-evoked release, while in contrast the R-(-)-isomer at this concentration produced no reduction, and rather a trend towards a further enhancement of the potentiating effect of substance P. In conclusion, we have found and characterized an effect of gabapentin that is of possible mechanistic relevance to the anti-hyperalgesic/allodynic actions of this compound.     

LLC-PK(1) cells stably expressing the human norepinephrine transporter: A functional model of carrier-mediated norepinephrine release in protracted myocardial ischemia.

In myocardial ischemia, adrenergic terminals undergo ATP depletion, hypoxia, and intracellular pH reduction, causing the accumulation of axoplasmic norepinephrine (NE) and intracellular Na(+) [via the Na(+)-H(+) exchanger (NHE)]. This forces the reversal of the Na(+)- and Cl(-)-dependent NE transporter (NET), triggering massive carrier-mediated NE release and, thus, arrhythmias. We have now developed a cellular model of carrier-mediated NE release using an LLC-PK(1) cell line stably transfected with human NET cDNA (LLC-NET). LLC-NET cells transported [(3)H]NE and [(3)H]N-methyl-4-phenylpyridinium ([(3)H]MPP(+)) in an inward direction. This uptake was abolished by the NET inhibitors desipramine (100 nM) and mazindol (300 nM) and by extracellular Na(+) removal. Na(+)-gradient reversal induced an efflux of (3)H-substrate from preloaded LLC-NET cells. Desipramine and mazindol blocked this efflux. Because of its greater intracellular stability and higher sensitivity to Na(+)-gradient reversal, [(3)H]MPP(+) proved preferable to [(3)H]NE as an NET substrate; therefore, only [(3)H]MPP(+) was used for subsequent studies. The K(+)/H(+) ionophore nigericin (10 microM) evoked a large efflux of [(3)H]MPP(+). This efflux was potentiated by the Na(+),K(+)-ATPase inhibitor ouabain (100 microM), was sensitive to desipramine, and was blocked by the NHE inhibitor 5-(N-ethyl-N-isopropyl)-amiloride (EIPA; 10 microM). In contrast, EIPA failed to inhibit the [(3)H]MPP(+) efflux elicited by the Na(+) ionophore gramicidin (10 microM). Furthermore, [(3)H]MPP(+) efflux induced by the NHE-stimulant proprionate (25 mM) was negatively modulated by imidazoline receptor activation. Our findings suggest that LLC-NET cells are a sensitive model for studying transductional processes of carrier-mediated NE release associated with myocardial ischemia.     

Antidepressant drug-induced alterations in neuron-localized tumor necrosis factor-alpha mRNA and alpha(2)-adrenergic receptor sensitivity

The pleiotropic cytokine tumor necrosis factor-alpha (TNF) and alpha(2)-adrenergic receptor activation regulate norepinephrine (NE) release from neurons in the central nervous system. The present study substantiates the role of TNF as a neuromodulator and demonstrates a reciprocally permissive relationship between the biological effects of TNF and alpha(2)-adrenergic receptor activation as a mechanism of action of antidepressant drugs. Immunohistochemical analysis and in situ hybridization reveal that administration of the antidepressant drug desipramine decreases the accumulation of constitutively expressed TNF mRNA in neurons of the rat brain. Superfusion and electrical field stimulation were applied to a series of rat hippocampal brain slices to study the regulation of [(3)H]NE release. Superfusion of hippocampal slices obtained from rats chronically administered the antidepressant drug zimelidine demonstrates that TNF-mediated inhibition of [(3)H]NE release is transformed, such that [(3)H]NE release is potentiated in the presence of TNF, an effect that occurs in association with alpha(2)-adrenergic receptor activation. However, chronic zimelidine administration does not alter stimulation-evoked [(3)H]NE release, whereas chronic desipramine administration increases stimulation-evoked [(3)H]NE release and concomitantly decreases alpha(2)-adrenergic autoreceptor sensitivity. Collectively, these data support the hypothesis that chronic antidepressant drug administration alters alpha(2)-adrenergic receptor-dependent regulation of NE release. Additionally, these data demonstrate that administration of dissimilar antidepressant drugs similarly transform alpha(2)-adrenergic autoreceptors that are functionally associated with the neuromodulatory effects of TNF, suggesting a possible mechanism of action of antidepressant drugs.     

