In vivo histamine release from brain cortex: the effects of modulating cellular and extracellular sodium and calcium channels

The in vivo mechanisms underlying the actions of modulating Na(+)- and Ca(2+)-sensitive channels and its effect on basal histamine release in the cerebral cortex of freely-moving unanesthetized rats was investigated. Basal histamine release in the cerebral cortex was determined by in vivo microdialysis coupled with high-performance liquid chromatography (HPLC) fluorometry detection. Basal levels of histamine were 0.67+/-0.02 pmol/10 microl of dialysate. Diltiazem, a Ca(2+) channel antagonist, produced a dose-dependent decrease in dialysate basal histamine concentration. Elevated K(+) (100 mM) in the perfusion medium increased basal histamine to a maximum of 223% of the baseline value. Similarly, diltiazem (60 mM) reduced the K(+), veratridine (100 microg/ml) and ouabain (100 microM)-evoked increase in dialysate histamine. Basal histamine decreased by 48% when the perfusate contained 3 microM of voltage dependent Na(+) antagonist tetrodotoxin. The results of these studies indicate that the release of histamine in rat cerebral cortex can be induced by modulating Na(+) and Ca(2+) channels and that the L-type voltage-dependent sensitive Ca(2+) channels are involved in this release process.     

Enhancement of acetylcholine release by homoanatoxin-a from Oscillatoria formosa

The strain NIVA-CYA 92 of Oscillatoria formosa Bory ex Gormont produces phycotoxins with neurotoxic properties. Chemical analysis by gas chromatography/mass spectrometry of a water extract of lyophilized material of the organism showed the presence of only homoanatoxin-a. The mechanism of action of homoanatoxin-a on peripheral cholinergic nerves is so far not known. The neurotoxicity of O. formosa containing homoanatoxin-a was investigated in rat bronchi, rat brain synaptosomes and in GH(4)C(1) cells. The water extract of lyophilized material of the organism produced a concentration-dependent reversible increase in the release of [(3)H]acetylcholine from both K(+) (51 mM) depolarised and non-depolarised cholinergic nerves of the rat bronchial smooth muscle. The K(+)-evoked release of [(3)H]acetylcholine was enhanced by about 75% by a water extract from 15-20 mg/ml of lyophilized algal material. The enhanced release of [(3)H]acetylcholine was substantially reduced by the L-type Ca(2+)-channel blocker verapamil (100 μM) and not by the N-type Ca(2+)-channel blocker ω-conotoxin GVIA (1.0 μM) or the P-type Ca(2+)-channel blocker ω-agatoxin IV-A (0.2 μM). Chelation of intra-cellular Ca(2+) by 1,2-bis-(aminofenoxi)etan-N,N,N',N'-tetraacidic acid/acetoxymethyl (BAPTA/AM) (30 μM) had no effect on the phycotoxin-induced release of [(3)H]acetylcholine, indicating that an extracellular pool of Ca(2+) was important for the action of the phycotoxin on the release of [(3)H]acetylcholine from peripheral cholinergic nerves. In rat brain synaptosomes the algal extract enhanced the influx of (45)Ca(2+) in a tetrodotoxin (1.0 μM) and ω-conotoxin MVIIC (blocker of N-, P- and Q-type Ca(2+) channels) (1.0 μM) insensitive manner. Patch-clamp studies showed that the phycotoxin opened endogenous voltage dependent L-type Ca(2+) channels in neuronal GH(4)C(1) cells. These Ca(2+) channels and the effect of the toxin on the channels were blocked by the L-type Ca(2+)-channel antagonist gallopamil (200 μM). The present results suggest, therefore, that the investigated strain of O. formosa contains homoanatoxin-a, which enhances the release of acetylcholine from peripheral cholinergic nerves through opening of endogenous voltage dependent neuronal L-type Ca(2-) channels.     

