Non-neuronal cardiac acetylcholine system playing indispensable roles in cardiac homeostasis confers resiliency to the heart.

BACKGROUND: We previously established that the non-neuronal cardiac cholinergic system (NNCCS) is equipped with cardiomyocytes synthesizes acetylcholine (ACh), which is an indispensable endogenous system, sustaining cardiac homeostasis and regulating an inflammatory status, by transgenic mice overexpressing choline acetyltransferase (ChAT) gene in the heart. However, whole body biological significances of NNCCS remain to be fully elucidated. METHODS AND RESULTS: To consolidate the features, we developed heart-specific ChAT knockdown (ChATKD) mice using 3 ChAT-specific siRNAs. The mice developed cardiac dysfunction. Factors causing it included the downregulation of cardiac glucose metabolism along with decreased signal transduction of Akt/HIF-1alpha/GLUT4, leading to poor glucose utilization, impairment of glycolytic metabolites entering the tricarboxylic (TCA) cycle, the upregulation of reactive oxygen species (ROS) production with an attenuated scavenging potency, and the downregulated nitric oxide (NO) production via NOS1. ChATKD mice revealed a decreased vagus nerve activity, accelerated aggression, more accentuated blood basal corticosterone levels with depression-like phenotypes, several features of which were accompanied by cardiac dysfunction. CONCLUSION: The NNCCS plays a crucial role in cardiac homeostasis by regulating the glucose metabolism, ROS synthesis, NO levels, and the cardiac vagus nerve activity. Thus, the NNCCS is suggested a fundamentally crucial system of the heart.

Stress-induced microglial activation occurs through ß-adrenergic receptor: noradrenaline as a key neurotransmitter in microglial activation.

BACKGROUND: The involvement of microglia in neuroinflammatory responses has been extensively demonstrated. Recent animal studies have shown that exposure to either acute or chronic stress induces robust microglial activation in the brain. In the present study, we investigated the underlying mechanism of brain microglial activation by acute stress. METHODS: We first looked at the spatial distribution of the noradrenaline (NA)-synthesizing enzyme, DBH (dopamine ß-hydroxylase), in comparison with NA receptors-ß1, ß2, and ß3 adrenergic receptors (ß1-AR, ß2-AR, and ß3-AR)-after which we examined the effects of the ß-blocker propranolol and α-blockers prazosin and yohimbine on stress-induced microglial activation. Finally, we compared stress-induced microglial activation between wild-type (WT) mice and double-knockout (DKO) mice lacking ß1-AR and ß2-AR. RESULTS: The results demonstrated that (1) microglial activation occurred in most studied brain regions, including the hippocampus (HC), thalamus (TM), and hypothalamus (HT); (2) within these three brain regions, the NA-synthesizing enzyme DBH was densely stained in the neuronal fibers; (3) ß1-AR and ß2-AR, but not ß3-AR, are detected in the whole brain, and ß1-AR and ß2-AR are co-localized with microglial cells, as observed by laser scanning microscopy; (4) ß-blocker treatment inhibited microglial activation in terms of morphology and count through the whole brain; α-blockers did not show such effect; (5) unlike WT mice, DKO mice exhibited substantial inhibition of stress-induced microglial activation in the brain. CONCLUSIONS: We demonstrate that neurons/microglia may interact with NA via ß1-AR and ß2-AR.

Various Regulatory Modes for Circadian Rhythmicity and Sexual Dimorphism in the Non-Neuronal Cardiac Cholinergic System.

Cardiomyocytes possess a non-neuronal cardiac cholinergic system (NNCCS) regulated by a positive feedback system; however, its other regulatory mechanisms remain to be elucidated, which include the epigenetic control or regulation by the female sex steroid, estrogen. Here, the NNCCS was shown to possess a circadian rhythm; its activity was upregulated in the light-off phase via histone acetyltransferase (HAT) activity and downregulated in the light-on phase. Disrupting the circadian rhythm altered the physiological choline acetyltransferase (ChAT) expression pattern. The NNCCS circadian rhythm may be regulated by miR-345, independently of HAT, causing decreased cardiac ChAT expression. Murine cardiac ChAT expression and ACh contents were increased more in female hearts than in male hearts. This upregulation was downregulated by treatment with the estrogen receptor antagonist tamoxifen, and in contrast, estrogen reciprocally regulated cardiac miR-345 expression. These results suggest that the NNCCS is regulated by the circadian rhythm and is affected by sexual dimorphism.

