Mechanism underlying the negative inotropic effect in rat left ventricle in hyperthermia: the role of TRPV1.

We have previously reported that the negative inotropic effects of hyperthermia (42 °C) on left ventricular (LV) mechanoenergetics using the excised, cross-circulated rat heart model. Here, we investigated the role of TRPV1 on LV mechanoenergetics in hyperthermia. We analyzed the LV end-systolic pressure-volume relation (ESPVR) and the linear relation between the myocardial oxygen consumption per beat (VO2) and the systolic pressure-volume area (PVA; a total mechanical energy per beat) during infusion of capsazepine (CPZ) in hyperthermia, or capsaicin (Cap) under 300 bpm pacing. LV ESP decreased in each LV volume and the resultant downward-shift of LV ESPVR was suppressed by CPZ infusion in hyperthermia-hearts. In Cap-treated hearts, LV ESPVR shifted downward from the control ESPVR, similar to hyperthermia-hearts. The slopes of VO2-PVA relationship were unchanged. The VO2 intercepts in hyperthermia-hearts did not decrease because of decreased E-C coupling VO2, and inversely increased basal metabolic VO2, which was suppressed by CPZ, though the VO2 intercepts in Cap-treated hearts significantly decreased. The levels of phosphorylated phospholamban at serine 16 decreased significantly in hyperthermia-hearts, as well as Cap-treated hearts. These results indicate that a Cap-induced decrease in the LV contractility, like in cases of hyperthermia, are due to the down-regulation of the total calcium handling in E-C coupling, suggesting that negative inotropic effect in hyperthermia-heart is, at least in part, mediated through TRPV1 signaling pathway.

The energy-saving effect of a new myosin activator, omecamtiv mecarbil, on LV mechanoenergetics in rat hearts with blood-perfused isovolumic contraction model.

A novel myosin activator, omecamtiv mecarbil (OM), is a cardiac inotropic agent with a unique new mechanism of action, which is thought to arise from an increase in the transition rate of myosin into the actin-bound force-generating state without increasing calcium (Ca2+) transient. There remains, however, considerable controversy about the effects of OM on cardiac contractility and energy expenditure. In the present study, we investigated the effects of OM on left ventricular (LV) mechanical work and energetics, i.e., mechanoenergetics in rat normal hearts (CTL) and failing hearts induced by chronic administration of isoproterenol (1.2 mg/kg/day) for 4 weeks (ISO-HF). We analyzed the LV end-systolic pressure-volume relation (ESPVR) and the linear relation between the myocardial oxygen consumption per beat (VO2) and systolic pressure-volume area (PVA; a total mechanical energy per beat) in isovolumically contracting rat hearts at 240- or 300-bpm pacing in the absence or presence of OM. OM did not change the ESPVR in CTL and ISO-HF. OM, however, significantly decreased the slope of VO2-PVA relationship in both CTL and ISO-HF, and significantly increased the mean VO2 intercept without changes in basal metabolism in ISO-HF. These results suggested that OM improved the oxygen cost of PVA (contractile efficiency) with the unchanged LV contractility in both CTL and ISO-HF but increased VO2 for Ca2+ handling in excitation-contraction (E-C) coupling in ISO-HF. We concluded that OM improves contractile efficiency in normal and failing hearts but increases O2 consumption of Ca2+ handling in failing hearts in isovolumically contracting rat model.

Left ventricular mechanoenergetics in excised, cross-circulated rat hearts under hypo-, normo-, and hyperthermic conditions.