Effects of L- and N-type Ca channel antagonists on excitatory amino acid-evoked dopamine release

In thc present study we tested the effect of dihydropyridine (DHP) Ca²⁺ channel antagonists and of ω-conotoxin GVIA on [³H]dopamine (DA) release evoked by the activation of excitatory amino acid (EAA) receptors in cultures of fetal rat ventral mesencephalon, in order to investigate the role of voltage-sensitive L- and N-type Ca²⁺ channels in these EAA-mediated processes. Micromolar concentrations (10–30 μM) of DHP L-type Ca²⁺ channel antagonists inhibited [³H]DA release evoked by N-methyl-D-aspartate (NMDA), kainate, quisqualate or veratridine. [³H]DA release evoked by the L-type Ca²⁺ channel agonist, Bay K 8644, was inhibited by lower concentrations (0.1–1 μM) of the DHP antagonist, nitrendipine, than was the release evoked by EAAs. The DHP antagonist, ( + )-PN 200-110, was more potent than ( − )-PN 200-110 in inhibiting [³H]DA release evoked by Bay K 8644, but the two stereoisomers were equipotent in inhibiting NMDA-evoked release. These results indicate that activation of L-type Ca²⁺ channels is able to evoke [³H]DA release. However activation of L-type channels is not involved in EAA-induced [³H]DA release and therefore inhibition of EAA-induccd [³H]DA release by micromolar concentrations of DHPs must be mediated by actions other than inhibition of L-type Ca²⁺ channels. ω-Conotoxin GVIA (3 μM) had no effect on [³H]DA release evoked by Bay K 8644, indicating that the toxin may selectively inhibit N-type channels in this preparation. ω-Conotoxin GVIA (3 μM) partially inhibited [³H]DA release evoked by NMDA or kainate, suggesting that N-type Ca²⁺ channels could possibly play a role in FAA-mediated responses in these cells.     

Omega-conotoxin GVIA and dihydropyridines discriminate two types of Ca2+ channels involved in GABA release from striatal neurons in culture

We previously reported that the opening of the L-type Ca2+ channel was only partly involved in the K(+)-evoked Ca2(+)-dependent gamma-aminobutyric acid (GABA) release from striatal neurons, suggesting that probably different types of voltage-sensitive Ca2+ channels were implicated in this physiological process. Here we demonstrate that omega-conotoxin GVIA, which has been reported to block L- and N-type neuronal Ca2+ channels, also partly inhibits the Ca2(+)-dependent GABA release. The maximal effects of omega-conotoxin GVIA and nifedipine, a highly specific antagonist of the L-type channels, were additive, a total inhibition of the Ca2(+)-dependent GABA release being obtained in the presence of both drugs. We therefore propose that omega-conotoxin GVIA and nifedipine block two different types of Ca2+ channels, both involved in the GABA release process.     

The phosphatidylinositol 4-kinase inhibitor phenylarsine oxide blocks evoked neurotransmitter release by reducing calcium entry through N-type calcium channels

The effects of the phosphatidylinositol 4-kinase inhibitor, phenylarsine oxide (PAO), on acetylcholine (ACh) release and on prejunctional Ca(2+) currents were studied at the frog neuromuscular junction using electrophysiological recording techniques. Application of PAO (30 microM) increased both spontaneous ACh release reflected as miniature end-plate potential (mepp) frequencies and evoked ACh release reflected as end-plate potential (epp) amplitudes with a similar time course. Following the initial increase in epp amplitudes produced by PAO, epps slowly declined and were eventually abolished after approximately 20 min. However, mepp frequencies remained elevated over this time period. PAO (30 microM) also inhibited the perineural voltage change associated with Ca(2+) currents through N-type Ca(2+) channels (prejunctional Ca(2+) currents) at motor nerve endings. Addition of British anti-lewisite (BAL, 1 mM), an inactivator of PAO, partially reversed both the inhibition of epps and the inhibition of the prejunctional Ca(2+) current. The effects of PAO on N-type Ca(2+) channels were investigated more directly using the whole cell patch clamp technique on acutely dissociated sympathetic neurons. Application of PAO (30 - 40 microM) to these neurons decreased the voltage-activated calcium currents through N-type Ca(2+) channels, an effect that was partially reversible by BAL. In combination, these results suggest that inhibition of neurotransmitter release by PAO occurs as a consequence of the inhibition of Ca(2+) entry via N-type calcium channels. The relationship between the effects of PAO on N-type Ca(2+) channels in motor nerve endings and in neuronal soma is discussed.     

