PDGFRα+ subepithelial interstitial cells act as a pacemaker to drive smooth muscle of the guinea pig seminal vesicle.

Smooth muscle cells (SMCs) of the guinea pig seminal vesicle (SV) develop spontaneous phasic contractions, Ca2+ flashes and electrical slow waves in a mucosa-dependent manner, and thus it was envisaged that pacemaker cells reside in the mucosa. Here, we aimed to identify the pacemaker cells in SV mucosa using intracellular microelectrode and fluorescence Ca2+ imaging techniques. Morphological characteristics of the mucosal pacemaker cells were also investigated using focused ion beam/scanning electron microscopy tomography and fluorescence immunohistochemistry. Two populations of mucosal cells developed spontaneous Ca2+ transients and electrical activity, namely basal epithelial cells (BECs) and subepithelial interstitial cells (SICs). Pancytokeratin-immunoreactive BECs were located on the apical side of the basement membrane (BM) and generated asynchronous, irregular spontaneous Ca2+ transients and spontaneous transient depolarisations (STDs). The spontaneous Ca2+ transients and STDs were not diminished by 10 µM nifedipine but abolished by 10 µM cyclopiazonic acid (CPA). Platelet-derived growth factor receptor α (PDGFRα)-immunoreactive SICs were distributed just beneath the basal side of the BM and developed synchronous Ca2+ oscillations and electrical slow waves, which were suppressed by 3 µM nifedipine and abolished by 10 µM CPA. In SV mucosal preparations in which some smooth muscle bundles remained attached, SICs and residual SMCs developed temporally correlated spontaneous Ca2+ transients. Neurobiotin injected into SICs spread not only to neighbouring SICs but also to neighbouring SMCs or vice versa. These results suggest that PDGFRα+ SICs electrotonically drive the spontaneous contractions of SV smooth muscle. KEY POINTS: In many visceral smooth muscle organs, spontaneous contractions are electrically driven by non-muscular pacemaker cells. In guinea pig seminal vesicles (SVs), as yet unidentified mucosal cells appear to drive neighbouring smooth muscle cells (SMCs). Two populations of spontaneously active cells are distributed in the SV mucosa. Basal epithelial cells (BECs) generate asynchronous, irregular spontaneous Ca2+ transients and spontaneous transient depolarisations (STDs). In contrast, subepithelial interstitial cells (SICs) develop synchronous Ca2+ oscillations and electrical slow waves. Pancytokeratin-immunoreactive (IR) BECs are located on the apical side of the basement membrane (BM), while platelet-derived growth factor receptor α (PDGFRα)-IR SICs are located on the basal side of the BM. Spontaneous Ca2+ transients in SICs are synchronised with those in SV SMCs. Dye-coupling between SICs and SMCs suggests that SICs act as pacemaker cells to drive the spontaneous contractions of SV smooth muscle.

Clinical trials of Cry j 1 and Cry j 2 T-cell epitope peptide-expressing rice in patients with Japanese cedar pollinosis.

BACKGROUND: In allergic models, administration of rice that expresses a hybrid peptide consisting of 7 major T cell epitopes of Cry j 1 and Cry j 2 (7Crp), suppressed allergic symptoms, IgE elevation and specific T cell response to Japanese cedar pollen. OBJECTIVE: To evaluate the efficacy and safety of 7Crp-expressing rice in patients with Japanese cedar pollinosis. METHODS: A 24-week randomized, double-blind, placebo-controlled study was performed to see the efficacy of 7Crp on allergic symptoms using scoring systems, in which 45 patients were assigned to take either 5 g, 20 g test rice, or placebo daily. A 96-week open study was also conducted to determine its inhibitory effect on serum IgE and T cell proliferative response for Japanese cedar pollen, in which 10 patients consumed 5 g test rice daily. RESULTS: No adverse events associated with the test rice occurred, and the intake rate was more than 96%. The test rice did not show suppression of symptoms related to Japanese cedar pollinosis within 24 weeks. However, intake of 5 g test rice led to a significant decrease in T cell response to Japanese cedar pollen during and after the second disperse season in a 96-week open trial, whereas the specific IgE titer remained unchanged. CONCLUSIONS: Tolerability and safety of 7Crp-expressing rice was accepted. Daily intake of up to 20 g transgenic rice did not provide beneficial effects on Japanese cedar pollinosis within 24 weeks, however, continuous intake of 5 g rice might reduce allergen specific T cell response.

