Voltage-gated Calcium Channel Antagonist Research Articles

Inosine induces presynaptic inhibition of acetylcholine release by activation of A3 adenosine receptors at the mouse neuromuscular junction

The role of inosine at the mammalian neuromuscular junction (NMJ) has not been clearly defined. Moreover, inosine was classically considered to be the inactive metabolite of adenosine. Hence, we investigated the effect of inosine on spontaneous and evoked ACh release, the mechanism underlying its modulatory action and the receptor type and signal transduction pathway involved. End-plate potentials (EPPs) and miniature end-plate potentials (MEPPs) were recorded from the mouse phrenic-nerve diaphragm preparations using conventional intracellular electrophysiological techniques. Inosine (100 μM) reduced MEPP frequency and the amplitude and quantal content of EPPs; effects inhibited by the selective A3 receptor antagonist MRS-1191. Immunohistochemical assays confirmed the presence of A3 receptors at mammalian NMJ. The voltage-gated calcium channel (VGCC) blocker Cd(2+) , the removal of extracellular Ca(2+) and the L-type and P/Q-type VGCC antagonists, nitrendipine and ω-agatoxin IVA, respectively, all prevented inosine-induced inhibition. In the absence of endogenous adenosine, inosine decreased the hypertonic response. The effects of inosine on ACh release were prevented by the Gi/o protein inhibitor N-ethylmaleimide, PKC antagonist chelerytrine and calmodulin antagonist W-7, but not by PKA antagonists, H-89 and KT-5720, or the inhibitor of CaMKII KN-62. Our results suggest that, at motor nerve terminals, inosine induces presynaptic inhibition of spontaneous and evoked ACh release by activating A3 receptors through a mechanism that involves L-type and P/Q-type VGCCs and the secretory machinery downstream of calcium influx. A3 receptors appear to be coupled to Gi/o protein. PKC and calmodulin may be involved in these effects of inosine.

Transient Receptor Potential Vanilloid 4 Mediates Hypotonicity‐Induced Enhancement of Synaptic Transmission in Hippocampal Slices

Changes in cerebrospinal fluid osmotic pressure modulate brain excitability. Transient receptor potential vanilloid 4 (TRPV4), which is sensitive to hypotonic stimulation, is expressed in hippocampus. The present study investigated the effect of hypotonic stimulation on hippocampal synaptic transmission and the role of TRPV4 in hypotonicity-action using electrophysiological recording and pharmacological technique. Accompanied with the decrease in paired pulse facilitation, field excitatory postsynaptic potential (fEPSP) was enhanced by hypotonicity and TRPV4 agonist 4α-PDD in hippocampal slices, which was sensitive to TRPV4 antagonist HC-067047. Hypotonicity-induced increase in fEPSP was blocked by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist, but not N-methyl-d-aspartate receptor or N- or P/Q-type voltage-gated calcium channel antagonist. High voltage-gated calcium current (ICa ) in hippocampal CA3 pyramidal neurons was not affected by hypotonicity. AMPA-activated current (IAMPA ) in hippocampal CA1 pyramidal neurons was increased by hypotonicity and 4α-PDD, which was attenuated by HC-067047. Inhibition of protein kinase C or protein kinase A markedly attenuated hypotonicity-increased IAMPA , whereas antagonism of calcium/calmodulin-dependent protein kinase II had no such effect. TRPV4 is involved in hypotonicity-induced enhancement of hippocampal synaptic transmission, which may be mediated through promoting presynaptic glutamate release and increasing postsynaptic AMPA receptor function.