Opposite effects of T- and L-type Ca(2+) channels blockers in generalized absence epilepsy

The role of the T-type Ca(2+) channel blocker, ethosuximide, the L-type Ca(2+) channel blocker, nimodipine and L-type Ca(2+) channel opener, BAY K8644 (1,4 Dihydro-2, 6-dimethyl-5-nitro-4-[trifluoromethyl)-phenyl]-3-pyridine carboxylic acid methyl ester), was investigated on spike-wave discharges in WAG/Rij rats. This strain is considered as a genetic model for generalized absence epilepsy. A dose-dependent decrease in the number of spike-wave discharges was found after i.c.v. ethosuximide, an increase after i.p. nimodipine and a decrease after i.c.v. BAY K8644. BAY K8644 was also able to antagonise the effects of nimodipine. Preliminary data were obtained with two conotoxins, MVIIC and GVIA, which block P/Q-type and N-type Ca(2+) channels, respectively. Only after i.c.v. administration of omega-conotoxin GVIA were the number and duration of spike-wave discharges reduced, but animals showed knock-out lying. The latter suggests behavioural or toxic effects and that the decrease in spike-wave activity cannot unequivocally be attributed to blockade of N-type Ca(2+) channels.It can be concluded that T- and L-type Ca(2+) channel blockers show opposite effects on spike-wave discharges. Furthermore, these effects are difficult to explain in terms of a model for spindle burst activity in thalamic relay cells proposed by McCormick and Bal [Sleep and arousal: thalamocortical mechanisms.     

Role of voltage-gated cation channels and axon reflexes in the release of sensory neuropeptides by capsaicin from isolated rat trachea

In order to reveal the role of axon reflexes and sensory receptors in sensory neuropeptide release in response to capsaicin, liberation of substance P, calcitonin gene-related peptide and somatostatin from isolated rat tracheae was investigated in the presence of voltage-sensitive Na(+) and Ca(2+) channel blocking agents. Neuropeptide release induced by capsaicin (10 nM) remained unchanged in the presence of 25 mM lidocaine, 1 microM tetrodotoxin or the N-type Ca(2+) channel inhibitor, omega-conotoxin GVIA (100-300 nM). Peptide release by 100 pulses of 2 Hz field stimulation was prevented by lidocaine or tetrodotoxin. Omega-agatoxin TK (250 nM) significantly inhibited and Cd(2+) (200 microM) prevented capsaicin-induced neuropeptide release. These results suggest that chemical stimulation-induced neuropeptide release does not involve activation of fast Na(+) channels or N- and P-type voltage-dependent Ca(2+) channels, but contribution of Q-type Ca(2+) channels is possible. Sensory neuropeptides are released by capsaicin from sensory receptors without axon reflexes.     

Algogen-specific pain processing in mouse spinal cord: differential involvement of voltage-dependent Ca(2+) channels in synaptic transmission

1. The effects of intrathecal (i.t.) administration of N-, P/Q- or L-type voltage-dependent Ca(2+)-channel blockers were tested in two pain models involving bradykinin (BK)- and alpha,beta-methylene ATP (alpha,beta meATP)-induced activation of primary afferent neurons in mice. 2. The nociceptive response (amount of time spent licking and biting the hindpaw) induced by intraplantar injection of BK (500 pmol mouse(-1)) was significantly attenuated by both omega-conotoxin GVIA (N-type blocker) and calciseptine (L-type) but not by omega-agatoxin IVA (P/Q-type). 3. The nociceptive response induced in a similar way by alpha,beta meATP (100 nmol) was significantly inhibited by both the above N- and P/Q-type Ca(2+)-channel blockers but not by the L-type blocker. 4. The nociceptive responses elicited by BK and alpha,beta meATP were dose-dependently inhibited by a tachykinin-NK1-receptor antagonist (L-703,606) and an N-methyl-D-aspartate (NMDA)-receptor antagonist (D-AP5), respectively. 5. Intrathecal administration of substance P (SP) (1.8 nmol) or NMDA (350 pmol) elicited algesic responses, such as licking, biting and scratching of the hindquarters. The SP-induced algesic behaviour was significantly inhibited by the L-type blocker but not by the N-type. The NMDA-induced response was not affected by either the N- or the P/Q-type blocker. 6. These findings suggest that BK and ATP most likely excite different types of sensory neurons in the periphery and that within the spinal cord the former stimulates peptidergic transmission regulated by presynaptic N- and postsynaptic L-type Ca(2+) channels, while the latter stimulates glutamatergic transmission regulated by presynaptic N- and P/Q-type channels.     