We investigated the effects of altering cardiac temperature on left ventricular (LV) myocardial mechanical work and energetics using the excised, cross-circulated rat heart model. We analyzed the LV end-systolic pressure-volume relationship (ESPVR) and linear relationship between myocardial oxygen consumption per beat (VO2) and systolic pressure-volume area (PVA; total mechanical energy per beat) in isovolumically contracting rat hearts during hypo- (32 °C), normo- (37 °C), and hyperthermia (42 °C) under a 300-beats per minute pacing. LV ESPVR shifted downward with increasing cardiac temperature. The VO2-PVA relationship was superimposable in these different thermal conditions; however, each data point of VO2-PVA shifted left-downward during increasing cardiac temperature on the superimposable VO2-PVA relationship line. VO2 for Ca2+ handling in excitation-contraction coupling decreased, which was associated with increasing cardiac temperature, during which sarcoplasmic reticulum Ca2+-ATPase (SERCA) activity was suppressed, due to phospholamban phosphorylation inhibition, and instead, O2 consumption for basal metabolism was increased. The O2 cost of LV contractility for Ca2+ also increased with increasing cardiac temperature. Logistic time constants evaluating LV relaxation time were significantly shortened with increasing cardiac temperature related to the acceleration of the detachment in cross-bridge (CB) cycling, indicating increased myosin ATPase activity. The results suggested that increasing cardiac temperature induced a negative inotropic action related to SERCA activity suppression in Ca2+ handling and increased myosin ATPase activity in CB cycling. We concluded that thermal intervention could modulate cardiac inotropism by changing CB cycling, Ca2+ handling, and basal metabolism in rat hearts.

Methods for the Preparation of an Excised, Cross-Circulated Rat Heart.

The Emax-Pressure-Volume Area (PVA)-VO2 framework proposed by Dr. Suga for canine hearts has dramatically advanced the field of cardiac mechanical work and energetics, i.e., mechanoenergetics. He and his collaborators investigated mechanoenergetics in the left ventricle (LV) of excised, cross-circulated canine heart preparations. We instituted the excised cross-circulated rat whole heart preparations and found a curvilinear end-systolic pressure-volume relation (ESPVR) in the rat LV, in contrast to the linear ESPVR in canine, rabbit, and human LVs. Although Emax, the slope of the linear ESPVR, could be used as an index of LV contractility, it was not applicable for evaluating LV contractility in the rat LV. Thus, we proposed a new index of contractility, equivalent Emax (eEmax) in the rat LV. We also found a linear VO2-PVA relationship in the rat LV. Here, we introduce the methods for the preparation of excised, cross-circulated rat whole hearts and the eEmax-PVA-VO2 framework in the rat LV. Using this method, we can obtain accurate LV volume and myocardial O2 consumption in real time for estimating cardiac mechanoenergetics, which is very challenging in in vivo experiments.

Role of follistatin in muscle and bone alterations induced by gravity change in mice.

Interactions between muscle and bone have been recently noted. We reported that the vestibular system plays crucial roles in the changes in muscle and bone induced by hypergravity in mice. However, the details of the mechanisms by which gravity change affects muscle and bone through the vestibular system still remain unknown. Here, we investigated the roles of humoral factors linking muscle to bone and myostatin-related factors in the hypergravity-induced changes in muscle and bone in mice with vestibular lesions (VL). Hypergravity elevated serum and mRNA levels of follistatin, an endogenous inhibitor of myostatin, in the soleus muscle of mice. VL blunted the hypergravity-enhanced levels of follistatin in the soleus muscle of mice. Simulated microgravity decreased follistatin mRNA level in mouse myoblastic C2C12 cells. Follistatin elevated the mRNA levels of myogenic genes as well as the phosphorylation of Akt and p70S6 kinase in C2C12 cells. As for bone metabolism, follistatin antagonized the mRNA levels of osteogenic genes suppressed by activin A during the differentiation of mesenchymal cells into osteoblastic cells. Moreover, follistatin attenuated osteoclast formation enhanced by myostatin in the presence of receptor activator of nuclear factor-κB ligand in RAW 264.7 cells. Serum follistatin levels were positively related to bone mass in mouse tibia. In conclusion, the present study provides novel evidence that hypergravity affects follistatin levels in muscle through the vestibular system in mice. Follistatin may play some roles in the interactions between muscle and bone metabolism in response to gravity change.

Effects of hypergravity on gene levels in anti-gravity muscle and bone through the vestibular system in mice.

We recently reported that hypergravity with 3 g for 4 weeks affects muscle and bone through the vestibular system in mice. The purpose of this study was to investigate the effects of hypergravity with 2 g, which had no influence on circulating glucocorticoid level, on the gene levels in muscle and bone, as well as the roles of the vestibular system in those changes using vestibular lesioned (VL) mice. Hypergravity for 2 and 8 weeks or VL exerted little effects on the mRNA levels of muscle differentiation factors and myokines in the soleus muscle. Although hypergravity for 2 weeks significantly elevated alkaline phosphatase (ALP) and type I collagen mRNA levels in the tibia, VL significantly attenuated the levels of ALP mRNA enhanced by hypergravity. In conclusion, the present study suggests that a 2-g load for 2 weeks enhances osteoblast differentiation partly through the vestibular system in mice.

