This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Niinomi, K. Articles by Dohi, S. Search for Related Content PubMed PubMed Citation Articles by Niinomi, K. Articles by Dohi, S. Related Collections Mechanisms Preclinical Pharmacology Pharmacology

---

ANESTHETIC PHARMACOLOGY

Nicorandil, an Adenosine Triphosphate-Sensitive Potassium Channel Opener, Inhibits Muscarinic Acetylcholine Receptor-Mediated Activation of Extracellular Signal-Regulated Kinases in PC12 Cells

Kazumi Niinomi, MD^*, Yoshiko Banno, PhD, Hiroki Iida, MD, PhD^*, and Shuji Dohi, MD, PhD^*

From the *Department of Anesthesiology and Pain Medicine, Gifu University Graduate School of Medicine, Gifu, Japan; and Department of Cell Signaling, Gifu University Graduate School of Medicine, Gifu, Japan.

Address correspondence and reprint requests to: Shuji Dohi, MD, Department of Anesthesiology and Pain Medicine, Gifu University Graduate School of Medicine, 1-1 Yanagido, Gifu 501-1194, Japan. Address e-mail to shu-dohi{at}gifu-u.ac.jp.

	Abstract

Top Abstract Introduction METHODS RESULTS DISCUSSION REFERENCES

 
BACKGROUND: Nicorandil, an adenosine triphosphate-sensitive potassium channel opener, is reported to have an antinociceptive effect by hyperpolarization through the K⁺ channel. The activation of extracellular signal-regulated kinase (ERK), a family of mitogen-activated protein kinases, plays an important role in synaptic plasticity and noxious stimulation in the dorsal root ganglion, and spinal neurons have been reported to induce its activation. To understand the biological mechanisms of nicorandil, we examined the effects of nicorandil on muscarinic acetylcholine (ACh) receptor-mediated activation of ERK in a neuronal model cell, rat pheochromocytoma PC12 cells.

METHODS: PC12 cells were stimulated with ACh in the presence or absence of nicorandil, and phosphorylation of ERK was examined by a Western blot analysis. We also examined the effects of nicorandil on the ERK activation induced by 4β-phorbol 12-myristate 13-acetate, an activator of protein kinase C, or ionomycin, a calcium ionophore. Intracellular Ca²⁺ increase was visualized in fluo-3-loaded PC12 cells using fluorescence microscopy.

RESULTS: Nicorandil inhibited ACh-induced ERK activation in a concentration-dependent manner. The inhibition was abolished by glibenclamide, an adenosine triphosphate-sensitive potassium channel blocker. Nicorandil suppressed the ERK activation induced by ionomycin but not 4β-phorbol 12-myristate 13-acetate. Pretreatment of PC12 cells with nicorandil reduced the intracellular Ca²⁺ concentration stimulated by ACh.

CONCLUSIONS: Nicorandil inhibits muscarinic activation of the ERK signaling pathway by reducing the intracellular Ca²⁺ concentration.

	Introduction

Top Abstract Introduction METHODS RESULTS DISCUSSION REFERENCES

 
Nicorandil is an adenosine triphosphate sensitive potassium (K⁺_ATP) channel opener. Previous studies demonstrate that the opening of central K⁺_ATP channels may elicit the antinociceptive effect,1 and enhance the analgesic action of morphine and dexmedetomidine.2 The K⁺_ATP channel openers induce cell hyperpolarization, thus, resulting in a decrease in the intracellular calcium ion (Ca²⁺) level and neurotransmitter release (calcitonin gene-related peptide and substance P) which account for antinociception.3,4 However, the mechanism involving intracellular signal transduction underlying the antinociception in neurons is not yet clearly understood.

