Neuronal Voltage-sensitive Calcium Channels Research Articles

Psychiatric Predisposition to Autonomic and Abnormal Perception Side-Effects of Ziconotide: A Case Series Study

Ziconotide is a reversible blocker of the N-type neuronal voltage-sensitive calcium channels with analgesic effects. The main adverse effects of ziconotide are ataxia, dizziness, gait disorder, confusion, hallucinations, and gastrointestinal symptoms. Eighteen chronic pain patients with intrathecal ziconotide treatment were investigated using the Mini International Neuropsychiatric Interview for psychiatric disorders according to the DSM IV. Ten patients showed good pain relief (ΔVAS ≥ 50%) after one year of treatment. Patients without psychiatric comorbidity exhibited better outcomes, without autonomic side-effects. Eight patients with panic disorder (always comorbid with other psychiatric disorders) showed the greatest number of side-effects during treatment with ziconotide. Emotional and cognitive symptoms of panic disorder are associated with autonomic symptoms resulting from parasympathetic activation. Dysfunction of both cholinergic and N-type calcium channel activity was found. A psychiatric disorder with cholinergic-noradrenergic system impairment could increase some side-effects of treatment with N-type calcium channel blockers.

Spinal antinociception of synthetic omega-conotoxin SO-3, a selective N-type neuronal voltage-sensitive calcium channel blocker, and its effects on morphine analgesia in chemical stimulus tests in rodent

SO-3, a novel Omega-superfamily conotoxin derived from Conus striatus, selectively inhibits N-type neuronal voltage-sensitive calcium channels. In current study, antinociception of SO-3 compared with MVIIA or morphine and its effects on morphine analgesia were investigated in rodent chemical stimulus tests after acute or repeated intrathecal administration. In mice acetic acid writhing test, similar to MVIIA, SO-3 caused dose- and time-dependent spinal antinociception with ED(50) of 0.25 microg/kg and t(1/2) of 4h, which was more potent and longer-acting than morphine. In rat formalin test after intrathecal bolus injection, SO-3 produced dose- and time-dependent antinociception by suppressing acute (ED(50), 1.79 microg/kg) and tonic phases (ED(50), 0.41 microg/kg), which was similar to MVIIA and approximately 10-fold potency and twice longer-acting of morphine in blocking tonic phase responses. After repeated intrathecal injections twice daily for 5 consecutive days, SO-3 produced analgesia without loss of potency whereas morphine produced analgesia tolerance in rat formalin test; further, SO-3 still produced potent analgesia in morphine-tolerant rats. SO-3 co-administered with morphine left-shift the dose-response curve of morphine in mice acetic acid writhing test and significantly potentiated morphine analgesia in rat formalin test. No changes in motor function were seen in mice or rats receiving antinociceptive doses of SO-3 whereas MVIIA caused motor dysfunction at doses of 1.0-2.0 microg/kg in rats. This study showed that (1) novel SO-3 produced potent and long-acting spinal antinociception without observable motor dysfunction, (2) SO-3 significantly potentiated morphine analgesia, (3) After repeated intrathecal administration, SO-3 produced neither tolerance nor cross-tolerance to morphine analgesia.

CNSB004 (Leconotide) Causes Antihyperalgesia Without Side Effects When Given Intravenously: A Comparison with Ziconotide in a Rat Model of Diabetic Neuropathic Pain

