Accumulating evidence has so far indicated that cross-talk between the nervous and immune systems plays a pivotal role in the pathophysiology of various diseases. As a prototypic demonstration of neuro-immune systems, the interaction between nerves and mast cells has been examined intensively. Anatomically, mast cells are often located in close proximity to nerves. Functionally, both cells communicate with each other in a bi-directional manner. Substance P released from nerves and proteases and cytokines from mast cells have proved to be important mediators in such communication. On the other hand, the molecules involved in membrane-membrane contacts between nerves and mast cells were largely unknown. In 2003, both cells were found to express the identical adhesion molecule, named SynCAM (synaptic cell adhesion molecule) or SgIGSF (spermatogenic immunoglobulin superfamily). Since SgIGSF/SynCAM binds homophilically, its involvement in nerve-mast cell interaction was examined in vitro. Superior cervical ganglia expressed SgIGSF/SynCAM along their neurites. Adhesion to these neurites of mast cells lacking SgIGSF/SynCAM was poor, and this was normalized by ectopic expression of SgIGSF/SynCAM. Moreover, SgIGSF/SynCAM-expressing mast cells were more competent in communicating with the neurites. Further understanding of the adhesion molecule-dependent interaction will be expected to open a new avenue in the field of neuro-immune cross-talk.

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Using the whole-cell voltage clamp, we examined acute effects of various agents on Na⁺/Ca²⁺ exchange current (I_NCX) in guinea-pig cardiac ventricular cells and transfected cells. Among the antiarrhythmic drugs, amiodarone, bepridil, dronedarone, cibenzoline, azimilide, and aprindine inhibited I_NCX in a concentration-dependent manner. We also investigated the effects on NCX of 2,3-buanedione monoxim (BDM) and selective NCX inhibitors such as KB-R7943, SEA0400, and SN-6. The presence of trypsin in the pipette solution attenuated the inhibitory effects on NCX of amiodarone, bepridil, and BDM, suggesting that these drugs inhibit NCX from the cytosolic side. In contrast, the trypsin-insensitive NCX inhibitors were aprindine, azimilide, dronedarone, cibenzoline, KB-R7943, SEA0400, and SN-6. KB-R7943, SEA0400, and SN-6 suppressed the uni-directional outward I_NCX more potently than the uni-directional inward I_NCX. The mechanism of this mode-dependency is unknown, but is suggested to be related to intracellular Na⁺ concentration.

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Sodium-calcium exchange (NCX) is one of the major regulators of intracellular Ca²⁺ concentration in cardiac myocytes. The bi-directional and electrogenic property of NCX raises a question about whether NCX is involved in arrhythmias. We reviewed the role of NCX in cardiac triggered activity in limited experimental conditions: the digitalis-induced arrhythmia, the arrhythmia caused from sustained opening of sodium channel, and the arrhythmia caused from the inhibition of inwardly rectifying potassium current. Effects of NCX inhibitors on ventricular arrhythmias recorded on ECG or the delayed afterdepolarizations and triggered activity recorded by the current clamp method were evaluated. As an NCX inhibitor, we preferred to use SEA0400 instead of KB-R 7943. For a precise analysis, a computational reconstruction of action potential with the Luo and Rudy model was applied. The cardiac NCX system seems to play a role only in the digitalis-induced arrhythmia and may not be involved in other arrhythmias. This review highlights the relationship between triggered activity and an NCX system and also suggests the physiologic and pathologic aspect of the NCX system in cardiac arrhythmias.

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The Na⁺/Ca²⁺ exchanger (NCX) plays a role in regulation of intracellular Ca²⁺ levels, but little is known about the functional role of NCX in microglia. To clarify the role of NCX in microglia, we studied the responses of NCX to pathological conditions such as interferon-γ or nitric oxide (NO) exposure. Treatment with interferon-γ caused a biphasic increase in NCX activity. The delayed increase in NCX activity was accompanied by increases in the mRNA and protein levels. Pharmacological studies show that protein kinase C and tyrosine kinase are involved in the transient and delayed increases in NCX activity, and the extracellular signal-regulated protein kinase is involved in the delayed increase in NCX activity. On the other hand, NO causes apoptotic cell death in cultured microglia. We observed, using the specific NCX inhibitor SEA0400, that NO activates NCX activity and NCX is involved in NO-induced depletion of Ca²⁺ in the endoplasmic reticulum (ER), leading to ER stress. These results suggest that NCX is involved in the regulation of Ca²⁺ levels in the ER. The responses of NCX to interferon-γ and NO implies that NCX plays a key role in microglial function.

