N-type Calcium Channel Antagonist Research Articles

Microneedle-mediated delivery of Ziconotide-loaded liposomes fused with exosomes for analgesia.

Ziconotide (ZIC) is an N-type calcium channel antagonist for treating severe chronic pain that is intolerable, or responds poorly to the administration of other drugs, such as intrathecal morphine and systemic analgesics. As it can only work in the brain and cerebrospinal fluid, intrathecal injection is the only administration route for ZIC. In this study, borneol (BOR)-modified liposomes (LIPs) were fused with exosomes from mesenchymal stem cells (MSCs) and loaded with ZIC to prepare microneedles (MNs) to improve the efficiency of ZIC across the blood-brain barrier. To evaluate local analgesic effects of MNs, the sensitivity of behavioral pain to thermal and mechanical stimuli was tested in animal models of peripheral nerve injury, diabetes-induced neuropathy pain, chemotherapy-induced pain, and ultraviolet-B (UV-B) radiation-induced neurogenic inflammatory pain. BOR-modified LIPs loaded with ZIC were spherical or nearly spherical, with a particle size of about 95nm and a Zeta potential of -7.8mV. After fusion with MSC exosomes, the particle sizes of LIPs increased to 175nm, and their Zeta potential increased to -3.8mV. The nano-MNs constructed based on BOR-modified LIPs had good mechanical properties and could effectively penetrate the skin to release drugs. The results of analgesic experiments showed that ZIC had a significant analgesic effect in different pain models. In conclusion, the BOR-modified LIP membrane-fused exosome MNs constructed in this study for delivering ZIC provide a safe and effective administration for chronic pain treatment, as well as great potential for clinical application of ZIC.

Intracerebroventricular administration of N-type calcium channel blocker ziconotide displays anticonvulsant, anxiolytic, and sedative effects in rats: A preclinical and pilot study

ObjectiveZiconotide (ω-conotoxin MVIIA peptide) is a novel analgesic agent acting on voltage-gated calcium channels and is administered intrathecally for neuropathic pain. While antiepileptic activities of other types of calcium channel blockers (T- or L-type) are well established, there is no information regarding the effect of ziconotide as an N-type calcium channel antagonist in pentylenetetrazol-induced seizures or its anxiolytic and sedative activities. The present study is the first to report on these effects. MethodsTo evaluate the anticonvulsant activity of ziconotide in the pentylenetetrazol (60 mg/kg) seizure model, ziconotide was administered intracerebroventricular (i.c.v.) as a single dose (1 μg/rat) or repeatedly (chronic administration: 0.1, 0.3, or 1 μg/rat once a day for seven days). The anxiolytic and sedative actions of ziconotide were evaluated with the elevated plus maze, light/dark (LD) box, and pentobarbital-induced sleep tests. Immediately after behavioral testing, the amygdala was completely removed bilaterally to determine corticosterone levels by immunoassay. ResultsIn all dosing regimens, ziconotide significantly decreased the seizure frequency and also delayed the latency period compared with control. Chronic administration affected the percentage of mortality protection, while a single dose of ziconotide did not. In behavioral tests, ziconotide significantly increased both the number of entries and the percentage of time spent in the open arms of the elevated plus maze. Furthermore, ziconotide significantly increased the latency period and the number of entries into the light compartment during the LD box examination. Chronic administration of ziconotide significantly reduced the latency to sleep and increased sleeping time, whereas these parameters were not affected by a single dose. Additionally, amygdala corticosterone levels were significantly decreased in rats treated with ziconotide compared with control. ConclusionZiconotide displays beneficial neurobehavioral effects in a model of epilepsy with anxiety as its comorbid event. It seems that at least one of the mechanisms involved in these effects is associated with a decrease in brain corticosterone levels. The main advantage of ziconotide over benzodiazepines (routine anxiolytic and sedative drugs) is that it does not cause tolerance, dependency, and addiction. Therefore, more than ever, it is necessary to improve the convenience of drug delivery protocols and attenuate the adverse effects associated with ziconotide-based therapies.

Phα1β Spider Toxin Reverses Glial Structural Plasticity Upon Peripheral Inflammation.

