CD146/MCAM links doxorubicin-induced epigenetic dysregulation to the impaired fatty acid transportation in H9c2 cardiomyoblasts.

CD146/MCAM has garnered significant attention for its potential contribution to cardiovascular disease; however, the transcriptional regulation and functions remain unclear. To explore these processes regarding cardiomyopathy, we employed doxorubicin, a widely used stressor for cardiomyocytes. Our in vitro study on H9c2 cardiomyoblasts highlights that, besides impairing the fatty acid uptake in the cells, doxorubicin suppressed the expression of fatty acid binding protein 4 (Fabp4) along with the histone deacetylase 9 (Hdac9), bromodomain and extra-terminal domain proteins (BETs: Brd2 and Brd4), while augmented the production of CD146/MCAM. Silencing and chemical inhibition of Hdac9 further augmented CD146/MCAM and deteriorated fatty acid uptake. In contrast, chemical inhibition of BETs as well as silencing of MCAM/CD146 ameliorated fatty acid uptake. Moreover, protein kinase C (PKC) inhibition abrogated CD146/MCAM, particularly in the nucleus. Taken together, our results suggest that epigenetic dysregulation of Hdac9, Brd2, and Brd4 alters CD146/MCAM expression, deteriorating fatty acid uptake by downregulating Fabp4. This process depends on the PKC-mediated nuclear translocation of CD146. Thus, this study highlights a pivotal role of CD146/MCAM in doxorubicin-induced cardiomyopathy.

Relationship between Ca2+ and cAMP as second messengers in ACTH-induced cortisol production in bovine adrenal fasciculata cells.

In adrenal fasciculata cells stimulated by ACTH, Ca2+ and cAMP play indispensable roles as second messengers in cortisol production. However, whether their second messengers cooperatively or independently participate in steroid production remains unclear. We focused on the roles of Ca2+ and cAMP in cortisol production in bovine adrenal fasciculata cells stimulated by ACTH for a relatively short period (1 h). Incubation of the cells with 100 pM ACTH in Ca2+-containing (normal) medium for 1 h increased cortisol production without affecting cAMP content. In contrast, treatment of the cells with the peptide at a higher concentration (1 nM) significantly augmented both cortisol production and cAMP content. However, ACTH did not increase either of them in the Ca2+-free medium. ACTH rapidly increased the intracellular free Ca2+ concentration ([Ca2+]i) in the normal medium, but did not influence [Ca2+]i in the Ca2+-free medium, indicating that ACTH caused Ca2+ influx into the cells. ACTH-induced Ca2+ influx and cortisol production were suppressed by a voltage-sensitive L-type Ca2+ channel blocker but not by a T-type, N-type, or P-type Ca2+ channel blocker. In contrast, dibutyryl cAMP, a cell-permeable cAMP analog, greatly enhanced cortisol production in the normal or Ca2+-free medium and slowly caused Ca2+ influx into the cells. These results strongly suggest that Ca2+, as a second messenger, is more critical than cAMP for cortisol production. However, both second messengers jointly participate in the production in adrenal fasciculata cells stimulated by ACTH.

Mizagliflozin, a selective SGLT1 inhibitor, improves vascular cognitive impairment in a mouse model of small vessel disease.

Previously, we showed that sodium/glucose cotransporter 1 (SGLT1) participates in vascular cognitive impairment in small vessel disease. We hypothesized that SGLT1 inhibitors can improve the small vessel disease induced-vascular cognitive impairment. We examined the effects of mizagliflozin, a selective SGLT1 inhibitor, and phlorizin, a non-selective SGLT inhibitor, on vascular cognitive impairment in a mouse model of small vessel disease. Small vessel disease was created using a mouse model of asymmetric common carotid artery surgery (ACAS). Two and/or 4 weeks after ACAS, all experiments were performed. Cerebral blood flow (CBF) was decreased in ACAS compared with sham-operated mice. Phlorizin but not mizagliflozin reversed the decreased CBF of ACAS mice. Both mizagliflozin and phlorizin reversed the ACAS-induced decrease in the latency to fall in a wire hang test of ACAS mice. Moreover, they reversed the ACAS-induced longer escape latencies in the Morris water maze test of ACAS mice. ACAS increased SGLT1 and proinflammatory cytokine gene expressions in mouse brains and phlorizin but not mizagliflozin normalized all gene expressions in ACAS mice. Hematoxylin/eosin staining demonstrated that they inhibited pyknotic cell death in the ACAS mouse hippocampus. In PC12HS cells, IL-1ß increased SGLT1 expression and decreased survival rates of cells. Both mizagliflozin and phlorizin increased the survival rates of IL-1ß-treated PC12HS cells. These results suggest that mizagliflozin and phlorizin can improve vascular cognitive impairment through the inhibition of neural SGLT1 and phlorizin also does so through the improvement of CBF in a mouse model of small vessel disease.