Immunological and Symptomatic Effects of Oral Intake of Transgenic Rice Containing 7 Linked Major T-Cell Epitopes from Japanese Cedar Pollen Allergens.

BACKGROUND: A rice-based peptide vaccine containing 7 linked human predominant T-cell epitopes (7Crp) derived from Japanese cedar (JC) pollen allergens, Cry j 1 and Cry j 2, was developed. Here, we examined the efficacy and safety of this transgenic rice in JC pollinosis patients. METHODS: Transgenic rice (5, 20, and 80 g) was administered orally. We measured the T-cell proliferative activity against 7Crp, Cry j 1, and Cry j 2; the cytokine expression levels; and specific IgE and IgG4 production levels. In addition, the symptom and medication scores were monitored during the pollen season, and quality of life (QOL) was evaluated. RESULTS: T-cell proliferative activities to Cry j 1, Cry j 2, and 7Crp were significantly depressed in a dose-dependent manner. Oral intake of 80 g transgenic rice for 20 weeks resulted in significant suppression of allergen-specific T-cell proliferation with downregulation of IL-13 and upregulation of IL-10 levels but no changes to specific IgE and IgG4 levels. The QOL symptom scores for allergic rhinitis were not significantly improved. CONCLUSIONS: Allergen-specific T-cell responses were significantly reduced by oral intake of transgenic rice in a dose-dependent manner. However, neither medication score nor QOL symptom scores could be improved during the JC pollen season with oral intake of transgenic rice for 20 weeks.

Long-term oral administration of transgenic rice containing cedar pollen T-cell epitopes potentially improves medication- and allergy-related quality-of-life scores.

Background: We previously developed a transgenic rice that contains seven linked human predominant T-cell epitopes (7Crp) derived from Japanese cedar (JC) pollen allergens Cry j 1 and Cry j 2. Oral administration of 80 g of transgenic rice for 20 weeks suppressed allergen-specific T-cell proliferation in participants with JC pollinosis, but their clinical symptoms did not improve. Objective: We examined the clinical efficacy of low-dose (5 g and 20 g) intake of the transgenic rice administered for two successive seasons. Methods: In this randomized, double-blind, placebo controlled study, transgenic rice seeds (5 g or 20 g) were orally administered to the participants for 24 weeks in each of two successive JC pollen seasons. We analyzed T-cell proliferation and cytokine expression, and monitored symptom and medication scores during the pollen season. Quality of life (QOL) was evaluated by using the Japanese Allergic Rhinitis Quality of Life Standard Questionnaire (JRQLQ). Results: Specific T-cell proliferation after stimulation with 7Crp, Cry j 1, and Cry j 2 was significantly suppressed in the second JC pollen season. No significant differences were found among the three groups (5 g, 20 g, and placebo) with regard to clinical symptoms or medication scores in the first season. However, the medication scores and face scale for overall condition of JRQLQ improved in the 5-g transgenic rice group in the second season, although careful re-examination with a large sample size is necessary to confirm the results. Conclusion: Low-dose oral administration of transgenic rice that contains 7Crp significantly reduced allergen-specific T-cell responses and improved medication scores during the second season of administration. Thus, oral intake of the transgenic rice has the potential to induce immune tolerance to JC pollen allergens when administered for at least two successive seasons.

Olfactory marker protein elevates basal cAMP concentration.

Olfactory marker protein (OMP), which is expressed abundantly in mature olfactory receptor neurons, operates as a cAMP-binding protein. OMP captures phasic cAMP surges induced by sensory stimuli and punctuates the downstream signalling in the cilia. On the other hand, OMP is also abundant in the soma. At equilibrium, OMP should exhibit association/dissociation reactions with cAMP. To examine the steady-state function of OMP, we expressed OMP in an HEK293 heterologous expression system and measured the activity of cAMP-dependent protein kinase (PKA) using a cAMP response element/luciferase reporter assay. In the presence of OMP, the basal activity level of PKA was elevated to approximately twice as much as that in the absence of OMP. Upon tonic stimulation by membrane-permeable cAMP, the PKA activity increased in a dose-dependent manner and was greater in the presence of OMP at all doses until saturation. These results indicate that OMP, a cytosolic cAMP-binding protein, operates as a cAMP reservoir by increases the basal cAMP concentration and enhances tonic cAMP actions. Together with the previous finding that OMP acutely sequesters cAMP-related responses, these results indicate that OMP can buffer acute surges in cAMP and tonic production, which stabilizes the basal cAMP pool in the long run.