The incoming signals from injured sensory neurons upon peripheral inflammation are processed in the dorsal horn of spinal cord, where glial cells accumulate and play a critical role in initiating allodynia (increased pain in response to light-touch). However, how painful stimuli in the periphery engage glial reactivity in the spinal cord remains unclear. Here, we found that a hind paw inflammation induced by CFA produces robust morphological changes in spinal astrocytes and microglia compatible with the reactive phenotype. Strikingly, we discovered that a single intrathecal injection with venom peptides that inhibit calcium channels reversed all the glial pathological features of the peripheral inflammation. These effects were more apparent in rats treated with the Phα1β spider toxin (non-specific calcium channel antagonist) than ω-MVIIA cone snail toxin (selective N-type calcium channel antagonist). These data reveal for the first time a venom peptide acting on glial structural remodeling in vivo. We, therefore, suggest that calcium-dependent plasticity is an essential trigger for glial cells to initiate reactivity, which may represent a new target for the antinociceptive effects of Phα1β and ω-MVIIA toxins in inflammatory pain conditions.

Valepotriates From the Roots and Rhizomes of Valeriana jatamansi Jones as Novel N-Type Calcium Channel Antagonists.

The roots and rhizomes of Valeriana jatamansi have long been used as folk medicine in Asia and usually named as “Zhizhuxiang” in Chinese for the treatment of abdominal distention and pain. However, its active ingredients and molecular targets for treatment of abdominal pain remain unrevealed. Inhibitors of Cav2.2 N-type voltage-gated calcium channels (VGCCs) are actively sought after for their potential in treating pain, especially chronic pain. As far as we know, the method used for seeking analgesic active ingredient from plant material has rarely been reported. The analgesic potentials of the EtOH extract (0.01 mg/ml) of the roots and rhizomes of V. jatamansi and its EtOAc, n-BuOH and H2O soluble parts (0.01 mg/ml, respectively) were tested herein on Cav2.2, using whole-oocyte recordings in vitro by tow-electrode voltage clamp. The results indicated that the EtOAc-soluble part exhibited the most potent inhibition of Cav2.2 peak current (20 mv). The EtOAc-soluble part was then subjected to silica gel column chromatography (CC) and giving 9 fractions. Phytochemical studies were carried out by repeated CC and extensive spectroscopic analyses after the fraction (0.01 mg/ml) was identified to be active and got seventeen compounds (1–17). All isolates were then sent for further bioactive verification (1 and 3 at concentration of 10 μM, others at 30 μM). In addition, the selectivity of the active compounds 1 and 3 were tested on various ion channels including Cav1.2, Cav2.1 and Cav3.1 VGCCs and Kv1.2, Kv2.1, Kv3.1 and BK potassium channels. The results indicated that compound 1 and 3 (an abundant compound) inhibited Cav2.2 with an EC50 of 3.3 and 4.8 μM, respectively, and had weaker or no effect on Cav1.2, Cav2.1 and Cav3.1 VGCCs and Kv1.2, Kv2.1, Kv3.1 and BK potassium channels. Compounds 1 and 3 appear to act as allosteric modulators rather than pore blockers of Cav2.2, which may play crucial role in attenuating nociception. The results of present research indicated that the ethnopharmacological utilization of V. jatamansi for relieving the abdominal distention and pain may mediate through Cav2.2 channel. Our work is the first demonstration of inhibition of Cav2.2 by iridoids, which may provide a fresh source for finding new analgesics.

Huwentoxin-XVI, an analgesic, highly reversible mammalian N-type calcium channel antagonist from Chinese tarantula Ornithoctonus huwena