Chronic HDAC6 Activation Induces Atrial Fibrillation Through Atrial Electrical and Structural Remodeling in Transgenic Mice.

Atrial fibrillation (AF) is a relatively common complication of hypertension. Chronic hypertension induces cardiac HDAC6 catalytic activity. However, whether HDAC6 activation contributes to hypertension-induced AF is still uncertain. We examined whether chronic cardiac HDAC6 activation-induced atrial remodeling, leading to AF induction.The HDAC6 constitutively active transgenic (TG) (HDAC6 active TG) mouse overexpressing the active HDAC6 protein, specifically in cardiomyocytes, was created to examine the effects of chronic HDAC6 activation on atrial electrical and structural remodeling and AF induction in HDAC6 active TG and non-transgenic (NTG) mice. Left atrial burst pacing (S1S1 = 30 msec) for 15-30 sec significantly increased the frequency of sustained AF in HDAC6 active-TG mice compared with NTG mice. Left steady-state atrial pacing (S1S1 = 80 msec) decreased the atrial conduction velocity in isolated HDAC6 active TG compared with NTG mouse atria. The atrial size was similar between HDAC6 active TG and NTG mice. In contrast, atrial interstitial fibrosis increased in HDAC6 active TG compared with that of NTG mouse atria. While protein expression levels of both CX40 and CX43 were similar between HDAC6 active TG and NTG mouse atria, a heterogeneous distribution of CX40 and CX43 occurred in HDAC6 active-TG mouse atria but not in NTG mouse atria. Gene expression of interleukin 6 increased in HDAC6 active TG compared with NTG mouse atria.Chronic cardiac HDAC6 activation induced atrial electrical and structural remodeling, and sustained AF. Hypertension-induced cardiac HDAC6 catalytic activity may play important roles in the development of AF.

SGLT1 participates in the development of vascular cognitive impairment in a mouse model of small vessel disease.

Sodium/glucose cotransporter 1 (SGLT1) participates in ischemia-reperfusion-induced cerebral injury. However, whether SGLT1 participates in the development of small vessel disease induced-vascular cognitive impairment is unknown. We examined the roles of SGLT1 in the development of vascular cognitive impairment in a mouse model of small vessel disease. Small vessel disease was created by placement of an ameroid constrictor around the right common carotid artery (CCA) and placement of a microcoil around the left CCA (ACAS) in wild-type (WT) and SGLT1-knock out (KO) mice. Two and/or 4 weeks after ACAS, all experiments were performed. Hematoxylin/eosin staining demonstrated that the number of pyknotic cell deaths was greater in the ACAS WT than ACAS SGLT1-KO hippocampus. The latency to fall in a wire hang test was significantly shorter in ACAS than sham-operated WT mice, whereas it was similar between ACAS and sham-operated SGLT1-KO mice. The Morris water maze test revealed that ACAS WT mice exhibited longer escape latencies than ACAS SGLT1-KO mice. ACAS significantly increased SGLT1 gene expression in WT mouse brains. Gene expressions of MCP-1, IL-1ß, TNF-α, and IL-6 were increased in ACAS WT compared with sham-operated WT mouse brains. Their increased gene expressions were significantly decreased in ACAS SGLT1-KO compared with ACAS WT mice. These results suggest that SGLT1 plays important roles in the development of small vessel dementia.

Pretreatment with KGA-2727, a selective SGLT1 inhibitor, is protective against myocardial infarction-induced ventricular remodeling and heart failure in mice.