Olfactory marker protein captures cAMP produced via Gαs-protein-coupled receptor activation.

Olfactory marker protein (OMP) labels the matured stage of olfactory receptor neurons (ORN) and has promoted the investigation on the physiology of olfaction. OMP regulates olfactory sensitivity and axonal projection of ORNs, both of which are under the control of the olfactory signaling mediator cAMP. Recently, it has been reported that OMP contains cAMP-binding sites. OMP directly captures the photo-uncaged cAMP in the cytosol and rapidly terminates the olfactory cyclic nucleotide-gated (CNG) channels activity to sharpen the olfactory responses. Here, we investigate the contribution of OMP to cAMP acutely produced via activation of Gαs-protein coupled receptors (GPCR). We expressed OMP and non-desensitizing CNGA2 channels in HEK293T cells together with ß1-adrenergic receptors (ADRB1) or photo-sensitive ß2-adrenergic receptors (opto-ß2). Continuous puff of adrenergic agonist isoproterenol to HEK29T cells with ADRB1 induced the lasting CNGA2 currents in the absence of OMP, while OMP rapidly deactivated the CNGA2 channel activity with residual currents. Photo-activation of opto-ß2 in the absence of OMP induced the CNGA2 currents with a prolonged increase, while OMP swiftly deactivated the CNGA2 channels after the initial surge. Therefore, cytosolic OMP rapidly uncouples CNGA2 channels and cAMP-signaling produced via GPCRs in the submembrane compartment.

Transgenic rice seeds expressing altered peptide ligands against the M3 muscarinic acetylcholine receptor suppress experimental sialadenitis-like Sjögren's syndrome.

Objective: We previously reported that Rag1-/- mice inoculated with splenocytes from M3 muscarinic acetylcholine receptor (M3R) knockout mice immunized with an M3R peptide mixture developed sialadenitis-like Sjögren's syndrome (M3R-induced sialadenitis [MIS]). We also found that intravenous administration of altered peptide ligand (APL) of N-terminal 1 (N1), which is one of the T-cell epitopes of M3R, suppressed MIS. In this study, we aimed to evaluate the suppressive ability and its mechanisms of rice seeds expressing N1-APL7 against MIS.Methods: Rice seeds expressing N1 and N1-APL7 were orally administered to MIS mice for 2 weeks. The changes in saliva flow and sialadenitis (salivary gland inflammation) were analyzed. The M3R-specific T-cell response in the spleen and the expression of regulatory molecules in the cervical lymph nodes and mesenteric lymph nodes were also analyzed.Results: Oral administration of N1-APL7-expressing rice seeds significantly recovered reduction in saliva flow and suppressed sialadenitis when compared with treatment with nontransgenic rice seeds and N1 rice seeds. IFNγ production from M3R-reactive T cells tended to decline in the N1-APL7 rice-treated group as compared with those in the other groups. In the N1-APL7 rice-treated group, the mRNA expression levels of Foxp3 in the cervical-lymph-node CD4+ T cells were higher than those in the other groups.Conclusion: Oral administration of N1-APL7-expressing rice suppressed MIS via suppression of M3R-specific IFNγ and IL-17 production and via enhancement of regulatory molecule expression.Key messagesWe generated N1-peptide- or N1-APL7-expressing rice seeds. Oral administration of N1-APL7-expressing rice seeds significantly recovered the reduction of saliva flow and suppressed sialadenitis via the suppression of M3R specific IFNγ and IL-17 production and via enhancement of regulatory T (Treg) cells.

Novel Device-Based Algorithm Provides Optimal Hemodynamics During Exercise in Patients With Cardiac Resynchronization Therapy.

