Urotensin II promotes vagal-mediated bradycardia by activating cardiac-projecting parasympathetic neurons of nucleus ambiguus.

Urotensin II (U-II) is a cyclic undecapeptide that regulates cardiovascular function at central and peripheral sites. The functional role of U-II nucleus ambiguus, a key site controlling cardiac tone, has not been established, despite the identification of U-II and its receptor at this level. We report here that U-II produces an increase in cytosolic Ca(2+) concentration in retrogradely labeled cardiac vagal neurons of nucleus ambiguus via two pathways: (i) Ca(2+) release from the endoplasmic reticulum via inositol 1,4,5-trisphosphate receptor; and (ii) Ca(2+) influx through P/Q-type Ca(2+) channels. In addition, U-II depolarizes cultured cardiac parasympathetic neurons. Microinjection of increasing concentrations of U-II into nucleus ambiguus elicits dose-dependent bradycardia in conscious rats, indicating the in vivo activation of the cholinergic pathway controlling the heart rate. Both the in vitro and in vivo effects were abolished by the urotensin receptor antagonist, urantide. Our findings suggest that, in addition, to the previously reported increase in sympathetic outflow, U-II activates cardiac vagal neurons of nucleus ambiguus, which may contribute to cardioprotection.

Etomidate evokes synaptic vesicle exocytosis without increasing miniature endplate potentials frequency at the mice neuromuscular junction.

Etomidate is an intravenous anesthetic used during anesthesia induction. This agent induces spontaneous movements, especially myoclonus after its administration suggesting a putative primary effect at the central nervous system or the periphery. Therefore, the aim of this study was to investigate the presynaptic and postsynaptic effects of etomidate at the mouse neuromuscular junction (NMJ). Diaphragm nerve muscle preparations were isolated and stained with the styryl dye FM1-43, a fluorescent tool that tracks synaptic vesicles exo-endocytosis that are key steps for neurotransmission. We observed that etomidate induced synaptic vesicle exocytosis in a dose-dependent fashion, an effect that was independent of voltage-gated Na(+) channels. By contrast, etomidate-evoked exocytosis was dependent on extracellular Ca(2+) because its effect was abolished in Ca(2+)-free medium and also inhibited by omega-Agatoxin IVA (30 and 200nM) suggesting the participation of P/Q-subtype Ca(2+) channels. Interestingly, even though etomidate induced synaptic vesicle exocytosis, we did not observe any significant difference in the frequency and amplitude of miniature end-plate potentials (MEPPs) in the presence of the anesthetic. We therefore investigated whether etomidate could act on nicotinic acetylcholine receptors labeled with α-bungarotoxin-Alexa 594 and we observed less fluorescence in preparations exposed to the anesthetic. In conclusion, our results suggest that etomidate exerts a presynaptic effect at the NMJ inducing synaptic vesicle exocytosis, likely through the activation of P-subtype voltage gated Ca(2+) channels without interfering with MEPPs frequency. The present data contribute to a better understanding about the effect of etomidate at the neuromuscular synapse and may help to explain some clinical effects of this agent.

Nesfatin-1 activates cardiac vagal neurons of nucleus ambiguus and elicits bradycardia in conscious rats.