The vestibular system is critical for the changes in muscle and bone induced by hypergravity in mice.

Gravity changes concurrently affect muscle and bone as well as induce alterations in vestibular signals. However, the role of vestibular signals in the changes in muscle and bone induced by gravity changes remains unknown. We therefore investigated the effects of vestibular lesions (VL) on the changes in muscle and bone induced by 3 g hypergravity for 4 weeks in C57BL/6J mice. Quantitative computed tomography analysis revealed that hypergravity increased muscle mass surrounding the tibia and trabecular bone mineral content, adjusting for body weight in mice. Hypergravity did not affect cortical bone and fat masses surrounding the tibia. Vestibular lesions blunted the increases in muscle and bone masses induced by hypergravity. Histological analysis showed that hypergravity elevated the cross-sectional area of myofiber in the soleus muscle. The mRNA levels of myogenic genes such as MyoD, Myf6, and myogenin in the soleus muscle were elevated in mice exposed to hypergravity. Vestibular lesions attenuated myofiber size and the mRNA levels of myogenic differentiation markers enhanced by hypergravity in the soleus muscle. Propranolol, a ß-blocker, antagonized the changes in muscle induced by hypergravity. In conclusion, this study is the first to demonstrate that gravity changes affect muscle and bone through vestibular signals and subsequent sympathetic outflow in mice.

Hypergravity Provokes a Temporary Reduction in CD4+CD8+ Thymocyte Number and a Persistent Decrease in Medullary Thymic Epithelial Cell Frequency in Mice.

Gravity change affects many immunological systems. We investigated the effects of hypergravity (2G) on murine thymic cells. Exposure of mice to 2G for three days reduced the frequency of CD4+CD8+ thymocytes (DP) and mature medullary thymic epithelial cells (mTECs), accompanied by an increment of keratin-5 and keratin-8 double-positive (K5+K8+) TECs that reportedly contain TEC progenitors. Whereas the reduction of DP was recovered by a 14-day exposure to 2G, the reduction of mature mTECs and the increment of K5+K8+ TEC persisted. Interestingly, a surgical lesion of the inner ear's vestibular apparatus inhibited these hypergravity effects. Quantitative PCR analysis revealed that the gene expression of Aire and RANK that are critical for mTEC function and development were up-regulated by the 3-day exposure and subsequently down-regulated by the 14-day exposure to 2G. Unexpectedly, this dynamic change in mTEC gene expression was independent of the vestibular apparatus. Overall, data suggest that 2G causes a temporary reduction of DP and a persistent reduction of mature mTECs in a vestibular system-dependent manner, and also dysregulates mTEC gene expression without involving the vestibular system. These data might provide insight on the impact of gravity change on thymic functions during spaceflight and living.

Feasibility of a Short-Arm Centrifuge for Mouse Hypergravity Experiments.

To elucidate the pure impact of microgravity on small mammals despite uncontrolled factors that exist in the International Space Station, it is necessary to construct a 1 g environment in space. The Japan Aerospace Exploration Agency has developed a novel mouse habitat cage unit that can be installed in the Cell Biology Experiment Facility in the Kibo module of the International Space Station. The Cell Biology Experiment Facility has a short-arm centrifuge to produce artificial 1 g gravity in space for mouse experiments. However, the gravitational gradient formed inside the rearing cage is larger when the radius of gyration is shorter; this may have some impact on mice. Accordingly, biological responses to hypergravity induced by a short-arm centrifuge were examined and compared with those induced by a long-arm centrifuge. Hypergravity induced a significant Fos expression in the central nervous system, a suppression of body mass growth, an acute and transient reduction in food intake, and impaired vestibulomotor coordination. There was no difference in these responses between mice raised in a short-arm centrifuge and those in a long-arm centrifuge. These results demonstrate the feasibility of using a short-arm centrifuge for mouse experiments.

A thin-film microprocessor with inkjet print-programmable memory.