Recent studies demonstrated that sodium-glucose co-transporter 1 (SGLT1) is associated with human ischemic cardiomyopathy. However, whether SGLT1 blockade is effective against ischemic cardiomyopathy is still uncertain. We examined the effects of KGA-2727, a selective SGLT1 inhibitor, on myocardial infarction (MI)-induced ischemic cardiomyopathy. To create MI, left anterior descending coronary artery (LAD) ligation with or without KGA-2727 administration was performed in C57BL/6J mice. Four weeks after the operation, all mice were investigated. Left ventricular fractional shortening (LVFS) was reduced and KGA-2727 significantly improved it in LAD-ligated MI mice. The cardiomyocyte diameter, and ANP, BNP, ß-MHC, and IL-18 gene expressions significantly increased in LAD-ligated mouse left ventricles compared with those of sham-operated mouse left ventricles, and KGA-2727 inhibited increases in them. Myocardial fibrosis and upregulation of CTGF and MMP-3 gene expressions in the left ventricle were increased in LAD-ligated mice compared with sham-operated mice, and KGA-2727 decreased them in the LAD-ligated left ventricles. SGLT1 protein expression level was significantly higher in LAD-ligated compared with sham-operated mouse ventricles regardless of KGA-2727 treatment. These results suggest that KGA-2727 pretreatment protects against MI-induced left ventricular remodeling through SGLT1 blockade and that it may become a new pharmacological therapy for ischemia-induced cardiomyopathy.

IL-1ß Plays an Important Role in Pressure Overload-Induced Atrial Fibrillation in Mice.

Hypertension is one risk for atrial fibrillation (AF) and induces cardiac inflammation. Recent evidence indicates that pressure overload-induced ventricular structural remodeling is associated with the activation of nucleotide binding-oligomerization domain (NOD)-like receptor P3 (NLRP3) inflammasomes, including an apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC). We hypothesized that NLRP3 inflammasomes are an initial sensor for danger signals in pressure overload-induced atrial remodeling, leading to AF. Transverse aortic constriction (TAC) or a sham procedure was performed in mice deficient for ASC-/- and interleukin-1ß (IL-1ß-/-). One week after the procedure, electrical left atrial burst pacing from the esophagus was performed for 30 s to induce AF. IL-1ß, monocyte chemotactic protein 1 (MCP-1), connective tissue growth factor (CTGF), and collagen 1 gene expression were also examined. The electrical burst pacing induced AF in TAC-operated wild-type (WT) (p < 0.001) and ASC-/- (p < 0.05) mice, compared to no AF in the sham-operated WT and ASC-/- mice, respectively. In contrast, the number of mice in which sustained AF was induced was similar between TAC-operated IL-1ß-/- and sham-operated IL-1ß-/- mice (p > 0.05). The expression of all genes tested was increased in TAC-operated WT and ASC-/- mice compared with sham-operated WT and ASC-/- mouse atria, respectively. CTGF and collagen 1, but not MCP-1, gene expressions were increased in TAC-operated IL-1ß-/- mouse atria compared with sham-operated WT and IL-1ß-/- mouse atria. In contrast, the IL-1ß gene was not detected in either TAC-operated or sham-operated IL-1ß-/- mouse atria. These results suggest that an IL-1ß activation pathway, different from NLRP3 inflammasomes, plays an important role in pressure overload-induced sustained AF.

Chronic Pressure Overload Induces Cardiac Hypertrophy and Fibrosis via Increases in SGLT1 and IL-18 Gene Expression in Mice.

Increased gene expression levels of sodium-glucose cotransporter 1 (SGLT1) are associated with hypertrophic and ischemic cardiomyopathy. However, it remains unclear whether chronic pressure overload increases SGLT1 expression, which in turn induces hypertrophic cardiomyopathy. We hypothesized that pressure overload could increase SGLT1 gene expression, leading to the development of hypertrophic cardiomyopathy.To create pressure overload-induced cardiomyopathy, transverse aortic constriction (TAC) was performed in SGLT1-deficient (SGLT1-/-) and wild-type (WT) mice. Six weeks after surgery, all mice were investigated. We observed a reduction of left ventricular fractional shortening and left ventricular dilatation in TAC-operated WT but not in TAC-operated SGLT1-/- mice. SGLT1, interleukin 18, connective tissue growth factor, and collagen type 1 gene expression levels were increased in TAC-operated WT mouse hearts compared with that of sham-operated WT mouse hearts. Moreover, heart/body weight ratio and ventricular interstitial fibrosis were increased in TAC-operated WT mice compared with that of sham-operated WT mice. Interestingly, these factors did not increase in TAC-operated SGLT1-/- mice compared with that of sham-operated WT and SGLT1-/- mice. Phenylephrine, an adrenergic α1 receptor agonist, caused cardiomyocyte hypertrophy in neonatal WT mouse hearts to a significantly larger extent than in neonatal SGLT1-/- mouse hearts.In conclusion, the results indicate that chronic pressure overload increases SGLT1 and IL-18 gene expressions, leading to the development of hypertrophic cardiomyopathy. These results make SGLT1 a potential candidate for the therapeutic target for hypertension-induced cardiomyopathy.

