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Complex regional pain syndrome (CRPS) is an intractable chronic pain syndrome with various signs and symptoms including allodynia/hyperalgesia, edema, swelling, and skin abnormalities. However, a definitive therapeutic treatment for CRPS has not been established. In CRPS patients, inflammatory cytokines such as TNF-α and IL-1β have been shown to increase in affected areas, suggesting that these molecules may be potential therapeutic targets for CRPS. Here, we first created a novel CRPS mouse model (CRPS-II-like) via sciatic nerve injury and cast immobilization, which was characterized by mechanical allodynia, local edema, and skin abnormalities, to evaluate the pathophysiology and pharmacotherapy of CRPS. When an anti-TNF-α antibody was consecutively administered near the injured sciatic nerve of CRPS model mice, persistent allodynia and CRPS-related signs in the ipsilateral hindpaw were markedly attenuated to control levels. Perineural administration of anti-TNF-α antibody also suppressed the upregulation of inflammatory cytokines as well as the activation of macrophages and Schwann cells in the injured sciatic nerve. These findings indicate that persistent allodynia and CRPS-related signs in CRPS models are primarily associated with TNF-α-mediated immune responses in injured peripheral nerves, suggesting that perineural treatment with anti-TNF-α antibody might be therapeutically useful.

Magnesium (Mg2+) plays an important role in various cellular functions such as protein synthesis, DNA stability, energy metabolism, enzyme and channel activities, and muscle contractility. Therefore, intracellular Mg2+ concentration is tightly regulated by multiple Mg2+ transporters and channels. So far, various candidate genes of Mg2+ transporters have been identified, and the research on their structure and function is currently in progress. The Solute Carrier 41 (SLC41) family, which is related to the bacterial Mg2+ transporter/channel MgtE, comprises three isoforms of SLC41A1, SLC41A2, and SLC41A3. Based on recent studies, SLC41A1 is thought to mediate Mg2+ influx or Na+-dependent Mg2+ efflux across the plasma membrane, whereas SLC41A2 and SLC41A3 may mediate Mg2+ fluxes across either the plasma membrane or organellar membranes. Intriguingly, SLC41A1 variants have been identified in patients with Parkinson's disease (PD) and nephronophthisis-related ciliopathies. Further genetic analyses reveal the association of SLC41A1 polymorphisms with PD risks. This review highlights the recent advances in the understanding of the molecular and functional characteristics of SLC41 family towards its therapeutic and diagnostic applications.

In this study, we assessed the regulation of transient receptor potential vanilloid 4 (TRPV4) promoting lymphangio/angiogenesis to improve the ischemic hindlimb animal model, and revealed that (1) a TRPV4 agonist improved the blood flow of ischemic hindlimbs by inducing both angiogenesis and lymphangiogenesis; (2) excessive TRPV4 expression was detected on lymphatic endothelial cells (LECs) in the ischemic hindlimb; and (3) hypoxic conditions promoted Ca2+ influx into LECs via TRPV4. It is considered that the upregulation of both lymphatic and blood vessels by activating TRPV4 would be a promising therapeutic strategy for peripheral artery disease.

Hydrogen sulfide (H2S) is a major physiologic gastrotransmitter. Its role in the regulation of the lower esophageal sphincter (LES) function remains unknown. The present study addresses this question. Methods: Isometric contraction was monitored in circular smooth muscle strips of porcine LES. Changes in cytosolic Ca2+ concentration ([Ca2+]i) and force were simultaneously monitored in fura-2-loaded strips with front-surface fluorometry. The contribution of endogenous H2S to LES contractility was investigated by examining the effects of inhibitors of H2S-generating enzymes, including cystathionine-β-synthase, cystathionine-γ-lyase, and 3-mercaptopyruvate sulfurtransferase, on the LES function. Results: Porcine LES strips myogenically maintained a tetrodotoxin-resistant basal tone. Application of AOA (cystathionine-β-synthase inhibitor) or L-aspartic acid (L-Asp; 3-mercaptopyruvate sulfurtransferase inhibitor) but not DL-PAG (cystathionine-γ-lyase inhibitor), decreased this basal tone. The relaxant effects of AOA and L-Asp were additive. Maximum relaxation was obtained by combination of 1 mM AOA and 3 mM L-Asp. Immunohistochemical analyses revealed that cystathionine-β-synthase and 3-mercaptopyruvate sulfurtransferase, but not cystathionine-γ-lyase, were expressed in porcine LES. AOA+L-Asp–induced relaxation was accompanied by a decrease in [Ca2+]i and inversely correlated with the extracellular Na+ concentration ([Na+]o) (25-137.4 mM), indicating involvement of an Na+/Ca2+ exchanger. The reduction in the basal [Ca2+]i level by AOA was significantly augmented in the antral smooth muscle sheets of Na+/Ca2+ exchanger transgenic mice compared with wild-type mice. Conclusions: Endogenous H2S regulates the LES myogenic tone by maintaining the basal [Ca2+]i via Na+/Ca2+ exchanger. H2S-generating enzymes may be a potential therapeutic target for esophageal motility disorders, such as achalasia.