Voltage-Gated Calcium Channel Antagonists and Traumatic Brain Injury

Traumatic brain injury (TBI) is a leading cause of death and disability in the United States. Despite more than 30 years of research, no pharmacological agents have been identified that improve neurological function following TBI. However, several lines of research described in this review provide support for further development of voltage gated calcium channel (VGCC) antagonists as potential therapeutic agents. Following TBI, neurons and astrocytes experience a rapid and sometimes enduring increase in intracellular calcium ([Ca2+]i). These fluxes in [Ca2+]i drive not only apoptotic and necrotic cell death, but also can lead to long-term cell dysfunction in surviving cells. In a limited number of in vitro experiments, both L-type and N-type VGCC antagonists successfully reduced calcium loads as well as neuronal and astrocytic cell death following mechanical injury. In rodent models of TBI, administration of VGCC antagonists reduced cell death and improved cognitive function. It is clear that there is a critical need to find effective therapeutics and rational drug delivery strategies for the management and treatment of TBI, and we believe that further investigation of VGCC antagonists should be pursued before ruling out the possibility of successful translation to the clinic.

Reactivation enables memory updating, precision-keeping and strengthening: Exploring the possible biological roles of reconsolidation

Although much has been learned regarding the molecular and cellular mechanisms of memory reconsolidation, its actual biological function remains unclear. In this work we investigate the possibility that three different mnemonic processes – updating, precision-keeping and trace strengthening – are mediated by reconsolidation in contextual fear conditioning. Reconsolidation involves the activation of calcium channels for the destabilization during the reactivation. Our results show that when memory is reactivated in a situation that does not match the original information, content is modified, i.e., “updated”. However, when the contextual condition matches the original one, memory reactivation contributes either to its strengthening or to the maintenance of its precision content over time. Since the L-type voltage-gated calcium channel antagonist nimodipine blocked these effects, we suggest that reconsolidation is the mechanism supporting these processes.

Non-Hebbian plasticity at C-fiber synapses in rat spinal cord lamina I neurons

Current concepts of memory storage are largely based on Hebbian-type synaptic long-term potentiation induced by concurrent activity of pre- and postsynaptic neurons. Little is known about non-Hebbian synaptic plasticity, which, if present in nociceptive pathways, could resolve a number of unexplained findings. We performed whole-cell patch-clamp recordings in rat spinal cord slices and found that a rise in postsynaptic [Ca2+]i due to postsynaptic depolarization was sufficient to induce synaptic long-term potentiation (LTP) in the absence of any presynaptic conditioning stimulation. LTP induction could be prevented by postsynaptic application of the Ca2+ chelator BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid), the L-type voltage-gated calcium channel (VGCC) antagonist nifedipine, and by postsynaptic application of the NMDA receptor antagonist MK801. This indicates that synaptic potentiation was induced postsynaptically by Ca2+ entry likely via L-type voltage-gated Ca2+ channels (VGCC) and via NMDA receptor channels. The paired pulse ratio and the coefficient of variation remained unchanged in neurons expressing LTP, suggesting that this form of synaptic potentiation was not only induced, but also expressed postsynaptically. Postsynaptic depolarization had no influence on firing patterns, action potential shape, or neuronal excitability. An increase in [Ca2+]i in spinal lamina I neurons induces a non-Hebbian form of synaptic plasticity in spinal nociceptive pathways without affecting neuronal active and passive membrane properties.

The role of magnesium in the management of cerebral vasospasm.

Subarachnoid hemorrhage (SAH) is characterized by bleeding into the subarachnoid space, often caused by ruptured aneurysm. Aneurysmal rupture occurs in 700,000 individuals per year worldwide, with 40,000 cases taking place in the United States. Beyond the high mortality associated with SAH alone, morbidity and mortality are further increased with the occurrence of cerebral vasospasm, a pathologic constriction of blood vessels that can lead to delayed ischemic neurologic deficits (DIND). Treatment of cerebral vasospasm is a source of contention. One extensively studied therapy is Magnesium (Mg) as both a competitive antagonist of calcium at the N-methyl D-aspartate (NMDA) receptor, and a noncompetitive antagonist of both IP3 and voltage-gated calcium channels, leading to smooth muscle relaxation. In our literature review, several animal and human studies are summarized in addition to two Phase III trials assessing the use of intravenous Mg in the treatment of SAH (IMASH and MASH-2). Though many studies have shown promise for the use of Mg in SAH, there has been inconsistency in study design and outcomes. Furthermore, the results of the recently completed clinical trials have shown no significant benefit from using intravenous Mg as adjuvant therapy in the treatment of cerebral vasospasm.

