Conotoxin GVIA Research Articles

Venoconstrictor responses to activation of bradykinin-sensitive pericardial afferents involve the region of the hypothalamic paraventricular nucleus.

Veins are important in the control of venous return, cardiac output, and cardiovascular homeostasis. However, the effector systems modulating venous function remain to be fully elucidated. We demonstrated that activation of bradykinin‐sensitive pericardial afferents elicited systemic venoconstriction. The hypothalamic paraventricular nucleus (PVN) is an important site modulating autonomic outflow to the venous compartment. We tested the hypothesis that the PVN region is involved in the venoconstrictor response to pericardial injection of bradykinin. Rats were anesthetized with urethane/alpha chloralose and instrumented for recording arterial pressure, vena caval pressure, and mean circulatory filling pressure (MCFP), an index of venous tone. The rats were fitted with a pericardial catheter and PVN injector guide tubes. Mean arterial pressure (MAP), heart rate (HR), and MCFP responses to pericardial injection of bradykinin (1, 10 µg/kg) were recorded before and after PVN injection of omega conotoxin GVIA (200 ng/200 nl). Pericardial injection of saline produced no systematic effects on MAP, HR, or MCFP. In contrast, pericardial injection of bradykinin was associated with short latency increases in MAP (16 ± 4 to 18 ± 2 mm Hg) and MCFP 0.35 ± 0.19 to 1.01 ± 0.27 mm Hg. Heart rate responses to pericardial BK were highly variable, but HR was significantly increased (15 ± 9 bpm) at the higher BK dose. Conotoxin injection in the PVN region did not affect baseline values for these variables. However, injection of conotoxin into the area of the PVN largely attenuated the pressor (−1 ± 3 to 6 ± 3 mm Hg), MCFP (−0.19 ± 0.07 to 0.20 ± 0.18 mm Hg), and HR (4 ± 14 bpm) responses to pericardial bradykinin injection. We conclude that the PVN region is involved in the venoconstrictor responses to pericardial bradykinin injection.

Specificity in the interaction of high-voltage-activated Ca2+ channel types with Ca2+-dependent afterhyperpolarizations in magnocellular supraoptic neurons.

Magnocellular oxytocin (OT) and vasopressin (VP) neurons express an afterhyperpolarization (AHP) following spike trains that attenuates firing rate and contributes to burst patterning. This AHP includes contributions from an apamin-sensitive, medium-duration AHP (mAHP) and from an apamin-insensitive, slow-duration AHP (sAHP). These AHPs are Ca2+ dependent and activated by Ca2+ influx through voltage-gated Ca2+ channels. Across central nervous system neurons that generate Ca2+-dependent AHPs, the Ca2+ channels that couple to the mAHP and sAHP differ greatly, but for magnocellular neurosecretory cells this relationship is unknown. Using simultaneous whole cell recording and Ca2+ imaging, we evaluated the effect of specific high-voltage-activated (HVA) Ca2+ channel blockers on the mAHP and sAHP. Block of all HVA channels via 400 μM Cd2+ inhibited almost the entire AHP. We tested nifedipine, conotoxin GVIA, agatoxin IVA, and SNX-482, specific blockers of L-, N-, P/Q-, and R-type channels, respectively. The N-type channel blocker conotoxin GVIA (1 μM) was the only toxin that inhibited the mAHP in either OT or VP neurons although the effect on VP neurons was weaker by comparison. The sAHP was significantly inhibited by N-type block in OT neurons and by R-type block in VP neurons although neither accounted for the entirety of the sAHP. Thus the mAHP appears to be elicited by Ca2+ from mostly N-type channels in both OT and VP neurons, but the contributions of specific Ca2+ channel types to the sAHP in each cell type are different. Alternative sources to HVA channels may contribute Ca2+ for the sAHP. NEW & NOTEWORTHY Despite the importance of afterhyperpolarization (AHP) mechanisms for regulating firing behavior of oxytocin (OT) and vasopressin (VP) neurons of supraoptic nucleus, which types of high-voltage-activated Ca2+ channels elicit AHPs in these cells was unknown. We found that N-type channels couple to the medium AHP in both cell types. For the slow AHP, N-type channels contribute in OT neurons, whereas R-type contribute in VP neurons. No single Ca2+ channel blocker abolished the entire AHP, suggesting that additional Ca2+ sources are involved.