BACKGROUND: An adaptive cardiac resynchronization therapy (aCRT) algorithm has been described for synchronized left ventricular (LV) pacing and continuous optimization of cardiac resynchronization therapy (CRT). However, there are few algorithmic data on the effect of changes during exercise.Methods and Results:We enrolled 27 patients with availability of the aCRT algorithm. Eligible patients were manually programmed to optimal atrioventricular (AV) and interventricular (VV) delays by using echocardiograms at rest or during 2 stages of supine bicycle exercise. We compared the maximum cardiac output between manual echo-optimization and aCRT-on during each phase. After initiating exercise, the optimal AV delay progressively shortened (P<0.05) with incremental exercise levels. The manual-optimized settings and aCRT resulted in similar cardiac performance, as demonstrated by a high concordance correlation coefficient between the LV outflow tract velocity time integral (LVOT-VTI) during each exercise stage (Ex.1: r=0.94 P<0.0008, Ex.2: r=0.88 P<0.001, respectively). Synchronized LV-only pacing in patients with normal AV conduction could provide a higher LVOT-VTI as compared with manual-optimized conventional biventricular pacing at peak exercise (P<0.05). CONCLUSIONS: The aCRT algorithm was physiologically sound during exercise by patients.

Mucosa-Dependent, Stretch-Sensitive Spontaneous Activity in Seminal Vesicle.

Seminal vesicles (SVs), a pair of male accessory glands, contract upon sympathetic nerve excitation during ejaculation while developing spontaneous phasic constrictions in the inter-ejaculatory storage phase. Recently, the fundamental role of the mucosa in generating spontaneous activity in SV of the guinea pig has been revealed. Stretching the mucosa-intact but not mucosa-denuded SV smooth muscle evokes spontaneous phasic contractions arising from action potential firing triggered by electrical slow waves and associated Ca2+ flashes. These spontaneous events primarily depend on sarco-endoplasmic reticulum (SR/ER) Ca2+ handling linked with the opening of Ca2+-activated chloride channels (CaCCs) resulting in the generation of slow waves. Slow waves in mucosa-intact SV smooth muscle are abolished upon blockade of gap junctions, suggesting that seminal smooth muscle cells are driven by cells distributed in the mucosa. In the SV mucosal preparations dissected free from the smooth muscle layer, a population of cells located just beneath the epithelium develop spontaneous Ca2+ transients relying on SR/ER Ca2+ handling. In the lamina propria of the SV mucosa, vimentin-immunoreactive interstitial cells including platelet-derived growth factor receptor α (PDGFRα)-immunoreactive cells are distributed, while known pacemaker cells in other smooth muscle tissues, e.g. c-Kit-positive interstitial cells or α-smooth muscle actin-positive atypical smooth muscle cells, are absent. The spontaneously-active subepithelial cells appear to drive spontaneous activity in SV smooth muscle either by sending depolarizing signals or by releasing humoral substances. Interstitial cells in the lamina propria may act as intermediaries of signal transmission from the subepithelial cells to the smooth muscle cells.

HCN4 pacemaker channels attenuate the parasympathetic response and stabilize the spontaneous firing of the sinoatrial node.

KEY POINTS: The contribution of HCN4 pacemaker channels in the autonomic regulation of the sino-atrial node (SAN) has been a matter of debate. The transgenic overexpression of HCN4 did not induce tachycardia, but reduced heart rate variability, while the conditional knockdown of HCN4 gave rise to sinus arrhythmia. The response of the SAN to ß-adrenergic stimulation was not affected by overexpression or knockdown of HCN4 channels. When HCN4 channels were knocked down, the parasympathetic response examined by cervical vagus nerve stimulation (CVNS) was enhanced; the CVNS induced complete sinus pause. The overexpression of HCN4 attenuated bradycardia induced by CVNS only during ß-adrenergic stimulation. We concluded that HCN4 pacemaker channels stabilize the spontaneous firing by attenuating the parasympathetic response of the SAN. ABSTRACT: The heart rate is dynamically controlled by the sympathetic and parasympathetic nervous systems that regulate the sinoatrial node (SAN). HCN4 pacemaker channels are the well-known causative molecule of congenital sick sinus syndrome. Although HCN4 channels are activated by cAMP, the sympathetic response of the SAN was preserved in patients carrying loss-of-function mutations of the HCN4 gene. In order to clarify the contribution of HCN4 channels in the autonomic regulation of the SAN, we developed novel gain-of-function mutant mice in which the expression level of HCN4 channels could be reversibly changed from zero to ∼3 times that in wild-type mice, using tetracycline transactivator and the tetracycline responsive element. We recorded telemetric ECGs in freely moving conscious mice and analysed the heart rate variability. We also evaluated the response of the SAN to cervical vagus nerve stimulation (CVNS). The conditional overexpression of HCN4 did not induce tachycardia, but reduced heart rate variability. The HCN4 overexpression also attenuated bradycardia induced by the CVNS only during the ß-adrenergic stimulation. In contrast, the knockdown of HCN4 gave rise to sinus arrhythmia, and enhanced the parasympathetic response; complete sinus pause was induced by the CVNS. In vitro, we compared the effects of acetylcholine on the spontaneous action potentials of single pacemaker cells, and found that similar phenotypic changes were induced by genetic manipulation of HCN4 expression both in the presence and absence of ß-adrenergic stimulation. Our study suggests that HCN4 channels attenuate the vagal response of the SAN, and thereby stabilize the spontaneous firing of the SAN.