In gastric smooth muscles, the released Ca2+ activates the contractile proteins and Ca2+ taken up from the cytosol cause relaxation. The Na+/Ca2+ exchanger (NCX) is an antiporter membrane protein that controls Ca2+ influx and efflux across the membrane. However, the possible relation of NCX in gastric fundus motility is largely unknown. Here, we investigated electric field stimulation (EFS)-induced relaxations in the circular muscles of the gastric fundus in smooth muscle-specific NCX1 transgenic mice (Tg). EFS caused a bi-phasic response, transient and sustained relaxation. The sustained relaxation prolonged for an extended period after the end of the stimulus. EFS-induced transient relaxation and sustained relaxation were greater in Tg than in wild-type mice (WT). Disruption of nitric oxide component by N-nitro-l-arginine, EFS-induced transient and sustained relaxations caused still marked in Tg compared to WT. Inhibition of PACAP by antagonist, EFS-induced sustained relaxation in Tg was not seen, similar to WT. Nevertheless, transient relaxation remained more pronounced in Tg than in WT. Next, we examined responses to NO and PACAP in smooth muscles. The magnitudes of NOR-1, which generates NO, and PACAP-induced relaxations were greater in Tg than in WT. In this study, we demonstrate that NCX1 regulates gastric fundus motility.

The Na+/Ca2+ exchanger (NCX) is a plasma membrane transporter involved in regulating intracellular Ca2+ concentrations. NCX is critical for Ca2+ regulation in cardiac muscle, vascular smooth muscle, and nerve fibers. To determine the role of NCX1 and NCX2 in gastrointestinal tissues, we examined electric field stimulation (EFS)-induced responses in the longitudinal smooth muscle of the distal colon in NCX1 and NCX2 double-heterozygote knockout mice (Double HET). We found that the amplitudes of EFS-induced relaxation that persisted during EFS were greater in Double HET than in wild-type mice (WT). Under the non-adrenergic, non-cholinergic (NANC) condition, EFS-induced relaxation in Double HET was similar in amplitude to that of WT. In the experiments in which l-NNA was added under NANC conditions following the EFS, the magnitudes of EFS-induced relaxation were smaller in Double HET than those in WT. In addition, an NCX inhibitor, SN-6, enhanced EFS-induced relaxation but did not affect EFS-induced relaxation under NANC condition, as in Double HET. Moreover, the magnitudes of relaxation induced by NOR-1, which generates NO, were greater in Double HET compared with WT. Similarly, SN-6 potentiated the magnitudes of NOR-1–induced relaxation. In this study, we demonstrate that NCX regulate colonic motility by altering the sensitivity of the inhibitory component. Keywords:: Na+/Ca2+ exchanger, smooth muscle, relaxation, nitric oxide, gastrointestinal motility