The mitogen-activated protein kinase (MAPK) is a family of serine or threonine protein kinases that transduce extracellular stimuli into intracellular posttranslational and transcriptional responses.5–7 The MAPK family includes extracellular signal-regulated kinase (ERK), p38 MAPK, c-Jun N-terminal kinase/stress-activated protein kinase and ERK5. The activation of ERK by MAP kinase (MEK)8 is involved in neuronal plasticity, such as long-term enhancement,9 long-term facilitation,10 and long-term spatial memory.11,12 In addition, the phosphorylation of ERK in primary afferent neurons occurs after noxious stimulation, and a MEK inhibitor U0126 dose-dependently attenuates thermal hyperalgesia after capsaicin injection.13 These studies suggest that the intracellular signal transduction pathway involving ERK activation in neurons may therefore play a crucial role in pain hypersensitivity.

Rat pheochromocytoma (PC12) cells contain Na⁺, K⁺_, and Ca²⁺ channels and several membrane-bound receptors, including muscarinic (mAChR) and nicotinic acetylcholine (ACh) receptors.14 They exhibit a number of properties characteristic of sympathetic neurons,15 and have been used widely to investigate signal transduction and are often used as a model system of neuronal proliferation and differentiation. We previously studied and described the inhibitory effect of local anesthetics on muscarinic receptor-mediated activation of ERK by using PC12 cells.16

mAChR are widely distributed in the peripheral and central nervous systems. They play a role in motor function and the processing of sensory information in the human spinal cord17 and activate many downstream signaling pathways, including the ERK activating pathway.18 Therefore, to examine whether the K⁺_ATP channel opener elucidates some of the mechanisms, including the intracellular ERK activation pathway, we investigated the effect of nicorandil on mAChR-mediated ERK activation using the PC12 cells as a neuronal model.

	METHODS

Top Abstract Introduction METHODS RESULTS DISCUSSION REFERENCES

 
Nicorandil and glibenclamide were purchased from Wako Pure Chemical Industries (Osaka, Japan). ACh, atropine sulfate salt, and 4β-phorbol 12-myristate 13-acetate (PMA) were from Nacalai Tesque (Kyoto, Japan). Ionomycin was purchased from Calbiochem (Nottingham, UK). Fluo-3 AM, albumin, Dullbecco modified Eagle's medium (DMEM) were purchased from Sigma Chemical Company (St. Louis, MO). Rabbit polyclonal antibodies against Tyr²⁰⁴ phosphorylated ERK1/2 (p-ERK1/2) and ERK1/2 (total-ERK1/2) were from Cell Signaling Technology (Danvers, MA). Anti-rabbit immunoglobulin G horseradish peroxidase linked whole antibody (from donkey) was purchased from Amersham Bioscience (Buckinghamshire, UK). The Western blot analysis was performed using the ECL Western Blotting Detection System (GE Healthcare Bio-Sciences Corp., NJ).

PC12 cells were a kind gift of Dr. Y. Sugimoto (Shirakawa Institute of Animal Genetics, Fukushima, Japan). Monolayer cultures of the cells were maintained in 100-mm-diameter tissue culture dishes in DMEM supplemented with 10% (vol/vol) fetal bovine serum and 5% (vol/vol) horse serum in a humidified atmosphere containing 5% carbon dioxide at 37°C. The stock cultures were subcultured routinely at a cell density of 2–3 x 10⁶/dish at least once a week, and the culture media were renewed every 2 d.

PC12 cells were subcultured in 60-mm-diameter tissue culture dishes at 8 x 10⁵ cells/dish and grown for 3 d. The cells were washed twice with 2 mL buffer A (1 mM CaCl₂, 1 mM MgCl₂, 1 mg/mL albumin, 5 mM glucose in Hanks' Balanced Salt Solutions) and preincubated in 3 mL buffer A with or without nicorandil or atropine at 37°C for 10 min. The cells were stimulated with 0.1 mM ACh for 2 min, 100 nM PMA for 5 min, 100 nM ionomycin for 15 min, or 10 mM NaF plus 10 µM AlCl₃ for 30 min at 37°C. The reaction was terminated by aspiration of the reaction buffer and washed twice with 2 mL ice-cold phosphate-buffered saline. The washed cells were scraped quickly into 100 µL RIPA buffer (10 mM Tris-HCl, pH 7.4, 0.5% deoxycholic acid, 0.1% sodium dodecyl sulfate, 150 mM NaCl, 1% NP-40), and lysed by two freeze-thaw cycles. The cell suspension was then centrifuged at 1500 rpm for 5 min to obtain the cell extract. The soluble extract (20 µg of protein of each sample) was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10%) and electrophoretically transferred to a polyvinylidene difluoride membrane. After the membranes were blocked with TBS-T (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween-20) containing 2% albumin, membranes were probed with antibodies against phospho-specific or total -ERK1/2 (1:2000) for 90 min at room temperature and then with the donkey Anti-rabbit immunoglobulin G horseradish peroxidase linked whole antibody for 60 min at room temperature. The bound antibodies were detected using the ECL Western blotting detection system. The density of protein bands was analyzed using a densitometer (Densitograph; Atto Corporation, Tokyo, Japan).

