Diabetic patients have shorter life span and poorer Quality of Life mainly due to diabetic vascular complications. Recent in vitro and in vivo studies have shown that advanced glycation endproducts (AGE) account for diabetic vascular complications through their engagement of the receptor for AGE (RAGE). In this review, we summarize our recent studies on the roles of the AGE-RAGE system in diabetes-induced vascular injury. In vitro experiments showed that AGE engagement of RAGE leads to changes in endothelial cells (EC) and pericytes, which are characteristic of diabetic microangiopathy. Diabetic RAGE transgenic mice that overexpress RAGE in vascular cells exhibited the exacerbation of the indices of nephropathy and retinopathy, and this was prevented by the inhibition of AGE formation. RAGE overexpression also caused calcium handling impairment in cardiac myocytes. In contrast to the RAGE-overexpressing mice, diabetic RAGE knockout mice showed marked improvement of nephropathy. We found that human vascular cells express a novel splice variant coding for a soluble RAGE protein and named it endogenous secretory RAGE (esRAGE). The esRAGE neutralizes AGE actions on EC and is present in human sera. Individual variations in circulating esRAGE could be a determinant for individual differences in susceptibility or resistance to the development of diabetic vascular complications. The AGE-RAGE system should be, therefore, a candidate molecular target for overcoming diabetic vascular complications.

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Alzheimer’s disease (AD) is the most common neurodegenerative disorder of late life characterized by insidious, chronic, and progressive memory impairment in association with the accumulation of senile plaques, neurofibrillary tangles, and massive loss of neurons. Apoptosis is believed to be an important contributor to progression and pathology of neurodegeneration in AD. There is considerable evidence that amyloid β-peptide, a major component of senile plaques, has the capacity to activate intracellular apoptosis pathways leading to neuronal cell death. AD-related mutations in genes coding presenilins are also shown to cause neuronal apoptosis, by directly and indirectly regulating apoptotic signaling cascades. Recent evidence suggests that two intrinsic pathways, mitochondrial dysfunction and endoplasmic reticulum stress, are central in the execution of apoptosis in AD. This review summarizes recent progress of research in this field focused on the molecular mechanisms involved in neuronal apoptosis mediated by organelle dysfunction.

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Behavioral models used to test potential antidepressants have shown that ligands that bind to sigma receptors possess “antidepressant-like” properties. The focus of this review is to discuss the literature concerning sigma receptors and their ligands, with respect to their antidepressants properties. In addition to the behavioral data, we discuss electrophysiological and biochemical models demonstrating sigma receptors’ ability to modulate important factors in the pathophysiology of depression and/or the mechanisms of action of antidepressants such as the serotonergic neurotransmission in the dorsal raphe nucleus (DRN) and the glutamatergic transmission in the hippocampus. We also discuss the significance of these two systems in the mechanism of action of antidepressants. Sigma ligands have potential as antidepressant medications with a fast onset of action as they produce a rapid modulation of the serotonergic system in the DRN and the glutamatergic transmission in the hippocampus. As these effects of sigma ligands may produce antidepressant properties by completely novel mechanisms of action, they may provide an alternative to the antidepressants currently available and may prove to be beneficial for treatment-resistant depressed patients.

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The Na⁺-Ca²⁺ exchanger (NCX) is involved in regulation of intracellular Ca²⁺ concentration. A specific inhibitor of NCX has been required for clarification of the physiological and pathological roles of NCX. We have developed 2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline (SEA0400), a highly potent and selective inhibitor of NCX. SEA0400 in the concentration range that inhibits NCX exhibits negligible affinities for the Ca²⁺ channels, Na⁺ channels, K⁺ channels, noradrenaline transporter, and 14 receptors; and it does not affect the activities of the store-operated Ca²⁺ channel, Na⁺-H⁺ exchanger, and several enzymes including Na⁺,K⁺-ATPase and Ca²⁺-ATPase. Furthermore, recent studies show that SEA0400 attenuates ischemia-reperfusion injury in the brain, heart, and kidney and radiofrequency lesion-induced edema in rat brain. These findings suggest that NCX plays a key role in ischemia-reperfusion injury and may be a target molecule for treatment of reperfusion injury-related diseases.