Estrogen and G protein-coupled estrogen receptor agonist G-1 cause relaxation of human gallbladder

ObjectiveEstrogen interacts with a membrane receptor, G protein-coupled estrogen receptor (GPER). It was reported that 17β-estradiol was able to inhibit contraction of the human colon and cause relaxation of the guinea pig gallbladder, however, the involvement of GPER was not clarified. The aim of the present study was to investigate the effect of estrogen on human gallbladder motility and the possible role of GPER. Materials and MethodsRelaxation of human gallbladder strips were measured using isometric transducers. Expression of GPER was evaluated by reverse transcription polymerase chain reaction (PCR), real-time PCR, and immunohistochemistry. ResultsIn human gallbladder strips, 17β-estradiol and G-1 elicited marked and rapid relaxation, whereas tamoxifen produced mild concentration-dependent relaxation. The relative efficacies to cause relaxation were as follows: 17β-estradiol = G-1 > tamoxifen. The relaxant response of 17β-estradiol was not attenuated by tetrodotoxin or conotoxin GVIA. This implies that nerve stimulation was not involved in the 17β-estradiol-induced gallbladder relaxation. Analysis by reverse transcription PCR and real-time PCR showed that GPER was expressed in the human gallbladder. Further analysis by immunohistochemistry revealed that GPER was expressed in the gallbladder muscle. This suggests that 17β-estradiol relaxes the human gallbladder via GPER. ConclusionThese results demonstrate for the first time that 17β-estradiol and GPER agonist G-1 cause relaxation of the human gallbladder, probably through GPER. Estrogen might play an important role in the control of human gallbladder motility.

Melanocortin 4 receptor activation inhibits presynaptic N-type calcium channels in amygdaloid complex neurons.

The melanocortin 4 receptor (MC4R) is a G protein-coupled receptor involved in food intake and energy expenditure regulation. MC4R activation modifies neuronal activity but the molecular mechanisms by which this regulation occurs remain unclear. Here, we tested the hypothesis that MC4R activation regulates the activity of voltage-gated calcium channels and, as a consequence, synaptic activity. We also tested whether the proposed effect occurs in the amygdala, a brain area known to mediate the anorexigenic actions of MC4R signaling. Using the patch-clamp technique, we found that the activation of MC4R with its agonist melanotan II specifically inhibited 34.5 ± 1.5% of N-type calcium currents in transiently transfected HEK293 cells. This inhibition was concentration-dependent, voltage-independent and occluded by the Gαs pathway inhibitor cholera toxin. Moreover, we found that melanotan II specifically inhibited 25.9 ± 2.0% of native N-type calcium currents and 55.4 ± 14.4% of evoked inhibitory postsynaptic currents in mouse cultured amygdala neurons. In vivo, we found that the MC4R agonist RO27-3225 increased the marker of cellular activity c-Fos in several components of the amygdala, whereas the N-type channel blocker ω conotoxin GVIA increased c-Fos expression exclusively in the central subdivision of the amygdala. Thus, MC4R specifically inhibited the presynaptic N-type channel subtype, and this inhibition may be important for the effects of melanocortin in the central subdivision of the amygdala.

Calcium channels in rat horizontal cells regulate feedback inhibition of photoreceptors through an unconventional GABA‐ and pH‐sensitive mechanism

Horizontal cells send inhibitory feedback to photoreceptors, helping form antagonistic receptive fields in the retina, but the neurotransmitter and the mechanisms underlying this signalling are not known. Since the proteins responsible for conventional Ca(2+)-dependent release of GABAergic synaptic vesicles are present in mammalian horizontal cells, we investigated this conventional mechanism as the means by which horizontal cells inhibit photoreceptors. Using Ca(2+) imaging in rat retinal slices, we confirm that horizontal cell depolarization with kainate inhibits and horizontal cell hyperpolarization with NBQX disinhibits the Ca(2+) signals produced by pH-sensitive activation of voltage-gated calcium channels (Ca channels) in photoreceptors. We show that while 100 μm Co(2+) reduces photoreceptor Ca(2+) signals, it disinhibits them at 10 μm, an effect reminiscent of earlier studies where low [Co(2+)] eliminated feedback. The low [Co(2+)] disinhibition is pH sensitive. We localized L-, N- and P/Q-type Ca channels in rat horizontal cells, and showed that both the N-type Ca channel blocker -conotoxin GVIA and the P/Q-type Ca channel blocker -agatoxin IVA increased Ca(2+) signals in photoreceptors in a pH-sensitive manner. Pronounced actions of GABAergic agents on feedback signals to photoreceptors were observed, and are pH sensitive, but are inconsistent with direct inhibition by GABA of photoreceptor [Ca(2+)]. Patch-clamp studies revealed that GABA activates a conductance having high bicarbonate permeability in isolated horizontal cells, suggesting that the commonality of pH sensitivity throughout the results could arise from a GABA autofeedback action in horizontal cells. This could change cleft pH with concomitant inhibitory influences on photoreceptor Ca channels.