Nesfatin-1, a peptide whose receptor is yet to be identified, has been involved in the modulation of feeding, stress, and metabolic responses. More recently, increasing evidence supports a modulatory role for nesfatin-1 in autonomic and cardiovascular activity. This study was undertaken to test if the expression of nesfatin-1 in the nucleus ambiguus, a key site for parasympathetic cardiac control, may be correlated with a functional role. As we have previously demonstrated that nesfatin-1 elicits Ca²âº signaling in hypothalamic neurons, we first assessed the effect of this peptide on cytosolic Ca²âº in cardiac pre-ganglionic neurons of nucleus ambiguus. We provide evidence that nesfatin-1 increases cytosolic Ca²âº concentration via a Gi/o-coupled mechanism. The nesfatin-1-induced Ca²âº rise is critically dependent on Ca²âº influx via P/Q-type voltage-activated Ca²âº channels. Repeated administration of nesfatin-1 leads to tachyphylaxis. Furthermore, nesfatin-1 produces a dose-dependent depolarization of cardiac vagal neurons via a Gi/o-coupled mechanism. In vivo studies, using telemetric and tail-cuff monitoring of heart rate and blood pressure, indicate that microinjection of nesfatin-1 into the nucleus ambiguus produces bradycardia not accompanied by a change in blood pressure in conscious rats. Taken together, our results identify for the first time that nesfatin-1 decreases heart rate by activating cardiac vagal neurons of nucleus ambiguus. Our results indicate that nesfatin-1, one of the most potent feeding peptides, increases cytosolic Ca²âº by promoting Ca²âº influx via P/Q channels and depolarizes nucleus ambiguus neurons; both effects are Gi/o-mediated. In vivo studies indicate that microinjection of nesfatin-1 into nucleus ambiguus produces bradycardia in conscious rats. This is the first report that nesfatin-1 increases the parasympathetic cardiac tone.

Differential responses to ω-agatoxin IVA in murine frontal cortex and spinal cord derived neuronal networks.

ω-Agatoxin-IVA is a well known P/Q-type Ca(2+) channel blocker and has been shown to affect presynaptic Ca(2+) currents as well postsynaptic potentials. P/Q-type voltage gated Ca(2+) channels play a vital role in presynaptic neurotransmitter release and thus play a role in action potential generation. Monitoring spontaneous activity of neuronal networks on microelectrode arrays (MEAs) provides an important tool for examining this neurotoxin. Changes in extracellular action potentials are readily observed and are dependent on synaptic function. Given the efficacy of murine frontal cortex and spinal cord networks to detect neuroactive substances, we investigated the effects of ω-agatoxin on spontaneous action potential firing within these networks. We found that networks derived from spinal cord are more sensitive to the toxin than those from frontal cortex; a concentration of only 10nM produced statistically significant effects on activity from spinal cord networks whereas 50 nM was required to alter activity in frontal cortex networks. Furthermore, the effects of the toxin on frontal cortex are more complex as unit specific responses were observed. These manifested as either a decrease or increase in action potential firing rate which could be statistically separated as unique clusters. Administration of bicuculline, a GABAA inhibitor, isolated a single response to ω-agatoxin, which was characterized by a reduction in network activity. These data support the notion that the two clusters detected with ω-agatoxin exposure represent differential responses from excitatory and inhibitory neuronal populations.

Dual actions of lindane (γ-hexachlorocyclohexane) on calcium homeostasis and exocytosis in rat PC12 cells.

The persistent organochlorine pesticide lindane is still abundantly found in the environment and in human and animal tissue samples. Lindane induces a wide range of adverse health effects, which are at least partially mediated via the known inhibition of GABA(A) and glycine receptors. Additionally, lindane has been reported to increase the basal intracellular Ca(2+) concentration ([Ca(2+)](i)). As Ca(2+) triggers many cellular processes, including cell death and vesicular neurotransmitter release (exocytosis), we investigated whether lindane affects exocytosis, Ca(2+) homeostasis, production of reactive oxygen species (ROS) and cytotoxicity in neuroendocrine PC12 cells. Amperometric recordings and [Ca(2+)](i) imaging experiments with fura-2 demonstrated that lindane (≥ 10 µM) rapidly increases basal exocytosis and basal [Ca(2+)](i). Additional imaging and electrophysiological recordings revealed that this increase was largely due to a lindane-induced membrane depolarization and subsequent opening of N- and P/Q-type voltage-gated Ca(2+) channels (VGCC). On the other hand, lindane (≥ 3 µM) induced a concentration-dependent but non-specific inhibition of VGCCs, thereby limiting the lindane-induced increase in basal [Ca(2+)](i) and exocytosis. Importantly, the non-specific inhibition of VGCCs also reduced stimulation-evoked exocytosis and Ca(2+) influx. Though lindane exposure concentration-dependently increased ROS production, cell viability was not affected indicating that the used concentrations were not acute cytotoxic. These combined findings indicate that lindane has two, partly counteracting effects. Lindane causes membrane depolarization, thereby increasing basal [Ca(2+)](i) and exocytosis. In parallel, lindane inhibits VGCCs, thereby limiting the basal effects and reducing stimulation-evoked [Ca(2+)](i) and exocytosis. This study further underlines the need to consider presynaptic, non-receptor-mediated effects in human risk assessment.