Ca2(+)-evoked [3H]dopamine release from synaptosomes is dependent on neuronal type Ca2+ channels and is not mediated by acetylcholine, glutamate or aspartate release

Elevation of potassium concentrations ([K+]) in the presence of Ca2+ is the most common method of evoking neurotransmitter release from synaptosomes. However, we have been investigating a method of releasing dopamine from synaptosomes that does not involve using elevated [K+]. In this paradigm of neurotransmitter release, dopamine is released from synaptosomes, previously exposed to micromolar or lower [Ca2+], by 1.25 mM Ca2+ in the presence of non-depolarizing [K+] (4.5 mM). The present experiments characterize the Ca2+ channel(s) involved in the Ca2(+)-evoked release of dopamine from synaptosomes, and determine whether the release is mediated by acetylcholine, glutamate or aspartate. omega-Conotoxin (10 nM), which blocks N-, L- and possibly T-type voltage-sensitive Ca2+ channels (VSCC), inhibited the Ca2(+)-evoked [3H]dopamine release from either striatal or olfactory tubercle synaptosomes to less than 50% of control. Neither 1 microM nifedipine nor 1 microM verapamil, which block L-type VSCC, affected Ca2(+)-evoked release. The N- and T-type VSCC blocker neomycin and the nonspecific Ca2+ antagonist, cobalt2+, inhibited release to a greater extent than omega-conotoxin. At 1 mM, both compounds inhibited release to approximately 30% of control. Neither the excitatory neurotransmitter glutamate nor aspartate (2mM) affected 1 microM LY-171555 (a dopamine D2 agonist) inhibition of Ca2(+)-evoked [3H]dopamine release. Also, the glutamate antagonist, glutamic acid diethyl ester, did not affect either Ca2(+)-evoked release or 1 microM LY-171555 inhibition thereof. The nicotinic antagonist hexamethonium (10 microM) and the muscarinic antagonist atropine (1 microM) were also ineffective in inhibiting Ca2(+)-evoked release or LY-171555 inhibition of release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple cellular mechanisms mediate the effect of lobeline on the release of norepinephrine

The complex effect of lobeline on [(3)H]norepinephrine ([(3)H]NE) release was investigated in this study. Lobeline-induced release of [(3)H]NE from the vas deferens was strictly concentration-dependent. In contrast, electrical stimulation-evoked release was characterized by diverse effects of lobeline depending on the concentration used: at lower concentration (10 microM), it increased the release and at high concentration (100 and 300 microM), the evoked release of [(3)H]NE was abolished. The effect of lobeline on the basal release was [Ca(2+)]-independent, insensitive to mecamylamine, a nicotinic acetylcholine receptor antagonist, and to desipramine, a noradrenaline uptake inhibitor. However, lobeline-induced release was temperature-dependent: at low temperature (12 degrees C), at which the membrane carrier proteins are inhibited, lobeline failed to increase the basal release. Lobeline dose dependently inhibited the uptake of [(3)H]NE into rat hippocampal synaptic vesicles and purified synaptosomes with IC(50) values of 1.19 +/- 0.11 and 6.53 +/- 1.37 microM, respectively. Lobeline also inhibited Ca(2+) influx induced by KCl depolarization in sympathetic neurons measured with the Fura-2 technique. In addition, phenylephrine, an alpha(1)-adrenoceptor agonist, contracted the smooth muscle of the vas deferens and enhanced stimulation-evoked contraction. Both effects were inhibited by lobeline. Our results can be best explained as a reversal of the monoamine uptake by lobeline that is facilitated by the increased intracellular NE level after lobeline blocks vesicular uptake. At high concentrations, lobeline acts as a nonselective Ca(2+) channel antagonist blocking pre- and postjunctional Ca(2+) channels serving as a counterbalance for the multiple transmitter releasing actions.     

Neurotensin modulates differently potassium, veratridine and 4-aminopyridine-evoked release of dopamine in rat striatal slices

Summary— We have studied the effects of neurotensin (NT) on the release of [³H]dopamine ([³H]DA) evoked by terminal depolarization with either K+, veratridine or 4-aminopyridine (4-AP). NT (1–1000 nM) induced a net potentiation (up to 170%) of the K+ (25 mM) -evoked release of [³H]DA. The capacity of NT to potentiate the effect of K+ ions decreased as the K+ concentration rose from 25 to 50 mM and totally disappeared at this high K+ concentration. NT (100 nM; 1000 nM) had no significant effect on the veratridine (1.5; 5μM) or 4-AP (20 μM) -evoked release of [³H]DA. The relevance of these experimental models of DA release to physiological transmitter release remains to be established. Those data highlight the complexity of the modulation of evoked neurotransmitter release by pharmacological agents.     