38 MOUSE VOIDING PATTERNS ARE STRAIN-SPECIFIC: A NOVEL ASSAY FOR AGE-RELATED DEVELOPMENT OF LOWER URINARY TRACT SYMPTOMS (LUTS)

They were divided into two groups; BOO and sham operated (SHAM) groups. BOO was created by a partial ligation of the urethra with a nylon suture, whereas SHAM group of rats underwent only exposure of the urethra without ligation. Six weeks after surgery, three experiments were performed: 1) mRNA expression of N-Ca in the bladder, 2) in vitro functional studies of detrusor strips focusing on the effects of -conotoxin GVIA (CTX; a selevctive N-Ca inhibitor, 10 6 M) on carbachol (CCh)-induced and electrical field stimulation (EFS)-induced contractions, and 3) cystometric investiogations (CMG) with continuous salineinstillation in a conscious state with intravenous administration of CTX (3 g/kg). RESULTS: N-Ca mRNA expression in the detrusor was about 50 times higher in BOO than SHAM group. CTX did not suppress CCh-induced contractions in either group. The EFS-induced contractions were weaker in the detrusor of BOO than SHAM group. CTX significantly suppressed the EFS-induced contractions in BOO group, but not in SHAM group (Figure 1 A, B). The suppressive effect of CTX on the EFS-induced contractions was more pronounced in the cholinergic component at 10 Hz. In CMG, the number of non-voiding contractions (NVCs) was significantly greater in BOO group than SHAM group. The number of NVCs was significantly decreased after CTXadministration in BOO rats (Figure 1 C). CONCLUSIONS: The results of the current study suggest that N-Ca may have an important role in facilitating release of excitatory transmitters including ACh from the efferent nerve terminals innervating the detrusor of the BOO rats, and this may link to the development of detrusor overactivity associated with BOO.

Stable Respiratory Activity Requires Both P/Q-Type and N-Type Voltage-Gated Calcium Channels

P/Q-type voltage-gated calcium channels (Ca(v)2.1) play critical presynaptic and postsynaptic roles throughout the nervous system and have been implicated in a variety of neurological disorders. Here we report that mice with a genetic ablation of the Ca(v)2.1 pore-forming α(1A) subunit (α(1A)⁻/⁻) encoded by CACNA1a (Jun et al., 1999) suffer during postnatal development from increasing breathing disturbances that lead ultimately to death. Breathing abnormalities include decreased minute ventilation and a specific loss of sighs, which was associated with lung atelectasis. Similar respiratory alterations were preserved in the isolated in vitro brainstem slice preparation containing the pre-Bötzinger complex. The loss of Ca(v)2.1 was associated with an alteration in the functional dependency on N-type calcium channels (Ca(v)2.2). Blocking N-type calcium channels with conotoxin GVIA had only minor effects on respiratory activity in slices from control (CT) littermates, but abolished respiratory activity in all slices from α(1A)⁻/⁻ mice. The amplitude of evoked EPSPs was smaller in inspiratory neurons from α(1A)⁻/⁻ mice compared with CTs. Conotoxin GVIA abolished all EPSPs in inspiratory neurons from α(1A)⁻/⁻ mice, while the EPSP amplitude was reduced by only 30% in CT mice. Moreover, neuromodulation was significantly altered as muscarine abolished respiratory network activity in α(1A)⁻/⁻ mice but not in CT mice. We conclude that excitatory synaptic transmission dependent on N-type and P/Q-type calcium channels is required for stable breathing and sighing. In the absence of P/Q-type calcium channels, breathing, sighing, and neuromodulation are severely compromised, leading to early mortality.