HCN4-Overexpressing Mouse Embryonic Stem Cell-Derived Cardiomyocytes Generate a New Rapid Rhythm in Rats with Bradycardia.

A biological pacemaker is expected to solve the persisting problems of an artificial cardiac pacemaker including short battery life, lead breaks, infection, and electromagnetic interference. We previously reported HCN4 overexpression enhances pacemaking ability of mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs) in vitro. However, the effect of these cells on bradycardia in vivo has remained unclear. Therefore, we transplanted HCN4-overexpressing mESC-CMs into bradycardia model animals and investigated whether they could function as a biological pacemaker. The rabbit Hcn4 gene was transfected into mouse embryonic stem cells and induced HCN4-overexpressing mESC-CMs. Non-cardiomyocytes were removed under serum/glucose-free and lactate-supplemented conditions. Cardiac balls containing 5 × 103 mESC-CMs were made by using the hanging drop method. One hundred cardiac balls were injected into the left ventricular free wall of complete atrioventricular block (CAVB) model rats. Heart beats were evaluated using an implantable telemetry system 7 to 30 days after cell transplantation. The result showed that ectopic ventricular beats that were faster than the intrinsic escape rhythm were often observed in CAVB model rats transplanted with HCN4-overexpressing mESC-CMs. On the other hand, the rats transplanted with non-overexpressing mESC-CMs showed sporadic single premature ventricular contraction but not sustained ectopic ventricular rhythms. These results indicated that HCN4-overexpressing mESC-CMs produce rapid ectopic ventricular rhythms as a biological pacemaker.

Role of mucosa in generating spontaneous activity in the guinea pig seminal vesicle.

KEY POINTS: The mucosa may have neuron-like functions as urinary bladder mucosa releases bioactive substances that modulate sensory nerve activity as well as detrusor muscle contractility. However, such mucosal function in other visceral organs remains to be established. The role of mucosa in generating spontaneous contractions in seminal vesicles (SVs), a paired organ in the male reproductive tract, was investigated. The intact mucosa is essential for the generation of spontaneous phasic contractions of SV smooth muscle arising from electrical slow waves and corresponding increases in intracellular Ca2+ . These spontaneous events primarily depend on Ca2+ handling by sarco-endoplasmic reticulum Ca2+ stores. A population of mucosal cells developed spontaneous rises in intracellular Ca2+ relying on sarco-endoplasmic reticulum Ca2+ handling. The spontaneously active cells in the SV mucosa appear to drive spontaneous activity in smooth muscle either by sending depolarizing signals and/or by releasing humoral substances. ABSTRACT: The role of the mucosa in generating the spontaneous activity of guinea-pig seminal vesicle (SV) was explored. Changes in contractility, membrane potential and intracellular Ca2+ dynamics of SV smooth muscle cells (SMCs) were recorded using isometric tension recording, intracellular microelectrode recording and epi-fluorescence Ca2+ imaging, respectively. Mucosa-intact but not mucosa-denuded SV preparations generated TTX- (1 µm) resistant spontaneous phasic contractions that were abolished by nifedipine (3 µm). Consistently, SMCs developed mucosa-dependent slow waves (SWs) that triggered action potentials and corresponding Ca2+ flashes. Nifedipine (10 µm) abolished the action potentials and spontaneous contractions, while suppressing the SWs and Ca2+ flashes. Both the residual SWs and spontaneous Ca2+ transients were abolished by cyclopiazonic acid (CPA, 10 µm), a sarco-endoplasmic reticulum Ca2+ -ATPase (SERCA) inhibitor. DIDS (300 µm) and niflumic acid (100 µm), blockers for Ca2+ -activated Cl- channels (CACCs), or low Cl- solution also slowed or prevented the generation of SWs. In SV mucosal preparations detached from the muscle layer, a population of mucosal cells generated spontaneous Ca2+ transients that were blocked by CPA but not nifedipine. These results suggested that spontaneous contractions and corresponding Ca2+ flashes in SV SMCs arise from action potential generation due to the opening of L-type voltage-dependent Ca2+ channels. Spontaneous Ca2+ transients appear to primarily result from Ca2+ release from sarco-endoplasmic reticulum Ca2+ stores to activate CACCs to develop SWs. The mucosal cells firing spontaneous Ca2+ transients may play a critical role in driving spontaneous activity of SV smooth muscle either by sending depolarizing signals or by releasing humoral substances.