Non-neuronal cardiac cholinergic system influences CNS via the vagus nerve to acquire a stress-refractory propensity.

We previously developed cardiac ventricle-specific choline acetyltransferase (ChAT) gene-overexpressing transgenic mice (ChAT tgm), i.e. an in vivo model of the cardiac non-neuronal acetylcholine (NNA) system or non-neuronal cardiac cholinergic system (NNCCS). By using this murine model, we determined that this system was responsible for characteristics of resistance to ischaemia, or hypoxia, via the modulation of cellular energy metabolism and angiogenesis. In line with our previous study, neuronal ChAT-immunoreactivity in the ChAT tgm brains was not altered from that in the wild-type (WT) mice brains; in contrast, the ChAT tgm hearts were the organs with the highest expression of the ChAT transgene. ChAT tgm showed specific traits in a central nervous system (CNS) phenotype, including decreased response to restraint stress, less depressive-like and anxiety-like behaviours and anti-convulsive effects, all of which may benefit the heart. These phenotypes, induced by the activation of cardiac NNCCS, were dependent on the vagus nerve, because vagus nerve stimulation (VS) in WT mice also evoked phenotypes similar to those of ChAT tgm, which display higher vagus nerve discharge frequency; in contrast, lateral vagotomy attenuated these traits in ChAT tgm to levels observed in WT mice. Furthermore, ChAT tgm induced several biomarkers of VS responsible for anti-convulsive and anti-depressive-like effects. These results suggest that the augmentation of the NNCCS transduces an effective and beneficial signal to the afferent pathway, which mimics VS. Therefore, the present study supports our hypothesis that activation of the NNCCS modifies CNS to a more stress-resistant state through vagus nerve activity.

Change in soluble epoxide hydrolase (sEH) during cisplatin-induced acute renal failure in mice.

Cisplatin is one of the most effective chemotherapeutic agents against various types of cancers; however, it is also associated with nephrotoxicity. Recently, it was reported that inflammatory mechanisms play a key role in the development of nephrotoxicity. Epoxyeicosatrienoic acids (EETs) have an anti-inflammatory effect and are metabolized by soluble epoxide hydrolase (sEH: encoded by EPHX2 gene). Here, we determined the change in sEH activity and EPHX2 expression in renal tissue associated with the development of cisplatin-induced nephrotoxicity. Cisplatin administration decreased hydrolase activity accompanied by down-regulation of sEH and EPHX2 expression. The down-regulation occurred prior to the elevation of blood urea nitrogen (BUN) and tumor necrosis factor-α (TNF-α) gene expression or at treatment with low dose cisplatin. In addition, a negative correlation was found between EPHX2 expression and renal thiobarbituric acid reactive substance (TBARS), and edaravone, a radical scavenger, administration did not down-regulate expression of this gene. The results of this study suggest that cisplatin decreased sEH activity through the down-regulation of sEH and EPHX2 expression, and this down-regulation was involved in a negative feedback loop to protect renal tissue from further damage. Thus, sEH is a potential therapeutic target of cisplatin-induced nephrotoxicity.

Altered gene expression in an embolic stroke model after thrombolysis with tissue plasminogen activator and Stachybotrys microspora triprenyl phenol-7.