Pharmacological evidence for an omega-conotoxin, dihydropyridine-insensitive neuronal Ca2+ channel.

Inactivation of N-type voltage-sensitive Ca2+ channels (VSCC) with omega-conotoxin (omega-CgTx) in tissue obtained from chicken brain produces a concentration dependent (0.01-0.1 microM) inhibition of K(+)-stimulated Ca2+ influx (delta K+), the rise in [Ca2+]i and acetylcholine (ACh) release. In identical preparations from rat brain, Ca2+ influx and the rise in [Ca2+]i were only marginally affected by much higher (1-10 microM) concentrations of omega-CgTx. The release of ACh, however, was inhibited to the same degree with similar amounts of omega-CgTx as those used in chicken brain. An L-type VSCC inhibitor failed to affect any of these parameters alone, or to augment the effect of omega-CgTx. The results suggest that almost all the VSCC in chicken brain are of the N type and that these channels regulate neurotransmitter release. In rat brain, on the other hand, Ca2+ channels resistant to N- or L-type blockers account for almost 75% of the measurable Ca2+ influx and rise in [Ca2+]i. The conspicuous dissociation between the regulation of Ca2+ influx and ACh release demonstrated in rat brain by using omega-CgTx, suggest that neurotransmitter release is governed by only a small proportion of strategically located N-type, omega-CgTx sensitive, VSCC in the presynaptic terminal.     

High affinity interaction of mibefradil with voltage-gated calcium and sodium channels

Mibefradil is a novel Ca(2+) antagonist which blocks both high-voltage activated and low voltage-activated Ca(2+) channels. Although L-type Ca(2+) channel block was demonstrated in functional experiments its molecular interaction with the channel has not yet been studied. We therefore investigated the binding of [(3)H]-mibefradil and a series of mibefradil analogues to L-type Ca(2+) channels in different tissues. [(3)H]-Mibefradil labelled a single class of high affinity sites on skeletal muscle L-type Ca(2+) channels (K(D) of 2.5+/-0.4 nM, B(max)=56.4+/-2.3 pmol mg(-1) of protein). Mibefradil (and a series of analogues) partially inhibited (+)-[(3)H]-isradipine binding to skeletal muscle membranes but stimulated binding to brain L-type Ca(2+) channels and alpha1C-subunits expressed in tsA201 cells indicating a tissue-specific, non-competitive interaction between the dihydropyridine and mibefradil binding domain. [(3)H]-Mibefradil also labelled a heterogenous population of high affinity sites in rabbit brain which was inhibited by a series of nonspecific Ca(2+) and Na(+)-channel blockers. Mibefradil and its analogue RO40-6040 had high affinity for neuronal voltage-gated Na(+)-channels as confirmed in binding (apparent K(i) values of 17 and 1.0 nM, respectively) and functional experiments (40% use-dependent inhibition of Na(+)-channel current by 1 microM mibefradil in GH3 cells). Our data demonstrate that mibefradil binds to voltage-gated L-type Ca(2+) channels with very high affinity and is also a potent blocker of voltage-gated neuronal Na(+)-channels. More lipophilic mibefradil analogues may possess neuroprotective properties like other nonselective Ca(2+)-/Na(+)-channel blockers.     

Calcium channels involved in noradrenaline release from sympathetic neurones in rabbit carotid artery