Leconotide is an omega-conotoxin that blocks neuronal voltage sensitive calcium channels. This study compared the antihyperalgesic potencies of leconotide and ziconotide given intravenously alone and in combinations with a potassium channel modulator flupirtine, given intraperitoneally, in a rat model of diabetic neuropathic pain. Rats were given streptozotocin (150 mg/kg ip) to induce diabetic neuropathy and hyperalgesia. Experiments were performed on diabetic rats with >or=30% hyperalgesia to noxious heat. Rats were given each conopeptide alone and with flupirtine. Open field activity monitoring and non-invasive blood pressure measurements were used to define the maximum doses and combinations that caused no side effects. Doses in a range up to maximum no side effect doses were tested for antihyperalgesic effects in rats with hyperalgesia. The maximum no side effect dose of leconotide (2 mg/kg intravenously) caused 51.7% reversal of hyperalgesia compared with 0.4% for the highest no side effect dose of ziconotide (0.02 mg/kg; P < 0.001, one-way anova). Leconotide caused dose-related antihyperalgesic effects that were potentiated by coadministration with flupirtine at doses that were ineffective when given alone. Leconotide (0.02 mg/kg) and flupirtine (5 mg/kg) caused 25.3 +/- 7.6 and -6 +/- 9.5% reversal of hyperalgesia, respectively when given alone but in combination they caused 84.1 +/- 7.2% reversal of hyperalgesia (P < 0.01; one-way anova). No such interaction occurred with ziconotide. Leconotide could have wider clinical applications than ziconotide. Unlike ziconotide, powerful antihyperalgesia without side effects can be achieved by intravenous administration of leconotide thus avoiding the need for an intrathecal injection.

Analgesic effect in rodents of native and recombinant Phα1β toxin, a high-voltage-activated calcium channel blocker isolated from armed spider venom

Calcium influx through neuronal voltage-sensitive calcium channels (VSCC S) mediates nociceptive information in the spinal dorsal horn. In fact, spinally administered VSCC S blockers, such as ω-conotoxin MVIIA, have analgesic effect apart of their low therapeutic index and many side effects. Here we study the analgesic potential of Phα1β, a calcium channel blocker, in rodent models of acute and persistent pain. Spinally administered Phα1β showed higher efficacy and long-lasting analgesia in a thermal model of pain, when compared with ω-conotoxin MVIIA. Moreover, Phα1β was more effective and potent than ω-conotoxin MVIIA not only to prevent, but especially to reverse, previously installed persistent chemical and neuropathic pain. Furthermore, the analgesic action of both toxins are related with the inhibition of Ca 2+-evoked release of pro-nociceptive neurotransmitter, glutamate, from rat spinal cord synaptosomes and decrease of glutamate overflow in cerebrospinal fluid. When side effects were assessed, we found that Phα1β had a therapeutic index wider than ω- conotoxin MVIIA. Finally, recombinant Phα1β expressed in Escherichia coli showed marked analgesic activity similar to the native toxin. Taken together, the present study demonstrates that native and recombinant Phα1β have analgesic effects in rodent models of pain, suggesting that this toxin may have potential to be used as a drug in the control of persistent pathological pain.

Neuroprotection afforded by a combination of eliprodil and a thrombolytic agent, rt-PA, in a rat thromboembolic stroke model

In the present study, we have assessed the efficacy of eliprodil, a neuroprotective agent which blocks both the modulatory polyamine site of the NMDA receptor and neuronal voltage-sensitive calcium channels, alone or in combination with the thrombolytic agent, rt-PA, in a rat embolic stroke model using a neurological score and the volume of the infarct as endpoints. Embolization was induced by intracarotid injection of an arterial blood clot. Eliprodil, administered at the dose of 1 mg/kg, iv, 10 min and 2 h 30 after embolization, reduced the neurological deficit by 54% ( P<0.01) and the total volume of the brain lesion by 49%. Thrombolysis with rt-PA (2.5 mg/kg, as a 30 min iv infusion beginning 1 h after embolization) decreased the neurological deficit by 48% ( P<0.05) and the size of the total infarct by 55% ( P<0.05). Combined therapy greatly improved the degree of neuroprotection as assessed by neurological and histological outcomes (70% ( P<0.001) and 89% ( P<0.01) neuroprotection, respectively). These results demonstrate that the administration of a neuroprotective drug (eliprodil) or a thrombolytic agent (rt-PA) similarly reduce the volume of brain damage and the neurological deficit in a rat embolic stroke model. Combined cytoprotective therapy and thrombolysis markedly improved the degree of neuroprotection and may, thus, represent a valuable approach for the treatment of stroke in humans.

