Orai1 forms a signal complex with SK3 channel in gallbladder smooth muscle.

Orai1 is one of the key components of store-operated Ca(2+) entry (SOCE) involved in diverse physiological functions. Orai1 may associate with other proteins to form a signaling complex. In the present study, we investigated the interaction between Orai1 and small conductance Ca(2+)-activated potassium channel 3 (SK3). With the use of RNA interference technique, we found that the SOCE and its associated membrane hyperpolarization were reduced while Orai1 was knocked down by a specific Orai1 siRNA in guinea pig gallbladder smooth muscle. However, with the use of isometric tension measurements, our results revealed that agonist-induced muscle contractility was significantly enhanced after Orai1 protein was knocked down or the tissue was treated by SK3 inhibitor apamin, but not affected by larger conductance Ca(2+)-activated potassium channel inhibitor iberiotoxin or intermediate conductance Ca(2+)-activated potassium channel inhibitor TRAM-34. In addition, in the presence of apamin, Orai1 siRNA had no additional effect on agonist-induced contraction. In coimmunoprecipitation experiment, SK3 and Orai1 pulled down each other. These data suggest that, Orai1 physically associated with SK3 to form a signaling complex in gallbladder smooth muscle. Ca(2+) entry via Orai1 activates SK3, resulting in membrane hyperpolarization in gallbladder smooth muscle. This hyperpolarizing effect of Orai1-SK3 coupling could serve to prevent excessive contraction of gallbladder smooth muscle in response to contractile agonists.

Investigation of the differential susceptibility of extraocular muscles in patients diagnosed with ocular myasthenia gravis based on the computerized diplopia test and the Ocular Motor Nerve Palsy Scale.

Introduction: The pattern of extraocular muscle involvement in ocular myasthenia gravis varies across different reports, diverging from our own observations. Thus, we employed two novel tools to discern this pattern. Methods: A retrospective analysis was conducted to collect and organize clinical data from 43 patients diagnosed with ocular myasthenia gravis. Each patient underwent both the computerized diplopia test and the Ocular Motor Nerve Palsy Scale assessment to evaluate the involvement of extraocular muscles. Results: Among the patients, there were 30 male and 13 female individuals, with a total of 113 affected extraocular muscles identified. Among all the affected extraocular muscles, the involvement of the levator palpebrae superioris muscle accounted for 35.40%, medial rectus muscle 7.7%, lateral rectus muscle 16.81%, superior rectus muscle 13.27%, inferior rectus muscle 12.39%, superior oblique muscle 1.77%, and inferior oblique muscle 2.65% of the total affected extraocular muscles. The positivity rates of the Neostigmine test were 89.19%, AChR antibody detection was 59.38%, and repetitive nerve stimulation was 34.38%. The AChR antibody positive rate among patients with only diplopia was 100%; among those with only ptosis, it was 80%; and among those with both diplopia and ptosis, it was 86.67%. Conclusion: The involvement of the extraocular muscles is not uniform. The levator palpebrae superioris exhibits the highest incidence rate, followed by the four rectus muscles and two oblique muscles. The inferior oblique involvement typically occurs when four or more EOMs are affected. Moreover, the levator palpebrae superioris and medial rectus show a higher tendency for bilateral involvement compared with other extraocular muscles.

Endothelium-derived hydrogen sulfide acts as a hyperpolarizing factor and exerts neuroprotective effects via activation of large-conductance Ca2+ -activated K+ channels.