Levetiracetam inhibits glutamate transmission through presynaptic P/Q-type calcium channels on the granule cells of the dentate gyrus.

BACKGROUND AND PURPOSE: Levetiracetam is an effective anti-epileptic drug in the treatment of partial and generalized seizure. The purpose of the present study was to investigate whether levetiracetam regulates AMPA and NMDA receptor-mediated excitatory synaptic transmission and to determine its site of action in the dentate gyrus (DG), the area of the hippocampus that regulates seizure activities. EXPERIMENTAL APPROACH: Whole-cell patch-clamp method was used to record the AMPA and NMDA receptor-mediated excitatory postsynaptic currents (EPSC(AMPA) and EPSC(NMDA)) in the presence of specific antagonists, from the granule cells in the DG in brain slice preparations from young Wistar rats (60-120 g). KEY RESULTS: Levetiracetam (100 microM) inhibited both evoked EPSC(AMPA) and EPSC(NMDA) to an equal extent (80%), altered the paired-pulse ratio (from 1.39 to 1.25), decreased the frequency of asynchronous EPSC and prolonged the inter-event interval of miniature EPSC(AMPA) (from 2.7 to 4.6 s) without changing the amplitude, suggesting a presynaptic action of levetiracetam. The inhibitory effect of levetiracetam on evoked EPSC(AMPA) was blocked by omega-agatoxin TK (100 nM), a selective P/Q-type voltage-dependent calcium channel blocker, but not by nimodipine (10 microM) or omega-conotoxin (400 nM). CONCLUSIONS AND IMPLICATIONS: These results suggest that levetiracetam modulated the presynaptic P/Q-type voltage-dependent calcium channel to reduce glutamate release and inhibited the amplitude of EPSC in DG. This effect is most likely to contribute to the anti-epileptic action of levetiracetam in patients.

Calcium control of gene regulation in rat hippocampal neuronal cultures.

Blockage of GABA-A receptors in hippocampal neuronal cultures triggers synchronous bursts of spikes initiating neuronal plasticity, partly mediated by changes of gene expression. By using specific pharmacological blockers, we have investigated which sources of Ca2+ entry primarily control changes of gene expression induced by 20 microM gabazine applied for 30 min (GabT). Intracellular Ca2+ transients were monitored with Ca2+ imaging while recording electrical activity with patch clamp microelectrodes. Concomitant transcription profiles were obtained using Affymetrix oligonucleotide microarrays and confirmed with quantitative RT-PCR. Blockage of NMDA receptors with 2-amino-5-phosphonovaleric acid (APV) did not reduce significantly somatic Ca2+ transients, which, on the contrary, were reduced by selective blockage of L, N, and P/Q types voltage gated calcium channels (VGCCs). Therefore, we investigated changes of gene expression in the presence of blockers of NMDA receptors and L, N, and P/Q VGCCs. Our results show that: (i) among genes upregulated by GabT, there are genes selectively dependent on NMDA activation, genes selectively dependent on L-type VGCCs and genes dependent on the activation of both channels; (ii) the majority of genes requires the concomitant activation of NMDA receptors and Ca2+ entry through VGCCs; (iii) blockage of N and P/Q VGCCs has an effect similar but not identical to blockage of L-type VGCCs.

Interaction between the second messengers cAMP and Ca2+ in mouse presynaptic taste cells.

