L-type calcium channel activation up-regulates the mRNAs for two different sodium channel α subunits (Nav1.2 and Nav1.3) in rat pituitary GH3 cells

Anesthesia &Amp; Analgesia

Gerner

Schmidt

et al. 2008

BACKGROUND Bulleyaconitine A (BLA) is an analgesic and antiinflammatory drug isolated from Aconitum plants. BLA has several potential targets, including voltage-gated Na+ channels. We tested whether BLA elicited long-lasting cutaneous analgesia, when co-injected with lidocaine and epinephrine, as a model for prolonged infiltration anesthesia. METHODS The local anesthetic properties of BLA were assessed by the patch-clamp technique in HEK293t cells expressing Nav1.7 and Nav1.8 neuronal Na+ channels, both crucial for nociception. Drug solutions (0.6 mL) were injected subcutaneously via rat shaved dorsal skin. Inhibition of the cutaneous trunci muscle reflex was evaluated by pinpricks. Skin cross-sections were stained with hematoxylin and eosin or with antibodies against PGP9.5. RESULTS BLA at 10 µM interacted minimally with resting or inactivated Nav1.7 and Nav1.8 Na+ channels when infrequently stimulated to +50 mV for 3 ms. However, when stimulated at 2 Hz for 1000 pulses, their peak Na+ currents were >90% reduced by BLA. This use-dependent inhibition was not significantly reversed after 15-min washing. Complete nociceptive blockade after injection of lidocaine (0.5%)/epinephrine (1:200,000) lasted for approximately 1 h in rats; full recovery occurred after approximately 6 h. Co-injection of 0.125 mM BLA with lidocaine/epinephrine increased the duration of complete nociceptive blockade to 24 h. Full recovery occurred after approximately 6 days. Skin histology including peripheral nerve fibers appeared unaffected by BLA. CONCLUSIONS BLA inhibits Nav1.7 and Nav1.8 Na+ currents in a use-dependent manner. Co-injection of BLA at ≤0.125 mM with lidocaine and epinephrine elicits complete cutaneous analgesia that lasts for up to 24 h without adverse effects.

Section: Discussionsupporting

confidence: 70%

Use of Bulleyaconitine A as an Adjuvant for Prolonged Cutaneous Analgesia in the Rat

Anesthesia &Amp; Analgesia

Gerner

Schmidt

et al. 2008

“…GH 3 cells are known to express neuronal Nav1.1, 1.2, 1.3, and 1.6 isoforms (Vega et al, 2003). Kinetics of these Na + currents have been extensively studied before (Cota and Armstrong, 1989).…”

Section: Block Of Neuronal Inactivation-deficient Na + Currents By Camentioning

confidence: 99%

“…These cultured cells were reported to express Nav1.1, 1.2. 1.3, and 1.6 Na + channel isoforms endogenously (Vega et al, 2003). Instead of using site-directed mutagenesis, we impaired neuronal Na + channel fast inactivation by two conventional techniques (chloramine-T and sea anemone toxin).…”

Section: Introductionmentioning

confidence: 99%

Preferential block of inactivation-deficient Na+ currents by capsaicin reveals a non-TRPV1 receptor within the Na+ channel

Mitchell

2007

Pain

Capsaicin elicits burning pain via the activation of the vanilloid receptor (TRPV1). Intriguingly, several reports showed that capsaicin also inhibits Na + currents but the mechanisms remain unclear. To explore this non-TRPV1 action we applied capsaicin to HEK293 cells stably expressing inactivation-deficient rat skeletal muscle Na + mutant channels (rNav1.4-WCW). Capsaicin elicited a conspicuous time-dependent block of inactivation-deficient Na + currents. The 50% inhibitory concentration (IC 50 ) of capsaicin for open Na + channels at +30 mV was measured 6.8 ± 0.6 μM (n = 5), a value that is 10-30 times lower than those for resting (218 μM) and inactivated (74 μM) wildtype Na + channels. On-rate and off-rate constants for capsaicin open-channel block at +30 mV were estimated to be 6.37 μM −1 s −1 and 34.4 s −1 , respectively, with a calculated dissociation constant (K D ) of 5.4 μM. Capsaicin at 30 μM produced ~70% additional use-dependent block of remaining rNav1.4-WCW Na + currents during repetitive pulses at 1 Hz. Site-directed mutagenesis showed that the local anesthetic receptor was not responsible for the capsaicin block of the inactivation-deficient Na + channel. Interestingly, capsaicin elicited little time-dependent block of batrachotoxin-modified rNav1.4-WCW Na + currents, indicating that batrachotoxin prevents capsaicin binding. Finally, neuronal open Na + channels endogenously expressed in GH 3 cells were as sensitive to capsaicin block as rNav1.4 counterparts. We conclude that capsaicin preferentially blocks persistent late Na + currents, probably via a receptor that overlaps the batrachotoxin receptor but not the local anesthetic receptor. Drugs that target such a non-TRPV1 receptor could be beneficial for patients with neuropathic pain.