The Internet of Things is driving extensive efforts to develop intelligent everyday objects. This requires seamless integration of relatively simple electronics, for example through 'stick-on' electronics labels. We believe the future evolution of this technology will be governed by Wright's Law, which was first proposed in 1936 and states that the cost of a product decreases with cumulative production. This implies that a generic electronic device that can be tailored for application-specific requirements during downstream integration would be a cornerstone in the development of the Internet of Things. We present an 8-bit thin-film microprocessor with a write-once, read-many (WORM) instruction generator that can be programmed after manufacture via inkjet printing. The processor combines organic p-type and soluble oxide n-type thin-film transistors in a new flavor of the familiar complementary transistor technology with the potential to be manufactured on a very thin polyimide film, enabling low-cost flexible electronics. It operates at 6.5 V and reaches clock frequencies up to 2.1 kHz. An instruction set of 16 code lines, each line providing a 9 bit instruction, is defined by means of inkjet printing of conductive silver inks.

Persistent overexpression of SERCA2a affects bladder functions under physiological conditions, but not in bladder outlet obstruction-induced sub-acute pathological conditions.

A functional impairment of the bladder and heart in a decompensated state caused by a pressure overload is accompanied by a decrease in the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2). The beneficial effects of SERCA2 overexpression in preserving cardiac functions have been previously reported. The aim of the present study was to investigate the effects of overexpressed SERCA2 on bladder functions under physiological and pathological conditions using partial bladder outlet obstruction (BOO) in SERCA2a transgenic Wistar rats (TG). Bladder cystometry and western blot analysis were performed using the wild-type Wistar rats (WT), TG, and BOO models (WTBOO and TGBOO). Persistent overexpression of SERCA2 induces reduced bladder compliance without hypertrophy in TG. BOO induces reduced bladder compliance and hypertrophy in WT and TG in the sub-acute phase, but persistent overexpression of SERCA2a in TG does not aggravate the bladder compliance and hypertrophy. In conclusion, SERCA2a overexpression affects bladder functions under physiological conditions, but not in BOO-induced sub-acute pathological conditions.

Possible peripheral mechanism for taste disorder in rats administered S-1.

BACKGROUND: Taste disorders are frequently observed in cancer patients undergoing chemotherapy and are serious adverse events which impair the quality of life (QoL) of the cancer patient. Nevertheless, taste disorder mechanisms in cancer patients undergoing chemotherapy have not yet been fully elucidated. The aim of this study was to reveal taste disorder-related peripheral mechanisms using the two-bottle preference test (TBPT) and histological examination of tongues by hematoxylin-eosin staining and immunohistochemistry with protein-gene product 9.5. METHODS: In the TBPT, one bottle was filled with the 0.01 mM quinine hydrochloride (quinine), as a bitter compound, and the other was filled with water. Doses of 50 and 100 mg kg(-1) day(-1) S-1 (tegafur/gimeracil/oteracil potassium) are lethal to Wistar rats. Therefore, doses ranging from 2-20 mg kg(-1) day(-1) were administered to the rats for 3 weeks. The S-1 dose of 2 mg kg(-1) day(-1) corresponds to the clinical dose administered to cancer patients. The part of the tongue containing the circumvallate papillae was excised the following TBPT. RESULTS: The rate of increase in terms of the average preference rate for the quinine vs. all intake (quinine plus water) was significant from the initial S-1 period to the final one, compared with that in control rats, suggesting the possibility of a worsening sensation for the bitter taste. In S-1 rats, the area of taste nerve fibers were significantly decreased and the rate of degeneration of intra-tongue ganglionic nerve cells was significantly increased. These changes were significantly correlated with the rate of increase in average preference rate of the quinine. CONCLUSION: Neuropathy of the gustatory nerve at the periphery may be involved in taste disorders induced by an anticancer drug.

Left ventricular mechanical and energetic changes in long-term isoproterenol-induced hypertrophied hearts of SERCA2a transgenic rats.