Abstract.: Cardiac hypertrophy is an increase in the muscle volume of the ventricle due to the enlargement of cardiac cells. Physiological cardiac hypertrophy is the normal response to healthy exercise, and pathological hypertrophy is the response to increased stress such as hypertension. Intracellular and extracellular aniosmotic conditions also change cell volume. Since persistent cell swelling or cell shrinkage during aniosmotic conditions results in cell death, the ability to regulate cell volume is important for the maintenance of cellular homeostasis. Cell swelling activates a regulatory volume decrease (RVD) response in which solute leakage pathways are stimulated and solute with water exits cells, reducing the cell volume towards the original value. In cardiac cells, one of the essential factors for cell-volume regulation is the volume-regulated anion channel (VRAC). However, the relationship between cardiac hypertrophy and cell-volume regulation is not clear. In this review, we introduce our recent findings showing that the impairment of VRAC current is exhibited in ventricular cells from mice with cardiac hypertrophy induced by transverse aortic constriction. Similar results were shown in caveolin-3–deficient mice, which develop cardiac hypertrophy without pressure overload. These results suggest that VRAC will be a new target for protection from the development of cardiac hypertrophy. Keywords:: hypertrophy, volume regulation, cardiac cell, anion channel, Cl− current, cardiovascular disease

The rise of Ca2+ concentration ([Ca2+] i ) by reducing external Na+ in urinary bladder smooth muscle cells (UBSMCs) from transgenic mice overexpressing Na+/Ca2+ exchanger type-1.3 (NCX1.3tg/tg) was about 4 times as large as that in the wild-type (WT). NCX1 protein expression in UB increased about 4-fold in NCX1.3tg/tg. The Ca2+ release by caffeine in UBSMCs was comparable between NCX1.3tg/tg and WT, but [Ca2+]i decay was faster in NCX1.3tg/tg. Contractions induced by acetylcholine, 60 mM K+, or electrical stimulation were significantly smaller in UB segments of NCX1.3tg/tg. NCX worked in Ca2+-extrusion mode during these contractions in UBSMCs of both WT and NCX1.3tg/tg. Keywords:: Na+-Ca2+ exchanger, urinary bladder smooth muscle, transgenic mouse

The expression levels of the Na+/Ca2+ exchanger type 1 (NCX1) change under various cardiac pathophysiological conditions, but the mechanism is unknown. We previously demonstrated that lysophosphatidylcholine (LPC) increased NCX1 expression by activating RhoB in H9c2 cardiomyoblasts. Conversely, fluvastatin (Flv), a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, decreased NCX1 mRNA and protein expression by inhibiting RhoB. RhoB can be isoprenylated by either geranylgeranylpyrophosphate (GGPP) or farnesylpyrophosphate (FPP). Here we investigated which of GGPP or FPP is involved in the NCX1-increasing effect of LPC. When LPC was added with GGPP to the Flv-treated H9c2 cells, NCX1 mRNA was increased to a level significantly higher than that in the control cells. Only GGPP, but not FPP, allowed LPC to increase NCX1 mRNA in the presence of Flv. Furthermore, geranylgeranyltransferase 1 inhibitor (GGTI), but not farnesyltransferase inhibitor (FTI), inhibited the LPC-induced NCX1 mRNA increase. We conclude that geranylgeranylation, but not farnesylation, of RhoB mediates LPC-induced NCX1 mRNA increase in H9c2 cells. Keywords:: lysophosphatidylcholine, Na+/Ca2+ exchanger, H9c2 cell, RhoB, geranylgeranylation

Using the whole-cell voltage clamp, we examined the mechanism of activation of the Na+/Ca2+ exchanger (NCX) by hydrogen peroxide (H2O2) in isolated guinea-pig cardiac ventricular myocytes. Exposure to H2O2 increased the NCX current. The effect was inhibited by cariporide, an inhibitor of the Na+/H+ exchanger (NHE), suggesting that there are NHE-dependent and -independent pathways in the effect of H2O2 on NCX. In addition, both pathways were blocked by edaravone, a hydroxyl radical (•OH) scavenger; pertussis toxin, a Gαi/o protein inhibitor; and U0126, an inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK). On the other hand, wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, inhibited only the NHE-dependent pathway, while PP2, a Src family protein tyrosine kinase inhibitor, inhibited only the NHE-independent pathway. Taken together, our data suggest that H2O2 increases the NCX current via two signal transduction pathways. The common pathway is the conversion of H2O2 to •OH, which activates Gαi/o protein and a mitogen-activated protein (MAP) kinase signaling pathway. Then, one pathway activates NHE with a PI3K-dependent mechanism and indirectly increases the NCX current. Another pathway involves activation of a Src family tyrosine kinase. Keywords:: Na+/Ca2+ exchanger (NCX), hydrogen peroxide (H2O2), Na+/H+ exchanger (NHE), cardiac myocyte