Presynaptic voltage-dependent calcium channels and regulation of the transmitter release in the peripheral nervous system

Calcium ions entering into the nerve terminal through voltage-dependent calcium channels during action potential initiate neurotransmitter release and thereby play an important role in the transmission of excitation. In addition to the amount of released neurotransmitter quanta (the quantal content of the endplate potential), another important characteristic of exocytosis is the kinetics of release of individual quanta that form total multiquantal response. Previously, we showed the dependence of the temporal parameters of neurotransmitter quantum release on the extracellular calcium ion concentration, but it remained unclear which calcium channel types are involved in the modulation of the quantal content and release kinetics. It is commonly accepted that, in contrast to the central nervous system, wherein the secretion take part various types of voltage-dependent calcium channels, exocytosis in the peripheral nervous system of adult animals mainly occurs owing to one type of calcium channels. This is the P/Q type (Ca V 2.1 in the latest classification) blocked with FTX toxin and ω -agatoxin IVA in mammalian neuromuscular junction. In frog synapses, this is the N type channels (Ca V 2.2), which are blocked by the ω -conotoxin GVIA. At the same time, the ability of FTX, a specifc P/Q channel blocker, to decrease the quantal content in frog synapses and effects of dihydropyridines that block L type (Ca V 1.2) channels in the developing Xenopus synapses, and L type (Ca V 1.2) channels activator effects in frog synapses, taken together, indicate the possibility of participation of other types of calcium channels in the exocytosis regulation in peripheral synapses. The possible role of changes in the activity of not only P/Q channels, but also other calcium channel types, in modulation of the temporal parameters of neurotransmitter release was shown in peripheral synapses of mouse and crayfish. In order to identify the type of voltage-dependent calcium channels modulating of the quanta secretion in frog and mouse synapses, the effects of specific blockers of various channel types on the endplate potentials quantal content and transmitter release kinetics were analyzed. On isolated Rana ridibunda neuromuscular preparation of m. cutaneus pectoris and on the mouse diaphragm the evoked uniquantal endplate currents were registered by the extracellular electrodes and the fluctuations of the real synaptic latency were estimated. Blocking of the P/Q-, N-, and L-channels in nerve-muscle junction of the frog by the appropriate specific blockers led to a decrease in the amount of released quanta, as well as in the synaptic latency variance, that indicate the synchronization of the process of synaptic vesicles exocytosis. Obtained results indicate that in low-calcium solution the kinetics of quanta secretion in the frog synapse may be regulated by N-, P/Q-, and L-calcium channels. In the mouse nerve muscle junctions at low calcium concentration and infrequent stimulation, when asynchrony of the secretion was more pronounced, the block of the P/Q-channels led to a decrease in the amount of released quanta without changing the kinetics of their secretion, while blocking the L-type channels only decreased variance of synaptic latency. The fact that the number of released quanta and the time course of their secretion may change independently when various types of voltage-dependent calcium channels are blocked witnesses that the role of N-, P/Q-, and L-channels in modulation of the process of synaptic vesicles exocytosis is different.

ω-conotoxin GVIA sensitive calcium channels on preganglionic nerve terminals in mouse pelvic and celiac ganglia

Release of acetylcholine (ACh) from preganglionic nerve terminals requires calcium entry through voltage-gated calcium channels. The calcium channel subtype required for ACh release varies depending on the particular ganglionic synapse. I have investigated the functional role of calcium channels in transmitter release from parasympathetic and sympathetic preganglionic terminals in pelvic and celiac ganglia of female mice. Single electrode voltage clamp was used to measure EPSC amplitude in the absence and presence of selective calcium channel antagonists. In pelvic ganglia ω- conotoxin GVIA, a selective N-type calcium channel antagonist, reduced the amplitude of EPSCs evoked by pelvic nerve stimulation by 46 ± 5% ( n = 8, P = 0.015). In contrast, in the celiac ganglion, ω- conotoxin GVIA had no effect on the amplitude of EPSCs evoked by splanchnic nerve stimulation ( P = 0.09, n = 7). EPSCs in both pelvic and celiac ganglia were resistant to the P-type calcium channel antagonist agatoxin (50 nM, n = 5 for both ganglia) and the R-type calcium channel antagonist SNX482 (100 nM, n = 4 for both ganglia). These results indicate that in female mice, release of ACh in sympathetic pathways to prevertebral ganglia does not require calcium entry from N-type calcium channels. However, release of ACh from sacral parasympathetic preganglionic neurons requires calcium entry from both N-type and toxin-resistant calcium channels.