Overexpression of cardiac sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) has been suggested as a strategic intervention for cardiac failure. However, its benefit in wild-type (WT) rats with normal SERCA2a levels seems to be small. To investigate whether it would be beneficial in a cardiac failure model with down-regulated SERCA2a levels, we made a cardiac hypertrophy model using isoproterenol infusion (1.2mgkg(-1)day(-1) for 1 or 4weeks; TG-ISO1w and TG-ISO4w, respectively) in SERCA2a transgenic (TG) rats and compared these rats with littermate WT rats that underwent the same treatments (WT-ISO1w and WT-ISO4w). We analyzed the left ventricular (LV) mechanoenergetics in the excised heart using our original cross-circulation system. The downward shift of curvilinear LV end-systolic pressure-volume relations (ESPVRs) observed in WT-ISO4w rats was abolished in TG-ISO4w rats. The slope and VO2 intercept of the VO2 (myocardial oxygen consumption per beat)-PVA (systolic pressure-volume area: total mechanical energy per beat) linear relation did not differ in any of the groups. The most important finding was a significantly smaller O2 cost of LV contractility in the TG-ISO4w group, which means that less O2 is needed to exert the same LV contractility, compared with the other groups. The increased ratio of SERCA2a/phospholamban returned to the level of the WT-control group only in the TG-ISO4w group. Longer-term up-regulation of mitochondrial transcription factor A for genes of mitochondrial enzymes producing ATP was observed in TG rats. In conclusion, longer-term overexpression of SERCA2a will be beneficial in the present cardiac failure model with down-regulated SERCA2a levels.

In vivo imaging of enteric neurogenesis in the deep tissue of mouse small intestine.

One of the challenges of using imaging techniques as a tool to study cellular physiology has been the inability to resolve structures that are not located near the surface of the preparation. Nonlinear optical microscopy, in particular two photon-excited fluorescence microscopy (2PM), has overcome this limitation, providing deeper optical penetration (several hundred µm) in ex vivo and in vivo preparations. We have used this approach in the gut to achieve the first in vivo imaging of enteric neurons and nerve fibers in the mucosa, submucosa, submucosal and myenteric plexuses, and circular and longitudinal muscles of the small intestine in H-line: Thy1 promoter GFP mice. Moreover, we obtained clear three-dimensional imaging of enteric neurons that were newly generated after gut transection and reanastomosis. Neurogenesis was promoted by oral application of the 5-HT(4)-receptor agonist, mosapride citrate (MOS). The number of newly generated neurons observed in mice treated with MOS for one week was 421±89 per 864,900 µm(2) (n = 5), which was significantly greater than that observed in preparations treated with MOS plus an antagonist (113±76 per 864,900 µm(2)) or in 4 week vehicle controls (100±34 per 864,900 µm(2)) (n = 4 both). Most neurons were located within 100 µm of the surface. These results confirm that activation of enteric neural 5-HT(4)-receptor by MOS promotes formation of new enteric neurons. We conclude that in vivo 2PM imaging made it possible to perform high-resolution deep imaging of the living mouse whole gut and reveal formation of new enteric neurons promoted by 5-HT(4)-receptor activation.

Evaluation of left ventricular mechanical work and energetics of normal hearts in SERCA2a transgenic rats.

Cardiac sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA2a) is responsible for most of the Ca(2+) removal during diastole and a larger Ca(2+) handling energy consumer in excitation-contraction (E-C) coupling. To understand the cardiac performance under long-term SERCA2a overexpression conditions, we established SERCA2a transgenic (TG) Wistar rats to analyze cardiac mechanical work and energetics in normal hearts during pacing at 300 beats/min. SERCA2a protein expression was increased in TGI and TGII rats (F2 and F3 of the same father and different mothers). Mean left ventricular (LV) end-systolic pressure (ESP) and systolic pressure-volume area (PVA; a total mechanical energy per beat) at midrange LV volume (mLVV) were significantly larger in TGI rats and were unchanged in TGII rats, compared to those in non-TG [wildtype (WT)] littermates. Mean myocardial oxygen consumption per minute for E-C coupling was significantly increased, and the mean slope of myocardial oxygen consumption per beat (VO(2))-PVA (systolic PVA) linear relation was smaller, but the overall O(2) cost of LV contractility for Ca(2+) is unchanged in all TG rats. Mean Ca(2+) concentration exerting maximal ESP(mLVV) in TGII rats was significantly higher than that in WT rats. The Ca(2+) overloading protocol did not elicit mitochondrial swelling in TGII rats. Tolerance to higher Ca(2+) concentrations may support the possibility for enhanced SERCA2a activity in TGII rats. In conclusion, long-term SERCA2a overexpression enhanced or maintained LV mechanics, improved contractile efficiency under higher energy expenditure for Ca(2+) handling, and improved Ca(2+) tolerance, but it did not change the overall O(2) cost of LV contractility for Ca(2+) in normal hearts of TG rats.