BACKGROUND AND PURPOSE: Endothelium-derived hyperpolarizing factor (EDHF) has been suggested as a therapeutic target for vascular protection against ischaemic brain injury. However, the molecular entity of EDHF and its action on neurons remains unclear. This study was undertaken to demonstrate whether the hydrogen sulfide (H2 S) acts as EDHF and exerts neuroprotective effect via large-conductance Ca2+ -activated K+ (BKCa /KCa 1.1) channels. EXPERIMENTAL APPROACH: The whole-cell patch-clamp technology was used to record the changes of BKCa currents in rat neurons induced by EDHF. The cerebral ischaemia/reperfusion model of mice and oxygen-glucose deprivation/reoxygenation (OGD/R) model of neurons were used to explore the neuroprotection of EDHF by activating BKCa channels in these neurons. KEY RESULTS: Increases of BKCa currents and membrane hyperpolarization in hippocampal neurons induced by EDHF could be markedly inhibited by BKCa channel inhibitor iberiotoxin or endothelial H2 S synthase inhibitor propargylglycine. The H2 S donor, NaHS-induced BKCa current and membrane hyperpolarization in neurons were also inhibited by iberiotoxin, suggesting that H2 S acts as EDHF and activates the neuronal BKCa channels. Besides, we found that the protective effect of endothelium-derived H2 S against mice cerebral ischaemia/reperfusion injury was disrupted by iberiotoxin. Importantly, the inhibitory effect of NaHS or BKCa channel opener on OGD/R-induced neuron injury and the increment of intracellular Ca2+ level could be inhibited by iberiotoxin but enhanced by co-application with L-type but not T-type calcium channel inhibitor. CONCLUSION AND IMPLICATIONS: Endothelium-derived H2 S acts as EDHF and exerts neuroprotective effects via activating the BKCa channels and then inhibiting the T-type calcium channels in hippocampal neurons.

Total Flavones of Rhododendron simsii Planch Flower Protect against Cerebral Ischemia-Reperfusion Injury via the Mechanism of Cystathionine-γ-Lyase-Produced H2S.

Total flavones of Rhododendron simsii Planch flower (TFR) have a significant protective effect against cerebral ischemia-reperfusion injury. However, its mechanism is unclear. This study investigated the protection of TFR against cerebral ischemia-reperfusion injury via cystathionine-γ-lyase- (CSE-) produced H2S mechanism. CSE-/- mice and CSE-siRNA-transfected rat were used. Relaxation of cerebral basilar artery (CBA), H2S, and CSE mRNA were measured. TFR significantly inhibited cerebral ischemia-reperfusion-induced abnormal neurological symptom and cerebral infarct in the normal rats and the CSE+/+ mice, but not in the CSE-/- mice, and the inhibition was markedly attenuated in CSE-siRNA-transfected rat; TFR elicited a significant vasorelaxation in rat CBA, and the relaxation was markedly attenuated by removal of endothelium or CSE-siRNA transfection or coapplication of NO synthase inhibitor L-NAME and PGI2 synthase inhibitor Indo. CSE inhibitor PPG drastically inhibited TFR-evoked vasodilatation resistant to L-NAME and Indo in endothelium-intact rat CBA. TFR significantly increased CSE mRNA expression in rat CBA endothelial cells and H2S production in rat endothelium-intact CBA. The increase of H2S production resistant to L-NAME and Indo was abolished by PPG. Our data indicate that TFR has a protective effect against the cerebral ischemia-reperfusion injury via CSE-produced H2S and endothelial NO and/or PGI2 to relax the cerebral artery.

Protective Effect and Mechanism of Total Flavones from Rhododendron simsii Planch Flower on Cultured Rat Cardiomyocytes with Anoxia and Reoxygenation.