Tx2‐6 toxin from Phoneutria nigriventer spider improves relaxation induced by electrical stimulation of rat cavernosum strips

Background: Accidents involving the spider P. nigriventer are characterized by different symptoms including priapism. Some toxins from poisonous animals exert their toxic effects by modifying the properties of ion channels. We have previously demonstrated that Tx2‐6, a peptide extracted from the venom of P. nigriventer, improves erectile function in anesthetized rats. Here we investigated the mechanism involved in the Tx2‐6 potentiation of penile erection.Methods: Rat cavernosum strips were incubated with bretylium (3 x 10–5M) and contracted with phenylephrine (10–5 M). Relaxation responses were evoked by electrical field stimulation (EFS) or sodium nitroprusside (SNP) before and after 5 min incubation with Tx2–6 (10–8 M). The effect of Tx2‐6 on relaxation induced by EFS was also tested in the presence of atropine (10–6 M), conotoxin GVIA (10–6 M) or nifedipine (10–7 M).Results: Relaxation induced by SNP was not affected by Tx2‐6. EFS‐induced relaxation was significantly potentiated by Tx2‐6 and this effect was not affected by atropine, whereas blockade of N‐ or L‐Type Ca+2 channels abolished the potentiation of the relaxation induced by Tx2‐6.Conclusion: Tx2‐6 potentiates the relaxation of cavernosum tissue via Na+ and Ca+2 channels, facilitating the nitric oxide/cGMP pathway. Tx2‐6 has great potential for development of drugs to treat erectile dysfunction.SUPPORT: CNPq, FAPEMIG, NIH

Synthesis and liposome encapsulation of a novel 18F‐conjugate of ω‐conotoxin GVIA for the potential imaging of N‐type Ca2+ channels in the brain by positron emission tomography

Abstractω‐Conotoxin GVIA is a potent, irreversible antagonist of N‐type voltage gated Ca2+ channels. A radiofluorinated analogue of GVIA could be useful in assessing regional synaptic density of the brain, in vivo, using positron emission tomography. N‐hydroxy succinimidyl 4‐[18F]fluorobenzoate was employed to site‐specifically label GVIA, preserving native binding affinity. The tracer was characterized with MALDI‐TOF mass spectrometry and colorimetric protein assay. Radiochemical decay‐corrected yield of the lysine‐24 labeled analogue of [18F]GVIA was 5%. Specific activity of this species was determined to be 1.2 × 105 Ci/mmol. Encapsulation of the tracer in sulfatide containing liposomes, a potential method for enhancing blood–brain penetrance, was accomplished with 40% efficiency. Copyright © 2006 John Wiley & Sons, Ltd.

Dosage et spécificité d’autoanticorps anti-canaux calcium dans le syndrome myasthénique de Lambert-Eaton

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune channelopathy in which patients produce autoantibodies directed against voltage-gated calcium channels. Autoantibodies down-regulate calcium channels resulting in reduced transmitter release, which in turn leads to muscular weakness and autonomic dysfunction. LEMS is paraneoplastic in 60-70% of patients, most frequently associated with small cell lung carcinoma (SCLC). SCLC lines express many neuronal and neuroendocrine proteins including neuronal calcium channels of the Cav2 family (P/Q and N-type channels). It is thus likely that the paraneoplastic form of LEMS is the consequence of an anti-tumoral immune response and the production of antibodies that cross-react with identical or homologous antigens in nerve terminals. Neurological symptoms generally appear several Months before detection of the tumor. Consequently correct diagnosis of LEMS is crucial as it can allow early treatment of a particularly aggressive carcinoma. Based on published studies, our laboratory has set-up serological assays for LEMS autoantibodies as an aid to diagnosis. Calcium channels in detergent extracts of rat brain or cerebellum membranes were labeled with radioligands specific for N-type (125I-omega conotoxin GVIA) or P/Q-type (125I-omega conotoxin MVIIC) calcium channels. Autoantibodies that immunoprecipitate the ligand/channel complex can thus be titrated. Analysis of 31 LEMS sera revealed the presence of anti-N type channel antibodies in 58% and anti-P/Q type channel antibodies in 74% of patients with titres ranging from 90 to 2950 pM. Only 5 patients were seronegative in both tests, thus a combination of the two assays reliably detected autoantibodies in 26/31 (84%) patients.