Clinical Concentrations of Local Anesthetics Bupivacaine and Lidocaine Differentially Inhibit Human Kir2.x Inward Rectifier K+ Channels.

BACKGROUND: Inward rectifier K channels of the Kir2.x subfamily are widely expressed in neuronal tissues, controlling neuronal excitability. Previous studies reported that local anesthetics (LAs) do not affect Kir2 channels. However, the effects have not been studied at large concentrations used in regional anesthesia. METHODS: This study used the patch-clamp technique to examine the effects of bupivacaine and lidocaine on Kir2.1, Kir2.2, and Kir2.3 channels expressed in human embryonic kidney 293 cells. RESULTS: When applied extracellularly in whole-cell recordings, both LAs inhibited Kir2.x currents in a voltage-independent manner. Inhibition with bupivacaine was slow and irreversible, whereas that with lidocaine was fast and reversible. Kir2.3 displayed a greater sensitivity to bupivacaine than Kir2.1 and Kir2.2 (50% inhibitory concentrations at approximately 5 minutes, 0.6 vs 8-10 mM), whereas their sensitivities to lidocaine were similar (50% inhibitory concentrations, 1.5-2.7 mM). Increases in the charged/neutral ratio of the LAs at an acidic extracellular pH attenuated their inhibitory effects, and a permanently charged lidocaine derivative QX-314 exhibited no effects when applied extracellularly. Inside-out experiments demonstrated that inhibition of Kir2.1 with cytoplasmic lidocaine and QX-314 was rapid and reversible, whereas that induced by bupivacaine was slow and irreversible. Furthermore, dose-inhibition relations for the charged form of bupivacaine and lidocaine obtained at different cytoplasmic pHs could be approximated by a single relation for each LA. CONCLUSIONS: The results indicate that both LAs at clinical concentrations equilibrated rapidly with the intracellular milieu, differentially inhibiting Kir2.x channel function from the cytoplasmic side.

Ectopic automaticity induced in ventricular myocytes by transgenic overexpression of HCN2.

Hyperpolarization-activated cyclic nucleotide-gated channels (HCNs) are expressed in the ventricles of fetal hearts but are normally down-regulated as development progresses. In the hypertrophied heart, however, these channels are re-expressed and generate a hyperpolarization-activated, nonselective cation current (Ih), which evidence suggests may increase susceptibility to arrhythmia. To test this hypothesis, we generated and analyzed transgenic mice overexpressing HCN2 specifically in their hearts (HCN2-Tg). Under physiological conditions, HCN2-Tg mice exhibited no discernible abnormalities. After the application of isoproterenol (ISO), however, ECG recordings from HCN2-Tg mice showed intermittent atrioventricular dissociation followed by idioventricular rhythm. Consistent with this observation, 0.3 µmol/L ISO-induced spontaneous action potentials (SAPs) in 76% of HCN2-Tg ventricular myocytes. In the remaining 24%, ISO significantly depolarized the resting membrane potential (RMP), and the late repolarization phase of evoked action potentials (APs) was significantly longer than in WT myocytes. Analysis of membrane currents revealed that these differences are attributable to the Ih tail current. These findings suggest HCN2 channel activity reduces the repolarization reserve of the ventricular action potential and increases ectopic automaticity under pathological conditions such as excessive ß-adrenergic stimulation.

Renal Nerve-Mediated Erythropoietin Release Confers Cardioprotection During Remote Ischemic Preconditioning.