The present study compares gene expression and infarct area in a mouse model of embolic stroke after thrombolysis with t-PA and SMTP-7. Embolic occlusion was induced by transfer of acetic acid-induced embolus into the brain. t-PA or SMTP-7 was administered 3 h after embolization. Changes in gene expression were evaluated using microarray and RT-PCR analysis. To determine the involvement of reactive oxygen species in the response to t-PA, the free radical scavenger edaravone was infused immediately before t-PA administration. The expressions of 459 genes involved in the inflammatory response, cell-to-cell signaling, cell movement, and inflammatory disease were altered by embolic occlusion. Twenty-two of those genes were upregulated after t-PA but not SMTP-7 administration. Differences between the t-PA- and SMTP-7-treated groups in the expression of genes including the proinflammatory genes Il6, Stat3, S100a8, and Mmp9 were confirmed with RT-PCR. Edaravone ameliorated the overexpression of these genes. Our data demonstrate differences in gene expression following treatment with SMTP-7 or t-PA that likely explain the difference in therapeutic time windows of the two drugs. ROS are involved in the overexpression of proinflammatory genes. The wide therapeutic time window may be achieved through an anti-oxidative effect and inhibition of proinflammatory gene overexpression.

Lysophosphatidic acid induces shear stress-dependent contraction in mouse aortic strip in situ.

We previously reported that lysophosphatidic acid (LPA) regulates Ca²âº influx of fluid flow in stimulated endothelial cells and that LPA and shear stress showed increment and suppressive effects on phenylephrine-induced vasoconstriction and acetylcholine-induced vasodilatation, respectively. However, a vasoconstrictive effect of LPA alone in the presence of shear stress was not found. The present study examined the effect of LPA alone in the presence of shear stress on Ca²âº responses in endothelial and smooth muscle cells and contraction in mouse aortic strip using real-time 2-photon laser scanning microscopy and a custom-made parallel-plate flow chamber. Application of micromolar LPA and high shear stress elicited movement of endothelial cells after Ca²âº responses. The endothelial cells moved along the major axis of smooth muscle cells, a direction that was identical to that found during vasoconstriction evoked by the application of phenylephrine. The frequency of Ca²âº oscillations in smooth muscle cells was highest according to endothelial movement. Vasoconstriction evoked by LPA and shear stress was significantly reduced by the application of a thromboxane A2 receptor antagonist, a cyclooxygenase inhibitor, and a thromboxane synthase inhibitor. These results suggest that micromolar LPA and high shear stress elicit vasoconstriction that is caused by Ca²âº-dependent contraction in medial smooth muscle cells. Thromboxane A2 may be involved in that response.

Vagus nerve stimulation induced long-lasting enhancement of synaptic transmission and decreased granule cell discharge in the hippocampal dentate gyrus of urethane-anesthetized rats.

Vagus nerve stimulation (VNS) ameliorates deficits of hippocampal functions, such as contextual learning and memory, probably through direct modulation of neuronal activity. Previous studies showed that VNS enhanced excitatory synaptic transmission in the hippocampal CA3 area via activation of ß-adrenergic receptors. However, effects of VNS on excitatory synaptic transmission and action potential (AP) discharge of granule cells (GCs) in the dentate gyrus have not been studied. Urethane-anesthetized rats were used to investigate whether VNS influences synaptic transmission efficacy at perforant path-GC synapses and population spike discharge in the dentate gyrus. Intermittent burst stimulation of the left vagus nerve (30Hz for 30s at an inter-train interval of 5min for 1h) significantly enhanced the perforant path-GC synaptic transmission efficacy in the dentate gyrus for at least 2h, indicating that VNS can induce a long-lasting enhancement of synaptic transmission in this area, similar to the situation observed in the CA3 area. In contrast, a 60-min period of VNS significantly reduced population spike amplitude (a parameter reflecting synchronized AP discharge of GCs) for a given excitatory postsynaptic potential. These findings suggest that acute VNS enhances the excitatory synaptic transmission and reduces synchronized AP discharge of GCs in the dentate gyrus. It is likely that enhancement of excitatory synaptic transmission and reduction of GC excitability contribute VNS treatment efficacy for learning deficits and intractable epilepsy, respectively.

Spatiotemporal dynamics of intracellular calcium in the middle cerebral artery isolated from stroke-prone spontaneously hypertensive rats.