Protein kinase C is involved in neurokinin receptor modulation of N- and L-type Ca2+ channels in DRG neurons of the adult rat.

Whole cell patch-clamp techniques were used to examine neurokinin receptor modulation of Ca2+ channels in small to medium size dorsal root ganglia neurons (<40 pF) that express mainly N- and L-type Ca2+ currents. Low concentrations of substance P enhanced Ca2+ currents (5-40%, <0.2 microM), while higher concentrations applied cumulatively reversed these enhancements (5-28% reductions, >0.5 microM). This apparent inhibition by high concentrations of substance P was blocked by the administration of the NK3 antagonist SB 235,375 (0.2 microM). The NK1 agonist, [Sar9,Met11]-substance P (0.05 to 1.0 microM) did not alter Ca2+ currents; whereas the NK2 agonist, [betaAla8]-neurokinin A (4-10), enhanced Ca2+ currents (5-36% increase, 0.05-0.5 microM). The enhancement was reversed by the NK2 antagonist MEN 10,376 (0.2 microM) but unaffected by the NK3 antagonist SB 235,375 (0.2 microM). The NK3 agonist [MePhe7]-neurokinin B (0.5-1.0 microM) inhibited Ca2+ currents (6-24% decrease). This inhibition was not prevented by the NK2 antagonist MEN 10,376 (0.2 microM) but was blocked by the NK3 antagonist SB 235,375 (0.2 microM). Both the enhancement and inhibition of Ca2+ currents by neurokinin agonists were reversed by the protein kinase C inhibitor bisindolylmaleimide I HCl (0.2-0.5 microM). Following inhibition of Ca2+ channels by [MePhe7]-neurokinin the facilitatory effect of BayK 8644 (5 microM) was increased and the inhibitory effect of the N-type Ca2+ channel blocker w -conotoxin GVIA (1 microM) was diminished, suggesting that the NK3 agonist inhibits N-type Ca2+ channels. Similarly, block of all but N-type Ca2+ channels, revealed that [betaAla8]-neurokinin A (4-10) enhanced the currents while [MePhe7]-neurokinin B inhibited the currents. Inhibition of all but L-type Ca2+ channels, revealed that [betaAla8]-neurokinin A (4-10) enhanced the currents while [MePhe7]-neurokinin B had no effect. Activation of protein kinase C with low concentrations of phorbol-12,13-dibutyrate enhanced Ca2+ currents, but high concentrations inhibited N- and L-type Ca2+ currents. In summary, these data suggest that in adult rat dorsal root ganglia neurons, NK2 receptors enhance both L- and N-type Ca2+ channels and NK3 receptors inhibit N-type Ca2+ channels and that these effects are mediated by protein kinase C phosphorylation of Ca2+ channels.

Role of L-type Ca2+ channels in transmitter release from mammalian inner hair cells. II. Single-neuron activity.

Previously reported changes in the gross sound-evoked cochlear potentials after intracochlear perfusion of nimodipine suggest that dihydropyridine-sensitive Ca2+ channels (L-type) control the sound-evoked release of transmitter from the inner hair cells of the mammalian cochlea. In the present study, we combined recording of the action potentials of single primary auditory afferent neurons with intracochlear perfusion to further investigate the role of voltage-gated Ca2+ channels at this synapse. Spontaneous action potential firing rates were depressed by the L-type channel blocker nimodipine, but were elevated by S(-) BAY K8644, an L-type channel agonist. Sound-evoked responses of single primary afferents were depressed by nimodipine in a manner that was consistent with a block at the inner hair cell-afferent dendrite synapse. Perfusions with solutions containing the N-type channel blocker conotoxin GVIA did not differ in their effects from control artificial perilymph perfusions. The results extend the conclusions of the earlier study by showing that L-type Ca2+ channels are primarily responsible for controlling both spontaneous and sound-evoked transmitter release from inner hair cells. In addition it was found that afferent neurons with widely different spontaneous firing rates were all sensitive to nimodipine and to BAY K8644, suggesting that the multiple synaptic outputs of each inner hair cell are under the control of only one major type of Ca2+ channel.