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Many kinds of vasodilators induce relaxation of the vascular smooth muscle cells (VSMCs) through the production of cyclic AMP (cAMP) or cyclic GMP (cGMP). The relaxant effects mediated by these second messengers are thought to be mainly due to the decrease in intracellular Ca²⁺ concentration ([Ca²⁺]_i), as well as the decrease in Ca²⁺ sensitivity of the contractile apparatus of VSMCs. To explain the cAMP- or cGMP-mediated decrease in [Ca²⁺]_i, several mechanisms have been proposed, including the inhibition of Ca²⁺ influx due to a hyperpolarization, a stimulation of Ca²⁺ uptake into the intracellular store, and an increase in Ca²⁺ extrusion from VSMCs by stimulation of sarcolemmal Ca²⁺-pump. VSMCs have two major systems for Ca²⁺ extrusion, namely, sarcolemmal Ca²⁺-pump and Na⁺/Ca²⁺ exchanger (NCX). However, the involvement of NCX in the vasodilator-induced relaxation of VSMCs has not been well established. In this article, the possible involvement of NCX in the vasodilator-induced relaxation of VSMCs will be reviewed.

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Excess salt intake is a major risk factor for hypertension. However, the molecular mechanisms underlying salt-dependent hypertension remain obscure. Our recent studies using selective Na⁺/Ca²⁺ exchange inhibitors and genetically engineered mice provide compelling evidence that salt-dependent hypertension is triggered by Ca²⁺ entry through Na⁺/Ca²⁺ exchanger type 1 (NCX1) in arterial smooth muscle. Endogenous cardiac glycosides, which may contribute to salt-dependent hypertension, seem to be necessary for NCX1-mediated hypertension. Intriguingly, recent studies by Dostanic-Larson et al. using knock-in mice with modified cardiac glycoside binding affinity of Na⁺,K⁺-ATPases demonstrate that this binding site plays an important physiological role in blood pressure control. Thus, when cardiac glycosides inhibit Na⁺,K⁺-ATPase in arterial smooth muscle cells, the elevation of local Na⁺ on the submembrane area is believed to facilitate Ca²⁺ entry through NCX1, resulting in vasoconstriction. This proposed pathway may have enabled us to explain how to link dietary salt to hypertension.

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Aldose reductase (AR) has been implicated in the pathogenesis of diabetic complications, although the clinical efficacy of AR inhibitors has not been clearly proven. To clarify the pathophysiological role of AR in the heart, we investigated effects of AR inhibitors applied either during the pre-ischemic phase, or during the post-ischemic reperfusion phase on ischemia-reperfusion injury in isolated heart from transgenic mice overexpressing human AR. On reperfusion following global ischemia, transgenic mouse hearts exhibited lower left developed pressure, increased release of creatine kinase, and lower ATP content compared with their littermates. When inhibitors of AR were applied during the pre-ischemic phase, they significantly improved deranged cardiac function, creatine kinase release, and ATP content. On the other hand, inhibition of AR during the post-ischemic reperfusion phase did not affect cardiac performance and ATP content, but it significantly attenuated creatine kinase release and the level of thiobarbiturate-reactive substances in transgenic mouse hearts. These results suggest a dual role of AR in ischemia-reperfusion injury. Inhibition of AR during ischemia preserved generation of ATP via glycolysis, whereas inhibition during the reperfusion phase reduced myocardial injury by attenuating oxidative stress elicited by ischemic insult and reoxygenation.

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Extracts of the medicinal plant St. John’s wort (Hypericum perforatum) are widely used for the treatment of affective disorders. Hyperforin, a constituent of St. John’s wort, is known to modulate the release and re-uptake of various neurotransmitters, an action that likely underlies its antidepressive activity. We now report that hyperforin also has N-methyl-D-aspartate (NMDA)-antagonistic effects. Hyperforin (10 μM) was found to inhibit the NMDA-induced calcium influx into cortical neurons. In rat hippocampal slices, hyperforin inhibited the NMDA-receptor-mediated release of choline from phospholipids. Hyperforin also antagonized the increase of water content in freshly isolated hippocampal slices, and it counteracted, at 3 and 10 μM, the increase of water content induced by NMDA. Hyperforin was inactive, however, in two in vivo models of brain edema formation, middle cerebral artery occlusion and water intoxication in mice. In conclusion, hyperforin has NMDA-receptor-antagonistic and potential neuroprotective effects in vitro. This effect may contribute to the therapeutic effectiveness of St. John’s wort extracts in some situations, for example, for relapse prevention in alcoholism.