Structure, function and therapeutic potential of omega conopeptides: Novel blockers of neuronal calcium channels

Recent molecular cloning strategies have revealed that the diversity of voltage-sensitive calcium channels (VSCC) in the nervous system is much larger than indicated by electrophysiological studies. Elucidation of the physiological functions of these novel classes of VSCC requires pharmacological tools such as the blockers which were highly useful in characterizing the L-type VSCC in skeletal, smooth and cardiac muscle. Omega conopeptides found in the venom of fish-eating marine snails are proving to be highly selective blockers of neuronal VSCC. Several omega conopeptides have been isolated from a variety ofConus, characterized and synthesized. Biochemical, electrophysiological and morphological studies with these synthetic conopeptides have shown that novel types of VSCC are located in discrete regions of the brain and control the release of neurotransmitters in a highly selective manner, hitherto unsuspected. Pharmacological studies in animal models of cerebral ischemia have shown that omega conopeptides which selectively block N-type VSCC are highly effective in preventing brain damage caused by the loss of oxygen supply to the brain during the ischemia episode.

Differential inhibition of neuronal calcium entry and [3H]-D-aspartate release by the quaternary derivatives of verapamil and emopamil.

1. Verapamil and emopamil are structurally related phenylalkylamine calcium channel/5-HT2 receptor antagonists that differ in their anti-ischaemic properties in experimental studies. The quaternary ammonium derivatives of these compounds were prepared and tested in assays of neuronal voltage-sensitive calcium channel (VSCC) function to determine whether the compounds act at intra- or extracellular sites. 2. The compounds were tested in K(+)-evoked: (1) rat brain synaptosomal 45Ca2+ influx, (2) release of [3H]-D-aspartate from rat hippocampal brain slices and (3) increase of intracellular calcium in rat cortical neurones in primary culture. 3. Verapamil, emopamil and the emopamil quaternary derivative caused concentration-dependent and comparable (IC50 values approximately 30 microM) inhibition of synaptosomal 45Ca2+ influx and [3H]-D-aspartate release. The verapamil quaternary derivative was considerably less active in these assays (IC50 > 300 microM). 4. The evoked increase of intracellular calcium in cortical neurones was inhibited with the following rank order of potency (IC50 value, microM): emopamil (3.6) > verapamil (17) > emopamil quaternary derivative (38) > verapamil quaternary derivative (200). 5. The results suggest that verapamil and emopamil inhibit nerve terminal VSCC function (synaptosomal 45Ca2+ influx and [3H]-D-aspartate release) by acting at distinct intracellular and extracellular sites, respectively. Verapamil and emopamil may inhibit cell body VSCC function (evoked increase of intracellular calcium in neocortical neurones) by acting at both intracellular and extracellular sites. 6. The different 'sidedness' of action of emopamil and verapamil on nerve terminal VSCC function and/or the preferential inhibition of cell body VSCC function by emopamil may at least partially explain the relatively greater neuroprotective efficacy of emopamil in experimental models of ischaemia.

Neuroanatomical distribution of receptors for a novel voltage-sensitive calcium-channel antagonist, SNX-230 (ω-conopeptide MVIIC)

Neuronal voltage-sensitive calcium channels (VSCCs) are a diverse family of proteins that regulate entry of Ca 2+ into neurons. Selective antagonists of VSCCs have proven to be powerful pharmacological tools for identifying and characterizing these channels. A new VSCC antagonist, SNX-230 (also known as ω-conopeptide MVIIC), binds with high affinity to receptors in rat brain and blocks one or more high-threshold VSCCs that are neither L- nor N-type. We have defined the neuroanatomical distribution of the high-affinity non-L, non-N VSCC receptors for SNX-230 using [ 125I]SNX-230 bound to rat brain sections and compared it with that of [ 125I]SNX-111, a reversible blocker of N-type VSCCs. Highest densities of binding for both ligands were seen in areas rich in synaptic connections, such as the oriens, radiatum and molecular layers of the hippocampus. In general, the density of [ 125I]SNX-230-binding was higher in cerebellum compared with that in forebrain. In contrast, this general distribution of density was reversed for [ 125I]SNX-111. In the glomeruli of the olfactory bulb, binding of [ 125I]SNX-230 was undetectable compared with the high density of [ 125I]SNX-111-binding. Differential localization of the two ligands was also seen in cervical spinal cord. The clearly different localization of [ 125I]SNX-230 compared with that of [ 125I]SNX-111 in the olfactory bulb and spinal cord suggested that the binding sites for [ 125I]SNX-230 in other brain regions, while co-localized macroscopically, are also distinct from those for [ 125I]SNX-111. This was confirmed when addition of saturating concentrations of SNX-111 did not affect the distribution pattern of [ 125I]SNX-230-binding. These observations demonstrate that the high-affinity [ 125I]SNX-230 receptors in the brain and those of the N-type VSCCs are distinct molecular entities. Thus, SNX-230 (ω-MVIIC) offers a useful ligand for investigating the distribution and functions of non-L-, non-N-type VSCCs in the brain.