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Neurochemical mechanisms underlying drug dependence and withdrawal syndrome remain unclear. In this review, we discuss how chronic nicotine exposure to neurons affects expression of diazepam binding inhibitor (DBI), an endogenous anxiogenic neuropeptide supposed to be a common substance participating drug dependence, and function of L-type high voltage-gated Ca²⁺ channels (HVCCs). We also discuss the functional interaction between DBI and L-type HVCCs in nicotine dependence. Both DBI levels and [⁴⁵Ca²⁺] influx significantly increased in the brain from mice treated with nicotine for long term, which was further enhanced after abrupt cessation of nicotine and was abolished by nicotinic acetylcholine receptor (nAChR) antagonists. Similar responses of DBI expression and L-type HVCC function were observed in cerebral cortical neurons after sustained exposure to nicotine. In addition, increased DBI expression was inhibited by antagonists of nAChR and L-type HVCCs. Sustained exposure of neurons to nicotine significantly enhanced expression of α₁ and α₂/δ₁ subunits for L-type HVCCs and caused an increase in the B_max value of [³H]verapamil binding to the particulate fractions. Therefore, it is concluded that the alterations in DBI expression is mediated via increased influx of Ca²⁺ through upregulated L-type HVCCs and these neurochemical changes have a close relationship with development of nicotine dependence and/or its withdrawal syndrome.

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N-Methyl-D-aspartate (NMDA) receptors are a principal subtype of excitatory ligand-gated ion channels with crucial roles in a variety of physiological and pathological processes in the mammalian central nervous system (CNS). In contrast to synaptic non-NMDA receptors that rapidly internalize, synaptic NMDA receptors have been shown to be rather static. However, recent accumulating evidence gives rise to the possibility that NMDA receptors may also undergo internalization. In our studies, repeated but not sustained stimulation by an agonist indeed induced desensitization of NMDA-receptor channels, which may be due to a decrease in the number of NMDA receptors expressed on cellular surfaces by internalization in a manner similar to the clathrin-induced endocytosis. Desensitization of NMDA-receptor channels provides a clue for the elucidation of fundamental mechanisms underlying dynamic regulation of the number of NMDA receptors at synapses, which is undoubtedly critical for the maintenance of both integrity and functionality in the CNS.

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Excessive elevation of intracellular calcium level seems to be a trigger of ischemic neuronal injury. Calcium/calmodulin (CaM)-dependent protein kinase kinase (CaM-KK) is an upstream kinase for CaM kinase IV (CaM-KIV) that was reported to prevent apoptosis through phosphorylation of CREB (cyclic AMP responsive element-binding protein). We here observed that CaM-KK could directly activate Akt, thereby preventing apoptosis in cultured cells. Then we examined changes in Akt and CaM-KIV activities in gerbil forebrain ischemia. In 5-min-ischemia-caused delayed neuronal death in hippocampal CA1 neurons, Akt and CaM-KIV activities were decreased after reperfusion. On the other hand, during induction of ischemic tolerance, Akt activity gradually and persistently increased in the CA1 neurons with transient increase in CREB phosphorylation. Inhibition of Akt activity with wortmannin or CREB-DNA binding with CRE-decoy injection resulted in failure of generation of ischemic tolerance. These results indicated activation of Akt and CaM-KIV play important roles in induction of the ischemic tolerance. Activation of CaM-KK may provide a new strategy for overcoming the ischemic stress.