The selective serotonin reuptake inhibitor citalopram induces the storage of serotonin in catecholaminergic terminals

We investigated whether selective inhibition of serotonin (5-hydroxytryptamine; 5-HT) transporter with citalopram leads to accumulation of 5-HT in catecholaminergic neurons. In the rabbit olfactory tubercle, citalopram (1-10 microM) inhibited [(3)H]5-HT uptake; however, the maximal degree of inhibition achieved was 70%. Addition of nomifensine (1-10 microM) was required for complete inhibition of [(3)H]5-HT uptake. In slices labeled with 0.1 microM [(3)H]5-HT, cold 5-HT (0.03-1 microM) induced a large increase in the efflux (release) of stored [(3)H]5-HT, an effect blocked by coperfusion with 1 microM citalopram. Similar concentrations (0.03-1 microM) of norepinephrine (NE) or dopamine (DA) failed to release [(3)H]5-HT. When labeling with 0.1 microM [(3)H]5-HT was carried out in the presence of citalopram, 1) low concentrations of 5-HT failed to release [(3)H]5-HT; 2) DA and NE were more potent and effective in releasing [(3)H]5-HT than in control slices; 3) coperfusion of NE, DA, or 5-HT with citalopram enhanced the release of [(3)H]5-HT induced by the catecholamines but not by 5-HT; and 4) coperfusion of NE or DA with nomifensine antagonized NE- and DA-evoked [(3)H]5-HT release, with a greater effect on NE than on DA. These results suggest that in the rabbit olfactory tubercle, where there is coexistence of 5-HT, NE, and DA neurons, inhibition of the 5-HT transporter led to accumulation of 5-HT in catecholaminergic terminals. Thus, during treatment with selective serotonin uptake inhibitors (SSRIs), 5-HT may be stored in catecholaminergic neurons acting as a false neurotransmitter and/or affecting the disposition of DA and/or NE. Transmitter relocation may be involved in the antidepressant action of SSRIs.     

Selective depression by general anesthetics of glutamate versus GABA release from isolated cortical nerve terminals

The role of presynaptic mechanisms in general anesthetic depression of excitatory glutamatergic neurotransmission and facilitation of GABA-mediated inhibitory neurotransmission is unclear. A dual isotope method allowed simultaneous comparisons of the effects of a representative volatile (isoflurane) and intravenous (propofol) anesthetic on the release of glutamate and GABA from isolated rat cerebrocortical nerve terminals (synaptosomes). Synaptosomes were prelabeled with L-[(3)H]glutamate and [(14)C]GABA, and release was determined by superfusion with pulses of 30 mM K(+) or 1 mM 4-aminopyridine (4AP) in the absence or presence of 1.9 mM free Ca(2+). Isoflurane maximally inhibited Ca(2+)-dependent 4AP-evoked L-[(3)H]glutamate release (99 +/- 8% inhibition) to a greater extent than [(14)C]GABA release (74 +/- 6% inhibition; P = 0.023). Greater inhibition of L-[(3)H]glutamate versus [(14)C]GABA release was also observed for the Na(+) channel antagonists tetrodotoxin (99 +/- 4 versus 63 +/- 5% inhibition; P < 0.001) and riluzole (84 +/- 5 versus 52 +/- 12% inhibition; P = 0.041). Propofol did not differ in its maximum inhibition of Ca(2+)-dependent 4AP-evoked L-[(3)H]glutamate release (76 +/- 12% inhibition) compared with [(14)C]GABA (84 +/- 31% inhibition; P = 0.99) release. Neither isoflurane (1 mM) nor propofol (15 microM) affected K(+)-evoked release, consistent with a molecular target upstream of the synaptic vesicle exocytotic machinery or voltage-gated Ca(2+) channels coupled to transmitter release. These findings support selective presynaptic depression of excitatory versus inhibitory neurotransmission by clinical concentrations of isoflurane, probably as a result of Na(+) channel blockade.     

Evidence that two stereochemically different alpha-2 adrenoceptors modulate norepinephrine release in rat cerebral cortex.