Novel ω-Conotoxins from C. Catus Reverse Signs of Mouse Inflammatory Pain after Systemic Administration

BackgroundAntagonists of N-type voltage-gated calcium channels (VGCC), Cav2.2, can manage severe chronic pain with intrathecal use and may be effective systemically. A series of novel ω-conotoxins that selectively inhibit N-type VGCCs was isolated from Conus catus. In the present study, the potency and reversibility of ω-conotoxins CVID, CVIE and CVIF to inhibit N-type calcium currents were investigated in mouse isolated dorsal root ganglion (DRG) neurons. The systemic potency of each ω-conotoxin to reverse signs of mouse chronic inflammatory pain was also compared.ResultsIn DRG neurons, the rank order of potency to inhibit N-type calcium currents was CVIE > CVIF > CVID. After subcutaneous administration, CVID and CVIE, but not CVIF, partially reversed impaired weight bearing in mice injected with Freund’s complete adjuvant (CFA) three days prior to testing. No side-effects associated with systemic administration of ω-conotoxins were observed.ConclusionsThe present study indicates a potential for CVID and CVIE to be developed as systemically active analgesics with no accompanying neurological side-effects.

The Plasma Membrane Ca2+-ATPase: A Novel Target of Parkinson’s Disease

Parkinson disease (PD) is a progressive neurological disease caused by selective degeneration of dopaminergic (DA) neurons in the substantia nigra. Although most cases of PD are sporadic cases, familial PD provides a versatile research model for basic mechanistic insights into the pathogenesis of PD. In this study, we generated DA neurons from PARK2 patient-specific, isogenic PARK2 null and PARK6 patient-specific induced pluripotent stem cells and found that these neurons exhibited more apoptosis and greater susceptibility to rotenone-induced mitochondrial stress. From phenotypic screening with an FDA-approved drug library, one voltage-gated calcium channel antagonist, benidipine, was found to suppress rotenone-induced apoptosis. Furthermore, we demonstrated the dysregulation of calcium homeostasis and increased susceptibility to rotenone-induced stress in PD, which is prevented by T-type calcium channel knockdown or antagonists. These findings suggest that calcium homeostasis in DA neurons might be a useful target for developing new drugs for PD patients.

Analgesic conotoxins: block and G protein‐coupled receptor modulation of N‐type (CaV2.2) calcium channels

Conotoxins (conopeptides) are small disulfide bonded peptides from the venom of marine cone snails. These peptides target a wide variety of membrane receptors, ion channels and transporters, and have enormous potential for a range of pharmaceutical applications. Structurally related ω-conotoxins bind directly to and selectively inhibit neuronal (N)-type voltage-gated calcium channels (VGCCs) of nociceptive primary afferent neurones. Among these, ω-conotoxin MVIIA (Prialt) is approved by the Food and Drug Administration (FDA) as an alternative intrathecal analgesic for the management of chronic intractable pain, particularly in patients refractory to opioids. A series of newly discovered ω-conotoxins from Conus catus, including CVID-F, are potent and selective antagonists of N-type VGCCs. In spinal cord slices, these peptides reversibly inhibit excitatory synaptic transmission between primary afferents and dorsal horn superficial lamina neurones, and in the rat partial sciatic nerve ligation model of neuropathic pain, significantly reduce allodynic behaviour. Another family of conotoxins, the α-conotoxins, are competitive antagonists of mammalian nicotinic acetylcholine receptors (nAChRs). α-Conotoxins Vc1.1 and RgIA possess two disulfide bonds and are currently in development as a treatment for neuropathic pain. It was initially proposed that the primary target of these peptides is the α9α10 neuronal nAChR. Surprisingly, however, α-conotoxins Vc1.1, RgIA and PeIA more potently inhibit N-type VGCC currents via a GABA(B) GPCR mechanism in rat sensory neurones. This inhibition is largely voltage-independent and involves complex intracellular signalling. Understanding the molecular mechanisms of conotoxin action will lead to new ways to regulate VGCC block and modulation in normal and diseased states of the nervous system.