To visualize the increase in the intracellular Ca²⁺ concentration, we loaded 10 µM Fluo-3 AM with serum-free DMEM for 16 h. The cells pretreated with or without nicorandil were stimulated with 0.1 mM ACh for 5 min. A fluorescence analysis was performed on digitized images of live cells taken with an OLYMPUS IX71 fluorescence microscope linked to a digital CCD camera. Images of three fields per well were taken with an average of 100 cells per field. The number of fluorescent cells was determined by a visual examination of the field.

The data are presented as the mean ± sd from three different experiments and three determinant analyses. The bands were quantified by scanning densitometry and the ratio of phosphorylated ERK to total ERK was calculated. The differences between the values were evaluated by Student's t-test. The results were considered statistically significant when P < 0.05.

	RESULTS

Top Abstract Introduction METHODS RESULTS DISCUSSION REFERENCES

 
Muscarinic Receptor Stimulation Induces ERK Phosphorylation in PC12 Cells
Treatment of PC12 cells with 0.1 mM ACh-induced rapid and transient increases in ERK1/2 phosphorylation as examined by a Western blot analysis with phospho-specific antibody against pERK1/2. The phosphorylation of ERK1/2 increased at a peak 2 min after stimulation with ACh and decreased to the basal level 10 min after stimulation (Fig. 1). The ACh-induced ERK1/2 activation was increased approximately nine times in comparison to the level without ACh stimulation.

View larger version (25K): [in this window] [in a new window]   Figure 1. Acetylcholine (ACh)-induced extracellular signal-regulated kinase (ERK) phosphorylation in PC12 cells. The cells were stimulated with 0.1 mM ACh for the indicated time. Western blots of PC12 cells probed with anti-pERK1/2 and anti-total ERK1/2 antibodies. The Western blots were representative of three different experiments. Bands were quantified by scanning densitometry and the ratio of phosphorylated ERK to total ERK was calculated. The data are expressed as -fold increase to without stimulated cells (control) as 1, and represent the mean ± sd of 3 different experiments. P < 0.05 was considered significant in comparison to the control. **P < 0.01 versus the control.

To determine if the ACh-mediated phosphorylation of ERK was due to mAChR activation, we examined the effect of pretreatment with the muscarinic-specific antagonist atropine on the ERK activation. The pretreatment of the cells with 5 µM atropine for 10 min completely abolished the ACh-induced increase of ERK1/2 phosphorylation (Fig. 2), suggesting that the ACh-induced ERK activation was mediated via mAChR.

View larger version (18K): [in this window] [in a new window]   Figure 2. The effect of pretreatment of muscarinic-specific antagonist atropine on ACh-induced ERK phosphorylation. The cells were pretreated for 10 min with or without atropine (5 µM) before the 2-min stimulation with ACh (0.1 mM). Western blots of PC12 cells probed with anti-pERK1/2 and anti-total ERK1/2 antibodies. The Western blots were representative of three different experiments. The bands were quantified by scanning densitometry and the ratio of phosphorylated ERK to total ERK was calculated. The data were expressed as fold increase to without stimulated cells (control) as one. The values represent the mean ± sd for three different experiments. **P < 0.01 versus the control.