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Circulating levels of calcium ion (Ca²⁺) are maintained within a narrow physiological range mainly by the action of parathyroid hormone (PTH) secreted from parathyroid cells. Parathyroid cells can sense small fluctuations in plasma Ca²⁺ levels by virtue of a cell surface Ca²⁺ receptor (CaR) that belongs to the superfamily of G-protein-coupled receptors. Calcimimetics are positive allosteric modulators that activate the CaR on parathyroid cells and thereby immediately suppress PTH secretion. Pre-clinical studies with NPS R-568, a first generation calcimimetic compound, have demonstrated that daily oral administration inhibits the elevation of plasma PTH levels and parathyroid gland hyperplasia and ameliorates impaired bone qualities in rats with chronic renal insufficiency. The results of clinical trials with cinacalcet hydrochloride, a second generation calcimimetic compound, have shown that calcimimetics possess lowering effects not only on serum PTH levels but also on serum calcium × phosphorus product levels, a hallmark of an increased risk for cardiovascular death in dialysis patients with end-stage renal disease (ESRD). Thus, calcimimetics have considerable potential as an innovative medical approach to manage secondary hyperparathyroidism associated with ESRD. Indeed, cinacalcet hydrochloride has been approved in several countries and is the first positive allosteric modulator of any G protein-coupled receptor to reach the market.

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Fluorescence resonance energy transfer (FRET) with green fluorescent protein (GFP) variants has become widely used for biochemical research. In order to expand the choice of fluorescent range in FRET analysis, we designed various color versions of the FRET-based probes for caspase activity, in which the substrate sequence of the caspase was sandwiched by donor and acceptor fluorescent proteins, and studied the potential of these color versions as fluorescent indicators. Six color versions were constructed by a combination of cyan fluorescent protein (CFP), GFP, yellow fluorescent protein (YFP), and DsRed. Real-time monitoring in single cells revealed that all probes could detect caspase activation during tumor necrosis factor (TNF)-α-induced cell death as a fluorescent change. GFP-DsRed and YFP-DsRed were as sensitive as CFP-YFP, and CFP-DsRed also showed a large fluorescent change. By using two probes, CFP-DsRed and YFP-DsRed, we carried out simultaneous multi-FRET analysis and revealed that the initiator- and effector-caspases were activated almost simultaneously in TNF-α-induced cell death. These findings may give experimental bases for the development of novel techniques to analyze multi-events simultaneously in single cells by using FRET probes in combination.

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To investigate whether nitric oxide (NO) induces apoptosis in myoblast cells, the effect of the sodium nitroprusside (SNP), NO donor, on the apoptosis of mouse C2C12 myoblast cells was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry, 4,6-diamidino-2-phenylindole (DAPI) staining, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, DNA fragmentation assay, reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis, and caspase-3 enzyme assay. Mouse C2C12 myoblast cells treated with SNP exhibited several apoptotic features. SNP increased p53 expression and bax expression. SNP also enhanced caspase-3 enzyme activity. The data show that NO may induce apoptotic cell death in myoblast cells through the activation of p53-, bax-, and caspase-dependent intracellular death-related pathways.

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Cannabinoids have been reported to have analgesic properties in animals of acute nociception or of inflammatory and neuropathic pain models, but the mechanisms by which they exert such alleviative effects are not yet fully understood. We investigated whether the CB₁-cannabinoid-receptor agonist HU210 modulates the capsaicin-induced ⁴⁵Ca²⁺ influx and substance P like-immunoreactivity (SPLI) release in cultured rat dorsal root ganglion (DRG) cells. HU210 attenuated the capsaicin-induced ⁴⁵Ca²⁺ influx and this effect was reversed by the CB₁ antagonist AM251. Treatment of DRG cells with 100 nM bradykinin for 3 h potentiated capsaicin-induced SPLI release accompanied with the induction of cyclooxygenase-2 mRNA expression. The potentiation of SPLI release by bradykinin was reversed by HU210 or the protein kinase A (PKA) inhibitor H-89. HU210 also reduced forskolin-induced cyclic AMP production and forskolin-induced potentiation of SPLI release. These results suggest that CB₁ could inhibit either the capsaicin-induced Ca²⁺ influx or the potentiation of capsaicin-induced SPLI release by a long-term treatment with bradykinin through involvement of a cyclic-AMP-dependent PKA pathway. In conclusion, CB₁-receptor stimulation modulates the activities of transient receptor potential vanilloid receptor 1 in cultured rat DRG cells.