The spatiotemporal dynamics of intracellular calcium within the middle cerebral artery (MCA) isolated from stroke-prone spontaneously hypertensive rats (SHR-SP) were investigated using real-time confocal laser microscopy. At 3 months of age (prestroke), rhythmical changes in the [Ca(2+)](i) during the tonic phase were found to precede vasomotion following application of 5-HT, but not other stimuli. These responses were not observed at 1 month of age; moreover, the MCA lost both responses post-stroke (5 months of age). When [Ca(2+)](i) was analysed in arteriolar smooth muscle cells, rhythmical changes in [Ca(2+)](i) occurred during the same cycle. Thus, these processes were synchronized. The synchronized rhythmical changes in [Ca(2+)](i) were abolished following application of 100 nM ketanserin and 10 µM nicardipine. Treatment with 60 nM charybdotoxin and 10 µM cyclopiazonic acid also significantly reduced rhythmical elevations in [Ca(2+)](i). In addition, rhythmical changes in [Ca(2+)](i) became unsynchronized following treatment with 100 µM carbenoxolone, a gap junction blocker. Connexin 45 mRNA and protein expression were both elevated in the MCA of SHR-SP. Taken together, these findings suggest that rhythmical changes in [Ca(2+)](i) of the MCA are dependent upon the 5-HT(2) receptor-mediated release of calcium from intracellular stores which, in turn, activates voltage-dependent calcium channels to enable an influx of calcium into smooth muscle cells. Subsequently, charybdotoxin-sensitive potassium channels are activated and provide a negative feedback pathway to regulate [Ca(2+)](i). Moreover, the co-ordinated synchronization of rhythmical changes in [Ca(2+)](i) across smooth muscle cells was found to be dependent upon gap junctions.

m-Calpain induction in vascular endothelial cells on human and mouse atheromas and its roles in VE-cadherin disorganization and atherosclerosis.

BACKGROUND: Although dysfunction of VE-cadherin-mediated adherence junctions in vascular endothelial cells (ECs) is thought to be one of the initial steps of atherosclerosis, little is known regarding how VE-cadherin is disrupted during atherogenic development. This study focused on the role of calpain, an intracellular cysteine protease, in the proteolytic disorganization of VE-cadherin and subsequent progression of atherosclerosis. METHODS AND RESULTS: Increased expression of m-calpain was observed in aortic ECs in atherosclerotic lesions in humans and low-density lipoprotein receptor-deficient (ldlr(-/-)) mice. Furthermore, proteolytic disorganization of VE-cadherin was shown in aortic ECs in ldlr(-/-) and apolipoprotein E-deficient (apoE(-/-)) mice. Long-term administration of calpain inhibitors into these mice attenuated atherosclerotic lesion development and proinflammatory responses, as well as VE-cadherin disorganization, without normalization of plasma lipid profiles. Furthermore, in vivo transfection of m-calpain siRNA to ldlr(-/-) mice prevented disorganization of VE-cadherin and proatherogenic hyperpermeability in aortic ECs. Treatment of cultured ECs with oxidized LDL, lysophosphatidylcholine, or LDL pretreated with secreted phospholipase A(2) led to the induction of m-calpain but not of µ-calpain, thereby eliciting selective m-calpain overactivation. These data suggest that lysophosphatidylcholine-induced m-calpain directly cleaves a juxtamembrane region of VE-cadherin, resulting in dissociation of ß-catenin from the VE-cadherin complex, disorganization of adherence junctions, and hyperpermeability in ECs. CONCLUSIONS: Subtype-selective induction of m-calpain in aortic ECs during atherosclerotic progression is associated with proteolytic disorganization of VE-cadherin and proatherogenic hyperpermeability in cells. Thus, a strategy to selectively inhibit m-calpain may be useful for the therapeutic treatment of patients with atherosclerosis.

Microinjection of different doses of corticotropin-releasing factor into the medial prefrontal cortex produces effects opposing anxiety-related behavior in rats.