Nicorandil Inhibits ACh-Induced ERK Phosphorylation in PC12 Cells
To examine the effect of nicorandil on ACh-induced ERK activation, PC12 cells were pretreated with 0.1–10 mM nicorandil for 10 min. As shown in Figure 3, the treatment with nicorandil inhibited ACh-induced ERK1/2 phosphorylation in a concentration-dependent manner, and it was completely abolished at 10 mM (Fig. 3).

View larger version (24K): [in this window] [in a new window]   Figure 3. The effect of nicorandil on ACh-induced ERK phosphorylation. The cells were pretreated with the indicated concentration of nicorandil for 10 min and then stimulated in the presence (+) or absence (–) of 0.1 mM ACh for 2 min. The Western blots of PC12 cells probed with anti-p ERK1/2 and antitotal ERK1/2 antibodies. The Western blots were representative of three different experiments. The bands were quantified by scanning densitometry and the ratio of phosphorylated ERK to total ERK was calculated. The data were expressed as percent of the expression ratio regarding without nicorandil as 100%. The values represent the mean ± sd for three different experiments and P < 0.05 was considered significant in comparison to the control. *P < 0.05, **P < 0.01, ***P < 0.001 versus the control.

Nicorandil is a vasodilator with the dual properties of a K⁺_ATP channel opener and a nitrate generator.19 To examine whether the inhibition of ACh-induced ERK1/2 activation by nicorandil was caused by opening the K⁺_ATP channel, we then pretreated PC12 cells with glibenclamide, a K⁺_ATP channel blocker, at 10 min before the nicorandil exposure. As shown in Figures 4 and 5 mM glibenclamide abolished the inhibition of ERK1/2 activation by 10 mM nicorandil. This result suggested that nicorandil inhibits the ACh-induced ERK activation by opening the K⁺_ATP channel.

View larger version (19K): [in this window] [in a new window]   Figure 4. The effect of K+ATP blocker glibenclamide. The cells were pretreated in the presence (+) or absence (–) of glibenclamide (5 mM) 10 min before the nicorandil (10 mM) exposure. After the treatment of glibenclamide and nicorandil, the cells were stimulated with (+) or without (–) 0.1 mM ACh for 2 min. Western blots of PC12 cells probed with anti-p ERK1/2 and antitotal ERK1/2 antibodies. The Western blots were representative of three different experiments. The bands were quantified by scanning densitometry and the ratio of phosphorylated ERK to total ERK was calculated. The data were expressed as fold increase to without stimulated cells (control) as one. The values represent the mean ± sd for 3 different experiments and P < 0.05 was considered significant in comparison to the control. **P < 0.01 vs the control.

View larger version (23K): [in this window] [in a new window]   Figure 5. The effect of nicorandil on ERK phosphorylation caused by other mitogen-activated protein kinases (MAPK) activators. The cells were pretreated with (+) or without (–) 10 mM nicorandil and stimulated in the presence (+) or absence (–) of 0.1 mM ACh for 2 min, 100 nM PMA for 5 min, 100 nM ionomycin for 15 min. Western blots of PC12 cells probed with anti-pERK1/2 and antitotal ERK1/2 antibodies. The Western blots were representative of three different experiments. The bands were quantified by scanning densitometry and the ratio of phosphorylated ERK to total ERK was calculated. The data were expressed as -fold increase to without stimulated cells (control) as one. The values represent the mean ± sd for three different experiments. *P < 0.05 versus the control.

Effect on PMA-Induced ERK Phosphorylation
Our previous study demonstrated that PMA, an activator of PKC which is often used as a receptor—bypass stimuli, causes the activation of ERK1/2 in PC12 cells.16 We thereafter examined whether nicorandil exerts an inhibitory effect on PMA-induced ERK phosphorylation. The treatment with 10 mM nicorandil did not have any effect on PMA-induced ERK1/2 phosphorylation (Fig. 4).