NHE-1 blockade reversed changes in calcium transient in myocardial slices from isoproterenol-induced hypertrophied rat left ventricle.

We previously reported that left ventricular (LV) slices from isoproterenol (ISO)-induced hypertrophied rat hearts showed an increase of energy expenditure due to remodeling of Ca(2+) handling in excitation-contraction coupling, i.e., suppressed SERCA2a activity and enhanced Na(+)/Ca(2+)exchanger-1 (NCX-1) activity. Na(+)/H(+) exchanger-1 (NHE-1) inhibitor (NHEI) has been demonstrated to exert beneficial effects in the development of cardiac remodeling. We hypothesized that a novel NHE-1 selective inhibitor, BIIB723 prevents remodeling of Ca(2+) handling in LV slices of ISO-induced hypertrophied rat hearts mediated by inhibiting NCX-1 activity. The significant shortening in duration of multi-cellular Ca(2+) transient in ISO group was normalized in ISO+BIIB723 group. The significant increase in amplitude of multi-cellular Ca(2+) waves (CaW) generated at high [Ca(2+)](o) of LV slices in ISO group was also normalized in ISO+BIIB723 group. However, the enhanced NCX-1 activity was not antagonized by BIIB723. We recently reported that ISO-induced down-regulation of a Ca(2+) handling protein, SERCA2a, was normalized by BIIB723. Therefore, it seems likely that BIIB723 normalized shortened multi-cellular Ca(2+) transient duration and increased CaW amplitude in LV slices mediated via normalization of SERCA2a activity. Furthermore, the results presented here suggest the multi-cellular Ca(2+) transient duration and CaW amplitude in LV slices might be better indices reflecting SERCA2a activity than SERCA2a protein expression level.

Age-related changes in afferent responses in sensory neurons to mechanical stimulation of osteoblasts in coculture system.

Bone homeostasis is regulated by mechanical stimulation (MS). The sensory mechanism of bone tissue for MS remains unknown in the maintenance of bone homeostasis. We aimed to investigate the sensory mechanism from osteoblasts to sensory neurons in a coculture system by MS of osteoblasts. Primary sensory neurons isolated from dorsal root ganglia (DRG) of neonatal, juvenile, and adult mice and osteoblasts isolated from calvaria of neonatal mice were cocultured for 24 h. The responses in DRG neurons elicited by MS of osteoblasts with a glass micropipette were detected by increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) with fluo 3-AM. In all developmental stages mice, [Ca(2+)](i)-increasing responses in osteoblasts were promptly elicited by MS. After a short delay, [Ca(2+)](i)-increasing responses were observed in neurites of DRG neurons. The osteoblastic response to second MS was largely attenuated by a stretch-activated Ca(2+) channel blocker, gadolinium. The increases of [Ca(2+)](i) in DRG neurons were abolished by a P2 receptor antagonist; suramin, a P2X receptor antagonist, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate; and an ATP-hydrolyzing enzyme, apyrase. Satellite cells were found around DRG neurons in cocultured cells of only neonatal and juvenile mice. After satellite cells were removed, excessive abnormal responses to MS of osteoblasts were observed in neonatal neurites with unchanged osteoblast responses. The present study indicated that MS of bone tissue elicited afferent P2X receptor-mediated purinergic transmission to sensory neurons in all stages mice. This transmission is modulated by satellite cells, which may have protective actions on sensory neurons.

Comparison of effects of a selective 5-HT reuptake inhibitor versus a 5-HT4 receptor agonist on in vivo neurogenesis at the rectal anastomosis in rats.