Cerebral cortex slices from the rat were loaded with [3H]norepinephrine ([3H]NE) and superfused in order to measure the release of radioactivity at rest and in response to electrical stimulation. The (-)-isomer and the (+)-isomer of CH-38083 ([7,8-(methylenedioxy)-14- alpha-hydroxyalloberbane HCl), a selective alpha-2-adrenoceptor antagonist with an alloberbane skeleton, increased the electrically induced release of [3H]NE in a concentration-dependent manner, and a similar effect was observed with racemic CH-38083 and idazoxan. The stereoisomers of CH-38083 applied in a concentration range of 10(-8) to 10(-6) mol/l were equipotent in facilitating stimulation-evoked [3H]NE release: concentrations needed to enhance tritium outflow by 50% were 1.3 X 10(-7) mol/l for (-)-CH-38083 and 1.4 X 10(-7) mol/l for (+)-CH-38083. Exogenous NE decreased the electrically stimulated release of [3H]NE, and the stereoisomers of CH-38083 antagonized this inhibition with different potencies: the dissociation constant (KB) values for (-)-isomer and for (+)-isomer of CH-38083 were 14.29 and 97.18 nmol/l. These data indicate that presynaptic alpha-2 adrenoceptors that are available for NE released from axon terminals do not show stereospecificity toward enantiomers of CH-38083, whereas those that are occupied by exogenous NE are much more sensitive toward (-)-CH-38083. The alpha-1 adrenoceptor antagonist prazosin also differentiated between the alpha-2 adrenoceptor subtypes: prazosin (10(-6) mol/l) did not alter the increase of electrically induced [3H]NE release evoked by (-)- and (+)-CH-38083; however, in its presence, the stereoisomers of CH-38083 failed to antagonize the inhibitory effect of exogenous NE on its own release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serotonergic modulation of the release of endogenous norepinephrine from rat hypothalamic slices

Ca+(+)-dependent release of endogenous norepinephrine (NE) and dopamine from superfused rat hypothalamic slices was stimulated by 40 mM K+. 20 mM K+ released only NE. Two consecutive exposures to 20 mM K+ (S1 and S2, respectively) produced NE release of similar magnitude (S2/S1 = 1.03 +/- 0.08). Serotonin (5-HT), 3 to 10 microM, in the presence of methylsergide or ritanserin (antagonists at 5-HT1-like and 5-HT2 receptors), caused a concentration-dependent decrease of K(+)-evoked NE release. 5-HT alone did not alter K(+)-evoked NE release. 2-Methyl-serotonin, 2-methyl-5-hydroxytryptamine, 3 to 10 microM (a selective 5-HT3 agonist), mimicked the 5-HT response in the presence and in the absence of ritanserin. A highly selective 5-HT3 antagonist, (3 alpha-tropanyl)1H-indole-3-carboxylic acid ester (ICS 205-930), 1 nM, inhibited the effect of both agonists. The isomers of another highly selective 5-HT3 antagonist, zacopride, inhibited the effect of 2-methyl-serotonin, 2-methyl-5-hydroxytryptamine, at a concentration range, 0.03 to 20 nM, characteristic of their interaction with 5-HT3 receptors. alpha-Methyl-serotonin, alpha-methyl-5-hydroxytryptamine, a selective 5-HT1-like/5-HT2 agonist, failed to affect the K(+)-evoked NE release, but antagonized the effect of 2-methyl-serotonin, 2-methyl-5-hydroxytryptamine. These observations provide direct evidence that, in rat hypothalamus, 5-HT modulates release of endogenous NE through activation of 5-HT3 and, possibly, 5-HT1C receptors.     

Mediation of highly concentrative uptake of pregabalin by L-type amino acid transport in Chinese hamster ovary and Caco-2 cells