Characterization of the Binding of Omega-Conopeptides to Different Classes of Non-L-Type Neuronal Calcium Channels

The interaction of two synthetic ω-conopeptides SNX111 (MVIIA) and SNX-230 (MVIIC) both derived from the marine snail Conus magus, with non-L-type neuronal voltage-sensitive calcium channels (VSCC) in rat brain synaptosomal preparations has been investigated with the aid of well-characterized 125I derivatives of the two peptides. To assess the effects of iodination on the binding characteristics of SNX-111 and SNX-230, the corresponding peptides containing monoiodotyrosine in place of tyrosine, namely, SNX-259 ([ 127I]SNX-111) and SNX260 ([ 127I]SNX-230), respectively, were prepared by solid-phase synthesis. Saturation analysis showed that [ 127I]SNX-111 and [ 127I]SNX-230 bound to two distinct classes of high-affinity sites with apparent K d's of 9 and 11 p M and B max's of 0.54 and 2.2 pmol/mg protein, respectively. Kinetic analysis revealed that both peptides exhibited high association rates as well as rapid dissociation rates in contrast to the 125I derivative of the synthetic ω-conopeptide from Conus geographus, GVIA (SNX- 124), which binds irreversibly to N-type channels in rat brain synaptosomes. Competition binding experiments with [ 125I]SNX-111 and [ 125I]SNX-124 established that both of them bind to the same site, namely, N-type VSCC. The site detected by the binding of [ 125I]SNX-230 is distinct from N-type VSCC since SNX-111 has very low affinity ( K i = 135 n M) in competition studies. Recent findings that a novel high-voltage-activated calcium channel in rat cerebellar granule neurons is resistant to blockers of L-, N-, and P-type VSCC but is highly sensitive to SNX-230 suggest that the [ 125I]SNX-230 binding site may represent this novel type of calcium channel or another, as yet undescribed, VSCC.

Solution structure of omega-conotoxin GVIA using 2-D NMR spectroscopy and relaxation matrix analysis.

We report here the solution structure of omega-conotoxin GVIA, a peptide antagonist of the N-type neuronal voltage-sensitive calcium channel. The structure was determined using two-dimensional NMR in combination with distance geometry and restrained molecular dynamics. The full relaxation matrix analysis program MARDIGRAS was used to generate maximum and minimum distance restraints from the crosspeak intensities in NOESY spectra. The 187 restraints obtained were used in conjunction with 23 angle restraints from vicinal coupling constants as input for the structure calculations. The backbones of the best 21 structures match with an average pairwise RMSD of 0.58 A. The structures contain a short segment of triple-stranded beta-sheet involving residues 6-8, 18-21, and 24-27, making this the smallest published peptide structure to contain a triple-stranded beta-sheet. Conotoxins have been shown to be effective neuroprotective agents in animal models of brain ischemia. Our results should aid in the design of novel nonpeptide compounds with potential therapeutic utility.

Actions of neomycin on neuronal L-, N-, and non-L/non-N-type voltage-sensitive calcium channel responses.