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The purpose of the present study was to examine 5-hydroxytryptamine (5-HT)_2A-receptor sarpogrelate interactions by site-directed mutagenesis. Based on molecular modeling studies, aspartic acid (Asp)155[3.32] and tryptophan (Trp)151[3.28] in transmembrane helix (TMH) III and Trp336[6.48] in TMH VI of the 5-HT_2A receptor were found to interact with sarpogrelate. All of these residues were mutated to alanine (Ala). The Asp3.32Ala mutant did not exhibit any affinity for [³H]ketanserin, whereas the Trp3.28Ala mutant showed a markedly decrease in the binding affinity for [³H]ketanserin (K_d >10,000 nM). Therefore, it was not possible to find any sarpogrelate affinity to the mutants using [³H]ketanserin. The mutation also abolished agonist-stimulated formation of [³H]inositol phosphates (IP) in both cases. On the other hand, the Trp6.48Ala mutant showed reduced binding affinity for [³H]ketanserin (K_d 2.0 nM vs 0.8 nM for the wild-type receptor) and had reduced affinity for sarpogrelate (pK_i value of 5.71 vs 9.06 for the wild-type receptor). The Trp6.48Ala mutant also showed the greatest decrease in sensitivity to sarpogrelate (pK_b value 8.81 for wild-type and 5.11 for mutant) in inhibiting agonist-stimulated IP formation. These results provide direct evidence that Asp3.32, Trp3.28, and less importantly, Trp6.48 are responsible for the interaction between the 5-HT_2A receptor and sarpogrelate. In addition, these results support the findings obtained from molecular modeling studies.

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Peripheral nerve injury leads to the establishment of a novel synaptic connection between afferent Aβ-fiber and lamina II neurons in spinal dorsal horn, which is hypothesized to underlie mechanical allodynia. However, how the novel synapses transmit nociceptive information is poorly understood. In the present study, the role of protein tyrosine kinases (PTKs) in Aβ-fiber-evoked excitatory postsynaptic currents (EPSCs) recorded in lamina II neurons in transverse spinal cord slices of rats was investigated using the whole-cell patch-clamp recording technique. In the slices from sciatic nerve transection (SNT) rats, genistein (50 μM), a broad-spectrum PTKs inhibitor, or PP2 (20 μM), a selective Src family tyrosine kinase inhibitor, significantly reduced the amplitude of Aβ-fiber EPSCs. In sham-operated rats, however, Aβ-fiber EPSCs were insensitive to genistein and PP2. The N-methyl-D-aspartate (NMDA) receptor antagonist AP-V (50 μM) suppressed Aβ-fiber EPSCs in slices from SNT rats but not from sham-operated rats. Following nerve injury, the slow inward currents elicited by bath application of NMDA (100 μM) significantly increased at −70 mV. In SNT rats, genistein and PP2 reduced Aβ-fiber-evoked EPSCs mediated by NMDA receptor; however, genistein produced little effect on Aβ-fiber EPSCs mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor. These data suggested that PTKs, especially Src family members, participated in Aβ-fiber-evoked synaptic transmission following sciatic nerve injury via potentiation of NMDA receptor function.

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Major local adverse reactions in the nicotine patches are skin reactions. To assess the skin reaction of PHK-301p, a newly developed nicotine patch, we conducted a phase I study that consisted of 2 parts: a skin irritation test (48-h closed patch test) and a photosensitivity test (24-h closed patch test + Ultraviolet A irradiation). Twenty healthy men were treated with PHK-301p and placebo. Both preparations were punched out to a circle of 6-mm diameter and were applied simultaneously to each participant. Skin irritation and photosensitivity were assessed by a physician who was kept unaware of the treatment. In the skin irritation test, moderate and mild erythemas were observed in each participant 72 h after application (24 h after removal) for PHK-301p. Mild erythema was observed in one participant 49 h after application (1 h after removal) for placebo. The skin irritation index, which was calculated based on the skin reactions of participants, was 7.5 for PHK-301p and 2.5 for placebo. In the photosensitivity test, one participant had mild erythema (±) approximately 25 and 72 h after application of PHK-301p. No solar urticaria was observed. From these results, we concluded that PHK-301p is an acceptable product as a nicotine patch.

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The Publisher would like to announce that the above paper has been retracted.