Pregabalin (PGB) is a novel drug under development for the treatment of epilepsy, neuropathic pain, fibromyalgia, and generalized anxiety disorder. In this study, we investigated PGB transport in rats, mammalian cell lines, and Xenopus laevis oocytes. In contrast to gabapentin (GBP), PGB absorption in rats showed unique linear pharmacokinetics. PGB entered CHO and Caco-2 cells predominately via Na(+)-independent processes. Uptake of PGB was mutually exclusive with leucine, GBP and 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid, the substrates preferential for system L. The preloaded PGB in CHO cells was exchangeable with leucine, but at a lower exchange rate than that of leucine and GBP. Dixon plots showed competitive inhibition of leucine uptake by PGB, with a K(i) value very close to the K(m) value for PGB uptake (377 versus 363 microM). At an extracellular concentration of 300 microM, the intracellular PGB concentration in CHO cells reached 1.5- and 23-fold higher than that of GBP and leucine, respectively. In contrast, at clinically relevant concentrations, PGB seemed not to interact with GABA transport in GAT1, GAT2, and GAT3 cell lines, system y(+), b(0,+), B(0,+), and B(0) transport activities in Caco-2 and NBL-1 cells, and the b(0,+)-like transport activity in rBAT cRNA-injected X. laevis oocytes. Taken together, these results suggest that L-type transport is the major transport route for PGB and GBP uptake in mammalian cells. The differential affinity of PGB and GBP at L-type system leads to more concentrative accumulation of PGB than GBP, which may facilitate PGB transmembrane absorption in vivo.     

Inhibitory effects of amphetamine on potassium-stimulated release of [3H]dopamine from striatal slices and synaptosomes

Amphetamine, 10(-7) M or greater, evoked the release of [3H]dopamine ([3H]DA) and inhibited subsequent K+-evoked [3H]DA release from striatal synaptosomes superfused at a flow rate (1 ml/min) that prevented reuptake. Amphetamine inhibited the K+-evoked release of [3H]DA to a lesser extent in striatal slices or in synaptosomes superfused at a flow rate (0.35 ml/min) that allowed reuptake. The observed decrease in amphetamine inhibition of K+-evoked release was primarily due to amphetamine blocking [3H]DA reuptake. Interneuronal interactions may account for some of the inhibitory effects of amphetamine on K+-evoked release in the slice. Inhibition of K+-evoked release from either slices or synaptosomes was still evident when 10(-6) M amphetamine was removed from the superfusion buffer and the spontaneous release had returned to control levels. The presence of Ca++ during amphetamine exposure was required for subsequent inhibition of K+-evoked release in synaptosomes. Amphetamine in the presence of Ca++ did not affect the subsequent release of [3H]DA evoked by the Ca++ ionophore, A23187. Therefore, amphetamine inhibition of the K+-evoked release of [3H]DA cannot be explained by prior depletion of Ca++-releasable pools. Nifedipine, 1 microM, failed to block either the Ca++-dependent release of [3H]DA or the inhibition of K+-evoked release by amphetamine. However, 1 mM cobalt inhibited the Ca++-dependent release of [3H]DA by amphetamine and antagonized the inhibition of K+-evoked release after amphetamine exposure. This suggests that amphetamine may open voltage-dependent Ca++ channels sensitive to cobalt but not nifedipine. Amphetamine may desensitize these voltage-dependent Ca++ channels and inhibit their activation by K+ depolarization.     

Endothelin (ET)-1-induced inhibition of ATP release from PC-12 cells is mediated by the ETB receptor: differential response to ET-1 on ATP, neuropeptide Y, and dopamine levels