The second messenger, 3',5'-cyclic adenosine monophosphate (cAMP), is known to be modulated in taste buds following exposure to gustatory and other stimuli. Which taste cell type(s) (Type I/glial-like cells, Type II/receptor cells, or Type III/presynaptic cells) undergo taste-evoked changes of cAMP and what the functional consequences of such changes are remain unknown. Using Fura-2 imaging of isolated mouse vallate taste cells, we explored how elevating cAMP alters Ca(2+) levels in identified taste cells. Stimulating taste buds with forskolin (Fsk; 1 microm) + isobutylmethylxanthine (IBMX; 100 microm), which elevates cellular cAMP, triggered Ca(2+) transients in 38% of presynaptic cells (n = 128). We used transgenic GAD-GFP mice to show that cAMP-triggered Ca(2+) responses occur only in the subset of presynaptic cells that lack glutamic acid decarboxylase 67 (GAD). We never observed cAMP-stimulated responses in receptor cells, glial-like cells or GAD-expressing presynaptic cells. The response to cAMP was blocked by the protein kinase A inhibitor H89 and by removing extracellular Ca(2+). Thus, the response to elevated cAMP is a PKA-dependent influx of Ca(2+). This Ca(2+) influx was blocked by nifedipine (an inhibitor of L-type voltage-gated Ca(2+) channels) but was unperturbed by omega-agatoxin IVA and omega-conotoxin GVIA (P/Q-type and N-type channel inhibitors, respectively). Single-cell RT-PCR on functionally identified presynaptic cells from GAD-GFP mice confirmed the pharmacological analyses: Ca(v)1.2 (an L-type subunit) is expressed in cells that display cAMP-triggered Ca(2+) influx, while Ca(v)2.1 (a P/Q subunit) is expressed in all presynaptic cells, and underlies depolarization-triggered Ca(2+) influx. Collectively, these data demonstrate cross-talk between cAMP and Ca(2+) signalling in a subclass of taste cells that form synapses with gustatory fibres and may integrate tastant-evoked signals.

Membrane depolarization inhibits spiral ganglion neurite growth via activation of multiple types of voltage sensitive calcium channels and calpain.

The effect of membrane electrical activity on spiral ganglion neuron (SGN) neurite growth remains unknown despite its relevance to cochlear implant technology. We demonstrate that membrane depolarization delays the initial formation and inhibits the subsequent extension of cultured SGN neurites. This inhibition depends directly on the level of depolarization with higher levels of depolarization causing retraction of existing neurites. Cultured SGNs express subunits for L-type, N-type, and P/Q type voltage-gated calcium channels (VGCCs) and removal of extracellular Ca(2+) or treatment with a combination of L-type, N-type, and P/Q-type VGCC antagonists rescues SGN neurite growth under depolarizing conditions. By measuring the fluorescence intensity of SGNs loaded with the fluorogenic calpain substrate t-butoxy carbonyl-Leu-Met-chloromethylaminocoumarin (20 microM), we demonstrate that depolarization activates calpains. Calpeptin (15 microM), a calpain inhibitor, prevents calpain activation by depolarization and rescues neurite growth in depolarized SGNs suggesting that calpain activation contributes to the inhibition of neurite growth by depolarization.

mGluR7 inhibits glutamate release through a PKC-independent decrease in the activity of P/Q-type Ca2+ channels and by diminishing cAMP in hippocampal nerve terminals.

The modulation of calcium channels by metabotropic glutamate receptors (mGluRs) is a key event in the fine-tuning of neurotransmitter release. Here we report that, in hippocampal nerve terminals from adult rats, the inhibition of glutamate release by the group III mGluR agonist L-2-amino-4-phosphonobutyrate (L-AP4) is largely mediated by mGluR7. In this preparation, P/Q-type Ca(2+) channels support the major component of glutamate release while the remaining release is supported by N-type Ca(2+) channels. The release associated with P/Q channels was modulated by mGluR7, either in the presence of omega-conotoxin-GVIA or after decreasing the extracellular Ca(2+) concentration [Ca(2+)](o) to abolish the contribution of N-type Ca(2+) channels. Under these conditions, L-AP4 (1 mm) reduced the evoked glutamate release by 35 +/- 2%. This inhibition was largely prevented by pertussis toxin, but it was insensitive to inhibitors of protein kinase C (bisindolylmaleimide) and protein kinase A (H-89). Furthermore, this inhibition was associated with a reduction in the Ca(2+) influx mediated by P/Q channels in the absence of any detectable change in cAMP levels. However, L-AP4 decreased the levels of cAMP in the presence of forskolin. The activation of this additional signalling pathway was very efficient in counteracting the facilitation of glutamate release induced by forskolin. Thus, mGluR7 mediates the inhibition of glutamate release at hippocampal nerve terminals primarily by inhibiting P/Q-type Ca(2+) channels, although augmenting the levels of cAMP reveals the ability of the receptor to decrease cAMP.