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The effects of SEA0400, a selective inhibitor of the Na⁺/Ca²⁺ exchanger (NCX), on Na⁺-dependent Ca²⁺ uptake and catecholamine (CA) release were examined in bovine adrenal chromaffin cells that were loaded with Na⁺ by treatment with ouabain and veratridine. SEA0400 inhibited Na⁺-dependent ⁴⁵Ca²⁺ uptake and CA release, with the IC₅₀ values of 40 and 100 nM, respectively. The IC₅₀ values of another NCX inhibitor KB-R7943 were 1.8 and 3.7 μM, respectively. These results indicate that SEA0400 is about 40 times more potent than KB-R7943 in inhibiting NCX working in the reverse mode. In intact cells, SEA0400 and KB-R7943 inhibited CA release induced by acetylcholine and DMPP. The IC₅₀ values of SEA0400 were 5.1 and 4.5 μM and the values of KB-R7943 were 2.6 and 2.1 μM against the release induced by acetylcholine and DMPP, respectively, indicating that the potency of SEA0400 is about a half of that of KB-R7943 in inhibiting the nicotinic receptor-mediated CA release. The binding of [³H]nicotine with nicotinic receptors was inhibited by SEA0400 (IC₅₀ = 90 μM) and KB-R7943 (IC₅₀ = 12 μM). From these results, it is concluded that unlike KB-R7943, SEA0400 has a potent and selective action on NCX in bovine adrenal chromaffin cells.

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The circulating endothelial progenitor cells (EPCs) have an important role in angiogenesis, and the smooth muscle progenitor cells (SMPCs) participate in atherosclerosis. However, little is known about the effects of treatment of diabetes mellitus (DM) on EPCs and SMPCs. Therefore, we investigated the relations between the number of circulating vasucular progenitor cells before and after the treatment for DM. Ten previously untreated DM patients were enrolled in this study. Blood samples were collected before and after treatment. The peripheral mononuclear cells were purified and cultured to differentiate them into EPCs and SMPCs. After two weeks, the number of EPCs was determined by Dil-labeled acetylated low density lipoprotein and lectin binding. The number of SMPCs was evaluated by immunocytochemical staining of α-smooth muscle actin. Before treatment, the number of EPCs and SMPCs was significantly related to hemoglobin A1c and blood sugar. Serial examination revealed that improvement of glycemic control significantly increased the number of both EPCs and SMPCs. DM reduces the number of circulating EPCs and SMPCs according to its severity, and treatment of DM significantly increases the number of EPCs and SMPCs, which may be involved in angiogenesis and atherosclerosis in diabetes.

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The release of excessive Zn²⁺ from presynaptic boutons into extracellular regions contributes to neuronal apoptotic events, which result in neuronal cell death. However, the mechanisms of Zn²⁺-induced neuronal cell death are still unclear. Therefore, we investigated the dynamics of intracellular Zn²⁺, calcium, and reactive oxygen species in PC12 cells. The addition of Zn²⁺ produced cell death in a concentration- and time-dependent manner. ⁴⁵Ca²⁺ influx occurred just after the treatment with Zn²⁺, although subsequent hydroxyl radical (^•OH) production did not begin until 3 h after Zn²⁺ exposure. ^•OH production was significantly attenuated in Ca²⁺-free medium or by L-type Ca²⁺ channel antagonist treatment, but it was independent of the intracellular Zn²⁺ content. Dantrolene treatment had no protective effects against Zn²⁺-induced cell death. Treatment with N-acetyl-L-cysteine blocked ^•OH generation and subsequent cell death. These data indicate that Ca²⁺ influx and subsequent ^•OH production are critical events in Zn²⁺-induced toxicity in PC12 cells.

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M475271, 4-quinazolinamine, N-(2-chloro-5-methoxyphenyl)-6-methoxy-7-[(1-methyl-4-piperidinyl) methoxy]-(9Cl), is a new anilinoquinazoline derivative that displays selective inhibition of Src kinase activity and tumor growth in vivo. Vascular endothelial growth factor (VEGF)-induced angiogenesis plays a pivotal role in tumor growth and metastasis. Vascular endothelial (VE)-cadherin is an endothelial cell-specific adhesion molecule that can interact with the cytoskeleton via several anchoring molecules such as β-catenin. Here, we examined the effect of M475271 on VE-cadherin and β-catenin phosphorylation and association. We also examined its effect on VEGF-induced human umbilical vein endothelial cell (HUVEC) proliferation, migration, and tube formation. The findings reveal pretreatment with M475271 significantly inhibits VEGF-induced VE-cadherin and β-catenin phosphorylation. However, M475271 significantly increases VE-cadherin and β-catenin association compared to the VEGF-treated group. Confocal laser microscopic examination confirmed the augmentation effect of M475271 on VE-cadherin and β-catenin association. Finally, M475271 was shown to have inhibitory effects comparable to those of PP2 and Herbimycin A on VEGF-induced HUVEC proliferation, migration, and tube formation. These findings suggest that M475271 attenuates VEGF-induced angiogenesis by maintaining cell-cell junction stability. Although the involvement of other signaling molecules cannot be ruled out, M475271 has potential as a drug for the inhibition of the angiogenesis needed for tumor growth and metastasis.