The effects of neomycin on neuronal voltage-sensitive calcium channel (VSCC) responses were investigated by evaluating its effects on calcium-dependent neuronal responses that are sensitive and insensitive to the N-type voltage-sensitive calcium channel antagonist omega-conotoxin GVIA and the L-type VSCC antagonist nitrendipine. Chick synaptosomal 45Ca2+ influx and K(+)-evoked release of [3H]norepinephrine from chick cortical brain slices were omega-conotoxin GVIA sensitive and nitrendipine insensitive, suggesting that these responses are mediated predominantly by N-type VSCC. The K(+)-evoked increase of intracellular calcium in cortical neurons and the K(+)-evoked release of [3H]norepinephrine from rat brain cortical slices was partially sensitive to omega-conotoxin GVIA and nitrendipine, suggesting that these responses are mediated by N-, L- and non-L/non-N-type VSCC. Rat synaptosomal 45Ca2+ influx and the K(+)-evoked release of [3H]D-aspartate from rat hippocampal slices were completely insensitive to omega-conotoxin GVIA and nitrendipine, suggesting that these responses were mediated predominantly by non-L/non-N-type VSCC. Neomycin caused a concentration-dependent and virtually complete inhibition of all response parameters, with IC50 values ranging from 90 to 400 microM. The results suggest that neomycin is a nonselective inhibitor of neuronal responses mediated by L-, N-, and non-L/non-N-type VSCC.

A goldfish model for evaluation of the neurotoxicity of ω-conotoxin GVI A and screening of monoclonal antibodies

The neurotoxicity of ω-conotoxin (ω-CgTx), a potent neuronal voltage-sensitive calcium channel blocker, was measured using a new bioassay. ω-CgTx was administered intraperitoneally (ip) to goldfish weighing approximately 1.6 g, and dose-related changes were observed over a 2-hr period. ω-CgTx induced time- and dose-dependent abnormal swimming behavior (ASB) and mortality. The antitoxin activity of the antibodies was investigated in vivo by either (1) preincubation of the antibody with ω-CgTx at 4°C overnight, or (2) pretreatment with antibody, 30 min before ω-CgTx injection in a 10:1 antibody/ω-CgTx molar ratio. The LD50 dose of ω-CgTx in goldfish was 5 nmol/kg ip, and preincubation of monoclonal antibody (50 nmol/kg ip) with ω-CgTx (5 nmol/kg ip) significantly ( p < 0.05) reduced mortality, ASB, and toxicity time. The antitoxin activity of the monoclonal antibodies evidenced in the goldfish bioassay was further tested in the conscious rat. In the rat, the increases in mean arterial pressure and heart rate induced by ω-CgTx (0.03 nmol/rat icv) were significantly ( p < 0.02 and p < 0.01, respectively) attenuated by preincubation of the toxin with the antibody (0.3 nmol/rat). We conclude that the goldfish bioassay provides a simple, accurate, and inexpensive in vivo model for the study of the toxicity of ω-CgTx.

125I]-Omega conotoxin binding to human frontal cortex from normal, Alzheimer's and non-Alzheimer's dementia patients

We studied the binding of the calcium antagonist neurotoxin [ 125I]-omega conotoxin (GVIA) in age-matched human brains from normal, Alzheimer's disease and non-Alzheimer's dementia patients. Crude preparations of plasmalemmal membranes from frontal cortex were utilized. Saturation isotherms were subjected to Scatchard analysis to determine maximal binding capacity (B max) and binding affinity (K d). In all brain samples tested, [ 125I]-GVIA binding was homogenous to a single class of high affinity binding sites. Scatchard analysis of saturation isotherms gave the following estimates for normal brains (mean ± S.D., n = 7): B max = .630 ± .200 pmol/mg and K d = .177 ± .054 nM. No significant change was observed in the K d or B max estimates for [ 125I]-omega conotoxin binding in Alzheimer's disease or non-Alzheimer's dementia brains when compared to normal brains. Although these findings do not rule out the existence of localized changes in calcium channel receptor binding in the frontal cortex of Alzheimer's disease patients, the results do suggest that the neuronal voltage sensitive calcium channel may be unaltered in Alzheimer's disease.