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SART (specific alternation of rhythm in temperature)-stressed rats are an animal model of autonomic imbalance created by exposing animals to repeated cold stress. The SART-stressed rats have been shown to easily develop orthostatic hypotension (OH). In this study, effects of AF-DX116, a selective M₂ antagonist, and other muscarinic receptor antagonists on OH were investigated in SART-stressed and unstressed rats. Each anesthetized rat was canulated into the left common carotid artery, and blood pressure (BP) and heart rate were measured. Stimulation for postural change was initiated by head-up tilting. As the indices of OH, the maximum fall of BP, % reflex (recovery from maximum fall), and the area enclosed between the baseline and the recovery curve for BP (AUC) were used. Large AUC and small % reflex in SART-stressed rats were changed, becoming similar to those of the unstressed rats by AF-DX116 and methoctoramine. Atropine and methylatropine had similar effects to AF-DX116. However, the effects of methoctoramine, atropine, and methylatropine were less than that of AF-DX116. Pirenzepine was not effective. In conclusion, it was suggested in SART-stressed rats that OH was related to hyperactivity in the parasympathetic nerve and the M₂ receptor played the major role in OH.

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Amiodarone, which is generally classified as class III antiarrhythmic drug in the Vaughan Williams classification, is widely used for the treatments of refractory arrhythmias. However, we previously reported that intravenous infusion of amiodarone (6.67 mg/kg per hour) did not suppress arrhythmias induced by coronary ligation/reperfusion in dogs. In this study, we examined effects of a prior oral loading of amiodarone on arrhythmias induced by coronary ligation/reperfusion. Sixteen female beagle dogs (8.5 – 12.5 kg) were divided into two groups; one group was given amiodarone (40 mg/kg, orally, n = 8), and the other was given empty gelatin capsules (n = 8) 2 h before the operation. Dogs were anesthetized with pentobarbital and artificially ventilated. The left chest was opened, and the left anterior descending coronary artery was ligated for 30 min and then reperfused. The mean plasma concentration of amiodarone was over 1.3 μg/ml. Although the prior oral loading of amiodarone did not change the QT interval, amiodarone suppressed the number of ectopic beats during coronary ligation and the incidence of ventricular fibrillation during coronary ligation and reperfusion periods (P<0.05 vs control group). In conclusion, a prior oral loading of amiodarone suppressed arrhythmias induced by coronary ligation/reperfusion with a dose that did not prolong the QT interval. This antiarrhythmic property of amiodarone is different from those of the other class III drugs in that antiarrhythmic effects were accompanied by QT prolongation in our all previous studies.

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Stricture of coronary arteries is closely related to ischemic heart disease. The purpose of this study was to examine whether changes in pH caused contraction of rat coronary arteries, as determined using Langendorff perfused hearts. Changing the pH of the perfusate increased perfusion pressure as an indication of the contractile state of coronary arteries. Alkaline pH-induced increase of perfusion pressure in Wistar Kyoto rats (WKY) was almost identical to that of spontaneously hypertensive rats (SHR), whereas acidic pH-induced increase in SHR was much greater than that in WKY. Acidic pH-induced increase in perfusion pressure was inhibited by verapamil, cromakalim, and adenosine. Feeding WKY with N^G-nitro-L-arginine resulted in hypertension followed by enhanced acidic pH-induced increase in perfusion pressure. These results suggest that acidic-pH induced contraction of rat coronary arteries is caused by Ca²⁺ influx through voltage-dependent Ca²⁺ channels and the contraction is enhanced by hypertension.