Cardiac overexpression of constitutively active Galpha q causes angiotensin II type1 receptor activation, leading to progressive heart failure and ventricular arrhythmias in transgenic mice.

BACKGROUND: Transgenic mice with transient cardiac expression of constitutively active Galpha q (Gαq-TG) exhibt progressive heart failure and ventricular arrhythmias after the initiating stimulus of transfected constitutively active Gαq becomes undetectable. However, the mechanisms are still unknown. We examined the effects of chronic administration of olmesartan on heart failure and ventricular arrhythmia in Gαq-TG mice. METHODOLOGY/PRINCIPAL FINDINGS: Olmesartan (1 mg/kg/day) or vehicle was chronically administered to Gαq-TG from 6 to 32 weeks of age, and all experiments were performed in mice at the age of 32 weeks. Chronic olmesartan administration prevented the severe reduction of left ventricular fractional shortening, and inhibited ventricular interstitial fibrosis and ventricular myocyte hypertrophy in Gαq-TG. Electrocardiogram demonstrated that premature ventricular contraction (PVC) was frequently (more than 20 beats/min) observed in 9 of 10 vehicle-treated Gαq-TG but in none of 10 olmesartan-treated Gαq-TG. The collected QT interval and monophasic action potential duration in the left ventricle were significantly shorter in olmesartan-treated Gαq-TG than in vehicle-treated Gαq-TG. CTGF, collagen type 1, ANP, BNP, and ß-MHC gene expression was increased and olmesartan significantly decreased the expression of these genes in Gαq-TG mouse ventricles. The expression of canonical transient receptor potential (TRPC) 3 and 6 channel and angiotensin converting enzyme (ACE) proteins but not angiotensin II type 1 (AT1) receptor was increased in Gαq-TG ventricles compared with NTG mouse ventricles. Olmesartan significantly decreased TRPC6 and tended to decrease ACE expressions in Gαq-TG. Moreover, it increased AT1 receptor in Gαq-TG. CONCLUSIONS/SIGNIFICANCE: These findings suggest that angiotensin II type 1 receptor activation plays an important role in the development of heart failure and ventricular arrhythmia in Gαq-TG mouse model of heart failure.

Phlorizin prevents electrically-induced ventricular tachyarrhythmia during ischemia in langendorff-perfused guinea-pig hearts.

Phlorizin is a type of flavonoids and has a peroxynitrite scavenging effect. This study aimed to elucidate the effects of phlorizin on ischemia-induced ventricular tachyarrhythmia (VT). Optical signals from the epicardial surface of the ventricle or left ventricular end diastolic pressure (LVEDP) were recorded during acute global ischemia in 42 Langendorff-perfused guinea pig hearts. Experiments were performed in the control condition and in the presence of phlorizin or N-2-mercaptopropionylglycine (2-MPG), a peroxynitrite scavenger, respectively. Mean action potential duration at 20 min of ischemia did not differ among the three interventions. Impulse conduction time-dependently slowed during 20 min of ischemia in the control. Phlorizin but not 2-MPG improved the ischemic conduction slowing at 15 and 20 min of ischemia. Programmed stimulation induced VT at 20 min of ischemia in the control and in the presence of 2-MPG but not in the presence of phlorizin (p<0.05). LVEDP was increased during 30 min of ischemia in the control and in the presence of 2-MPG but not in the presence of phlorizin. These results indicate that phlorizin prevents VT through the improvement of impulse conduction slowing during ischemia. Phlorizin may be more useful for ischemia-induced VT than 2-MPG.