Effect on Ionomycin-Induced ERK Phosphorylation
Ca²⁺ is one of the second messengers of the MAPK pathway, and activation of mAChR causes the rapid release of Ca²⁺ from intracellular stores and a sustained influx of external Ca²⁺ in PC12 cells.21,22 In this relation, we examined whether nicorandil has an effect on ERK activation by an increase of the influx of Ca²⁺ using calcium ionophore ionomycin. As shown in Figure 5, ionomycin (100 nM) induced ERK1/2 phosphorylation at 80% of ACh stimulation in PC12 cells, and the pretreatment with 10 mM nicorandil inhibited ionomycin-induced ERK1/2 phosphorylation by approximately 60%. This result suggests that nicorandil partially inhibits Ca²⁺-induced ERK1/2 phosphorylation in PC12 cells.

Nicorandil Suppresses the Increase of Intracellular Ca²⁺ Concentration
We then examined the effect of nicorandil on the ACh-induced increase of intracellular Ca²⁺ concentration. The increase of intracellular Ca²⁺ concentration was measured by visualizing, using fluorescence microscopy, PC12 cells pretreated with or without 10 mM nicorandil before 0.1 mM ACh stimulation. As shown in Figure 6, ACh stimulation induced many luminous cells in comparison to the control cells. In the nicorandil-treated cells, only few luminous cells were observed. This result suggests that nicorandil inhibits the ACh-induced increase of intracellular Ca²⁺ concentration.

View larger version (19K): [in this window] [in a new window]   Figure 6. The fluorescence microscopy on the Fluo-3 AM loaded PC12 cells. Before the drug treatment, the cells were loaded 10 µM Fluo-3 AM with serum-free Dullbecco modified Eagle's medium for the night. After removing the medium and washing twice, the cells were pretreated with (+) or without (–) nicorandil (10 mM) and then stimulated with 0.1 mM ACh for 5 min. A fluorescence analysis was performed on digitized images of live cells taken with a fluorescence microscope linked to a digital CCD camera. The first picture is a control (without nicorandil/without ACh stimulation). The next picture shows the condition after the stimulation of ACh without nicorandil pretreatment. The final picture shows the condition after the ACh stimulation with nicorandil pretreatment. Images of three fields per well were taken with an average of 100 cells per field. The number of fluorescent cells was determined by visual examination of the field and represents the mean ± sd for three different experiments, P < 0.05 was considered significant in comparison to the control. **P < 0.01 versus the control.

	DISCUSSION

Top Abstract Introduction METHODS RESULTS DISCUSSION REFERENCES

 
The present study demonstrated that nicorandil suppressed mAChR-mediated ERK phosphorylation of PC12 cells. Because the effect of nicorandil was abolished by a K⁺_ATP channel blocker, glibenclamide, this suppression is intended to be due to its opening effect of K⁺_ATP channels.

Previous studies suggest that the mAChR stimulation increases the intracellular Ca²⁺ concentration by 1) redistribution of Ca²⁺ from cytoplasmic stores to the cytosol, mediated by generation of inositol 1,4,5-triphosphate (IP₃) as a consequence of the muscarinic-receptor-coupled hydrolysis of phosphatidylinositol 4,5-bisphosphate and 2) increased Ca²⁺ influx through a pathway of the plasma membrane.22 In addition, it has also been well documented that the 3) emptying of intracellular Ca²⁺ stores often stimulates the influx of extracellular Ca²⁺.21 Nicorandil causes the cell membrane hyperpolarization induced by K⁺_ATP channel opener to inhibit Ca²⁺ influx by blocking voltage-dependent Ca²⁺ channels23 and Ca²⁺ release from intracellular stores by IP₃ production.24 In the rat myocytes, nicorandil attenuates the mitochondrial Ca²⁺ overload induced by depolarization of the mitochondrial membrane.25 These results suggest that nicorandil suppresses the intracellular Ca²⁺ concentration. In our study, we demonstrated that nicorandil partially inhibited the ionomycin-induced ERK phosphorylation, suggesting that nicorandil may abolish the increase of intracellular Ca²⁺ concentration, by not only the cell hyperpolarization, but also by inhibiting the redistribution of Ca²⁺ from the cytoplasmic stores to the cytosol and/or the inhibition of the following Ca²⁺ influx via activation of Ca²⁺ store-operated channels.