During sympathetic neurotransmitter release, there is evidence for differential modulation of cotransmitter release by endothelin (ET)-1. Using nerve growth factor (NGF)-differentiated PC12 cells, the effects of ET-1 on K(+)-stimulated release of ATP, dopamine (DA), and neuropeptide Y (NPY) were quantified using high-pressure liquid chromatography or radioimmunoassay. ET-1, in a concentration-dependent manner, inhibited the release of ATP, but not DA and NPY. Preincubation with the ET(A/B) antagonist, PD 142893 (N-acetyl-beta-phenyl-D-Phe-Leu-Asp-Ile-Ile-Trp), reversed the inhibitory effect of ET-1 on ATP release, which remained unaffected in the presence of the ET(A)-specific antagonist BQ123 [cyclo(D-Asp-Pro-D-Val-Leu-D-Trp)]. The ET(B) agonists, sarafotoxin 6c (Cys-Thr-Cys-Asn-Asp-Met-Thr-Asp-Glu-Glu-Cys-Leu-Asn-Phe-Cys-His-Gln-Asp-Val-Ile-Trp), BQ 3020 (N-acetyl-[Ala(11,15)]-endothelin 1 fragment 6-21Ac-Leu-Met-Asp-Lys-Glu-Ala-Val-Tyr-Phe-Ala-His-Leu-Asp-IIe-IIe-Trp), and IRL 1620 (N-succinyl-[Glu(9), Ala(11,15)]-endothelin 1 fragment 8-21Suc-Asp-Glu-Glu-Ala-Val-Tyr-Phe-Ala-His-Leu-Asp-Ile-Ile-Trp), decreased K(+)-stimulated release of ATP in a dose-dependent manner, and this effect was reversed by the ET(B) antagonists RES 701-1 [cyclic (Gly1-Asp9) (Gly-Asn-Trp-His-Gly-Thr-Ala-Pro-Asp-Trp-Phe-Phe-Asn-Tyr-Tyr-Trp)] and BQ 788 (N-[N-[N-[(2,6-dimethyl-1-piperidinyl)carbonyl]-4-methyl-l-leucyl]-1-(methoxycarbonyl)-D-tryptophyl]-D-norleucine sodium salt). Preincubation of PC12 cells with pertussis toxin reversed the ET-1-induced inhibition of the K(+)-evoked ATP release. Real-time intracellular calcium level recordings were performed on PC-12 cell suspensions, and ET-1 induced a dose-dependent decrease in the K(+)-evoked calcium levels. Nifedipine, the L-type voltage-dependent Ca(2+) channel antagonist, caused inhibition of the K(+)-stimulated ATP release, but the N-type Ca(2+) channel antagonist, omega-conotoxin GVIA, did not reverse the effect on ATP release. These data suggest that ET-1 modulates the release of ATP via the ET(B) receptor and its associated G(i/o) G-protein through attenuation of the influx of extracellular Ca(2+) through L-type channels.     

Inhibition of [3H] γ-aminobutyric acid release from guinea-pig hippocampal synaptosomes by serotonergic agents

We studied the effects of (m-trifluoromethyl-phenyl)piperazine (TFMPP) and quipazine on the K(+)-evoked [3H]GABA release from guinea-pig hippocampal synaptosomes loaded with [3H]GABA.TFMPP and quipazine inhibited the K(+)-evoked release of [3H]GABA dose-dependently (IC50 = 153 and 123 microM, respectively). Serotonergic antagonists such as methiothepin (0.1, 0.3 and 1 microM), ketanserin (0.1, 0.3 and 1 microM), dihydroergotamine (0.1 microM), metergoline (0.1 and 0.3 microM), methysergide (0.3 microM), propranolol (1 microM) and yohimbine (1 microM) did not significantly alter the inhibitory effect of TFMPP on [3H]GABA release suggesting that neither 5-HT1 nor 5-HT2 receptors are involved in this process. By contrast, the effect of TFMPP was diminished by selective 5-HT3 receptor antagonist: MDL 72222 (0.3 microM), tropisetron (0.3 and 1 microM), ondansetron (0.3 microM) and metoclopramide (1 microM). Tropisetron (1 microM) and ondansetron (0.3 microM) also inhibited significantly the quipazine effect whereas methiothepin (1 microM), dihydroergotamine (0.1 microM), yohimbine (1 microM) and ketanserin (1 microM) were ineffective on the quipazine inhibition of [3H]GABA release. Our results show a serotonergic modulatory effect on the K(+)-evoked [3H]GABA release from guinea-pig hippocampal synaptosomes by receptors which are neither 5-HT1, 5-HT2 or 5-HT4. They appear to be pharmacologically related to the 5-HT3 type but different from the 5-HT3 ionic channel receptors.     

Alpha-2 adrenergic inhibition of Ca(++)-evoked [3H]norepinephrine release from synaptosomes is blocked by depolarization