N-type calcium channels play important roles in the control of neurotransmission release and transmission of pain signals to the central nervous system. Their selective inhibitors are believed to be potential drugs for treating chronic pain. In this study, a novel neurotoxin named Huwentoxin-XVI (HWTX-XVI) specific for N-type calcium channels was purified and characterized from the venom of Chinese tarantula Ornithoctonus huwena. HWTX-XVI is composed of 39 amino acid residues including six cysteines that constitute three disulfide bridges. HWTX-XVI could almost completely block the twitch response of rat vas deferens to low-frequency electrical stimulation. Electrophysiological assay indicated that HWTX-XVI specifically inhibited N-type calcium channels in rat dorsal root ganglion cells (IC50 ∼60 nM). The inhibitory effect of HWTX-XVI on N-type calcium channel currents was dose-dependent and similar to that of CTx-GVIA and CTx-MVIIA. However, the three peptides exhibited markedly different degrees of reversibility after block. The toxin had no effect on voltage-gated T-type calcium channels, potassium channels or sodium channels. Intraperitoneal injection of the toxin HWTX-XVI to rats elicited significant analgesic responses to formalin-induced inflammation pain. Toxin treatment also changed withdrawal latency in hot plate tests. Intriguingly, we found that intramuscular injection of the toxin reduced mechanical allodynia induced by incisional injury in Von Frey test. Thus, our findings suggest that the analgesic potency of HWTX-XVI and its greater reversibility could contribute to the design of a novel potential analgesic agent with high potency and low side effects.

Loss of Calcium/Calmodulin-dependent Protein Kinase II Activity in Cortical Astrocytes Decreases Glutamate Uptake and Induces Neurotoxic Release of ATP

The extent of calcium/calmodulin-dependent protein kinase II (CaMKII) inactivation in the brain after ischemia correlates with the extent of damage. We have previously shown that a loss of CaMKII activity in neurons is detrimental to neuronal viability by inducing excitotoxic glutamate release. In the current study we extend these findings to show that the ability of astrocytes to buffer extracellular glutamate is reduced when CaMKII is inhibited. Furthermore, CaMKII inhibition in astrocytes is associated with the rapid onset of intracellular calcium oscillations. Surprisingly, this rapid calcium influx is blocked by the N-type calcium channel antagonist, ω-conotoxin. Although the function of N-type calcium channels within astrocytes is controversial, these voltage-gated calcium channels have been linked to calcium-dependent vesicular gliotransmitter release. When extracellular glutamate and ATP levels are measured after CaMKII inhibition within our enriched astrocyte cultures, no alterations in glutamate levels are observed, whereas ATP levels in the extracellular environment significantly increase. Extracellular ATP accumulation associated with CaMKII inhibition contributes both to calcium oscillations within astrocytes and ultimately cortical neuron toxicity. Thus, a loss of CaMKII signaling within astrocytes dysregulates glutamate uptake and supports ATP release, two processes that would compromise neuronal survival after ischemic/excitotoxic insults.

Ziconotide: a clinical update and pharmacologic review

Introduction: Ziconotide is an N-type calcium channel antagonist to treat chronic pain that is delivered intrathecally. It is the only intrathecal, FDA-approved, non-opioid analgesic and is recommended as first-line therapy. Despite these advantages, a small therapeutic window limits ziconotide's clinical utility, with adverse event (AE) challenges that include, but are not limited to, dizziness, nausea, and somulence.Areas covered: Pharmacokinetics, pharmacodynamics, efficacy, safety, trialing, and chronic infusion after searching EMBASE, PubMed, and Cochrane Database of Systemic Reviews were used to search published literature from 1966 to January 1, 2013 to identify studies related to the intrathecal delivery of ziconotide.Expert opinion: Ziconotide is a safe and effective strategy to treat chronic pain, although limitations remain, including a small therapeutic window. Low starting doses and slow incremental increases and long titration intervals may improve tolerability. AEs may be mitigated by also employing combination therapy, although further study is needed. Concomitant use of ziconotide and morphine is an option when considering use of FDA-labeled intrathecal drugs in those resistant to monotherapy.