There are five mAChR subtypes, m1m5, which classically couple to 2 G proteins, Gi and Gq. As summarized in Figure 7, MAPK has been shown to be activated via Gq-coupled receptor m1 in PC12 cells.18 When the mAChRs are stimulated, the subunit of Gq dissociated from the β subunits stimulates phospholipase C activation. Phospholipase C activation, in turn, hydrolyzes phosphatidylinositol 4,5-bisphosphate into IP₃ and diacylglycerol, both of which activate PKC. PKC could be involved in the ERK activation by Raf phosphorylation on serine residues and initiates a sequence of activation of Ras, Raf and MEK that, in turn, activates ERK.16 IP₃ redistributes Ca²⁺ from intracellular stores to the cytosol, and free cytosolic Ca²⁺ activate Ca²⁺-dependent tyrosine kinase 2 (Pyk2). Pyk2, together with src, has been shown to recruit Grb2-SOS to the plasma membrane and in this way induces Ras activation.26

View larger version (19K): [in this window] [in a new window]   Figure 7. A hypothetical scheme of the effect of nicorandil on muscarinic receptor-mediated ERK phosphorylation in PC12 cells. The muscarinic acetylcholine receptor stimulation increases intracellular Ca2+ concentration ([Ca2+]i) by redistribution of Ca2+ from cytoplasmic stores to the cytosol, mediated by generation of inositol 1,4,5-triphosphate as a consequence of the muscarinic-receptor-coupled hydrolysis of phosphatidylinositol 4,5-bisphosphate, and increased Ca2+ influx from the outside of the cells. This increase of [Ca2+]i initiates a sequence of activation of protein kinase C (PKC), Ras-ERK pathway. PLC = phospholipase C; DG = diacylglycerol; ER = endoplasmic reticulum; Pyk-2 = Ca2+-dependent tyrosine kinase-2; CaM = calmodulin; CaMK II = calmodulin-dependent protein kinase II; MEK = MAPK/ERK kinase.

PMA-induced ERK phosphorylation was not affected by the presence of nicorandil. This led us to the conclusion that nicorandil inhibits muscarinic activation of ERK via the action on the upstream of PKC or independent of PKC at the target sites.

The intracellular signal transduction pathways in neurons after noxious stimulation are not well understood. However, several studies have reported ERK phosphorylation to occur in dorsal root ganglion neurons and dorsal horn neurons in response to noxious stimuli of the peripheral tissue or electrical stimulation to the peripheral nerve, i.e., activity-dependent activation of ERK in nociceptive neurons.13,27,28 Under conditions of tissue and nerve damage, ERK can be activated by nociceptive activity and inflammatory mediators in primary sensory neurons in the peripheral nervous system, and spinal cord neurons and glia in the central nervous system.29 The activation of ERK in the peripheral and central nervous systems is nociceptive-specific and suppressed by several analgesics. Therefore, examination of ERK activation has potential for the development of an assay to test the efficacy of new analgesics.

Asano et al.2 reported the epidural administration of K⁺_ATP channel openers (levcromakalim and nicorandil) to enhance the analgesic action of both morphine and dexmedetomidine, probably via the activation of K⁺_ATP channels and modified morphine's pharmacokinetics at the spinal cord level, and that they could modulate the activities of intracellular second messengers, such as Ca²⁺. Because another study suggested that K⁺_ATP channel-opening plays a role in capsaicin-evoked acute nociception mechanisms,30 the K⁺_ATP channel opener may therefore modulate Ca²⁺ influx. We therefore examined whether µ-opioid and ₂-agonists have any effect on the ERK phosphorylation and K⁺_ATP channel opener to modify the effect of µ-opioid and ₂-agonists in PC12 cells. In addition, it was also thus considered worthwhile to examine the effect of K⁺_ATP channel opener on the ERK phosphorylation in the dorsal root ganglion neurons or dorsal horn neurons under conditions of tissue and nerve damage based on the present findings.