Many flavonoids have cardioprotection against myocardial ischemia/reperfusion (I/R) injury. Total flavones from Rhododendron simsii Planch flower (TFR) can protect myocardial ischemic injuries. However, its protective mechanism is still unknown. The present study was designed to investigate the mechanism of TFR on myocardial I/R and anoxia/reoxygenation (A/R) injuries. Rat model of myocardial I/R injury was made, and myocardial infarction was determined. A/R injury was induced in cultured rat cardiomyocytes; cellular damage was evaluated by measuring cell viability, LDH and cTnT releases, and MDA content. Expressions of ROCK1 and ROCK2 protein were examined by Western blot analysis, and K(+) currents were recorded by using whole-cell patch clamp technique. TFR 20~80 mg/kg markedly reduced I/R-induced myocardial infarction. TFR 3.7~300 mg/L significantly inhibited A/R-induced reduction of cell viability, LDH and cTnT releases, and MDA production. Exposure to A/R significantly increased ROCK1 and ROCK2 expressions in rat cardiomyocytes, but TFR 33.3~300 mg/L obviously inhibited this increase. 300 mg/L TFR significantly augmented inward rectifier K(+) current and other K(+) currents in rat cardiomyocytes. These results indicate that TFR has a protective effect on rat cardiomyocytes A/R damage, and the protective mechanism may be engaged with the inhibition of ROCK1 and ROCK2 and activation of K(+) channels.

Production and identification of mannosylerythritol lipid-A homologs from the ustilaginomycetous yeast Pseudozyma aphidis ZJUDM34.

Mannosylerythritol lipids (MELs) are mainly produced by strains of the genus Pseudozyma and by Ustilago maydis. These glycolipid biosurfactants exhibit not only excellent surface-active properties but also versatile bioactivities. Mannosylerythritol lipid-A (MEL-A) is worth investigating due to its self-assembling property. In this work, crude MELs were produced by resting Pseudozyma aphidis ZJUDM34 cells using different culture media. MEL-A fractions were isolated and identified using high-performance liquid chromatography combined with mass spectrometry (HPLC-MS) and gas chromatography combined with mass spectrometry (GC-MS). The results showed that MEL-A homologs had long unsaturated fatty acid chains, and the chain lengths range from C8 to C20. Nuclear magnetic resonance (NMR) was employed to confirm the chemical structures of the MEL-A homologs. Fermentation medium without NaNO3 and medium with manganese ions enhanced MEL-A production by Pseudozyma aphidis ZJUDM34.

Cellular mechanisms underlying Hyperin-induced relaxation of rat basilar artery.

BACKGROUND AND AIM: Hyperin, a flavonol compound extracted from the Chinese herb Abelmoschus manihot L. Medic, is reported to exert protective actions in cerebral ischemic injury. The specific aim of the present study was to study the relaxation of Hyperin in rat isolated basilar artery and identify the underlying cellular mechanisms. METHODS: Rat isolated basilar artery segments were cannulated and perfused while being superfused with PSS solution. Vessel images were recorded by video microscopy and diameters measured. Membrane potential was recorded using glass microelectrodes to evaluate the basilar artery smooth muscle cell hyperpolarization. RESULTS: Perfusion of Hyperin (1~100 µM) elicited a concentration-dependent relaxation of basilar artery segments preconstricted with 0.1 µM U46619. The response was significantly inhibited by the removal of the endothelium. Hyperin also elicited marked and concentration-dependent hyperpolarization of smooth muscle cells. 30 µM nitro-L-arginine (an inhibitor of nitric oxide synthase) and indomethacin (an inhibitor of cyclooxygenase), partially inhibited Hyperin-induced relaxation and hyperpolarization leaving an attenuated, but significant, endothelium-dependent relaxation and hyperpolarization. This remaining effect was almost completely blocked by 1mM tetraethylammonium (an inhibitor of Ca(2+)-activated K(+) channels), or by 100 µM DL-propargylglycine, an inhibitor of cystathionine-γ-lyase (a synthase of the endogenous H(2)S). CONCLUSION: These findings show that Hyperin produces significant hyperpolarization in rat basilar artery smooth muscle cells and relaxation through both endothelium-dependent and endothelium-independent mechanisms. The underlying mechanisms appeared to be multi-factorial involving nitric oxide, prostacyclin, and endothelium-derived hyperpolarizing factor (EDHF). Our data further suggest that endogenous H(2)S is a component of the EDHF-mediated hyperpolarization and relaxation to Hyperin.