Intrathecal Ziconotide for Cancer Pain Relief: When, How and Why

channels via a G protein–coupled mechanism. Ziconotide, an N-type calcium channel antagonist, effective in the treatment of neuropathic, nociceptive, and mixed neuropathic/nociceptive pain [1,2] blocks calcium influx into the presynaptic nerve terminal, preventing the release of neurotransmitters into the synapse. Unlike morphine, which acts indirectly and only partially (because some of the µ-opioid receptors are not linked to N-type calcium channels), ziconotide directly inhibits the N-type calcium channel. The phenomenon of tolerance observed with opioids, given the lack of coupling of µ-opioid receptors with calcium channels, has not been observed with ziconotide because it acts directly on calcium channels.An algorithm allowing identification of the “

Antioxidant treatment restores prejunctional regulation of purinergic transmission in mesenteric arteries of deoxycorticosterone acetate-salt hypertensive rats

Norepinephrine (NE) and ATP are co-released by periarterial sympathetic nerves. In mesenteric arteries (MA) from deoxycorticosterone-acetate (DOCA)-salt hypertensive rats, ATP, but not norepinephrine, release is impaired suggesting that their release may be regulated differently. We tested the hypothesis that different calcium channels contribute to ATP and norepinephrine release from sympathetic nerves in vitro in MA from normotensive and DOCA-salt hypertensive rats and that oxidative stress disrupts prejunctional regulation of co-transmission. Excitatory junction potentials (EJPs) were used to measure ATP release. Norepinephrine release was measured amperometrically with carbon-fiber microelectrodes. CdCl 2 (30 μM) inhibited norepinephrine release in sham and DOCA-salt arteries by 78% and 85%, respectively. The N-type calcium channel antagonist, ω-conotoxin GVIA (CTX, 0.1 μM) inhibited norepinephrine release by 50% and 67% in normotensive and DOCA-salt arteries, respectively while CTX blocked EJPs. The P/Q-type calcium channel antagonist ω-agatoxin IVA (ATX; 0.03 μM) reduced norepinephrine release in sham but not DOCA-salt arteries and increased EJPs in sham but not DOCA-salt arteries. ATX did not increase EJPs in sham arteries in the presence of the α 2-adrenergic receptor antagonist, yohimbine (1 μM). α 2-Autoreceptor-sensitive EJP facilitation is impaired in DOCA-salt hypertension but this response is restored in DOCA-salt rats treated chronically with the antioxidant, apocynin. Apocynin restored α 2-autoreceptor regulation of norepinephrine release. We conclude that ATP released from periarterial sympathetic nerves is controlled directly by N-type calcium channels. Norepinephrine release is controlled by N and P/Q type calcium channels. Norepinephrine release controlled by P/Q channels acts at α 2-adrenergic receptors to inhibit norepinephrine release suggesting that there may be multiple pools of norepinephrine in periarterial sympathetic nerves. Regulation of norepinephrine release by α 2-autoreceptors and P/Q-type channels is impaired in DOCA-salt hypertension and α 2-autoreceptor function is disrupted by oxidative stress.

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.

A Blocker of N- and T-type Voltage-Gated Calcium Channels Attenuates Ethanol-Induced Intoxication, Place Preference, Self-Administration, and Reinstatement

There is a clear need for new therapeutics to treat alcoholism. Here, we test our hypothesis that selective inhibitors of neuronal calcium channels will reduce ethanol consumption and intoxication, based on our previous studies using knock-out mice and cell culture systems. We demonstrate that pretreatment with the novel mixed N-type and T-type calcium channel antagonist 1-(6,6-bis(4-fluorophenyl)hexyl)-4-(3,4,5-trimethoxybenzyl)piperazine (NP078585) reduced ethanol intoxication. NP078585 also attenuated the reinforcing and rewarding properties of ethanol, measured by operant self-administration and the expression of an ethanol conditioned place preference, and abolished stress-induced reinstatement of ethanol seeking. NP078585 did not affect alcohol responses in mice lacking N-type calcium channels. These results suggest that selective calcium channel inhibitors may be useful in reducing acute ethanol intoxication and alcohol consumption by human alcoholics.