Corticotropin-releasing factor (CRF) in the medial prefrontal cortex (mPFC) is suggested to play an important role in mediating fear, anxiety, and depression. The results of the studies of the actions of CRF in the mPFC regarding anxiety-related behavior, however, seem contradictory. In one study, microinjection of CRF into the mPFC produced an increase in anxiety-related behavior on the elevated plus maze, whereas in another study CRF produced an anxiolytic-like effect. To test whether the different doses of CRF used in these experiments are responsible for the differing results, we examined the dose-dependent effects of CRF (0.015, 0.05, 0.15, 0.5, and 1.0 µg/0.5 µL/site) microinjected into the bilateral mPFC of male Wistar rats on anxiety-related behavior in the elevated plus maze. We found that microinjection of 0.05 µg CRF significantly decreased the number of open-arm entries, whereas 1.0 µg CRF significantly increased the time spent on the open arms. The results indicate that CRF has effects opposing anxiety-related behavior in the elevated plus maze: anxiety-related behavior at a lower dose and an anxiolytic-like effect at a higher dose.

Corticotropin-releasing factor (CRF) receptor subtypes in mediating neuronal activation of brain areas involved in responses to intracerebroventricular CRF and stress in rats.

Corticotropin-releasing factor (CRF) plays an important role in stress responses through activation of its receptor subtypes, CRF1 receptor (CRF(1)) and CRF2 receptor (CRF(2)). The parvocellular paraventricular nucleus of the hypothalamus (PVNp), the central nucleus of the amygdala (CeA), and the oval nucleus of the bed nucleus of the stria terminalis (BNSTov), which are rich in CRF neurons with equivocal expression of CRF(1) and CRF(2), are involved in stress-related responses. In these areas, Fos expression is induced by various stimuli, although the functions of CRF receptor subtypes in stimuli-induced Fos expression are unknown. To elucidate this issue and to examine whether Fos is expressed in CRF or non-CRF neurons in these areas, the effects of antalarmin and antisauvagine-30 (AS-30), CRF(1)- and CRF(2)-specific antagonists, respectively, on intracerebroventricular (ICV) CRF- or 60min-restraint-induced Fos expression were examined in rats. ICV CRF increased the number of Fos-positive CRF and non-CRF neurons in the PVNp, with the increases being inhibited by antalarmin in CRF and non-CRF neurons and by AS-30 in CRF neurons. Restraint also increased Fos-positive CRF and non-CRF neurons in the PVNp, with the increases being inhibited by antalarmin in the CRF neurons. ICV CRF also increased Fos-positive non-CRF neurons in the CeA and the BNSTov, which was inhibited by AS-30 in both areas, and inhibited by antalarmin in the BNSTov only. Restraint increased Fos-positive non-CRF neurons in the CeA and BNSTov, with the increases being almost completely inhibited by either antagonist. These results indicate that both ICV CRF and restraint activate both CRF and non-CRF neurons in the PVNp and non-CRF neurons in the CeA and BNSTov, and that the activation is mediated by CRF(1) and/or CRF(2). However, the manner of involvement for CRF(1) and CRF(2) in ICV CRF- and restraint-induced activation of neurons differs with respect to the stimuli and brain areas; being roughly equivalent in the CeA and BNSTov, but different in the PVNp. Furthermore, the non-CRF(1&2)-mediated signals seem to primarily play a role in restraint-induced activation of non-CRF neurons in the PVNp since the activation was not inhibited by CRF receptor antagonists.

Involvement of CRF2 receptor in the brain regions in restraint-induced anorexia.

We have reported that corticotropin-releasing factor (CRF) receptor subtypes, CRF1 and CRF2, are involved in stress-induced anorexia. To clarify in which brain regions the CRF receptor is involved in mediating stress-induced anorexia, we examined the effect of microinjecting CRF1-selective or CRF2-selective antagonist into the lateral septum or the bed nucleus of the stria terminalis (BNST), which are implicated in regulating stress response. The results demonstrated that injecting antisauvagine-30 into the lateral septum or the BNST significantly attenuated restraint-induced anorexia, whereas injecting antalarmin into these regions did not affect anorexia. These results suggest that the CRF2 receptor in the lateral septum and the BNST is involved in the stress-induced inhibitory mechanism of feeding behavior.

Lysophosphatidic acid induces shear stress-dependent Ca2+ influx in mouse aortic endothelial cells in situ.