BACKGROUND: Remote ischemic preconditioning (RIPC) induced by transient limb ischemia is a powerful innate mechanism of cardioprotection against ischemia. Several described mechanisms explain how RIPC may act through neural pathways or humoral factors; however, the mechanistic pathway linking the remote organ to the heart has not yet been fully elucidated. This study aimed to investigate the mechanisms underlying the RIPC-induced production of Janus kinase (JAK)-signal transducer and activator of the transcription (STAT)-activating cytokines and cardioprotection by using mouse and human models of RIPC. METHODS AND RESULTS: Screened circulating cardioprotective JAK-STAT-activating cytokines in mice unexpectedly revealed increased serum erythropoietin (EPO) levels after RIP induced by transient ischemia. In mice, RIPC rapidly upregulated EPO mRNA and its main transcriptional factor, hypoxia-inducible factor-1α (HIF1α), in the kidney. Laser Doppler blood flowmetry revealed a prompt reduction of renal blood flow (RBF) after RIPC. RIPC activated cardioprotective signaling pathways and the anti-apoptotic Bcl-xL pathway in the heart, and reduced infarct size. In mice, these effects were abolished by administration of an EPO-neutralizing antibody. Renal nerve denervation also abolished RIPC-induced RBF reduction, EPO production, and cardioprotection. In humans, transient limb ischemia of the upper arm reduced RBF and increased serum EPO levels. CONCLUSIONS: Based on the present data, we propose a novel RIPC mechanism in which inhibition of infarct size by RIPC is produced through the renal nerve-mediated reduction of RBF associated with activation of the HIF1α-EPO pathway.

Identification of rice Allene Oxide Cyclase mutants and the function of jasmonate for defence against Magnaporthe oryzae.

Two photomorphogenic mutants of rice, coleoptile photomorphogenesis 2 (cpm2) and hebiba, were found to be defective in the gene encoding allene oxide cyclase (OsAOC) by map-based cloning and complementation assays. Examination of the enzymatic activity of recombinant GST-OsAOC indicated that OsAOC is a functional enzyme that is involved in the biosynthesis of jasmonic acid and related compounds. The level of jasmonate was extremely low in both mutants, in agreement with the fact that rice has only one gene encoding allene oxide cyclase. Several flower-related mutant phenotypes were observed, including morphological abnormalities of the flower and early flowering. We used these mutants to investigate the function of jasmonate in the defence response to the blast fungus Magnaporthe oryzae. Inoculation assays with fungal spores revealed that both mutants are more susceptible than wild-type to an incompatible strain of M. oryzae, in such a way that hyphal growth was enhanced in mutant tissues. The level of jasmonate isoleucine, a bioactive form of jasmonate, increased in response to blast infection. Furthermore, blast-induced accumulation of phytoalexins, especially that of the flavonoid sakuranetin, was found to be severely impaired in cpm2 and hebiba. Together, the present study demonstrates that, in rice, jasmonate mediates the defence response against blast fungus.

Persistent α1-adrenergic receptor function in the nucleus locus coeruleus causes hyperexcitability in AD/HD model rats.

Spontaneously hypertensive rats (SHR) are widely used as a model of attention deficit hyperactivity disorder (ADHD) as their ADHD-like behaviors are restored by methylphenidate. However, a postnatal neural development in SHR is unknown. We performed whole cell patch clamp recordings from locus coeruleus (LC) neurons in neonatal [postnatal day (P) 3-5], juvenile (P21-28), and adult (P 49-56) SHR and age-matched Wistar rats to evaluate α1- and α2-adrenergic receptor (ARs) activities at each developmental period. LC neurons in neonatal Wistar rats and SHR showed no difference in resting membrane potential and spontaneous firing rate, while juvenile and adult SHR LC neurons showed depolarized resting membrane potential and faster spontaneous firing rate than in Wistar rats. Blockade of α1-AR activity by prazosin hyperpolarized the membrane and abolished spontaneous firings in all developmental periods in SHR LC neurons, but not in juvenile and adult Wistar rats. α1-AR stimulation by phenylephrine evoked an inward current in juvenile LC neurons in SHR, but not in juvenile Wistar rats. This phenylephrine-induced inward current was abolished by nonselective cation channel blockers. By contrast, α2-AR stimulation-induced outward currents in the presence of an α1-AR antagonist were equivalent in SHR and Wistar LC neurons. These data suggest that Wistar LC neurons lose α1-AR function during development, whereas α1-ARs remain functional in SHR LC neurons. Thus persistent intrinsic activity of α1-ARs may be a neural mechanism contributing to developmental disorders in juvenile SHRs.