Nicorandil improves electrical remodelling, leading to the prevention of electrically induced ventricular tachyarrhythmia in a mouse model of desmin-related cardiomyopathy.

1. Transgenic (TG) mice overexpressing an arg120gly missense mutation in heat shock protein B5 (HSPB5; i.e. R120G TG mice) exhibit desmin-related cardiomyopathy. Recently, the cardioprotective effect of nicorandil has been shown to prolong the survival of R120G TG mice. However, whether the TG mice exhibit ventricular arrhythmias and whether nicorandil can inhibit these arrhythmias remain unknown. In the present study we examined the effects of chronic nicorandil administration on ventricular electrical remodelling and arrhythmias in R120G TG mice. 2. Mice were administered nicorandil (15 mg/kg per day) or vehicle (water) orally from 5 to 30 weeks of age. Electrocardiograms (ECG) and optical action potentials were recorded from R120G TG mouse hearts. In addition, the expression of ventricular connexin 43 and the cardiac Na(+) channel Nav1.5 was examined in TG mice. 3. All ECG parameters tested were prolonged in R120G TG compared with non-transgenic (NTG) mice. Nicorandil improved the prolonged P, PQ and QRS intervals in R120G TG mice. Interestingly, impulse conduction slowing and increases in the expression of total and phosphorylated connexin 43 and Nav1.5 were observed in ventricles from R120G TG compared with NTG mice. Nicorandil improved ventricular impulse conduction slowing and normalized the increased protein expression levels of total and phosphorylated connexin 43, but not of Nav1.5, in R120G TG mouse hearts. Electrical rapid pacing at the ventricle induced ventricular tachyarrhythmias (VT) in six of eight R120G TG mouse hearts, but not in any of the eight nicorandil-treated R120G TG mouse hearts (P < 0.05). 4. These findings demonstrate that nicorandil inhibits cardiac electrical remodelling and that the prevention of VT by nicorandil is associated with normalization of connexin 43 expression in this model.

Nicorandil prevents Gαq-induced progressive heart failure and ventricular arrhythmias in transgenic mice.

BACKGROUND: Beneficial effects of nicorandil on the treatment of hypertensive heart failure (HF) and ischemic heart disease have been suggested. However, whether nicorandil has inhibitory effects on HF and ventricular arrhythmias caused by the activation of G protein alpha q (Gα(q)) -coupled receptor (GPCR) signaling still remains unknown. We investigated these inhibitory effects of nicorandil in transgenic mice with transient cardiac expression of activated Gα(q) (Gα(q)-TG). METHODOLOGY/PRINCIPAL FINDINGS: Nicorandil (6 mg/kg/day) or vehicle was chronically administered to Gα(q)-TG from 8 to 32 weeks of age, and all experiments were performed in mice at the age of 32 weeks. Chronic nicorandil administration prevented the severe reduction of left ventricular fractional shortening and inhibited ventricular interstitial fibrosis in Gα(q)-TG. SUR-2B and SERCA2 gene expression was decreased in vehicle-treated Gα(q)-TG but not in nicorandil-treated Gα(q)-TG. eNOS gene expression was also increased in nicorandil-treated Gα(q)-TG compared with vehicle-treated Gα(q)-TG. Electrocardiogram demonstrated that premature ventricular contraction (PVC) was frequently (more than 20 beats/min) observed in 7 of 10 vehicle-treated Gα(q)-TG but in none of 10 nicorandil-treated Gα(q)-TG. The QT interval was significantly shorter in nicorandil-treated Gα(q)-TG than vehicle-treated Gα(q)-TG. Acute nicorandil administration shortened ventricular monophasic action potential duration and reduced the number of PVCs in Langendorff-perfused Gα(q)-TG mouse hearts. Moreover, HMR1098, a blocker of cardiac sarcolemmal K(ATP) channels, significantly attenuated the shortening of MAP duration induced by nicorandil in the Gα(q)-TG heart. CONCLUSIONS/SIGNIFICANCE: These findings suggest that nicorandil can prevent the development of HF and ventricular arrhythmia caused by the activation of GPCR signaling through the shortening of the QT interval, action potential duration, the normalization of SERCA2 gene expression. Nicorandil may also improve the impaired coronary circulation during HF.