Using real-time two-photon laser scanning microscopy, we have demonstrated that lysophosphatidic acid (LPA), a bioactive lipid mediator, causes shear stress-dependent oscillatory local increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in fluo-4-loaded endothelial cells of isolated mouse aortic strips in situ. The increase in [Ca(2+)](i) occurred independently in the individual endothelial cells in a stepwise manner or repetitively during constant flow. The percentage of cells that responded and the averaged level of increase in [Ca(2+)](i) were dependent on both the concentration of LPA (0.3-10 µm) and the shear stress (10-80 dyn cm(-2)). The response was inhibited by removing extracellular Ca(2+), but not by thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase. The spatiotemporal properties of the [Ca(2+)](i) response were completely different from those of a Ca(2+) wave induced by ATP, a Ca(2+)-mobilizing agonist. These results were almost the same as those in the previous investigation using cultured bovine aortic endothelial cells, and suggest that LPA enhanced the shear stress-induced oscillatory Ca(2+) influx, termed 'Ca(2+) spot', in endothelial cells via activation of elementary Ca(2+) influx. In conclusion, the present study demonstrates, for the first time, that LPA functions as an endogenous sensitizer for mechanotransduction in endothelial cells in shear conditions in aortic strips in situ as well as in cultured cells. This indicates an important role for LPA as an endogenous factor in fluid flow-induced endothelial function.

Distinct effects of tissue-type plasminogen activator and SMTP-7 on cerebrovascular inflammation following thrombolytic reperfusion.

BACKGROUND AND PURPOSE: Thrombolysis therapy using tissue-type plasminogen activator (t-PA) is occasionally accompanied by harmful outcomes, including intracerebral hemorrhage. We have reported that Stachybotrys microspora triprenyl phenol-7 (SMTP-7), a candidate thrombolytic drug, has excellent therapeutic effect on cerebral infarction in embolic stroke model in mice; however, little is known regarding whether this agent influences cerebrovascular inflammation following thrombolytic reperfusion. The current study aimed to compare the effects of recombinant t-PA (rt-PA) and SMTP-7 on cerebrovascular inflammation. METHODS: The impact of rt-PA- and SMTP-7-induced thrombolytic reperfusion on leukocyte dynamics was investigated in a photochemically induced thrombotic middle cerebral artery occlusion (tMCAo) model in mice. RESULTS: Both rt-PA and SMTP-7 administration in tMCAo mice (each 10 mg/kg) resulted in thrombolytic reperfusion. The SMTP-7-administered mice showed relatively mild rolling and attachment of leukocytes to the vascular wall in the middle cerebral vein, with weak peroxynitrite reactions and proinflammatory gene expression (IL-1ß, TNF-α, ICAM-1, and VCAM-1); thus, a small infarct volume compared with rt-PA-administered mice. In vitro study suggested that rt-PA at 20 µg/mL, but not SMTP-7 at a similar concentration, promotes cytokine-induced reactive oxygen species generation in cultured endothelial cells; moreover, SMTP-7 suppressed cytokine-induced VCAM-1 induction in the cells and leukocyte/ endothelial cell adhesions. CONCLUSIONS: Relatively mild cerebrovascular inflammation and cerebral infarction in the SMTP-7 mice, compared with in rt-PA mice, is thought to be caused at least in part by direct antioxidative actions of SMTP-7 in ECs.

Shear stress-dependent effects of lysophosphatidic acid on agonist-induced vasomotor responses in rat mesenteric artery.

We have previously shown that lysophosphatidic acid (LPA), a bioactive plasma lysophospholipid, markedly accelerates shear stress-induced Ca2+ responses in cultured vascular endothelial cells (ECs). This study aimed to demonstrate the impact of LPA and luminal shear stress on vasomotor regulation in the isolated rat mesenteric artery (MA) using a videomicroscopic technique. Although the addition of LPA to the perfusate in a concentration range of 0.03-0.3 µM had no significant effect on the basal MA tone, LPA in a similar concentration range led to increased phenylephrine-induced MA contraction and reduced acetylcholine-induced MA relaxation under physiological shear conditions. These vasomodulatory actions of LPA, which vanished upon removal of ECs, were positively dependent on luminal shear stress levels and were markedly inhibited by the LPA receptor antagonist Ki16425, the cyclooxygenase inhibitor indomethacin, and the thromboxane A2 receptor antagonist SQ29548. These data thus suggest that LPA can modify the agonist-induced vasomotor responses in MAs in a shear stress-dependent manner. This effect of LPA was mediated through ECs, the LPA receptor, and cyclooxygenase/thromboxane A2 signaling.