ATP has broad functions as an autocrine/paracrine molecule. The mode of ATP release and its intracellular source, however, are little understood. Here we show that bradykinin via B2-receptor stimulation induces the extracellular release of ATP via the inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-signaling pathway in cultured taenia coli smooth muscle cells. It was found that bradykinin also increased the production of Ins(1,4,5)P3 and 2-APB-inhibitable [Ca2+]i. The evoked release of ATP was suppressed by the Ca2+-channel blockers, nifedipine, and verapamil. Moreover, the extracellular release of ATP was elicited by photoliberation of Ins(1,4,5)P3. Bradykinin caused a quick and transient accumulation of intracellular ATP from cells treated with 1% perchloric acid solution (PCA), but not with the cell lysis buffer. Peak accumulation was prevented by 2-APB and thapsigargin, but not by nifedipine or verapamil, inhibitors of extracellular release of ATP. These findings suggest that bradykinin elicits the extracellular release of ATP that is mediated by the Ins(1,4,5)P3-induced Ca2+ signaling and, finally, leads to a Ca2+-dependent export of ATP from the cells. Furthermore, the bradykinin-induced transient accumulation of ATP in the cells treated with PCA may imply a possible release of ATP from the endoplasmic reticulum. Keywords:: bradykinin B2 receptor, extracellular ATP release, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-mediated Ca2+ signaling, endoplasmic reticulum, cultured smooth muscle cell

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. Keywords:: salt-dependent hypertension, Na+/Ca2+ exchanger type 1 (NCX1), SEA0400, endogenous ouabain, Na+,K+-ATPase

The Na+/Ca2+ exchanger (NCX) is an ion transporter that exchanges Na+ and Ca2+ in either Ca2+ efflux or Ca2+ influx mode, depending on membrane potential and transmembrane ion gradients. In myocytes, neurons, and nephron cells, NCX is thought to play an important role in the regulation of intracellular Ca2+ concentration. Recently, the benzyloxyphenyl derivatives KB-R7943, SEA0400, and SN-6 have been developed as selective NCX inhibitors. Currently, SEA0400 is the most potent and selective inhibitor. These inhibitors possess different isoform-selectivities, although they have similar properties, such as Ca2+ influx mode-selectivity and I1 inactivation-dependence. Recent site-directed mutagenesis has revealed that these inhibitors possess some molecular determinants (Phe-213, Val-227, Tyr-228, Gly-833, and Asn-839) for interaction with NCX1. These benzyloxyphenyl derivatives are expected to be useful tools to study the physiological roles of NCX. Moreover, such inhibitors may have therapeutic potential as a new remedy for ischemic disease, arrhythmias, heart failure, and hypertension. Keywords:: Na+/Ca2+ exchanger (NCX), NCX inhibitor, inhibitory mechanism, ischemia/reperfusion injury

Sensitivities of the reverse-mode Na+/Ca2+ exchange activity measured as the Na+i-dependent Ca2+ uptake to extracellular monovalent cations K+, Li+, and Na+ were compared between the K+-dependent (NCKX2) and the K+-independent Na+/Ca2+ exchanger (NCX1) overexpressed in a fibroblast cell. Interestingly, the exchange activity of NCKX2 was not influenced by Li+ while it was increased by K+. On the contrary, the activity of NCX1 was increased by Li+. Thus, the cation sensitivities to K+ and Li+ markedly differed between NCKX2 and NCX1. In addition, Na+ exerted a significantly smaller inhibitory effect on the activity in NCKX2 than in NCX1. The Na+/Ca2+ exchange activities of NCKX2 and NCX1 are considered to be regulated differentially via the respective binding site domains that have distinct sensitivities to the external monovalent cations. Keywords:: Na+/Ca2+ exchanger (NCX), K+-dependent NCX (NCKX)2, NCX1