Blue-light regulation of ZmPHOT1 and ZmPHOT2 gene expression and the possible involvement of Zmphot1 in phototropism in maize coleoptiles.

MAIN CONCLUSION: ZmPHOT1 and ZmPHOT2 are expressed differentially in maize coleoptiles and leaves, with Zmphot1 possibly involved in first-positive phototropic curvature of red-light-adapted maize coleoptiles exposed to pulsed low-fluence blue light. Unilateral blue-light perception by phototropin(s) is the first event of phototropism, with the subsequent signal causing lateral transport of auxin at the coleoptile tip region of monocots. In this study, we analyzed the behavior of two maize phototropin genes: ZmPHOT1 and ZmPHOT2, the latter identified from the maize genome database and newly characterized. Quantitative real-time PCR analysis demonstrated that ZmPHOT1 was abundantly expressed in etiolated coleoptiles, while lower expressions of both ZmPHOT1 and ZmPHOT2 were observed in young leaves. Interestingly, these genes were not specifically expressed in the coleoptile tip region, a key position for photoperception in phototropism. Exposure to pulsed low-fluence blue light (LBL) (0.33 µmol m(-2) s(-1) × 8 s) and continuous high-fluence blue light (HBL) (10 µmol m(-2) s(-1)) rapidly decreased ZmPHOT1 gene expression in coleoptiles, with levels of ZmPHOT2 not significantly altered in that tissue. In young leaves, no drastic expression changes were induced in either ZmPHOT1 or ZmPHOT2 by LBL or HBL irradiation. The Zmphot1 protein was investigated by Western blot analysis with anti-Osphot1 antibodies. Zmphot1 was detected in microsomal fractions, with higher levels in coleoptiles than in leaves. HBL caused rapid phosphorylation of the protein, whereas no phot1 phosphorylation was induced by LBL. The involvement of Zmphot1 in LBL-induced phototropic curvature of maize coleoptiles is discussed.

Tissue-specific and light-dependent regulation of phytochrome gene expression in rice.

Phytochromes are red- and far red light photoreceptors in higher plants. Rice (Oryza sativa L.) has three phytochromes (phyA, phyB and phyC), which play distinct as well as cooperative roles in light perception. To gain a better understanding of individual phytochrome functions in rice, expression patterns of three phytochrome genes were characterized using promoter-GUS fusion constructs. The phytochrome genes PHYA and PHYB showed distinct patterns of tissue- and developmental stage-specific expression in rice. The PHYA promoter-GUS was expressed in all leaf tissues in etiolated seedlings, while its expression was restricted to vascular bundles in expanded leaves of light-grown seedlings. These observations suggest that light represses the expression of the PHYA gene in all cells except vascular bundle cells in rice seedlings. Red light was effective, but far red light was ineffective in gene repression, and red light-induced repression was not observed in phyB mutants. These results indicate that phyB is involved in light-dependent and tissue-specific repression of the PHYA gene in rice.

Light induces jasmonate-isoleucine conjugation via OsJAR1-dependent and -independent pathways in rice.

The bioactive form of jasmonate is the conjugate of the amino acid isoleucine (Ile) with jasmonic acid (JA), which is biosynthesized in a reaction catalysed by the GH3 enzyme JASMONATE RESISTANT 1 (JAR1). We examined the biochemical properties of OsJAR1 and its involvement in photomorphogenesis of rice (Oryza sativa). OsJAR1 has a similar substrate specificities as its orthologue in Arabidopsis. However, osjar1 loss-of-function mutants did not show as severe coleoptile phenotypes as the JA-deficient mutants coleoptile photomorphogenesis 2 (cpm2) and hebiba, which develop long coleoptiles in all light qualities we examined. Analysis of hormonal contents in the young seedling stage revealed that osjar1 mutants are still able to synthesize JA-Ile conjugate in response to blue light, suggesting that a redundantly active enzyme can conjugate JA and Ile in rice seedlings. A good candidate for this enzyme is OsJAR2, which was found to be able to catalyse the conjugation of JA with Ile as well as with some additional amino acids. In contrast, if plants in the vegetative stage were mechanically wounded, the content of JA-Ile was severely reduced in osjar1, demonstrating that OsJAR1 is the most important JA-Ile conjugating enzyme in the wounding response during the vegetative stage.