Effects of prolonged exposure to nanomolar concentrations of methylmercury on voltage-sensitive sodium and calcium currents in PC12 cells

The neurotoxicant methylmercury (CH 3Hg +) inhibits voltage-sensitive Na + and Ca 2+ currents in neuronal preparations following acute, in vitro, exposure. In the present study, effects on voltage-sensitive Na + ( I Na) and Ca 2+ ( I Ca) currents in pheochromocytoma (PC12) cells were examined following prolonged exposure to CH 3Hg +. When PC12 cells cultured in the presence of nerve growth factor (NGF) for 7 days (‘primed’) were replated in the presence of NGF and 30 nM CH 3Hg +, I Ca, but not I Na, amplitude was reduced (29%) significantly ∼24 h later. Quantitative assessment of morphology indicated that this ∼24 h exposure to CH 3Hg + significantly reduced neurite length. The N-type voltage-sensitive Ca 2+ channel (VSCC) antagonist ω-conotoxin GVIA (500 pM) was without significant effect on current amplitude or morphology in this exposure protocol. When undifferentiated cells were cultured in the presence of NGF and 10 nM CH 3Hg + for 6 days, I Ca and I Na amplitude were reduced by 36 and 52%, respectively. I Ca at the end of a 150 ms test pulse was also reduced by 40% in CH 3Hg +-treated cells. Thus, both inactivating and non-inactivating I Ca were reduced equally. There was no change in [ 3H]saxitoxin or ω-[ 125I]conotoxin GVIA binding, nor were there any morphological alterations in cells treated with CH 3Hg + for 6 days. Omega-conotoxin GVIA (500 pM, 6 days), reduced significantly I Ca, but not I Na, but was without effect on morphology. These results demonstrate that prolonged exposure to low concentrations of CH 3Hg + reduces cationic currents in differentiating PC12 cells, but that current reduction is not always associated with morphological alteration.

Oxytocin retrogradely inhibits evoked, but not miniature, EPSCs in the rat supraoptic nucleus: role of N- and P/Q-type calcium channels.

We previously reported that oxytocin (OXT), released from the dendrites of magnocellular neurons in the supraoptic nucleus (SON), acts retrogradely on presynaptic terminals to inhibit glutamatergic transmission. Here we test the hypothesis that oxytocin reduces calcium influx into the presynaptic terminal. We used nystatin perforated-patch recording in vitro to first identify the calcium channels involved in glutamatergic transmission in the SON. [omega]-Conotoxin GVIA ([omega]-CTx) and [omega]-Agatoxin TK ([omega]-Aga) both reduced evoked EPSC amplitude, while nicardipine and nickel had no effect. A combination of [omega]-CTx and [omega]-Aga completely abolished the evoked EPSCs. This depressant effect was accompanied by an increase in the paired pulse ratio with no change in the kinetics of the evoked EPSCs, AMPA currents or postsynaptic cell properties. These results suggest that presynaptic N- and P/Q-type calcium channels mediate glutamate release in the SON while L-, T- and R-type channels make little or no contribution. Oxytocin-induced reduction of the evoked EPSC was substantially occluded in the presence of [omega]-CTx but only partially in the presence of [omega]-Aga. Amastatin, an endopeptidase inhibitor that increases the level of endogenous OXT, also reduced the evoked EPSC. This amastatin effect was also occluded by [omega]-CTx and [omega]-Aga. Miniature EPSCs, which are independent of extracellular calcium, were unaffected by either [omega]-CTx or by OXT, thus further substantiating an action of both compounds on calcium channels. Therefore, dendritically released oxytocin acts mainly via a mechanism involving the N-type channel, and to a lesser extent the P/Q-type channel, to decrease excitatory transmission.

Presynaptic mechanisms underlying cannabinoid inhibition of excitatory synaptic transmission in rat striatal neurons.