Methamphetamine induces endoplasmic reticulum stress related gene CHOP/Gadd153/ddit3 in dopaminergic cells.

We examined the toxicity of methamphetamine and dopamine in CATH.a cells, which were derived from mouse dopamine-producing neural cells in the central nervous system. Use of the quantitative real-time polymerase chain reaction revealed that transcripts of the endoplasmic reticulum stress related gene (CHOP/Gadd153/ddit3) were considerably induced at 24-48 h after methamphetamine administration (but only under apoptotic conditions), whereas dopamine slightly induced CHOP/Gadd153/ddit3 transcripts at an early stage. We also found that dopamine and methamphetamine weakly induced transcripts for the glucose-regulated protein 78 gene (Grp78/Bip) at the early stage. Analysis by immunofluorescence microscopy demonstrated an increase of　CHOP/Gadd153/ddit3 and Grp78/Bip proteins at 24 h after methamphetamine administration. Treatment of CATH.a cells with methamphetamine caused a re-distribution of dopamine inside the cells, which mimicked the presynaptic activity of neurons with cell bodies located in the ventral tegmental area or the substantia nigra. Thus, we have demonstrated the existence of endoplasmic reticulum stress in a model of presynaptic dopaminergic neurons for the first time. Together with the recent evidence suggesting the importance of presynaptic toxicity, our findings provide new insights into the mechanisms of dopamine toxicity, which might represent one of the most important mechanisms of methamphetamine toxicity and addiction.

Involvement of gicerin, a cell adhesion molecule, in the portal metastasis of rat colorectal adenocarcinoma cells.

Gicerin, an Ig-superfamily cell adhesion molecule, has homophilic adhesion activity, thus leading to the formation of gicerin aggregates. Gicerin is highly expressed in various embryonic tissues, and it contributes to development through its adhesive activities. In contrast, the expression of the protein is limited to the muscular tissues and endothelial cells in the mature animals. In the liver, gicerin is constitutively expressed in sinusoidal endothelial cells. Interestingly, an overexpression of gicerin is found in a variety of tumors and may play a role in tumorigenesis. Previously, up-regulated expression of the gicerin protein was found in some sporadic cases of chicken colorectal adenocarcinomas and their hepatic metastasized lesions. In the present study, gicerin cDNA was introduced into endogenous gicerin negative ACL-15 cells, a rat colon adenocarcinoma cell line. The cells were subsequently evaluated for changes in their metastatic potentials in order to elucidate the possible role of gicerin in the hepatic metastasis of colorectal adenocarcinomas. The stable overexpression of gicerin in the cells enhanced the self-aggregation and migratory activities on the protein compared with the mock-transfectants. In addition, the gicerin- transfectants had enhanced metastatic potential to the liver compared with mock-transfected cells after implantation into the ileocolic vein of the cognate rats. These results suggest that gicerin might promote the interaction of tumor cells with a hepatic endothelium, thus leading to the hepatic metastasis of colon adenocarcinomas.

Involvement of gicerin, a cell adhesion molecule, in a dermal autograft chicken model.

Gicerin is a cell adhesion molecule in the immunoglobulin superfamily. This molecule has homophilic and heterophilic adhesive activities, binding to the neurite out-growth factor (NOF). We have previously reported that gicerin plays an important role in the development and regeneration as well as in the metastasis of tumors through its adhesive activities, mediating cell-cell and/or cell-extracellular matrix interactions. In this study, we investigated the involvement of gicerin in a dermal autograft chicken model. Gicerin and NOF were transiently present in the regenerating epithelia after the dermal graft transplantation. The treatment with an anti-gicerin polyclonal antibody, by placing drops onto the wounds, inhibited the adhesiveness of the grafts to the marginal skin. The chimeric protein of gicerin-IgG, gicerin-Fc, and NOF proteins promoted the regeneration of the grafts. These findings suggest the potential function of gicerin in dermal autografts, and gicerin and NOF proteins could help clinical improvement after transplantations.

Inflammatory cytokines decrease the expression of nicotinic acetylcholine receptor during the cell maturation.