It was recently reported that activation of enteric neural 5-HT(4) receptors (SR4) promotes reconstruction of enteric neural circuit injury in distal gut of guinea pigs and that this reconstruction involves neural stem cells. We aimed to explore a novel approach using a selective serotonin reuptake inhibitor (SSRI), which increases endogenous 5-HT, to repair enteric nerve fiber injury in the rat distal gut. Enteric nerve fiber injury was performed by rectal transection and subsequent end-to-end one-layer anastomosis. The SSRI fluvoxamine maleate (100 µmol/l) was applied locally at the anastomotic site to compare with the 5-HT(4) agonist mosapride citrate (100 µmol/l) (applied for patent) applied locally and orally. Unlike mosapride, fluvoxamine failed to promote the regeneration of the nerve fiber tract across the anastomosis. Furthermore, fluvoxamine did not generate anti-distal-less homeobox 2 (DLX2)- and anti-SR4-positive cells (neural stem cells) and/or anti-neurofilament (NF)-positive cells (neural cells) in newly formed granulation tissue at the anastomosis, whereas these cell types were observed in mosapride-treated preparations. In contrast to its effects in guinea pigs, mosapride generated 5-bromo-2'-deoxyuridine (BrdU)-positive neural cells in ganglia sites 3 mm oral and anal from the anastomosis 2 wk after nerve fiber injury. All actions of mosapride were observed after local and or oral applications. These findings indicate that local SSRI treatment does not induce in vivo nerve fiber tract growth across the anastomosis in the rat distal gut. Mosapride induces nerve fiber tract growth across the anastomosis, mediated through enteric neural stem cells possibly from neural crest-derived stem cells or mesenchymal stem cells in the bone marrow.

NHE-1 participates in isoproterenol-induced downregulation of SERCA2a and development of cardiac remodeling in rat hearts.

Impaired Ca(2+) handling is one of the main characteristics in heart failure patients. Recently, we reported abnormal expressions of Ca(2+)-handling proteins in isoproterenol (ISO)-induced hypertrophied rat hearts. On the other hand, Na(+)/H(+) exchanger (NHE)-1 inhibitor has been demonstrated to exert beneficial effects in ischemic-reperfusion injury and in the development of cardiac remodeling. The aims of the present study are to investigate the role of NHE-1 on Ca(2+) handling and development of cardiac hypertrophy in ISO-infused rats. Male Wistar rats were randomly divided into vehicle [control (CTL)] and ISO groups without or with pretreatment with a selective NHE-1 inhibitor, BIIB-723. ISO infusion for 1 wk significantly increased the ratios of heart to body weight and left ventricle (LV) to body weight and collagen accumulation. All of these increases were antagonized by coadministration with BIIB-723. The ISO-induced significant increase in LV wall thickness was suppressed significantly (P < 0.05) by BIIB-723. ISO-induced decreases in cardiac stroke volume and a total mechanical energy per beat index, systolic pressure-volume area at midrange LV volume, were normalized by BIIB-723. The markedly higher expression of NHE-1 protein in the ISO group than that in CTL group was suppressed (P < 0.05) by BIIB-723. Surprisingly, ISO induced downregulation of the important Ca(2+)-handling protein sarcoplasmic reticulum Ca(2+)-ATPase 2a, the expression of which was also normalized by BIIB-723 without changes in phosphorylated phospholamban (PLB)/PLB expression. We conclude that NHE-1 contributes to ISO-induced abnormal Ca(2+) handling associated with cardiac hypertrophy. Inhibition of NHE-1 ameliorates cardiac Ca(2+)-handling impairment and prevents the development of cardiac dysfunction in ISO-infused rats.

In vitro enhanced differentiation of neural networks in ES gut-like organ from mouse ES cells by a 5-HT4-receptor activation.

Using an embryoid body (EB) culture system, we developed a functional organ-like cluster, a "gut", from mouse embryonic stem (ES) cells (ES gut). Each ES gut exhibited various types of spontaneous movements. In these spontaneously contracting ES guts, dense distributions of interstitial cells of Cajal (ICC) (c-kit, a transmembrane receptor that has tyrosine kinase activity, positive cells; gut pacemaker cells) and smooth muscle cells were discernibly identified, but enteric neural networks were not identified. In the present study, we succeeded in forming dense enteric neural networks by a 5-HT(4)-receptor (SR4) agonist, mosapride citrate (1-10 µM) added only during EB formation. Addition of an SR4-antagonist, GR113808 (10 µM) abolished the SR4-agonist-induced formation of enteric neural networks. The SR4-agonist (1 µM) up-regulated the expression of mRNA of SR4 and the SR4-antagonist abolished this upregulation. 5-HT per se exerted similar effects to those of SR4-agonist, though less potent. These results suggest SR4-agonist differentiated enteric neural networks, mediated via activation of SR4 in the ES gut.