In conclusion, a K⁺_ATP channel opener nicorandil inhibits the mAChR-mediated activation of ERK, which is implicated in the suppression of the increase of intracellular Ca²⁺ concentration in PC12 cells.

	Footnotes

 
Accepted for publication July 24, 2008.

Supported by Ministry of Education, Culture, Sports, Science and Technology of Japan grant 19209050.

Presented at the IARS 81st Clinical and Scientific Congress, March 23–27, 2007, Orlando, FL.

	REFERENCES

Top Abstract Introduction METHODS RESULTS DISCUSSION REFERENCES

 

1. Narita M, Takamori K, Kawashima N, Funada M, Kamei J, Suzuki T, Misawa M, Nagase H. Activation of central ATP-sensitive potassium channels produces the antinociception and spinal noradrenaline turnover-enhancing effect in mice. Psychopharmacology (Berl) 1993;113:11–4[Medline]
2. Asano T, Dohi S, Iida H. Antinociceptive action of epidural K+(ATP) channel openers via interaction with morphine and an alpha(2)- adrenergic agonist in rats. Anesth Analg 2000;90: 1146–51[Medline]
3. Lohmann AB, Welch SP. ATP-gated K(+) channel openers enhance opioid antinociception: indirect evidence for the release of endogenous opioid peptides. Eur J Pharmacol 1999; 385:119–27[Medline]
4. Ocana M, Cendan CM, Cobos EJ, Entrena JM, Baeyens JM. Potassium channels and pain: present realities and future opportunities. Eur J Pharmacol 2004;500:203–19[Web of Science][Medline]
5. Seger R, Krebs EG. The MAPK signaling cascade. Faseb J 1995; 9:726–35[Abstract]
6. Lewis TS, Shapiro PS, Ahn NG. Signal transduction through MAP kinase cascades. Adv Cancer Res 1998;74:49–139[Web of Science][Medline]
7. Widmann C, Gibson S, Jarpe MB, Johnson GL. Mitogen-activated protein kinase: conservation of a three-kinase module from yeast to human. Physiol Rev 1999;79:143–80[Abstract/Free Full Text]
8. Rosen LB, Ginty DD, Weber MJ, Greenberg ME. Membrane depolarization and calcium influx stimulate MEK and MAP kinase via activation of Ras. Neuron 1994;12:1207–21[Web of Science][Medline]
9. English JD, Sweatt JD. A requirement for the mitogen-activated protein kinase cascade in hippocampal long term potentiation. J Biol Chem 1997;272:19103–6[Abstract/Free Full Text]
10. Martin KC, Michael D, Rose JC, Barad M, Casadio A, Zhu H, Kandel ER. MAP kinase translocates into the nucleus of the presynaptic cell and is required for long-term facilitation in Aplysia. Neuron 1997;18:899–912[Web of Science][Medline]
11. Blum S, Moore AN, Adams F, Dash PK. A mitogen-activated protein kinase cascade in the CA1/CA2 subfield of the dorsal hippocampus is essential for long-term spatial memory. J Neurosci 1999;19:3535–44[Abstract/Free Full Text]
12. Impey S, Obrietan K, Storm DR. Making new connections: role of ERK/MAP kinase signaling in neuronal plasticity. Neuron 1999;23:11–4[Web of Science][Medline]
13. Dai Y, Iwata K, Fukuoka T, Kondo E, Tokunaga A, Yamanaka H, Tachibana T, Liu Y, Noguchi K. Phosphorylation of extracellular signal-regulated kinase in primary afferent neurons by noxious stimuli and its involvement in peripheral sensitization. J Neurosci 2002;22:7737–45[Abstract/Free Full Text]
14. Shafer TJ, Atchison WD. Transmitter, ion channel and receptor properties of pheochromocytoma (PC12) cells: a model for neurotoxicological studies. Neurotoxicology 1991;12:473–92[Web of Science][Medline]
