HVA ICa Research Articles

Analysis of the long-term actions of gabapentin and pregabalin in dorsal root ganglia and substantia gelatinosa.

The α2δ-ligands pregabalin (PGB) and gabapentin (GBP) are used to treat neuropathic pain. We used whole cell recording to study their long-term effects on substantia gelatinosa and dorsal root ganglion (DRG) neurons. Spinal cord slices were prepared from embryonic day 13 rat embryos and maintained in organotypic culture for >5 wk (neuronal age equivalent to young adult rats). Exposure of similarly aged DRG neurons (dissociated and cultured from postnatal day 19 rats) to GBP or PGB for 5-6 days attenuated high-voltage-activated calcium channel currents (HVA ICa). Strong effects were seen in medium-sized and in small isolectin B4-negative (IB4-) DRG neurons, whereas large neurons and small neurons that bound isolectin B4 (IB4+) were hardly affected. GBP (100 μM) or PGB (10 μM) were less effective than 20 μM Mn(2+) in suppression of HVA ICa in small DRG neurons. By contrast, 5-6 days of exposure to these α2δ-ligands was more effective than 20 μM Mn(2+) in reducing spontaneous excitatory postsynaptic currents at synapses in substantia gelatinosa. Spinal actions of gabapentinoids cannot therefore be ascribed to decreased expression of HVA Ca(2+) channels in primary afferent nerve terminals. In substantia gelatinosa, 5-6 days of exposure to PGB was more effective in inhibiting excitatory synaptic drive to putative excitatory neurons than to putative inhibitory neurons. Although spontaneous inhibitory postsynaptic currents were also attenuated, the overall long-term effect of α2δ-ligands was to decrease network excitability as monitored by confocal Ca(2+) imaging. We suggest that selective actions of α2δ-ligands on populations of DRG neurons may predict their selective attenuation of excitatory transmission onto excitatory vs. inhibitory neurons in substantia gelatinosa.

Differential down‐regulation of voltage‐gated calcium channel currents by glutamate and BDNF in embryonic cortical neurons

In the embryonic brain, post-mitotic cortical neurons migrate from their place of origin to their final location. Various external factors such as hormones, neurotransmitters or peptides regulate their migration. To date, however, only a few studies have investigated the effects of these external factors on the electrical properties of the newly formed embryonic cortical neurons. The aim of the present study was to determine whether glutamate and brain-derived neurotrophic factor (BDNF), known to regulate neuronal cell migration, could modulate currents through voltage-gated calcium channels (ICa) in cortical neurons isolated from embryonic day 13 (E13) mouse foetuses. Whole cell recordings of ICa showed that E13 cortical cells kept 1 day in vitro expressed functional low- and high-voltage activated (LVA and HVA) Ca2+ channels of T-, L- and N-types. A 1-day glutamate treatment non-specifically inhibited LVA and HVA ICa whereas BDNF down-regulated HVA with N-type ICa being more depressed than L-type ICa. The glutamate-induced ICa inhibition was mimicked by NMDA. BDNF exerted its action by recruiting trkB receptors and SKF-96365-sensitive channels. BAPTA prevented the glutamate- and the BDNF-dependent inhibition of Ica, indicating a Ca2+-dependent mechanism of action. It is proposed that an influx of Ca2+ through NMDA receptors depresses the expression of LVA and HVA Ca2+ channels whereas a Ca2+ influx through SKF-96365-sensitive TRPC (transient receptor potential protein of C subtype) channels preferentially inhibits the expression of HVA Ca2+ channels. Glutamate and BDNF appear as potent modulators of the electrical properties of early post-mitotic neurons. By down-regulating ICa they could exert a neuroprotective action on embryonic cortical neurons.

Adrenergic regulation of the intracellular [Ca2+] and voltage-operated Ca2+ channel currents in the rat prostate neuroendocrine cells.

The prostate gland contains numerous neuroendocrine cells (PNECs) innervated by adrenergic neurons. PNECs are believed to influence the growth and physiological function of the prostate gland via paracrine release of hormones. Using fura-2 fluorescence measurement and patch-clamp techniques, we investigated the effects of adrenergic stimulation on cytosolic concentration of Ca2+ ([Ca2+]c) and high voltage-activated Ca2+ channel currents (HVA-I(Ca)) of the putative rat prostate neuroendocrine cells (RPNECs) freshly isolated by an enzymic digestion. Noradrenaline (NA, 1 microM) induced a sharp, transient increase of [Ca2+]c measured by the fura-2 fluorescence. Pharmacological studies showed that alpha1-adrenoceptors (alpha1-ARs) coupled with PLC/IP3 signaling pathway induce the release of stored Ca2+, which subsequently recruits store-operated Ca2+ entry pathways. In the whole-cell voltage clamp experiment, NA decreased the amplitude of HVA-I(Ca) by 40%, which was mimicked by an alpha2-AR agonist (UK14304) but not by an alpha1-AR agonist (phenyleprine). After selective blockade of N-type Ca2+ channels by omega-conotoxin GVIA, the addition of NA showed no further inhibition on the remaining L-type Ca2+ channel currents. The adrenergic inhibition of HVA-I(Ca) was partially prevented by the pretreatment with pertussis toxin (PTX) (5 microg/ml, 4 hr, 37 degrees C). RPNECs express both alpha1- and alpha2-ARs, signaling the release of stored Ca2+ and the inhibition of N-type Ca2+ channels, respectively.