Dopamine D(1) receptor facilitation of depolarization-induced release of gamma-amino-butyric acid in rat striatum is mediated by the cAMP/PKA pathway and involves P/Q-type calcium channels.

Transmission in the "direct" pathway through the basal ganglia, which has an important role in the control of motor movement, is markedly facilitated by the concurrent activation of dopamine D(1) receptors. Consistent with this, Ca(2+)-dependent, depolarization-induced release of [(3)H]-GABA from striatal slices from rats pretreated with reserpine was greatly increased in the presence of 1 microM SKF 38393, a dopamine D(1)-like receptor agonist. The effect of SKF 38393 was mimicked by 1 mM 8-bromo-cyclic AMP (Br-cAMP) and inhibited by the protein kinase A (PKA) inhibitor H-89, mean inhibition 92% +/- 4% with 10 microM H-89 (n = 3). The effects of SKF 38393 and Br-cAMP were not additive. The stimulatory effects of SKF 38393 and Br-cAMP were practically abolished in the presence of the histamine H(3) receptor agonist immepip (1 microM). The depolarization-induced release of [(3)H]-GABA in the presence of SKF 38393 was not significantly inhibited by 5 microM nimodipine, an L-type Ca(2+) channel blocker, or by 0.3 microM omega-conotoxin MVIIA, a selective blocker of N-type channels. However, preincubation of the slices with 0.95 microM omega-agatoxin TK, a P/Q-type channel blocker, followed by washing before changing to a depolarizing medium containing SKF 38393, resulted in a marked inhibition of the stimulated release of [(3)H]-GABA, mean 68% +/- 4% (n = 3). These observations provide evidence that dopamine D(1) agonist facilitation of the depolarization-induced release of GABA from striatal terminals is mediated by the cAMP/PKA pathway and involves mainly P/Q-type Ca(2+) channels.

D1-like dopamine receptors selectively block P/Q-type calcium channels to reduce glutamate release onto cholinergic basal forebrain neurones of immature rats.

Whole-cell patch-clamp recordings of non-NMDA glutamatergic EPSCs were made from identified cholinergic neurones in slices of basal forebrain (BF) of young rats (P13-P18), to investigate the subtypes of calcium channels involved in dopamine D(1)-like receptor-mediated presynaptic inhibition of the EPSCs. The BF cholinergic neurones were pre-labelled by intracerebroventricular injection of a fluorescent marker, Cy3-192IgG. A D(1)-like receptor agonist, SKF 81297 (30 microM) suppressed the EPSCs reversibly by about 30%, and this inhibition was reproducible. Calcium channel subtypes involved in the glutamatergic transmission were elucidated using selective Ca(2+) channel blockers. The N-type Ca(2+) channel blocker omega-conotoxin (omega-CgTX, 3 microM) suppressed the EPSCs by 57.5%, whereas the P/Q-type channel selective blocker omega-agatoxin-TK (omega-Aga-TK, 200 nM) suppressed the EPSCs by 68.9%. Simultaneous application of both blockers suppressed the EPSCs by 96.1%. The R-type Ca(2+) channel blocker SNX-482 (300 nM) suppressed the EPSCs by 18.4%, whereas nifedipine, the L-type Ca(2+) channel blocker (10 microM), had little effect. In the presence of omega-Aga-TK, SKF 81297, a dopamine D(1)-like receptor agonist, had no effect on the EPSCs. On the other hand, SKF 81297 could still inhibit the EPSCs in the presence of either omega-CgTX, SNX-482 or nifedipine. SKF 81297 had no further effect on the EPSCs when external Ca(2+) concentration was raised to 7.2 mM in the presence of omega-Aga-TK, but could still inhibit the EPSCs in high Ca(2+) solution after omega-CgTX application. Forskolin (FK, 10 microM), an activator of adenylyl cyclase pathway, suppressed the EPSCs, and the FK-induced effect was mostly blocked in the presence of omega-Aga-TK but not that of omega-CgTX. These results suggest that D(1)-like receptor activation selectively blocks P/Q-type calcium channels to reduce glutamate release onto BF cholinergic neurones.