Voltage-Dependent P/Q-Type Calcium Channels at the Frog Neuromuscular Junction

It is well known that antagonists of N-type voltage-gated calcium channels inhibit the evoked quantal release of acetylcholine in amphibian neuromuscular synapses. This, however, does not exclude the functional expression of other types of voltage-gated calcium channels in these nerve terminals. Using immunocytochemistry, we detected the expression of the alpha1A subunit of P/Q-type calcium channels (that is otherwise typical of mammalian motor nerve endings) in the frog neuromuscular junction. In addition, we demonstrated that the P/Q-type channel blocker omega-agatoxin IVA (20 nM) reduced the action potential-induced calcium transient and significantly decreased both spontaneous and evoked mediator release. Our data indicates the functional expression of P/Q-type calcium channels in the frog motor nerve ending which participate in acetylcholine release.

T-type voltage-gated calcium channels regulate the tone of mouse efferent arterioles

Voltage-gated calcium channels are important for the regulation of renal blood flow and the glomerular filtration rate. Excitation-contraction coupling in afferent arterioles is known to require activation of these channels and we studied their role in the regulation of cortical efferent arteriolar tone. We used microdissected perfused mouse efferent arterioles and found a transient vasoconstriction in response to depolarization with potassium; an effect abolished by removal of extracellular calcium. The T-type voltage-gated calcium channel antagonists mibefradil and nickel blocked this potassium-induced constriction. Further, constriction by the thromboxane analogue U46619 was significantly inhibited by mibefradil at a concentration specific for T-type channels. Using PCR, we found that two channel subtypes, Ca(v)3.1 and Ca(v)3.2, were expressed in microdissected efferent arterioles. Ca(v)3.1 was found by immunocytochemistry to be located in mouse efferent arterioles, human pre- and postglomerular vasculature, and Ca(v)3.2 in rat glomerular arterioles. Inhibition of endothelial nitric oxide synthase by L-NAME or its deletion by gene knockout changed the potassium-elicited transient constriction to a sustained response. Low concentrations of nickel, an agent that blocks Ca(v)3.2, had a similar effect. Thus, T-type voltage-gated calcium channels are functionally important for depolarization-induced vasoconstriction and subsequent dilatation in mouse cortical efferent arterioles.

Site-Specific Effects of Diselenide Bridges on the Oxidative Folding of a Cystine Knot Peptide, ω-Selenoconotoxin GVIA

Structural and functional studies of small, disulfide-rich peptides depend on their efficient chemical synthesis and folding. A large group of peptides derived from animals and plants contains the Cys pattern C-C-CC-C-C that forms the inhibitory cystine knot (ICK) or knottin motif. Here we report the effect of site-specific incorporation of pairs of selenocysteine residues on oxidative folding and the functional activity of omega-conotoxin GVIA, a well-characterized ICK-motif peptidic antagonist of voltage-gated calcium channels. Three selenoconotoxin GVIA analogues were chemically synthesized; all three folded significantly faster in the glutathione-based buffer compared to wild-type GVIA. One analogue, GVIA[C8U,C19U], exhibited significantly higher folding yields. A recently described NMR-based method was used for mapping the disulfide connectivities in the three selenoconotoxin analogues. The diselenide-directed oxidative folding of selenoconotoxins was predominantly driven by amino acid residue loop sizes formed by the resulting diselenide and disulfide cross-links. Both in vivo and in vitro activities of the analogues were assessed; the block of N-type calcium channels was comparable among the analogues and wild-type GVIA, suggesting that the diselenide replacement did not affect the bioactive conformation. Thus, diselenide substitution may facilitate oxidative folding of pharmacologically diverse ICK peptides. The diselenide replacement has been successfully applied to a growing number of bioactive peptides, including alpha-, mu-, and omega-conotoxins, suggesting that the integrated oxidative folding of selenopeptides described here may prove to be a general approach for efficient synthesis of diverse classes of disulfide-rich peptides.