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Growth hormone (GH) has been known to enhance immune responses directly or through insulin-like growth factor-I (IGF-I). The present study aimed to clarify the roles of GH in the differentiation of B-lineage precursors. In short-term bone marrow cultures, which contained stem cells and early B-lineage cells, GH (10 μg/L) treatment for one day decreased the percentages of stem cells (0.5-fold) and increased those of B-lineage cells (1.4-fold). Furthermore, GH changed the expressions of transcription factors for B cell progenitors differentiation such as paired box gene-5 (Pax-5), immunoglobulin-associated-α (Ig-α)/CD79a, Ig-β/CD79b, and IGF-I. Thus, a physiological concentration of GH stimulated the differentiation of B-lymphoid precursors from bone marrow stem cells. Since mRNAs of both GH and GH receptor were present in stem cells and B-cell precursors in bone marrow, GH may modulate B-lymphoid precursors development in an autocrine or paracrine manner in bone marrows.

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In canine ventricular myocardium, endothelin-1 (ET-1) alone induced only a weak transient negative inotropic effect (NIE). However, ET-1 induced a marked sustained positive inotropic effect (PIE) subsequent to a transient NIE in the presence of norepinephrine (NE) at low concentrations (0.1 – 1 nM) and elicited a pronounced sustained NIE in the presence of NE at high concentrations (around 100 nM). Thus, the extent of β-adrenoceptor stimulation induced by NE played a crucial role in determining the characteristics of the inotropic effects of ET-1. The characteristics of ET receptor subtypes involved in contractile regulation and Ca²⁺ signaling induced by ET-1 were determined. The ET-1-induced transient NIE and decrease in Ca²⁺ transients were abolished by the selective ET_A-receptor antagonist FR319317, but not by the selective ET_B-receptor antagonist BQ-788. The sustained PIE and the increase in Ca²⁺ transients induced by ET-1 were abolished by FR319317, but not inhibited by BQ-788. In contrast, the sustained NIE of ET-1 was abolished by the non-selective ET antagonist TAK-044, markedly attenuated by FR319317, and partially inhibited by BQ-788. ET-1 alone elicited a PIE in the presence of BQ-788, which indicates that the activation of ET_B-receptors counteracts the development of the PIE of ET-1. The current findings indicate that both ET_A and ET_B receptors are involved in the regulation of Ca²⁺ signaling and contractility in canine ventricular myocardium.

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Neurotropin^®, a non-protein extract from the inflamed skin of rabbits inoculated with vaccinia virus, has been clinically used as an analgesic drug for treatment of chronic pain. In this study, we investigated the analgesic mechanisms of Neurotropin in the adjuvant-induced arthritic rat, a chronic pain model with inflammation. Neurotropin caused dose-dependent inhibition of hyperalgesia in the adjuvant-induced arthritic rat after single intravenous (10 – 100 NU/kg) and oral (30 – 200 NU/kg) administration. The analgesic effect of Neurotropin (intravenous 100 NU/kg and oral 200 NU/kg) was significantly inhibited by intrathecal injections of the α₂-adrenoceptor antagonist yohimbine (30 nmol/animal) and the selective 5-HT₃ serotonin receptor antagonist MDL72222 (30 nmol/animal), and slightly inhibited by the non-selective serotonin receptor antagonist methysergide (100 nmol/animal). The results suggest that the analgesic action of Neurotropin is at least in part due to the enhancement of noradrenergic and serotonergic descending pain inhibitory pathways. Neurotropin may be useful for the clinical management of chronic pain diseases such as a rheumatoid arthritis and osteoarthritis.