It is known that the nervous system significantly attenuates systemic inflammatory responses through the parasympathetic nervous system. Furthermore, it has been reported that the alpha 7 subunit of a nicotinic acetylcholine receptor is required for a cholinergic inhibition against cytokine synthesis in a macrophage. As antigen-presenting cells (APCs) play a central role in the generation of primary T cell responses and the maintenance of immunity, in this study, we investigated the expression level of nicotinic receptors of a p53-deficient APC cell line (JawsII) derived from a mouse bone marrow. We showed that stimulation of the JawsII cells with lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNF-alpha) led increase of CD80 and CD86 expression while diminishment of the surface nicotinic receptor. On the other hand, stimulation of nicotinic receptor had no effect on these phenomena. Furthermore, we examined the ability of the cells to release cytokine when stimulated with both nicotine and LPS and showed that the stimulation with LPS augmented the secretion of IL-1a, IL-1b, IL-6, and TNF-alpha. These results suggested that nicotinic stimulation had no effect on the diminishment of alpha 7 nicotinic acetylcholine receptor on JawsII cells by LPS stimulation.

Involvement of transforming growth factor-beta in the expression of gicerin, a cell adhesion molecule, in the regeneration of hepatocytes.

Gicerin, an Ig-superfamily cell adhesion molecule, appears transiently in embryonic tissues including those of the nervous, urogenital, respiratory and digestive systems, and it promotes neurite extension, cell migration and epithelialization through its cell adhesive activities. In addition, gicerin also reappears in regenerating tissue after suffering either a traumatic injury or a viral infection. In the present study, we examined the expression pattern of gicerin in the regeneration of hepatocytes. Immunohistochemically, gicerin protein appeared in the regenerating hepatocytes of carbon tetrachloride (CCl4)-induced acute hepatitis, while it was scarcely expressed in the hepatocytes of normal mouse liver. Real-time PCR revealed the up-regulation of gicerin transcription in the regenerating process of CCl4-induced hepatitis. The expression of transforming growth factor (TGF)-beta1 was also increased during the regeneration. Furthermore, the gicerin mRNA expression increased during the process of an in vitro hepatocyte regeneration model using mouse primary hepatocytes and hepa 1-6 cells. To note, the mRNA levels of gicerin in these cells were enhanced by the presence of TGF-beta1. Collectively, these findings suggest that TGF-beta1 may therefore regulate gicerin expression in hepatocytes leading to liver regeneration by cell-cell or cell-ECM interactions.

Ca2+ channel activating action of maitotoxin in cultured brainstem neurons.

The actions of maitotoxin were studied using cultured brainstem cells and adrenal chromaffin cells. Maitotoxin induced a profound increase in the Ca2+ influx into cultured brainstem cells after a brief lag period. The maitotoxin-induced Ca2+ influx was suppressed by various voltage-dependent Ca2+ channel blockers such as Co2+, Mn2+, verapamil and diltiazem. Maitotoxin-catecholamine release in brainstem cells initiated to increase after a lag period of about 1 min and the increase continued even at 4 min after treatment, while in the adrenal chromaffin cells the release started after an about 1-min lag period to attain a maximum within first 2-min and gradually decrease thereafter. These results suggest that maitotoxin acts on Ca2+ channels to increase the Ca2+ influx, accompanied by enhancement of catecholamine release in the brainstem cells with a different temporal profile from that in the adrenal chromaffin cells.

Neurite extension of DRG neurons by gicerin expression is enhanced by nerve growth factor.

Gicerin, a cell adhesion molecule, is expressed in dorsal root ganglion (DRG) and sciatic nerves during chick development. This molecule re-appears in these tissues after an injury to the sciatic nerve. In the present study, we investigated the expression of nerve growth factor (NGF) in the regenerating sciatic nerve of chicks and the effects of NGF on the expression and neurite activities of gicerin in DRG. In the sciatic nerve after a crush injury, the expression of NGF and gicerin increased in the Schwann cells and in the nerve fibers, respectively. NGF promoted the neurite projections from in vitro DRG on the gicerin ligands, which were inhibited by anti-NGF antibody. The gicerin mRNA expression increased in the DRG with NGF, which was inhibited by the co-incubation with anti-NGF antibody. These results indicate that NGF might therefore enhance the expression of gicerin in DRG, thereby promoting the gicerin-dependent neurite extension during sciatic nerve regeneration.