Genetic suppression of ghrelin receptors activates brown adipocyte function and decreases fat storage in rats.

To clarify the role of ghrelin and its receptor (GHS-R) in the regulatory mechanism of energy metabolism, we analyzed transgenic (Tg) rats expressing an antisense GHS-R mRNA under the control of the tyrosine hydroxylase (TH) promoter. Tg rats showed lower visceral fat weight and higher O(2) consumption, CO(2) production, rectal temperature, dark-period locomotor activity, brown adipose tissue (BAT) weight and uncoupling protein 1 expression compared with wild-type (WT) rats on a standard diet. A high-fat diet for 14days significantly increased body weight, visceral fat weight, and the sizes of white and brown adipocytes in WT rats but not in Tg rats compared with the corresponding standard-diet groups. Antisense GHS-R mRNA was expressed and GHS-R expression was reduced in TH-expressing cells of the vagal nodose ganglion in Tg rats. Ghrelin administered intravenously suppressed noradrenaline release in the BAT of WT rats, but not in Tg rats. These results suggest that ghrelin/GHS-R plays an important role in energy storage by modifying BAT function and locomotor activity. As our previous study showed that peripheral ghrelin-induced noradrenaline release suppression in BAT is blocked by vagotomy, the present findings also suggest that vagal afferents transmit the peripheral ghrelin signal to the sympathetic nervous system innervating BAT.

m-Calpain antagonizes RhoA overactivation and endothelial barrier dysfunction under disturbed shear conditions.

AIMS: It has been reported that laminar shear flow (LF) improves barrier functions in vascular endothelial cells (ECs), whereas disturbed flow (DF) impairs the barrier. Our previous study showed that LF stimulus led to the activation of the cysteine protease, m-calpain, in ECs, which can influence RhoA activity. We hypothesized that m-calpain participates in the shear pattern-dependent EC barrier maintenance through RhoA signalling. METHODS AND RESULTS: m-Calpain expression levels in the intima in the inferior aspect of mouse aortic arch where DF dominates were higher than those in adjacent regions. Elevation in transendothelial albumin permeability, which was induced by administration of a calpain inhibitor (ALLM), was prominent in the inferior arch; moreover, this elevation was abolished by Rho kinase (ROCK) inhibitor (Y-27632). Similarly, short interfering RNA (siRNA)-induced silencing of m-calpain resulted in increased RhoA activity and hyperpermeability in the aortic arch, which was accompanied by ROCK inhibitor-sensitive phosphorylation of downstream effecter LIM kinase 2 (LIMK2), stress fibre accumulation in endothelium and enhanced interendothelial gaps. Exposure of human umbilical vein endothelial cells to LF diminished RhoA activity; in contrast, DF facilitated the activity. siRNA-induced m-calpain silencing further accelerated the DF-induced RhoA overactivation, phosphorylation of LIMK2, and cytoskeletal rearrangement, resulting in barrier dysfunction in the cells. CONCLUSION: Our findings revealed relatively high m-calpain expression levels in the inferior arch. The m-calpain activity antagonizes DF-induced overactivation of RhoA/ROCK/LIMK2 signalling and subsequent cytoskeletal rearrangement in ECs, which leads to barrier improvement.

Nitrogen oxide air pollutants interfere with the measurement of nitric oxide using 2,3-diaminonaphthalene: reduction of background interference.

Nitrite, a stable metabolite of nitric oxide (NO), is measured fluorometrically as an indicator of NO production using 2,3-diaminonaphthalene. In cultured cells, it has been believed that a longer period of incubation improves the detection sensitivity because of the accumulation of nitrite formed from NO in culture media. However, here we show that nitrite formed from nitrogen oxide air pollutants accumulates continuously in culture media during the incubation and interferes with the measurement of NO as nitrite. Thus, a proper period of incubation is important to allow maximum nitrite signals from NO with minimum background nitrite from the air.