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To examine the effect of the arginine-vasopressin fragment, [pGlu⁴,Cyt⁶]AVP_(4–9) (AVP4–9), on group II metabotropic glutamate receptor (mGluR2/3) agonist and antagonist induced impairment of passive avoidance (PA) task performance, AVP4–9 or phorbol 12-myristate 13-acetate (PMA) was administered in the presence of mGluR2/3-related drugs that induced the impairment of the step-through-type PA task performance. The PA task performance was evaluated in terms of the latency (the time that elapsed prior to entry into the dark compartment) at 24 h after the electrical stimulation. The subcutaneous injection of AVP4–9 at 1 μg/kg had the greatest facilitative effect on the performance, and the facilitative effect of AVP4–9 was inhibited by NPC-15437, a specific protein kinase C (PKC) inhibitor. The injection of AVP4–9 ameliorated PA task performance impairment induced by DCG-IV, an mGluR2/3 agonist. Intracisternal injection of PMA, a PKC activator, also ameliorated the DCG-IV-induced impairment. High doses of AVP4–9 exacerbated the PA task performance impairment induced by LY341495 (an mGluR2/3 antagonist), and PMA injection (1 μg) also exacerbated the impairment induced by the antagonist. These results suggest that an increase in the activity of the PKC-signaling pathway may not always facilitate PA task performance; therefore, AVP4–9 can either enhance or inhibit memory performance in mice.

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Sphingosine-1-phosphate (S1P) acts on a set of G protein-coupled receptors in the plasma membrane and also as a second messenger in certain cell types. There are two possible pathways to mobilize intracellular Ca²⁺ concentration by S1P. One is through phospholipase C, and the other is through intracellular Ca²⁺ channels operated by S1P. The Mn²⁺ quenching method was applied to elucidate the action mode of S1P-induced Ca²⁺ mobilization in rat hepatocytes. In permeabilized hepatocytes, inositol trisphosphate induced Mn²⁺ quenching, and it was blocked by heparin. However, S1P did not induce Mn²⁺ quenching. Results suggest that S1P did not mobilize Ca²⁺ through intracellular Ca²⁺ channels.

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The interleukin (IL)-18 level in plasma is elevated during the acute rejection after organ transplantation. IL-18 elicits adhesion molecule expression as well as interferon-γ/IL-12 production and T-cell proliferation in the human mixed lymphocyte reaction, an in vitro model of acute rejection. We examined whether antibodies (Abs) against intercellular adhesion molecule (ICAM)-1, B7, CD40, and CD40, ligand (CD40L) affect the cytokine production and T-cell proliferation. Anti-ICAM-1 and B7 Abs suppressed the cytokine production, while all Abs inhibited T-cell proliferation. ICAM-1 and B7 as well as CD40 may play different roles in the acute rejection.

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The N⁶-cyclohexyladenosine (CHA)-induced hypothermia in Syrian hamsters was characterized as follows: intracerebroventricular injection of CHA-induced hypothermia and the potency was increased by lowering the ambient temperature. CHA microinjection into the anterior hypothalamus (AH) elicited the most marked body temperature (T_b) decrease compared with other regions such as the preoptic area, dorsomedial hypothalamus, posterior hypothalamus, and hippocampus. In contrast, microinjected CHA into the medial septum, ventromedial hypothalamus, and lateral hypothalamus resulted in negligible changes in T_b. These results suggest that CHA-induced hypothermia was probably due to suppression of thermogenesis via the site(s) of CHA action, viz., the AH and medial hypothalamus.

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Parkinson’s disease (PD) is pathologically characterized by dopaminergic (DA) cell death and the presence of Lewy bodies (LB) in the brain. α-Synuclein (α-syn) and ubiquitin (Ub) are the major components of LB, however, the process of their accumulation and their relationship to DA cell loss has not yet been resolved. Now, in this journal, Inden et al. showed the protective effect of proteasome inhibitors (PSI) on DA cell death in the rat PD model using 6-hydroxyl dopamine (6-OHDA). Co-administration of PSI, lactacystin, or MG-132 significantly prevented the nigral degeneration and apomorphine-induced rotational asymmetry of the model with increased appearance of α-syn- and Ub-positive inclusions in the substantia nigra. This study indicates that in their model, accelerated formation of inclusions via proteasome inhibition protects against DA cell death. Previous literature linked the impairments or inhibitions of the ubiquitin-proteasome system (UPS) and DA cell death. However, this report implies that the relationship between the UPS and the pathogenesis of PD may be more complex than we thought.

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