The P/Q-type voltage-dependent calcium channel as pharmacological target in spinocerebellar ataxia type 6: gabapentin and pregabalin may be of therapeutic benefit.

Voltage-dependent calcium channels (VDCCs) are heteromultimeric complexes that mediate calcium influx into cells in response to changes in membrane potential. The alpha1A subunit, encoded by the CACNA1A gene, is the pore-forming subunit specific to the neuronal P/Q-type VDCCs. These are implicated in fast excitatory and inhibitory neurotransmission. Their highest levels of expression are found in the Purkinje cell layer of the cerebellum, and in the hippocampus. Spinocerebellar ataxia type 6 (SCA 6) is an autosomal dominant cerebellar degeneration that shares neuropathological findings with late-onset cortical cerebellar atrophy (CCA). It is caused by an abnormal expansion of a trinucleotide (CAG) repeat in exon 47 of CACNA1A, on chromosome 19p13. This translates into a polyglutamine (polyQ) tract of prolonged length in the carboxyl terminal of the alpha1A subunit. Heterologous expression of mutated alpha1A subunits results in increased channel inactivation in electrophysiological tests. No treatment is known to improve SCA 6 at present, as none of the available drugs is able to reverse alpha1A dysregulation, nor disturbed protein aggregation, transport and localization in this disease. The drugs gabapentin and pregabalin interact with the alpha2delta subunit of the P/Q-type VDCCs. Gabapentin and pregabalin slow the rate of inactivation in recombinant P/Q-type VDCCs, expressed in Xenopus oocytes. These drugs improve ataxia in cases of CCA, olivopontocerebellar atrophy and ataxia-telangiectasia. On the basis of the neuropathological identity of SCA 6 with CCA, and given the capacity of gabapentin and pregabalin to decrease P/Q-type VDCCs inactivation, in this paper the authors put forward the hypothesis that the administration of gabapentin and pregabalin might prove beneficial in SCA 6 as the ataxia caused by this disease would be expected to improve. The authors hope that researchers working with this illness will be inspired and encouraged to undertake the appropriate clinical and experimental work.

Pharmacological characterization of inhibitory effects of postsynaptic opioid and cannabinoid receptors on calcium currents in neonatal rat nucleus tractus solitarius.

1. The profile of opioid and cannabinoid receptors in neurons of the nucleus tractus solitarius (NTS) has been studied using the whole-cell configuration of the patch clamp technique. 2. Experiments with selective agonists and antagonists of opioid, ORL and cannabinoid receptors indicated that mu-opioid, kappa-opioid, ORL-1 and CB1, but not delta-opioid, receptors inhibit VDCCs in NTS. 3. Application of [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO; mu-opioid receptor agonist), Orphanin FQ (ORL-1 receptor agonist) and WIN55,122 (CB1 receptor agonist) caused inhibition of I(Ba) in a concentration-dependent manner, with IC50's of 390 nM, 220 nM and 2.2 microM, respectively. 4. Intracellular dialysis of the G(i)-protein antibody attenuated DAMGO-, Orphanin FQ- and WIN55,122-induced inhibition of I(Ba). 5. Both pretreatment with adenylate cyclase inhibitor and intracellular dialysis of the protein kinase A (PKA) inhibitor attenuated WIN55,122-induced inhibition of I(Ba) but not DAMGO- and Orphanin FQ-induced inhibition. 6. Mainly N- and P/Q-type VDCCs were inhibited by both DAMGO and Orphanin FQ, while L-type VDCCs were inhibited by WIN55,122. 7. These results suggest that mu- and kappa-opioid receptors and ORL-1 receptor inhibit N- and P/Q-type VDCCs via G alpha(i)-protein betagamma subunits, whereas CB1 receptors inhibit L-type VDCCs via G alpha(i)-proteins involving PKA in NTS.