Transmitter release from nerve terminals is dependent on the entry of Ca(2+) through neuronal voltage-gated calcium channels. In sympathetic neurones both N- and L-type calcium channels are present. Potassium channel blockade increases Ca(2+) entry into sympathetic neurones. We examined the participation of N- and L-type calcium channels in the stimulation-evoked release of noradrenaline from vascular sympathetic neurones. Rings of rabbit carotid artery were preincubated with [3H]-noradrenaline. Electrical field stimulation was used to evoke 3H overflow. The selective N-type calcium channel blocking agent omega-conotoxin GVIA (single concentrations: 3 x 10(-10)-10(-8) M) caused a slowly developing reduction of the stimulation-evoked 3H overflow. At 3 x 10(-8) M, omega-conotoxin GVIA caused an equilibrium block with a rapid (15 min.) onset. After 2 hr exposure to omega-conotoxin the inhibition was steady (pIC50 (-log M): 9.43; Emax: 91%). The selective L-type calcium blocking agents nifedipine (10(-7)-10(-5) M) and nimodipine (10(-8)-10(-5) M) had no effect on the stimulation-evoked 3H overflow. The calcium channel opener Bay K 8644 (10-6 M) likewise had no effect. The potassium channel blocking agent 4-aminopyridine (10-5-10-3 M) enhanced the stimulation-evoked 3H overflow up to 5 times. 4-Aminopyridine (10(-4) M) did not alter the inhibitory effect of omega-conotoxin GVIA (3 x 10(-8) M). In the presence of 4-aminopyridine (10(-4) M), nifedipine (10(-5) M) and nimodipine (10(-6) M) enhanced the 3H overflow. We conclude that the stimulation-evoked release of noradrenaline from sympathetic neurones in rabbit carotid artery is mediated by N-type calcium channels and that L-type channels are not involved even when potassium channels are blocked by 4-aminopyridine.     

Molecular and pharmacological bases for the gating regulation of L-type voltage-dependent Ca2+ channels

The voltage-dependent L-type Ca(2+) channel plays a key role in the spacial and temporal regulation of Ca(2+). In cardiac excitation-contraction coupling, Ca(2+)-induced Ca(2+) release (CICR) from ryanodine receptors (RyRs), triggered by Ca(2+) entry through the nearby L-type Ca(2+) channel, induces the Ca(2+)-dependent inactivation (CDI) of the Ca(2+) channel. We demonstrated that the CICR-dependent CDI of L-type Ca(2+) channels, under control of the privileged cross-signaling between L-type Ca(2+) channels and RyRs, plays important roles for monitoring and tuning the SR Ca(2+) content via changes of AP waveform and the amount of Ca(2+)-influx during AP in ventricular myocytes. L-type Ca(2+) channels are modulated by the binding of Ca(2+) channel antagonists and agonists to the pore-forming alpha(1C) subunit. We identified Phe(1112) and Ser(1115) in the pore-forming IIIS5-S6 linker region of the alpha(1C) subunit as critical determinants of the binding of dihydropyridines (DHP). Interestingly, double mutant Ca(2+) channel (F1112A/S1115A) failed to discriminate between a DHP Ca(2+) channel agonist and antagonist stereoisomers. We proposed that Phe(1112) and Ser(1115) in the pore-forming IIIS5-S6 linker region is required for the stabilization of the Ca(2+) channel in the open state by Ca(2+) channel agonists and further proposed a novel model for the DHP-binding pocket of the alpha(1C) subunit. These integrative studies on the gating regulation of cardiac L-type Ca(2+) channels will provide the molecular basis for the pharmacology of Ca(2+) channel modulators.     

Facilitation of L-type Ca2+ currents by fluid flow in rabbit cerebral artery myocytes

Blood vessels are receptive to hemodynamic forces, such as blood pressure and flow, which result in myogenic responses. The present study aimed to investigate the effect of mechanical stresses on L-type voltage-dependent Ca(2+) channels in rabbit cerebral artery myocytes. Cell swelling induced by the exposure to a 16% hypotonic solution increased peak values of whole-cell Ba(2+) currents (IBa). Similarly, an elevation of bath perfusion rate increased peak values of IBa. However, the response was reduced by the continued fluid flow stimulation and the current amplitude almost returned to the baseline. This reduction of the current was abolished by pretreatment with thapsigargin, implying the contribution of Ca(2+) release from the sarcoplasmic reticulum to the response. These results suggest that L-type Ca(2+) currents are facilitated not only by cell swelling but also by fluid flow in cerebral artery myocytes.     