Differential Inhibition of Ca2+ Channels by α2-Adrenoceptors in Three Functional Subclasses of Rat Sympathetic Neurons

A comparison of identified sympathetic neurons in the isolated intact superior cervical ganglion revealed that secretomotor, pilomotor, and vasoconstrictor cells differ in their action potential mechanisms and in their postsynaptic alpha(2)-adrenergic responses to 10 microM norepinephrine (NE). In normal saline, the half-width of the spike afterhyperpolarization (AHP) in secretomotor neurons (103.5 +/- 6.2 ms) was twofold that recorded in vasoconstrictor neurons (47.7 +/- 2.9 ms) and 1.5-fold that in pilomotor neurons (71.4 +/- 10.3 ms). Bath-applied NE reversibly inhibited the action potential repolarization shoulder, AHP amplitude, and AHP duration in secretomotor and pilomotor neurons to a similar extent, but had no effect on vasoconstrictor neurons. The insensitivity of vasomotor neurons to NE was not an artifact produced by microelectrode recording because all three cell groups were similar in terms of resting potential and input resistance. Moreover, NE insensitivity was not a natural consequence of briefer AHP duration in vasoconstrictor cells. Adding 10 mM TEA(+) caused marked accentuation of the shoulder and AHP duration in vasoconstrictor neurons and comparable changes in the other two cell types, but did not unmask any sign of NE sensitivity in the vasoconstrictors. However, the spike shoulder and AHP in vasoconstrictors were Cd(2+) sensitive, blocked by omega-conotoxin, an N-type calcium channel antagonist, and inhibited by oxotremorine-M, a muscarinic receptor agonist. These data show that NE can differentially modulate functional subsets of mammalian sympathetic neurons and that NE insensitivity can serve as a practical experimental criterion for identification of vasomotor neurons in the isolated ganglion.

Prolonged Attenuation of Amygdala-Kindled Seizure Measures in Rats by Convection-Enhanced Delivery of the N-Type Calcium Channel Antagonists ω-Conotoxin GVIA and ω-Conotoxin MVIIA

Convection-enhanced delivery (CED) permits the homogeneous distribution of therapeutic agents throughout localized regions of the brain parenchyma without causing tissue damage as occurs with bolus injection. Here, we examined whether CED infusion of the N-type calcium channel antagonists omega-conotoxin GVIA (omega-CTX-G) and omega-conotoxin MVIIA (omega-CTX-M) can attenuate kindling measures in fully amygdala-kindled rats. Rats were implanted with a combination infusion cannula-stimulating electrode assembly into the right basolateral amygdala. Fully kindled animals received infusions of vehicle, omega-CTX-G (0.005, 0.05, and 0.5 nmol), omega-CTX-M (0.05, 0.15, and 0.5 nmol), proteolytically inactivated omega-CTX-M (0.5 nmol), or carbamazepine (500 nmol) into the stimulation site. CED of omega-CTX-G and omega-CTX-M over a 20-min period resulted in a dose-dependent increase in the afterdischarge threshold and a decrease in the afterdischarge duration and behavioral seizure score and duration during a period of 20 min to 1 week after the infusion, indicating an inhibitory effect on the triggering and expression of kindled seizures. The protective effects of omega-conotoxins reached a maximum at 48 h postinfusion, and then they gradually resolved over the next 5 days. In contrast, carbamazepine was active at 20 min but not at 24 h after the infusion, whereas CED of vehicle or inactivated omega-CTX-M had no effect. Except for transient tremor in some rats receiving the highest toxin doses, no adverse effects were observed. These results indicate that local CED of high-molecular-weight presynaptic N-type calcium channel blockers can produce long-lasting inhibition of brain excitability and that they may provide prolonged seizure protection in focal seizure disorders.

Tumor necrosis factor alpha stimulates NMDA receptor activity in mouse cortical neurons resulting in ERK‐dependent death