Slowed N-type calcium channel (CaV2.2) deactivation by the cyclin-dependent kinase inhibitor roscovitine.

The lack of a calcium channel agonist (e.g., BayK8644) for CaV2 channels has impeded their investigation. Roscovitine, a potent inhibitor of cyclin-dependent kinases 1, 2, and 5, has recently been reported to slow the deactivation of P/Q-type calcium channels (CaV2.1). We show that roscovitine also slows deactivation (EC(50) approximately 53 microM) of N-type calcium channels (CaV2.2) and investigate gating alterations induced by roscovitine. The onset of slowed deactivation was rapid ( approximately 2 s), which contrasts with a slower effect of roscovitine to inhibit N-current (EC(50) approximately 300 microM). Slow deactivation was specific to roscovitine, since it could not be induced by a closely related cyclin-dependent kinase inhibitor, olomoucine (300 microM). Intracellularly applied roscovitine failed to slow deactivation, which implies an extracellular binding site. The roscovitine-induced slow deactivation was accompanied by a slight left shift in the activation-voltage relationship, slower activation at negative potentials, and increased inactivation. Additional data showed that roscovitine preferentially binds to the open channel to slow deactivation. A model where roscovitine reduced a backward rate constant between two open states was able to reproduce the effect of roscovitine on both activation and deactivation.

Spontaneous muscle action potentials are blocked by N-type and P/Q-calcium channels blockers in the rat spinal cord-muscle co-culture system.

This study investigated the effects of the calcium channel blockers nicardipine, calcicludine, omega-conotoxin GVIA, omega-agatoxin IVA, SNX-482, and NiCl on spontaneous muscle action potential of a rat spinal cord-muscle co-culture system. Spontaneous muscle action potential of the innervated muscle cells was blocked by d-tubocurarine (1 microM), but was not significantly affected by the L-type channel blocker nicardipine (100 nM). The neuronal L-type calcium channel blocker, calcicludine (50 and 100 nM), also had no effect on the frequency of spontaneous muscle action potential. However, nicardipine (100 nM) and calcicludine (100 nM) significantly increased the amplitude of muscle action potential. Application of the N-type calcium channel blocker, omega-conotoxin GVIA (50 and 100 nM), and the P/Q-type calcium channel blocker, omega-agatoxin IVA (10, 30, 50, and 100 nM), blocked the frequency and amplitude of spontaneous muscle action potential of the spinal cord-muscle co-cultured cells. In contrast, spontaneous muscle action potential was not affected by the R-type calcium channel blockers SNX-482 (100 nM) or NiCl (500 nM). These results indicate that blockers of N- and P/Q-type voltage-dependent calcium channels inhibit transmitter release at neuromuscular junctions in the rat spinal cord-muscle co-culture system.

Gabapentin fails to alter P/Q-type Ca2+ channel-mediated synaptic transmission in the hippocampus in vitro.

Gabapentin (Neurontin) has been successfully used in the treatment of both epilepsy and neuropathic pain. Despite its widespread clinical use, its mechanism of action is very poorly understood. Indeed, the only protein it is known to interact with is the alpha2delta subunit of the voltage-gated Ca(2+) channel complex. In a recent article, gabapentin was reported to inhibit synaptic transmission in the spinal cord through an inhibitory effect on presynaptic P/Q-type Ca(2+) channels in both glutamatergic primary afferents and glycinergic interneuones. To examine if such inhibition of P/Q-channel-mediated synaptic transmission by gabapentin generalised to other synaptic pathways, we tested the actions of gabapentin of P/Q-type Ca(2+) channel-mediated synaptic responses in the CA1 subfield of the hippocampus. We found that gabapentin was completely inactive on such synaptic responses even at 10 times the maximally effective concentration used in the spinal cord. A small ( approximately 10%) but consistent depression of control synaptic responses was elicited by 10 microM gabapentin. No greater response was observed at a 10 times higher concentration. From these data we conclude that gabapentin is not a generic inhibitor of presynaptic P/Q-type channels and its actions at the spinal level must represent a feature of the P/Q-type channel not present in the hippocampus. Given the known interactions of this compound, the best candidate for this is the presence, subtype, or state of the alpha2delta subunit.