The Ca(++)-evoked release of [3H]norepinephrine was used in these studies to investigate presynaptic regulation of norepinephrine release. In hippocampal synaptosomes, previously unexposed to Ca++ during isolation and superfusion, 1.25 mM Ca++ evoked a modest (4 to 7% of total stores) release of [3H]norepinephrine with 4.5 mM [K+] present. The alpha-2 adrenergic agonist clonidine inhibited 60% of the Ca(++)-evoked [3H]norepinephrine release. The alpha-2 adrenergic antagonists idazoxan and yohimbine reversed clonidine inhibition of release whereas the alpha-1 antagonist prazosin did not. Increasing the [K+] before Ca++ exposure increased [3H]norepinephrine release, and at 20 [K+] the release increased to over 20% of total stores. However, at [K+] above 9 mM, inhibition of Ca(++)-evoked release by clonidine decreased, and by 20 mM [K+] clonidine no longer inhibited release. Release was unaffected by 5 microM idazoxan or the opiate antagonist naloxone at 15 or 20 mM [K+]. The K+ channel blockers tetraethylammonium (5 mM) and 4-aminopyridine (0.1 mM) increased Ca(++)-evoked release almost 4-fold above control (4.5 mM [K+] present). Neither clonidine nor idazoxan affected Ca(++)-evoked release with the K+ channel blockers present. Therefore, even though K+ channel blockers and 20 mM [K+] increase neurotransmitter release, it is not autoreceptor activation by released endogenous norepinephrine that is responsible for blocking alpha-2 inhibition, but the depolarization produced by these treatments. The 20 mM [K+] blockade of alpha-2 inhibition was decreased by lowering the [Ca++] in the superfusion buffer. Therefore, synaptosomal accumulation of Ca++ may partially explain the loss of alpha-2 inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nicotinic acetylcholine receptor-mediated [3H]dopamine release from hippocampus

The mechanism of nicotinic acetylcholine receptor (nAChR)-induced hippocampal dopamine (DA) release was investigated using rat hippocampal slices. nAChRs involved in hippocampal DA and norepinephrine (NE) release were investigated using prototypical agonists and antagonists and several relatively novel compounds: ABT-594 [(R)-5-(2-azetidinylmethoxy)-2-chloropyridine], (+/-)-UB-165 [(2-chloro-5-pyridyl)-9-azabicyclo [4.2.1]non2-ene], and MG 624 [N,N,N-triethyl-2-[4-(2 phenylethenyl)phenoxy]-ethanaminium iodine]. (+/-)-Epibatidine, (+/-)-UB-165, anatoxin-a, ABT-594, (-)-nicotine, 1,1-dimethyl-4-phenyl-piperazinium iodide, and (-)-cytisine (in decreasing order of potency) evoked [(3)H]DA release in a mecamylamine-sensitive manner. Aside from (+/-)-UB-165, all the agonists displayed full efficacy relative to 100 microM (-)-nicotine in [(3)H]DA release. In contrast, (+/-)-UB-165 was a partial agonist, evoking 58% of 100 microM (-)-nicotine response. Mecamylamine, MG 624, hexamethonium, d-tubocurare, and dihydro-beta-erythroidine (in decreasing order of potency), but not alpha-conotoxin-MII, methyllycaconitine, alpha-conotoxin-ImI, or alpha-bungarotoxin, attenuated 100 microM (-)-nicotine-evoked [(3)H]DA release in a concentration-dependent manner. (+/-)-UB-165, ABT-594, and MG 624 exhibited different pharmacologic profiles in the [(3)H]NE release assay when compared with their effect on [(3)H]DA release. ABT-594 was 4.5-fold more potent, and (+/-)-UB-165 was a full agonist in contrast to its partial agonism in [(3)H]DA release. MG 624 potently and completely blocked NE release evoked by 100 microM (-)-nicotine and 10 microM (+/-)-UB-165, whereas it only partially inhibited (-)-nicotine-evoked [(3)H]DA release. In conclusion, we provide evidence that [(3)H]DA can be evoked from the hippocampus and that the pharmacologic profile for nAChR-evoked hippocampal [(3)H]DA release suggests the involvement of alpha3beta4(*) and at least one other nAChR subtype, thus distinguishing it from that of nAChR-evoked hippocampal [(3)H]NE release.     

Long-lasting facilitation of 4-amino-n-[2,3-(3)H]butyric acid ([(3)H]GABA) release from rat hippocampal slices by nicotinic receptor activation