Multiple cytokines are secreted in the brain during pro-inflammatory conditions and likely affect neuron survival. Previously, we demonstrated that glutamate and tumor necrosis factor alpha (TNFalpha) kill neurons via activation of the N-methyl-d-aspartate (NMDA) and TNFalpha receptors, respectively. This report continues characterizing the signaling cross-talk pathway initiated during this inflammation-related mechanism of death. Stimulation of mouse cortical neuron cultures with TNFalpha results in a transient increase in NMDA receptor-dependent calcium influx that is additive with NMDA stimulation and inhibited by pre-treatment with the NMDA receptor antagonist, DL-2-amino-5-phosphonovaleric acid, or the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainate receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione. Pre-treatment with N-type calcium channel antagonist, omega-conotoxin, or the voltage-gated sodium channel antagonist, tetrodotoxin, also prevents the TNFalpha-stimulated calcium influx. Combined TNFalpha and NMDA stimulation results in a transient increase in activity of extracellular signal-regulated kinases (ERKs) and c-Jun N-terminal kinases (JNKs). Specific inhibition of ERKs but not JNKs is protective against TNFalpha and NMDA-dependent death. Death is mediated via the low-affinity TNFalpha receptor, TNFRII, as agonist antibodies for TNFRII but not TNFRI stimulate NMDA receptor-dependent calcium influx and death. These data demonstrate how microglial pro-inflammatory secretions including TNFalpha can acutely facilitate glutamate-dependent neuron death.

Effects of nimodipine, an L-type calcium channel antagonist, on the chicken’s cochlear potentials

At most synapses in the brain, neurotransmitter release depends on N-type or P/Q-type calcium channels. However, available in vitro experimental data suggest that there exist almost exclusively L-type calcium channels in sensory hair cells of most species. To test whether chicken hair cells depend on L-type calcium channels for neurotransmitter release, we examined the effects of nimodipine, a selective L-type calcium channel antagonist, on acoustically evoked cochlear potentials in 10–15 week old chickens in vivo. Diffusion of nimodipine into scala tympani significantly elevated threshold, dramatically decreased the amplitude and increased the latency of the compound action potential within 20 min of drug application. The summating potential was also significantly reduced in amplitude, but the cochlear microphonic was relatively less affected. All the effects were reversible after nimodipine was washed out with artificial perilymph except that the cochlear microphonic amplitude remained decreased. Application of omega-conotoxin GVIA, an N-type calcium channel antagonist and agatoxin Tk, a P-type calcium channel antagonist had no observable effects on the cochlear potentials. These results suggest that L-type calcium channels control neurotransmitter release from avian hair cells.

The Magnitude of α7 Nicotinic Receptor Currents in Rat Hippocampal Neurons Is Dependent upon GABAergic Activity and Depolarization

Hippocampal alpha7(*) nicotinic acetylcholine receptors modulate the release of GABA and glutamate. The control of functional receptor pools by cell firing or synaptic activity could therefore allow for a local adjustment of the sensitivity to cholinergic input upon changes in neuronal activity. We first investigated whether tonic depolarization or cell firing affected the function of alpha7(*). The amplitude of alpha7(*)-gated whole-cell currents in cultured rat hippocampal neurons exposed to high-extracellular K(+) (40 mM KCl) for 24 to 48 h increased 1.3 to 5.5 times. The proportion of alpha7(*)-responsive neurons (99%), the potency of acetylcholine, and the sensitivity to nicotinic antagonists were all unaffected. In contrast, block of spontaneous cell firing with tetrodotoxin for 24 h led to a 37% reduction in mean current amplitude. Reduced alpha7(*) responses were seen after a 24-h blockade of N-type calcium channels but not of L-type calcium channels, N-methyl-d-aspartate (NMDA), or non-NMDA receptor channels, protein kinase C, or calcium-calmodulin kinases II and IV. The N-type or L-type calcium channel antagonists omega-conotoxin GVIA and nifedipine did not prevent the current-potentiating effect of KCl. The GABA(A) antagonist picrotoxin led to a 44% reduction of the currents, despite increasing action potential firing, and also reversed the potentiating effect of KCl. Treatment with GABA, midazolam, or a GABA uptake blocker led to increased currents. These data indicate that alpha7(*)-gated currents in hippocampal neurons are regulated by GABAergic activity and suggest that depolarization-induced GABA release may underlie the effect of increased extracellular KCl.