The inhibition of release by mGlu7 receptors is independent of the Ca2+ channel type but associated to GABAB and adenosine A1 receptors

Neurotransmitter release is inhibited by G-protein coupled receptors (GPCRs) through signalling pathways that are negatively coupled to Ca(2+) channels and adenylyl cyclase. Through Ca(2+) imaging and immunocytochemistry, we have recently shown that adenosine A(1), GABA(B) and the metabotropic glutamate type 7 receptors coexist in a subset of cerebrocortical nerve terminals. As these receptors inhibit glutamate release through common intracellular signalling pathways, their co-activation occluded each other responses. Here we have addressed whether the occlusion of receptor responses is restricted to the glutamate release mediated by N-type Ca(2+) channels by analysing this process in nerve terminals from mice lacking the alpha(1B) subunit (Ca(v) 2.2) of these channels. We found that glutamate release from cerebrocortical nerve terminals without these channels, in which release relies exclusively on P/Q type Ca(2+) channels, is not modulated by mGlu7 receptors. Furthermore, there is no occlusion of the release inhibition by GABA(B) and adenosine A(1). Hence, in the cerebrocortical preparation, these three receptors only appear to coexist in N-type channel containing nerve terminals. In contrast, in hippocampal nerve terminals lacking this subunit, where mGlu7 receptors modulate glutamate release via P/Q type channels, the occlusion of inhibitory responses by co-stimulation of adenosine A(1), GABA(B) and mGlu7 receptors was observed. Thus, occlusion of the responses by the three GPCRs is independent of the Ca(2+) channel type but rather, it is associated to functional mGlu7 receptors.     

Chronic morphine treatment decreases the Cav1.3 subunit of the L-type calcium channel

Voltage-gated L- and N-type calcium channels (VOCs) are implicated in the activity of morphine, but their contribution to the expression of opioid tolerance remains uncertain. L- and N-type VOCs are heteropentamers of alpha(1), alpha(2)delta, beta, and gamma subunits. The alpha(1) subunit forms both the ion pore and the binding site for ligands. The Ca(v)1.2 and Ca(v)1.3 are the neuronal dihydropyridine (DHP)-sensitive L-type channel subunit types. The Ca(v)2.2 subunit is found in omega conotoxin GVIA-sensitive N-type calcium channels. Ca(v)1.2 VOC gating properties are phosphorylation-dependent with many kinases implicated. We hypothesized that changes in channel subunit structure or phosphorylation state, induced by chronic opioid exposure, may in part explain changes in calcium regulation observed both in vivo and in vitro. Antibodies, specific for the Ca(v)1.2, Ca(v)1.3, and Ca(v)2.2 subunits of VOCs were employed with Western immunoassays to access whether chronic morphine treatment had an effect on receptor protein levels. The L-type channel Ca(v)1.3 protein, but not the Ca(v)1.2 protein or phosphorylation state, significantly decreased upon chronic morphine treatment. The Ca(v)2.2 subunit protein of the N-type channel of VOCs remained unchanged. The Ca(v)1.3 subunit modification may represent one of many potential adaptive changes in tolerance to morphine-induced changes in intracellular calcium.     

Antisympathetic and hemodynamic property of a dual L/N-type Ca(2+) channel blocker cilnidipine in rats.

The in vivo antisympathetic property of a dual L/N-type Ca(2+) channel blocker cilnidipine compared with that of typical N-type Ca(2+) channel blockers has never been clarified. We investigated the effects of the drug on a sympathetic nerve-mediated vascular response and vasodilating action in rats in comparison with those of an N-type Ca(2+) channel blocker omega-conotoxin MVIIA. In pithed rats, omega-conotoxin MVIIA preferentially suppressed the sympathetic nerve stimulation-induced pressor response, whereas cilnidipine suppressed the pressor response induced by sympathetic nerve stimulation and angiotensin II. In anesthetized rats, cilnidipine or omega-conotoxin MVIIA decreased mean blood pressure, while heart rate was decreased by omega-conotoxin MVIIA, but slightly increased by cilnidipine. These results suggest that cilnidipine can affect sympathetic N-type Ca(2+) channels in addition to vascular L-type Ca(2+) channels in antihypertensive doses in the rat in vivo. The antisympathetic activity of cilnidipine is not excessive for an antihypertensive drug in comparison with that of omega-conotoxin MVIIA.     