The identification of inhibitors of Schistosoma mansoni miracidial transformation by incorporating a medium-throughput small-molecule screen

In Schistosoma mansoni, the miracidium-to-primary sporocyst transformation process is associated with many physiological, morphological, transcriptional and biochemical changes. In the present study, we use a medium-throughput small-molecule screen to identify chemical compounds inhibiting or delaying the in vitro transformation of miracidia to the sporocyst stage. The Sigma–Aldrich Library of Pharmacologically Active Compounds (LOPAC) contains 1280 well-characterized chemical compounds with various modes of action including enzyme inhibitors, antibiotics, cell-cycle regulators, apoptosis inducers and GPCR ligands. We identified 47 compounds that greatly reduce or delay this transformation process during a primary screen of live miracidia. The majority of compounds inhibiting larval transformation were from dopaminergic, serotonergic, ion channel and phosphorylation classes. Specifically, we found that dopamine D2-type antagonists, serotonin reuptake inhibitors, voltage-gated calcium channel antagonists and a PKC activator significantly reduced in vitro miracidial transformation rates. Many of the targets of these compounds regulate adenylyl cyclase activity, with the inhibition or activation of these targets resulting in increased cAMP levels in miracidia and concomitant blocking/delaying of larval transformation.

Chronic Benzodiazepine Administration Potentiates High Voltage-Activated Calcium Currents in Hippocampal CA1 Neurons

Signs of physical dependence as a consequence of long-term drug use and a moderate abuse liability limit benzodiazepine clinical usefulness. Growing evidence suggests a role for voltage-gated calcium channel (VGCC) regulation in mediating a range of chronic drug effects from drug withdrawal phenomena to dependence on a variety of drugs of abuse. High voltage-activated (HVA) calcium currents were measured in whole-cell recordings from acutely isolated hippocampal CA1 neurons after a 1-week flurazepam (FZP) treatment that results in withdrawal-anxiety. An approximately 1.8-fold increase in Ca(2+) current density was detected immediately after and up to 2 days but not 3 or 4 days after drug withdrawal. Current density was unchanged after acute desalkyl-FZP treatment. A significant negative shift of the half-maximal potential of activation of HVA currents was also observed but steady-state inactivation remained unchanged. FZP and diazepam showed use- and concentration-dependent inhibition of Ca(2+) currents in hippocampal cultured cells following depolarizing trains (FZP, IC(50) = 1.8 microM; diazepam, IC(50) = 36 microM), pointing to an additional mechanism by which benzodiazepines modulate HVA Ca(2+) channels. Systemic preinjection of nimodipine (10 mg/kg), an L-type (L)-VGCC antagonist, prevented the benzodiazepine-induced increase in alpha-amino-3-hydroxy-5-methylisoxasole-4-propionic acid receptor (AMPAR)-mediated miniature excitatory postsynaptic current in CA1 neurons 2 days after FZP withdrawal, suggesting that AMPAR potentiation, previously linked to withdrawal-anxiety may require enhanced L-VGCC-mediated Ca(2+) influx. Taken together with prior work, these findings suggest that enhanced Ca(2+) entry through HVA Ca(2+) channels may contribute to hippocampal AMPAR plasticity and serve as a potential mechanism underlying benzodiazepine physical dependence.

Nerve growth factor inhibits Gd3+-sensitive calcium influx and reduces chemical anoxic neuronal death