Effects of the blockade of high voltage‐activated calcium channels on in vitro pineal melatonin synthesis

The presence of high voltage-activated calcium channels in the rat pineal gland is well known. However, their role in pineal metabolism is not completely understood and is even controversial. Better to understand this matter, we investigated the effects of L-, N- or P/Q-type calcium channel blockers (nifedipine, omega-conotoxin GVIA, omega-agatoxin IVA, respectively) on melatonin content and arylalkylamine-N-acetyltransferase activity of denervated rat pineal glands kept for 48 h in culture and stimulated with norepinephrine. Melatonin was measured by high performance liquid chromatography with electrochemical detection and arylalkylamine-N-acetyltransferase activity was quantified by radiometric assay. Pre-incubation with any of these high voltage-activated calcium channel blockers reduced the melatonin production induced by norepinephrine although arylalkylamine-N-acetyltransferase activity was reduced only by the N-type calcium channel antagonist, omega-conotoxin GVIA. The results indicate that calcium influx through L-, N- or P/Q-type of high voltage-activated calcium channels is necessary for the full expression of the metabolic process leading to melatonin synthesis in the rat pineal glands. However, the mechanisms involved in this process are different for the L- or P/Q- and N-type calcium channels.

Ziconotide, an Intrathecally Administered N-Type Calcium Channel Antagonist for the Treatment of Chronic Pain

Ziconotide is a novel peptide that blocks the entry of calcium into neuronal N-type voltage-sensitive calcium channels, preventing the conduction of nerve signals. N-type calcium channels are present in the superficial laminae of the dorsal horn of the spinal cord. In various animal models of pain, intrathecal administration of ziconotide blocked nerve transmission and nociception. The United States Food and Drug Administration recently approved ziconotide intrathecal infusion for the management of severe chronic pain in patients who require intrathecal therapy and who are intolerant of or refractory to other treatment, such as systemic analgesics, adjunctive therapies, or intrathecal morphine. The drug has a narrow therapeutic window and a lag time for the onset and offset of analgesia and adverse events. In early clinical trials, frequent and severe psychiatric and central nervous system adverse effects were associated with rapid intrathecal infusion (0.4 microg/hr) and frequent up-titration (every 12 hrs). Therefore, patients with psychiatric symptoms are not candidates for this drug. Drug trials of external intrathecal catheters and microinfusion devices demonstrated a 3% risk of meningitis. A low initial infusion rate of 0.1 microg/hour and limiting infusion rate increases to 2-3 times/week are now recommended. Patients responsive to intrathecal ziconotide require an implanted infusion system to receive long-term therapy.

Medullary N-type and P/Q-type calcium channels contribute to neuropathy-induced allodynia

The present study was designed to determine the contribution of N-type, P/Q-type and L-type calcium channels in the rostral ventromedial medulla to tactile allodynia following peripheral nerve injury. L5/L6 spinal nerve ligation in rats produced tactile allodynia, which was dose-dependently inhibited by intrarostral ventromedial medulla microinjection of the N-type calcium channel antagonist omega-conotoxin MVIIA. Similarly, intrarostral ventromedial medulla microinjection of the P/Q-type calcium channel antagonist omega-agatoxin IVA inhibited spinal nerve ligation-induced tactile allodynia, whereas intrarostral ventromedial medulla microinjection of the L-type calcium channel antagonist nimodipine had no effect. These results demonstrate that N-type and P/Q-type calcium channels in the rostral ventromedial medulla contribute to tactile allodynia following peripheral neuropathy, likely via neurotransmitter-mediated activation of descending facilitatory systems from the rostral ventromedial medulla.

Design, Synthesis, and Preliminary Pharmacological Evaluation of 4-Aminopiperidine Derivatives as N-Type Calcium Channel Blockers Active on Pain and Neuropathic Pain

Several compounds with a 4-aminopiperidine scaffold decorated on both nitrogen atoms by alkyl or acyl moieties containing the structural motifs of verapamil and of flunarizine, as well as those that are more frequent in known N-type calcium channel antagonists, have been synthesized. Antinociceptive activity on the mouse hot-plate test was used to select molecules to be submitted to further studies. Active compounds were tested in vitro on a PC12 rat pheochromocytoma clonal cell line, to evaluate their action on N-type calcium channels, and on a rat model of neuropathic pain. Two compounds that show N-type calcium channel antagonism and are endowed with potent action on pain and neuropathic pain (3 and 18) have been selected for further studies.