A study on the role of nitric oxide and iron in 3-morpholino-sydnonimine-induced increases in dopamine release in the striatum of freely moving rats

1. We showed previously that interaction between NO and iron (II), both released following the decomposition of sodium nitroprusside (SNP), accounted for the late SNP-induced dopamine (DA) increase in dialysates from the striatum of freely moving rats; in addition, we showed that co-infusion of iron (II) with the NO-donor S-nitroso-N-acetylpenicillamine mimicked SNP effects on striatal DA release. 2. In the present study, intrastriatal co-infusion of iron (II) (given as FeSO(4), 1 mM for 40 min) with the NO-donor and potential peroxynitrite generator 3-morpholinosydnonimine (SIN-1) (0.2, 0.5, 1.0 or 5.0 mM for 180 min), potentiated the SIN-1-induced increase in DA concentration in dialysates from the striatum of freely moving rats. Neither alone nor associated with iron (II) did SIN-1 induce changes in dialysate ascorbic acid or uric acid concentrations. 3. Neither co-infusion of a superoxide dismutase mimetic nor uric acid affected SIN-1-induced increases in dialysate DA concentration. 4. Infusion of the iron chelator deferoxamine (0.2 mM for 180 min) decreased dialysate DA and attenuated SIN-1-induced increases in dialysate DA concentrations. 5. These results suggest that iron plays a key role in SIN-1-induced release of striatal DA and do not support any role for either peroxynitrite or superoxide anion in SIN-1-induced release of striatal DA.     

Cellular mechanisms of acute decrease of glutamate release induced by raloxifene in rat cerebral cortex

Raloxifene, a selective estrogen receptor modulator, has been observed to offer a neuroprotective effect in several in?vitro models of neurotoxicity. An excessive release of glutamate is considered to be related to neuropathology of several neurological diseases. In this study, we investigated whether raloxifene could affect endogenous glutamate release in nerve terminals of rat cerebral cortex (synaptosomes) and explored the possible mechanism. Raloxifene exhibited a dose-dependent inhibition of 4-aminopyridine (4-AP)-evoked release of glutamate, and this effect was not blocked by the estrogen receptor antagonists. The effect of raloxifene on the evoked glutamate release was prevented by the chelating extracellular Ca(2+) ions, and by the vesicular transporter inhibitor bafilomycin A1, but was insensitive to the glutamate transporter inhibitor DL-TBOA. Raloxifene decreased the depolarization-induced increase in the cytosolic free Ca(2+) concentration ([Ca(2+)](C)), whereas it did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization. The effect of raloxifene on evoked glutamate release was prevented by blocking the Ca(v)2.2 (N-type) and Ca(v)2.1 (P/Q-type) channels, but not by blocking intracellular Ca(2+) release or Na(+)/Ca(2+) exchange. In addition, the inhibitory effect of raloxifene on evoked glutamate release was abolished by the mitogen-activated/extracellular signal-regulated kinase kinase (MEK) inhibitors, PD98059 and U0126. Furthermore, raloxifene significantly decreased the depolarization-induced phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase 1 and 2 (MAPK/ERK1/2) and synapsin I, the main presynaptic target of ERK. Thus, the effect of raloxifene on evoked glutamate release is linked to a decrease in [Ca(2+)](i) contributed by Ca(2+) entry through presynaptic voltage-dependent Ca(2+) channels and to the subsequent suppression of the ERK/synapsin I signaling cascade.     

尼卡地平抑制dbcAMP分化的神经母细胞瘤x神经胶质瘤杂种细胞（NG1 …

AIM: To investigate the possibility of dihydropyridine inhibition of N-type calcium channels. METHODS: Effects of nifedipine and nicardipine on the high K(+)-induced intracellular Ca2+ concentration ([Ca2+]i) increase were studied by measuring [Ca2+]i using the fluorescent indicator Fura-2. RESULTS: Pretreatment of cells with nifedipine 50 mumol.L-1 inhibited the high K(+)-induced [Ca2+]i transient by about 60% (n = 3); however, pretreatment of cells with nicardipine 10 mumol.L-1 completely prevented the high K(+)-evoked [Ca2+]i increase in dibutyryl cyclic AMP (dbcAMP)-differentiated NG 108-15 cells (n = 5). The high K(+)-induced [Ca2+]i increase was mediated by L- and N-type voltage-sensitive calcium channels (VSCC) in NG 108-15 cells. CONCLUSION: Nicardipine at micromolar range inhibited both L- and N-type VSCC in dbcAMP-differentiated NG 108-15 cells whereas nifedipine mainly inhibited L-type calcium channels.