The striatum is a crucial site of action for the motor effects of cannabinoids (CBs). However, the electrophysiological consequences of activation of CB receptors on the striatal neurons have not been established. Here we report for the first time that the cannabimimetic aminoalkylindole WIN 55,212-2 and the endogenous cannabinoid anandamide substantially depress corticostriatal glutamatergic synaptic transmission onto striatal neurons in the brain slice preparation. The selective CB1 receptor antagonist SR 141716 effectively reversed this inhibition. WIN 55,212-2 significantly increased the paired-pulse facilitation of synaptically evoked EPSCs, while having no effect on the sensitivity of postsynaptic neurons to [alpha]-amino-3-hydroxy-5-methylisoxazole-4-propionic acid. WIN 55,212-2 also reduced the frequency of spontaneous, action potential-dependent EPSCs (sEPSCs) without altering their amplitude distribution. Superfusion of WIN 55,212-2 elicited a membrane hyperpolarization accompanied by a decrease in input resistance. Both effects were blocked by intracellular caesium. In contrast, intracellular caesium failed to affect WIN 55,212-2-mediated synaptic inhibition. The WIN 55,212-2-mediated synaptic inhibition was blocked by the Gi/o protein inhibitor pertussis toxin (PTX), but not by the GABA(A) receptor antagonist bicuculline or GABA(B) receptor antagonist SCH 50911. Pretreatment with the N-type Ca2+ channel antagonist [omega]-conotoxin GVIA selectively abolished the WIN-55,212-2-mediated synaptic inhibition. These results suggest that cannabinoids depress the corticostriatal glutamatergic synaptic transmission through the activation of presynaptic CB1 receptors to inhibit N-type Ca2+ channel activity, which in turn reduces glutamate release. The presynaptic action of cannabinoids is mediated by a PTX-sensitive Gi/o protein-coupled signalling pathway.

Agatoxin-IVA-sensitive calcium channels mediate the presynaptic and postsynaptic nicotinic activation of cardiac vagal neurons.

Whole cell currents and miniature glutamatergic synaptic events (minis) were recorded in vitro from cardiac vagal neurons in the nucleus ambiguus using the patch-clamp technique. We examined whether voltage-dependent calcium channels were involved in the nicotinic excitation of cardiac vagal neurons. Nicotine evoked an inward current, increase in mini amplitude, and increase in mini frequency in cardiac vagal neurons. These responses were inhibited by the nonselective voltage-dependent calcium channel blocker Cd (100 microM). The P-type voltage-dependent calcium channel blocker agatoxin IVA (100 nM) abolished the nicotine-evoked responses. Nimodipine (2 microM), an antagonist of L-type calcium channels, inhibited the increase in mini amplitude and frequency but did not block the ligand gated inward current. The N- and Q-type voltage-dependent calcium channel antagonists conotoxin GVIA (1 microM) and conotoxin MVIIC (5 microM) had no effect. We conclude that the presynaptic and postsynaptic facilitation of glutamatergic neurotransmission to cardiac vagal neurons by nicotine involves activation of agatoxin-IVA-sensitive and possibly L-type voltage-dependent calcium channels. The postsynaptic inward current elicited by nicotine is dependent on activation of agatoxin-IVA-sensitive voltage-dependent calcium channels.

Functional expression of L-, N-, P/Q-, and R-type calcium channels in the human NT2-N cell line.

The biophysical and pharmacological properties of voltage-gated calcium channel currents in the human teratocarcinoma cell line NT2-N were studied using the whole cell patch-clamp technique. When held at -80 mV, barium currents (I(Ba)s) were evoked by voltage commands to above -35 mV that peaked at +5 mV. When holding potentials were reduced to -20 mV or 5 mM barium was substituted for 5 mM calcium, there was a reduction in peak currents and a right shift in the current-voltage curve. A steady-state inactivation curve for I(Ba) was fit with a Boltzmann curve (V(1/2) = -43.3 mV; slope = -17.7 mV). Maximal current amplitude increased from 1-wk (232 pA) to 9-wk (1025 pA) postdifferentiation. Whole cell I(Ba)s were partially blocked by specific channel blockers to a similar extent in 1- to 3-wk and 7- to 9-wk postdifferentiation NT2-N cells: 10 microM nifedipine (19 vs. 25%), 10 microM conotoxin GVIA (27 vs. 25%), 10 microM conotoxin MVIIC (15 vs. 16%), and 1.75 microM SNX-482 (31 vs. 33%). Currents were completely blocked by 300 microM cadmium. In the presence of nifedipine, GVIA, and MVIIC, approximately 35% of current remained, which was reduced further by SNX-482 (7-14% of current remained), consistent with functional expression of L-, N-, and P/Q-calcium channel types and one or more R-type channel. The presence of multiple calcium currents in this human neuronal-type cell line provides a potentially useful model for study of the regulation, expression and cellular function of human derived calcium channel currents; in particular the R-type current(s).

Isolation and Characterization of a Novel ConusPeptide with Apparent Antinociceptive Activity

Isolation and Characterization of a Novel ConusPeptide with Apparent Antinociceptive Activity