Embryonic rat motoneurons express a functional P-type voltage-dependent calcium channel

Only L- and N-type high voltage-activated calcium currents (HVA ICa) have been demonstrated in identified embryonic spinal motoneurons. However, pharmacological experiments suggest that other HVA ICa, including P-type, govern neurotransmitter release at the adult neuromuscular junction. We sought to analyse if embryonic motoneurons express these other ICa, using the whole-cell voltage-clamp method on motoneurons purified by a new metrizamide-panning technique from E15 rat embryos. In addition to L-type dihydropyridine-sensitive and N-type ω-GVIA-sensitive currents, motoneurons express two other HVA ICa. One has properties related to the P-type channel currents described in Purkinje cells: it is inhibited by the peptide ω-agatoxin-IVA with a maximal effect at 100–200 nM. The inhibited current has a characteristic sustained component during depolarizing test pulses. Furthermore, 50–100 nM concentration of ω-agatoxin-IVA reduce the increase in cytoplasmic calcium concentration observed after depolarization. The other HVA ICa is resistant to saturating concentrations of verapamil, ω-conotoxin GVIA and ω-agatoxin-IVA which block L, N and P-type HVAICa, respectively. These results suggest that it is now possible to dissect, using a simple method of purification, the properties of the ICa in embryonic mammalian motoneurons and to provide pharmacological evidence for multiple calcium channels which may be involved in regulation of their activity during development.

Local anesthetics depress the calcium current of rat sensory neurons in culture.

Local anesthetics are known to inhibit the voltage-gated sodium current (INa) of the nerve membrane, but it has not been fully studied whether anesthetic concentrations of local anesthetics depress the voltage-gated calcium current (ICa) of mammalian neurons. The effects of local anesthetics on ICa evoked in cultured rat dorsal root ganglion cells were studied. Whole cell patch clamp recordings were made from rat dorsal root ganglion cells cultured for 1-3 weeks. ICa was recorded using patch electrodes filled with Cs-aspartate in Na(+)-free external solution containing 5 mM-Ba2+. All drugs, including local anesthetics, were applied by miniperfusion from micropipettes by pressure ejection. Tetracaine (300 microM) depressed the peak amplitudes of high voltage-activated (HVA)-ICa to 22.6 +/- 8.8% of control values (n = 14) without affecting the current-voltage relation. A tetracaine dose-response curve for HVA-ICa indicated an apparent dissociation constant of 79.5 microM. Tetracaine (30 microM) depressed nicardipine-sensitive HVA-I(Ca) (L-type) to 14.3 +/- 6.7% (n = 6), omega-conotoxin-sensitive HVA-ICa (N-type) to 81.6 +/- 9.6% (n = 7), and low voltage-activated (LVA)-ICa (T-type) to 65.1 +/- 11.1% (n = 6) of their respective controls. Local anesthetics other than tetracaine also depressed HVA-ICa but were of different potency; the rank sequence was dibucaine > tetracaine > bupivacaine >> procaine = lidocaine. These results suggest that both HVA-ICa and LVA-ICa are depressed by tetracaine used at the concentrations required for spinal anesthesia and that the L-type Ca2+ channel among Ca2+ channel subtypes is the most susceptible to tetracaine. A good correlation between local anesthetic potencies to inhibit HVA-ICa and their anesthetic potencies implies that the inhibition of calcium influx through voltage-gated channels may contribute to spinal anesthetic mechanisms.

Enhancement of voltage-gated Ca2+ currents induced by daily stimulation of hippocampal neurons with glutamate

The regulation of calcium channel currents (ICa) induced by daily stimulation (1 hr) with 10 microM glutamate was studied in full differentiated hippocampal cells in culture. We report a specific enhancement of the high-voltage-activated current type (HVA ICa) ongoing over days. The density of HVA ICa increased about twofold after the second glutamate session, and this enhancement was still observed after the fifth day of treatment, while low-voltage-activated calcium currents (LVA ICa) remained unchanged. During glutamate application, a transient increase of intracellular calcium (Cai) was observed, followed by a slow decay within 2-3 min, and substantial recovery in about 10 min. Similarly, Cai transients induced by periodic membrane depolarization mimicked the long-term effect of glutamate on ICa. These results demonstrate for the first time an increase of ICa in a time frame of days. Since the effect of glutamate on ICa was prevented by cycloheximide, neosynthesis of channel proteins presumably supports this enhancement.

Calcium currents in pyramidal neurons acutely dissociated from the rat frontal cortex: a study by the nystatin perforated patch technique

We studied electrical and pharmacological properties of voltage-activated Ca 2+ currents (I Ca) in freshly dissoaciated rat frontal cortical pyramidal neurons by using nystatin perforated patch and conventional patch-clamp techniques. Within wither nystatin perofated technique or conventional patch-clamp technique, low voltage-activatedI Ca (LVAI Ca) and hihg voltage-activated Ca (HVAI Ca) were recorded. However, in conventional whole-cell recording, HVAI Ca ran down within 5–20 min, whereas the current remained stable for 50 min at least in nystatin perforated patch recording. The HVAI Ca recorded by nystatin perforated patch technique was further classified to L-type, N-type and a current component resistant to both nicardipine and ω-conotoxin by their current kinetics, voltage- and drug-sensitivities. Present results suggest that the perforated patch technique is a useful method for studying the properties of high voltage-activated Ca 2+ channels.