Presynaptic Cav2.1 and Cav2.2 differentially influence release dynamics at hippocampal excitatory synapses.

Presynaptic calcium influx at most excitatory central synapses is carried by both Cav2.1 and Cav2.2 channels. The kinetics and modulation of Cav2.1 and Cav2.2 channels differ and may affect presynaptic calcium influx. We compared release dynamics at CA3/CA1 synapses in rat hippocampus after selective blockade of either channel subtype and subsequent quantal content restoration. Selective blockade of Cav2.1 channels enhanced paired-pulse facilitation, whereas blockade of Cav2.2 channels decreased it. This effect was observed at short (50 msec) but not longer (500 msec) intervals and was maintained during prolonged bursts of presynaptic activity. It did not reflect differences in the distance of the channels from the calcium sensor. The suppression of this effect by preincubation with the G(o/i)-protein inhibitor pertussis toxin suggests instead that high-frequency stimulation relieves inhibition of Cav2.2 by G(o/i), thereby increasing the number of available channels.

Differential sensitivity of N- and P/Q-type Ca2+ channel currents to a mu opioid in isolectin B4-positive and -negative dorsal root ganglion neurons.

Opioids have a selective effect on nociception with little effect on other sensory modalities. However, the cellular mechanisms for this preferential effect are not fully known. Two broad classes of nociceptors can be distinguished based on their growth factor requirements and binding to isolectin B4(IB4). In this study, we determined the difference in the modulation of voltage-gated Ca2+ currents by [D-Ala2,N-Me-Phe4,Gly-ol5]-enkephalin (DAMGO, a specific mu opioid agonist) between IB4-positive and -negative small dorsal root ganglion (DRG) neurons. Whole-cell voltage-clamp recordings were performed in acutely isolated DRG neurons in adult rats. Both 1-10 microM DAMGO and 1 to 10 microM morphine had a greater effect on high voltage-activated Ca2+ currents in IB4-negative than IB4-positive cells. However, DAMGO had no significant effect on T-type Ca2+ currents in both groups. The N-type Ca2+ current was the major subtype of Ca2+ currents inhibited by DAMGO in both IB4-positive and -negative neurons. Although DAMGO had no effect on L-type and R-type Ca2+ currents in both groups, it produced a larger inhibition on N-type and P/Q-type Ca2+ currents in IB4-negative than IB4-positive neurons. Furthermore, double labeling revealed that there was a significantly higher mu opioid receptor immunoreactivity in IB4-negative than IB4-positive cells. Thus, these data suggest that N-and P/Q-type Ca2+ currents are more sensitive to inhibition by the mu opioids in IB4-negative than IB4-positive DRG neurons. The differential sensitivity of voltage-gated Ca2+ channels to the mu opioids in subsets of DRG neurons may constitute an important analgesic mechanism of mu opioids.

Synthesis and biological evaluation of nonpeptide mimetics of omega-conotoxin GVIA.

A benzothiazole-derived compound (4a) designed to mimic the C(alpha)-C(beta) bond vectors and terminal functionalities of Lys2, Tyr13 and Arg17 in omega-conotoxin GVIA was synthesised, together with analogues (4b-d), which had each side-chain mimic systematically truncated or eliminated. The affinity of these compounds for rat brain N-type and P/Q-type voltage gated calcium channels (VGCCs) was determined. In terms of N-type channel affinity and selectivity, two of these compounds (4a and 4d) were found to be highly promising, first generation mimetics of omega-conotoxin. The fully functionalised mimetic (4a) showed low microM binding affinity to N-type VGCCs (IC(50)=1.9 microM) and greater than 20-fold selectivity for this channel sub-type over P/Q-type VGCCs, whereas the mimetic in which the guanidine-type side chain was truncated back to an amine (4d, IC(50)= 4.1 microM) showed a greater than 25-fold selectivity for the N-type channel.