Biological & Pharmaceutical Bulletin

“…For instance, entry of Ca 2ϩ through L-type Ca 2ϩ channels increased the number of VSSCs via regulation of mRNAs for VSSCs in GH 3 cells. 19,20) In N1E-115 neuroblastoma cells, on the contrary, an increase in [Ca 2ϩ ]i reduced the expression of Na ϩ channel mRNA. 21) As for alterations in Ca 2ϩ channels in NG108-15 cells, our previous study has shown that the rate of KCl-stimulated 45 Ca uptake by NG108-15 cells became greater when the cells were treated with Bt 2 cAMP and that this 45 Ca uptake was selectively inhibited by various types of L-type channel blockers.…”

Section: Resultsmentioning

confidence: 95%

Enhancement of Veratridine-Induced Sodium Dynamics in NG108-15 Cells during Differentiation

Imanishi

Matsushima

Kawaguchi

et al. 2006

An NG108-15 cell line, a hybrid cell line, is artificially formed by the fusion of mouse neuroblastoma (N18TG-2) and rat glioma (C6-Bu1) cell lines. NG108-15 cells have been used as a suitable model system to investigate the mechanisms of neuronal development and differentiation. 1)When NG108-15 cells are differentiated in culture with inducing reagents such as dibutyryl cAMP (Bt 2 cAMP), prostaglandin E 1 (PG E 1 ) plus theophylline, forskolin, dexamethasone, retinoic acid etc., the cells exhibit the growth of neurites characteristic of neuronal differentiation. 2)We have reported that the rate of KCl-stimulated 45 Ca uptake in differentiated NG108-15 cells (treated with Bt 2 cAMP) was significantly higher than that in undifferentiated cells and that the 45 Ca uptake was selectively inhibited by various blockers of L-type voltage-sensitive Ca 2ϩ channels (VSCCs).3) The B max value for specific binding of (ϩ ] i ) induced by high-KCl application and the amplitude of Ca 2ϩ current were significantly augmented when NG108-15 cells were differentiated. 5-10)Voltage-sensitive sodium channels (VSSCs) have been existed not only in neuron, cardiac and skeletal muscle, but also in a variety of hormone secretory cells. VSSCs play a critical role in controlling the electrical excitability of animal cells, being primarily responsible for the rising phase of the action potential. 11,12) In mammalian nerve and muscle cells, VSSCs consist of a pore-forming a subunit in association with one or two auxiliary b subunits. a Subunits have been cloned nine isoforms (Na V 1.1-1.9). These isoforms have been classified as members of a single gene family, Na V 1, on the basis of amino acid sequences. Six isoforms (Na V 1.1-1.4, 1.6 and 1.7) are all considered 'tetrodetoxin (TTX)-sensitive' Na ϩ channels. On the other hand, the remaining three isoforms (Na V 1.5, 1.8 and 1.9) are all considered 'TTX-resistant' Na ϩ channels. In addition, b subunits have been identified three isoforms (b1-b3).In contrast, little is known about developmental changes in the dynamics of Na ϩ during differentiation of NG108-15 cells. The present work, therefore, aims to reveal any developmental alterations in intracellular Na ϩ concentration ([Na ϩ ] i ) evoked by the application of high-KCl solution or veratridine (Vtd), a VSSCs agonist. It is to be noted that voltage-sensitive Na ϩ channels (VSSCs) are characteristic of mammalian nerve cells, 11,12) and NG108-15 cells are thought to obtain neuronal features when differentiated. 1) MATERIALS AND METHODSCell Culture NG108-15 cells (passage number: less than 15) were grown in high glucose Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum and 0.1 mM hypoxanthine, 1 mM aminopterin, 16 mM thymidine (HAT) under a humidified atmosphere of 7.5% CO 2 at 37°C. The cells were harvested and subcultured by reseeding at a density of about 10 4 cells/ml, in culture dishes with quartz bottoms, which were precoated with poly-L-ornithine. On the following day, 0.75 mM Bt 2 cAMP was added to the...