15. Greene LA, Tischler, AS. PC12 pheochromocytoma cells in neurobiological research. Adv Cell Neurobiol 1982;3:373–414
16. Tan Z, Dohi S, Ohguchi K, Nakashima S, Nozawa Y. Local anesthetics inhibit muscarinic receptor-mediated activation of extracellular signal-regulated kinases in rat pheochromocytoma PC12 cells. Anesthesiology 1999;91:1014–24[Web of Science][Medline]
17. Scatton B, Dubois A, Javoy-Agid F, Camus A. Autoradiographic localization of muscarinic cholinergic receptors at various segmental levels of the human spinal cord. Neurosci Lett 1984;49:239–45[Web of Science][Medline]
18. Berkeley JL, Levey AI. Muscarinic activation of mitogen-activated protein kinase in PC12 cells. J Neurochem 2000;75:487–93[Web of Science][Medline]
19. Taira N. Nicorandil as a hybrid between nitrates and potassium channel activators. Am J Cardiol 1989;63:18J–24[Medline]
20. Blackmore PF, Exton JH. Studies on the hepatic calcium-mobilizing activity of aluminum fluoride and glucagon. Modulation by cAMP and phorbol myristate acetate. J Biol Chem 1986;261:11056–63[Abstract/Free Full Text]
21. Ebihara T, Guo F, Zhang L, Kim JY, Saffen D. Muscarinic acetylcholine receptors stimulate Ca2+ influx in PC12D cells predominantly via activation of Ca2+ store-operated channels. J Biochem (Tokyo) 2006;139:449–58[Abstract/Free Full Text]
22. Pozzan T, Di Virgilio F, Vicentini LM, Meldolesi J. Activation of muscarinic receptors in PC12 cells. Stimulation of Ca2+ influx and redistribution. Biochem J 1986;234:547–53[Web of Science][Medline]
23. Nelson MT, Quayle JM. Physiological roles and properties of potassium channels in arterial smooth muscle. Am J Physiol 1995;268:C799–822[Web of Science][Medline]
24. Yanagisawa T, Yamagishi T, Okada Y. Hyperpolarization induced by K+ channel openers inhibits Ca2+ influx and Ca2+ release in coronary artery. Cardiovasc Drugs Ther 1993;7(suppl 3):565–74
25. Ishida H, Higashijima N, Hirota Y, Genka C, Nakazawa H, Nakaya H, Sato T. Nicorandil attenuates the mitochondrial Ca2+ overload with accompanying depolarization of the mitochondrial membrane in the heart. Naunyn Schmiedebergs Arch Pharmakol 2004;369:192–7
26. Agell N, Bachs O, Rocamora N, Villalonga P. Modulation of the Ras/Raf/MEK/ERK pathway by Ca(2+), and calmodulin. Cell Signal 2002;14:649–54[Web of Science][Medline]
27. Ji RR, Baba H, Brenner GJ, Woolf CJ. Nociceptive-specific activation of ERK in spinal neurons contributes to pain hypersensitivity. Nat Neurosci 1999;2:1114–9[Web of Science][Medline]
28. Pezet S, Malcangio M, Lever IJ, Perkinton MS, Thompson SW, Williams RJ, McMahon SB. Noxious stimulation induces Trk receptor and downstream ERK phosphorylation in spinal dorsal horn. Mol Cell Neurosci 2002;21:684–95[Web of Science][Medline]
29. Ji RR. Mitogen-activated protein kinases as potential targets for pain killers. Curr Opin Investig Drugs 2004;5:71–5[Medline]
30. Maia JL, Lima-Junior RC, Melo CM, David JP, David JM, Campos AR, Santos FA, Rao VS. Oleanolic acid, a pentacyclic triterpene attenuates capsaicin-induced nociception in mice: possible mechanisms. Pharmacol Res 2006;54:282–6[Medline]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Niinomi, K. Articles by Dohi, S. Search for Related Content PubMed PubMed Citation Articles by Niinomi, K. Articles by Dohi, S. Related Collections Mechanisms Preclinical Pharmacology Pharmacology