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The effect of bifemelane hydrochloride (bifemelane) was examined on human origin astrocyte clonal cells (Kings-1). Bifemelane (125 – 1000 μM) induced a dose-dependent increase in the intracellular calcium concentration ([Ca²⁺]_i). In the highest concentration (1000 μM), the drug caused the second large increase in [Ca²⁺]_i during the washing. The increase that occurred during the administration partially remained in the Ca²⁺-free medium and was blocked by 2-aminoethoxydiphenyl borate (2-APB), an IP₃-receptor blocker, indicating that the source of Ca²⁺ for the increase could be ascribed to the intracellular store. The increase in [Ca²⁺]_i was not observed during washing with Ca²⁺-free medium, but was observed when the washing was performed with Ca²⁺-containing medium. Bifemelane caused a dose-dependent ATP release, but histamine and carbachol, which induced a large increase in [Ca²⁺]_i, had no effects on the ATP release. The effects on the [Ca²⁺]_i were blocked by pretreatment with pyridoxal phosphate-6-azophenyl-2',4' disulfonic acid, a P2-receptor antagonist. Although the mechanisms of ATP release induced by the drug have not been elucidated yet, the present results demonstrate that the increase in [Ca²⁺]_i induced by bifemelane is not due to its direct effect on the cells, but is dependent upon the ATP released from the cells.

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The anticancer agent genistein inhibits cell growth of tumor cell lines from various malignancies. In our study, we investigated the effectiveness of combined treatment of ionizing radiation (IR) with genistein on cervical HeLa cells and its possible mechanism. It was found that the inhibitory rate in cells with combined treatment was significantly higher than that of the cells treated with IR or genistein alone. After treatments of IR (4 Gy) combined with genistein (40 μmol/L), the apoptotic index of the cells was significantly increased and the cells were arrested in the G2/M phase. Survivin mRNA expression increased after IR (4 Gy), while it significantly decreased after combined treatment. These findings indicated that genistein enhanced the radiosensitivity of cervical cancer HeLa cells, and the mechanisms for this action might include increase of apoptosis, decrease of survivin expression, and prolongation of cell cycle arrest.

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d-Pseudoephedrine (PSE) is one of the main ingredients of Ephedrae herba. Although PSE is widely applied for patients with a common cold and upper respiratory inflammation as a decongestant, the effects of PSE on cough have never been reported. In this study, we investigated the antitussive effects of intraperitoneal injection of PSE on the cough reflex induced by microinjection of citric acid into the larynx of guinea pigs. PSE decreased the number of cough reflexes dose-dependently (−18.3 ± 5.0% at 20 mg/kg, P<0.05; −41.1 ± 7.2% at 60 mg/kg, P<0.01). Furthermore, PSE (60 mg/kg) increased the threshold intensity for inducing fictive cough by electrical micro-stimulation of the nucleus tractus solitarius (+72.7 ± 8.4%, P<0.01). On the afferent discharge of the superior laryngeal nerve, PSE suppressed the increases of amplitude and frequency when stimulated by citric acid at laryngeal mucosa. These results demonstrate that PSE possesses an antitussive effect that might be derived from both central and peripheral actions.

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Nicotine inhibited interleukin (IL)-18 and -12 production in lipopolysaccharide (LPS)-stimulated monocytes, and the action of nicotine was antagonized by a non-selective and a selective α7 nicotinic acetylcholine receptor (α7-nAChR) antagonist, suggesting that the stimulation of α7-nAChR may be involved in the action of nicotine. Nicotine is reported to induce prostaglandin E₂ (PGE₂) production in monocytes through the up-regulation of cyclooxygenase (COX)-2 expression. PGE₂ is known to increase cAMP levels and to activate protein kinase A (PKA). COX-2 and PKA inhibitors prevented the action of nicotine, indicating that the mechanism of action of nicotine may be via endogenous PGE₂ production.

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