To investigate whether glutamate and voltage-gated calcium channels-independent calcium influx exists during acute anoxic neuronal damage and its possible relationship to neuronal protective function of NGF. In in vitro model of acute anoxia, hippocampal cultures from newborn rats were exposed to 3 mmol/L KCN. Changes of intracellular Ca(2+) concentration ([Ca(2+)](i)) were monitored by con-focal imaging and cell viability was assayed by PI and cFDA staining. The results showed that after treatment with primary hippocampal cultures with 3 mmol/L KCN for 15 min, [Ca(2+)](i) was significantly increased 6.27-fold compared to pre-anoxia level and 73.3% of the cells died. When combination of 20 micromol/L MK-801 (glutamate receptor antagonist), 40 micromol/L CNQX (AMPA receptor antagonist) and 5 micromol/L nimodipine (voltage-gated calcium channel antagonist) (hereafter denoted as MCN) were administrated to hippocampal cultures, levels of [Ca(2+)](i) and cell death rate induced by KCN were partially reduced by 35.9% and 47.5% respectively. However, Gd(3+) (10 micromol/L) almost completely blocked KCN-mediated [Ca(2+)](i) elevation by 81.9% and reduced neuronal death by 88.8% in the presence of MCN. It is noteworthy that NGF, used in combination with MCN, inhibited KCN-induced [Ca(2+)](i) increase by 77.4% and reduced cell death by 87.1%. Only PLC inhibitor U73122 (10 micromol/L) abolished NGF effects. It is concluded that Gd(3+)-sensitive calcium influx, which is NMDA (glutamate receptor) and voltage-gated calcium channels-independent, is responsible for acute anoxic neuronal death. NGF can inhibit Gd(3+)-sensitive calcium influx and reduce anoxic neuronal death through activating PLC pathway.

ω-Conotoxin inhibition of excitatory synaptic transmission evoked by dorsal root stimulation in rat superficial dorsal horn

A number of ω-conotoxins are potent and selective antagonists of N-type voltage-gated calcium channels (VGCCs) and are potentially effective as analgesic agents. ω-Conotoxins CVID and CVIB, venom peptides from Conus catus, inhibit N-type and N/P/Q-type VGCCs, respectively, in rat dorsal root ganglion sensory neurons. In the present study, we tested the effects of five different ω-conotoxins, CVID, CVIB, MVIIA, MVIIC and GVIA, on excitatory synaptic transmission between primary afferents and dorsal horn superficial lamina neurons of rat spinal cord. The N-type VGCC antagonists CVID (200 nM) and MVIIA (500 nM) completely and irreversibly inhibited excitatory postsynaptic currents (EPSCs) in the dorsal horn superficial lamina. The N- and P/Q-type VGCC antagonist CVIB (200 nM) reversibly reduced evoked EPSC amplitude an average of 34 ± 8%, whereas MVIIC (200 nM) had no effect on excitatory synaptic transmission. In neurons receiving polysynaptic input, CVIB reduced both the EPSC amplitude and the “success rate” calculated as the relative number of primary afferent stimulations that resulted in postsynaptic responses. These results indicate that (i) the analgesic action of ω-conotoxins that antagonise N-type VGCCs may be attributed to inhibition of neurotransmission between primary afferents and superficial dorsal horn neurons, (ii) nociceptive synaptic transmission between primary afferents and superficial lamina neurons is mediated predominantly by N-type VGCCs, and (iii) in contrast to the irreversible inhibition by CVID, MVIIA and GVIA, the inhibition of excitatory monosynaptic transmission by CVIB is reversible.

Activation of group I metabotropic glutamate receptors induces long-term depression in the hippocampal CA1 region of adult rats in vitro

Previous studies have implicated that long-term depression (LTD) was developmentally regulated since LTD can be readily induced by low frequency stimulation (LFS) in acute hippocampal slices prepared from juvenile but not adult animals. Here, we have examined the LTD induced by LFS (1 Hz, 900 pulses) paired with a certain pattern at the Schaffer collateral-CAl synapse in adult hippocampal slices. We found that, in the 90-day-old rat hippocampus, LTD could be induced reliably by LFS paired with stronger stimulus intensity than that used during baseline recording. However, this synaptic depression could be completely abolished by application of metabotropic glutamate receptor (mGluR) antagonist ( S)-amethyl-4-carboxyphenylglycine (MCPG) which had no effect on that induced by the same protocol in the 16-day-old rat hippocampus. Furthermore, preincubation with group I mGluR antagonist, 2-methyl-6-(phenylethynyl)-pyridine (MPEP) and ( S)-2-methyl-4-carboxyphenylglycine (LY367385), also completely prevented the LFS-induced LTD. In contrast, group II mGluR antagonist (2 S)-a-ethylglutamic acid (EGLU), N-methyl- d-aspartate (NMDA) receptor antagonist APV and voltage-gated calcium channel antagonist nimodipine had no effect on the LFS-induced LTD. Taken together, these observations suggest that LFS paired with strong stimulus strength can efficiently induce group I mGluR-dependent LTD in the adult hippocampal CA1 region, proving insight into the functional significance of hippocampal mGluR-mediated LTD in learning and memory.