Evidence for a dihydropyridine‐sensitive and conotoxin‐insensitive release of noradrenaline and uptake of calcium in adrenal chromaffin cells

1. It has been suggested that neuronal voltage-sensitive calcium channels (VSCC) may be divided into dihydropyridine (DHP)-sensitive (L) and DHP-insensitive (N and T), and that both the L and the N type channels are attenuated by the peptide blocker omega-conotoxin. Here the effects of omega-conotoxin on release of noradrenaline and uptake of calcium in bovine adrenal chromaffin cells were investigated. 2. Release of noradrenaline in response to 25 mM K+, 65 mM K+, 10 nM bradykinin or 10 microM prostaglandin E1 was not affected by omega-conotoxin in the range 10 nM-1 microM. 3. 45Ca2+ uptake stimulated by high K+ and prostaglandin was attenuated by 1 microM nitrendipine and enhanced by 1 microM Bay K 8644; these calcium fluxes were not modified by 20 nM omega-conotoxin. 4. With superfused rat brain striatal slices in the same medium as the above cell studies, release of dopamine in response to 25 mM K+ was attenuated by 20 nM omega-conotoxin. 5. These results show that in these neurone-like cells, release may be effected by calcium influx through DHP-sensitive but omega-conotoxin-insensitive VSCC, a result inconsistent with the suggestion that omega-conotoxin blocks both L-type and N-type neuronal calcium channels.

Effect of ω-conotoxin GVIA on Ca 2+ influx and endogenous acetylcholine release from chicken brain preparations

ω-Conotoxin GVIA (ω-conotoxin) is an inhibitor of neuronal voltage sensitive calcium channels (VSCCs). We have examined the effects of various manipulations aimed at elucidating the effects of ω-conotoxin particularly on K + stimulated 45Ca 2+ influx and endogenous acetylcholine (ACh) release. The initial phase of K + stimulated 45Ca 2+ influx was accompanied by a large rapid release of ACh measured by a modification of the chemiluminescent technique of Israël and Lesbats ( J. Neurochem. 39, 248–250, 1982). ACh release was greatly reduced ( ⋍90%) in Ca 2+ free solution or by Cd 2+ (100 μM) period ω-conotoxin (1 μM) completely blocked the initial phase of 45Ca 2+ influx (1–3 s) from synaptosomes and reduced K + stimulated ACh release from tissue slices. ω-Conotoxin (0.1 μM) reduced ACh release which was reversed in the presence of high Ca 2+ concentrations. The dihydropyridine VSCC inhibitor (−)202–791 or agonist (+)202–791 had no effect on ACh release or 45Ca 2+ influx. The pattern of responses on 45Ca 2+ influx and endogenous ACh release was consistent with the suggestion that ω-conotoxin inhibits N-type VSCCs and that these channels are intimately related to ACh release.

The effect of dihydropyridine calcium agonists and antagonists on neuronal voltage sensitive calcium channels

The effect of dihydropyridine agonists and antagonists on neuronal voltage sensitive calcium channels was investigated. The resting intracellular calcium concentration of synaptosomes prepared from whole brain was 110±9nM, as assayed by the indicator quin 2. Depolarisation of the synaptosomes with K + produced an immediate increase in [Ca 2+] i. The calcium agonist Bay K 8644 and antagonist nifedipine did not affect [Ca 2+] i under resting or depolarising conditions. In addition, K + stimulated 45Ca 2+ uptake into synaptosomes prepared from the hippocampus was insensitive to Bay K 8644 and PY 108-068 in normal or Na + free conditions. In neuronally derived NG108-15 cells the enantiomers of the dihydropyridine derivative 202–791 showed opposite effects in modulating K + stimulated 45Ca 2+ uptake. (−)-R-202-791 inhibited K + induced 45Ca 2+ uptake with an IC 50 of 100nM and (+)-S-202-791 enhanced K + stimulated uptake with an EC 50 of 80nM. These results suggest that synaptosomal voltage sensitive calcium channels either are of a different type to those found in peripheral tissues and cells of neural origin or that expression of functional effects of dihydropyridines requires different experimental conditions to those used here.