In this study we explored the effect of the stimulation of nicotinic acetylcholine receptors located on interneurons by measuring 4-amino-n-[2,3-(3)H]butyric acid ([(3)H]GABA) release and monitoring [Ca (2+)](i) in superfused hippocampal slices. In the presence of 6-cyano-7-nitroquinoxaline-2,3-dione, (+/-)-2-amino-5-phosphonopentanoic acid, and atropine, i.e., under the blockade of N-methyl-D-aspartate and non-N-methyl-D-aspartate glutamate and muscarinic receptors, nicotine did not alter the spontaneous outflow of [(3)H]GABA, but significantly increased the stimulation-evoked [(3)H]GABA efflux. This effect of nicotine depended on the time interval between nicotine treatment and electrical stimulus, the concentration of nicotine (1-100 microM), and the parameters of electrical depolarization. Acetylcholine (0.03-3 mM), and the alpha 7 subtype-selective agonist choline (0.1-10 mM), also potentiated stimulus-evoked release of [(3)H]GABA, whereas 1,1-dimethyl-4-phenilpiperazinium iodide failed to increase the tritium outflow significantly. The effect of nicotine treatment was prevented by tetrodotoxin (1 microM) and by the nicotinic acetylcholine receptor antagonist mecamylamine (10 microM), and the alpha 7 subtype-selective antagonists alpha-bungarotoxin (100 nM) and methyllycaconitine (10 nM), whereas dihidro-beta-erythroidine (20 nM) was without effect. Perfusion of 100 microM nicotine caused a [Ca(2+)](i) transient in about one-third of the tested interneurons; however, the response to subsequent electrical stimulation remained unchanged. Inhibition of the GABA transporter system by nipecotic acid (1 mM) or by decreasing the bath temperature to 12 degrees C abolished completely the effect of nicotine to potentiate the stimulation-evoked release of GABA. These findings indicate that the activation of alpha 7-type nicotinic receptors of hippocampal interneurons results in a long-lasting ability of these cells to respond to depolarization with an increased release of GABA mediated by the transporter system.     

Muscarinic receptors mediating inhibition of gamma-aminobutyric acid release in rat corpus striatum and their pharmacological characterization

The effects of acetylcholine (ACh) and of cholinergic agonists on the release of tritiated gamma-aminobutyric acid ([3H]GABA) were studied in superfused synaptosomes prepared from rat corpus striatum and prelabeled with the radioactive amino acid. ACh, oxotremorine or (-)-nicotine, all tested at 100 microM had no effect on the spontaneous outflow of [3H]GABA. The depolarization-evoked overflow obtained by exposing the synaptosomes to 9 mM KCl was decreased in a concentration-dependent manner by ACh, oxotremorine, oxotremorine-M or carbachol. The maximal inhibition caused by ACh was 50%. The EC50 (agonist concentration causing half-maximal effect) amounted to 1 microM. Oxotremorine and oxotremorine-M were almost equipotent to ACh, whereas the concentration-response curve of carbachol was slightly (although not significantly) shifted to the right with respect to that of ACh. (-)-Nicotine (100 microM) did not affect the K(+)-evoked [3H]GABA overflow. ACh also inhibited the K(+)-evoked release of endogenous GABA. The inhibitory effect of 10 microM ACh on the release of [3H]GABA evoked by 9 mM KCl was insensitive to the nicotinic antagonist mecamylamine (10 microM) but it was potently blocked by the muscarinic antagonist atropine (IC50 = 5 nM) and weakly antagonized by pirenzepine, dicyclomine and AF-DX 116. The pharmacological profile of this receptor was very similar to that of the muscarinic autoreceptors regulating [3H]ACh release. The extent of [3H]GABA release inhibition caused by ACh did not differ between dorsal and ventral striatum. The inhibitory effect of ACh was much less pronounced in hippocampus and cortex than in the striatum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure-activity relationship between guanidine alkyl derivatives and norepinephrine release: site(s) and mechanism(s) of action

The effect of guanidine alkyl derivatives on the evoked release of [3H]norepinephrine [( 3H]NE) from spleen strips was examined. Guanidine, methyl guanidine and N,N-dimethyl guanidine all enhanced the field-stimulated release of [3H]NE 2- to 3-fold, whereas N,N'-dimethyl guanidine and propyl guanidine were without effect. The latter compound blocked the stimulatory effect of an equimolor concentration (4 mM) of guanidine. Guanidine enhanced moderately the field-stimulated release of [3H]NE from spleen strips pretreated with phenoxybenzamine. The efflux of [3H]NE from spleen slices induced by calcium ionophore A-23187 was not altered by guanidine incubation. The effect of guanidine on intracellular calcium movement was also tested by monitoring the effect of the drug on evoked secretion of ATP from human platelets. Guanidine did not modify this release. It is concluded that guanidine and its active structural derivatives augment [3H]NE release by increasing the influx of calcium through the voltage-sensitive calcium channels, but not by the mobilization of intracellular calcium pools. The biochemical basis for the action of the guanidinium cation is discussed.