Secondary hyperalgesia in the rat first degree burn model is independent of spinal cyclooxygenase and nitric oxide synthase

Various animal models of pain are dependent on activation of different glutamate receptor subtypes. First degree burn of the paw elicits a secondary hyperalgesia that is dependent on Ca 2++ permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), but not N-methyl- d-aspartate (NMDA) receptors. The present study takes advantage of that specificity by examining the effects of spinal pretreatments of agents on this secondary hyperalgesia. Rats with indwelling intrathecal catheters were pretreated with agents prior to paw injury. Mechanical withdrawal thresholds were measured before, and for three h after the injury. Spinal pretreatment with cyclooxygenase (10 and 30 μg (S)-(+)-ibuprofen; and 3 and 30 μg ketorolac) and nitric oxide synthase (33 and 100 μg N G Nitro- l-arginine methyl ester hydrochloride ( l-NAME) and 10 μg thiocitrulline) inhibitors resulted in no specific anti-allodynia. In contrast, ziconotide (0.3, 1.0 and 3 μg), the N-type voltage gated calcium channel antagonist was very effective in blocking burn-induced sensitivity at all doses used. l-type (Diltiazam 230 μg) and P-type (Agatoxin IVA 0.3 μg) calcium channel blockers produced intermediate effects. Thus, cyclooxygenase and nitric oxide synthase are assumed not to be downstream of Ca 2++ permeable AMPA receptors. Voltage gated calcium channels blockers could exert their effects either pre- or post-synaptically.

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

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

Benzodiazepine-induced hippocampal CA1 neuron α-amino-3-hydroxy-5-methylisoxasole-4-propionic acid (AMPA) receptor plasticity linked to severity of withdrawal anxiety: differential role of voltage-gated calcium channels and N-methyl-D-aspartic acid receptors

Withdrawal from 1-week oral administration of the benzodiazepine, flurazepam (FZP) is associated with increased alpha-amino-3-hydroxy-5-methylisoxasole-4-propionic acid (AMPA) receptor (AMPAR) miniature excitatory postsynaptic currents (mEPSCs) but reduction of N-methyl-D-aspartic acid (NMDA) receptor (NMDAR)-evoked (e)EPSCs in hippocampal CA1 neurons. A positive correlation was observed between increased AMPAR-mediated mEPSC amplitude and anxiety-like behavior in 1-day FZP-withdrawn rats. These effects were disrupted by systemic AMPAR antagonist administration (GYKI-52466, 0.5 mg/kg, intraperitoneal) at withdrawal onset, strengthening the hypothesis that CA1 neuron AMPAR-mediated hyperexcitability is a central component of a functional anatomic circuit associated with the expression of withdrawal anxiety. Abolition of AMPAR current upregulation in 2-day FZP withdrawn rats by GYKI-52466 injection also reversed the reduction in NMDAR-mediated eEPSC amplitude in CA1 neurons from the same rats, suggesting that downregulation of NMDAR function may serve a protective, negative-feedback role to prevent AMPAR-mediated neuronal overexcitation. NMDAR antagonist administration (MK-801, 0.25 mg/kg intraperitoneally) had no effect on modifying increased glutamatergic strength or on withdrawal anxiety, whereas injection of an L-type voltage-gated calcium channel antagonist, nimodipine (10 mg/kg, intraperitoneally) averted AMPAR current enhancement and anxiety-like behavior, suggesting that these manifestations may be initiated by a voltage-gated calcium channel-dependent signal transduction pathway. An evidence-based model of likely cellular mechanisms in the hippocampus contributing to benzodiazepine withdrawal anxiety was proposed implicating regulation of multiple CA1 neuron ion channels.