Mei-Fang Chen

Role of Perivascular Adipose Tissue–Derived Methyl Palmitate in Vascular Tone Regulation and Pathogenesis of Hypertension

Background— Perivascular adipose tissue (PVAT)–derived relaxing factor (PVATRF) significantly regulates vascular tone. Its chemical nature remains unknown. We determined whether palmitic acid methyl ester (PAME) was the PVATRF and whether its release and/or vasorelaxing activity decreased in hypertension. Methods and Results— Using superfusion bioassay cascade technique, tissue bath myography, and gas chromatography/mass spectrometry, we determined PVATRF and PAME release from aortic PVAT preparations of Wistar Kyoto rats and spontaneously hypertensive rats. The PVAT of Wistar Kyoto rats spontaneously and calcium dependently released PVATRF and PAME. Both induced aortic vasorelaxations, which were inhibited by 4-aminopyridine (2 mmol/L) and tetraethylammonium 5 and 10 mmol/L but were not affected by tetraethylammonium 1 or 3 mmol/L, glibenclamide (3 &mgr;mol/L), or iberiotoxin (100 nmol/L). Aortic vasorelaxations induced by PVATRF- and PAME-containing Krebs solutions were not affected after heating at 70°C but were equally attenuated after hexane extractions. Culture mediums of differentiated adipocytes, but not those of fibroblasts, contained significant PAME and caused aortic vasorelaxation. The PVAT of spontaneously hypertensive rats released significantly less PVATRF and PAME with an increased release of angiotensin II. In addition, PAME-induced relaxation of spontaneously hypertensive rats aortic smooth muscle diminished drastically, which was ameliorated significantly by losartan. Conclusions— We found that PAME is the PVATRF, causing vasorelaxation by opening voltage-dependent K+ channels on smooth muscle cells. Diminished PAME release and its vasorelaxing activity and increased release of angiotensin II in the PVAT suggest a noble role of PVAT in pathogenesis of hypertension. The antihypertensive effect of losartan is attributed partly to its reversing diminished PAME-induced vasorelaxation.

Alpha-bungarotoxin binding to target cell in a developing visual system by carboxylated nanodiamond

Biological molecules conjugating with nanoparticles are valuable for applications including bio-imaging, bio-detection, and bio-sensing. Nanometer-sized diamond particles have excellent electronic and chemical properties for bio-conjugation. In this study, we manipulated the carboxyl group produced on the surface of nanodiamond (carboxylated nanodiamond, cND) for conjugating with alpha-bungarotoxin (α-BTX), a neurotoxin derived from Bungarus multicinctus with specific blockade of alpha7-nicotinic acetylcholine receptor (α7-nAChR). The electrostatic binding of cND-α-BTX was mediated by the negative charge of the cND and the positive charge of the α-BTX in physiological pH conditions. Sodium dodecyl sulfate-polyacrylamide gel analysis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF-MS) spectra displayed that α-BTX proteins were conjugated with cND particles via non-covalent bindings. The green fluorescence of the cND particles combining with the red fluorescence of tetramethylrhodamine-labeled α-BTX presented a yellow color at the same location, which indicated that α-BTX proteins were conjugated with cND particles. Xenopus laeviss oocytes expressed the human α7-nAChR proteins by microinjection with α7-nAChR mRNA. The cND-α-BTX complexes were bound to α7-nAChR locating on the cell membrane of oocytes and human lung A549 cancer cells analyzed by laser scanning confocal microscopy. The choline-evoked α7-nAChR-mediated inward currents of the oocytes were blocked by cND-α-BTX complexes in a concentration-dependent manner using two-electrode voltage-clamp recording. Furthermore, the fluorescence intensity of cND-α-BTX binding on A549 cells could be quantified by flow cytometry. These results indicate that cND-conjugated α-BTX still preserves its biological activity in blocking the function of α7-nAChR, and provide a visual system showing the binding of α-BTX to α7-nAChR.

Cholinesterase Inhibitor Blockade and its Prevention by Statins of Sympathetic α7-nAChR-Mediated Cerebral Nitrergic Neurogenic Vasodilation

Cholinesterase inhibitors (ChEIs) have been used to treat Alzheimers disease (AD). The efficacy of these drugs, however, is less than satisfactory. The possibility that ChEIs may have effects unrelated to ChE activity, such as negatively modulate neuronal nicotinic acetylcholine receptors (nAChRs) was evaluated. Since α7-nAChRs on cerebral perivascular sympathetic neurons mediate cerebral parasympathetic-nitrergic vasodilation, effects of physostigmine, neostigmine, and galantamine on α7-nAChR-mediated dilation in isolated porcine basilar arterial rings denuded of endothelium was examined using in vitro tissue bath technique. The results indicated that these ChEIs blocked vasodilation induced by choline (0.3 mmol/L), nicotine (0.1 mmol/L), and transmural nerve stimulation (TNS). The ChEl inhibition of dilation induced by TNS but not by choline or nicotine was prevented by atropine (0.1 μmol/L) pretreatment. Furthermore, using confocal microscopy, significant calcium influx induced by choline and nicotine in cultured porcine superior cervical ganglion (SCG) cells was attenuated by ChEIs. In α7-nAChR-expressed Xenopus oocytes, nicotine-induced inward currents were attenuated by α-bungarotoxin and ChEIs. Moreover, ChEI inhibition of nicotine- and choline-induced dilation was prevented by pretreatment with mevastatin and lovastatin (10 μmol/L), which did not affect ChEI inhibition of TNS-induced relaxation. These findings suggest that ChEIs inhibit the α7-nAChRs located on postganglionic sympathetic nerve terminals of SCG origin, causing a decreased release of nitric oxide in the neighboring nitrergic nerves and cerebral vasodilation. Inhibition of α7-nAChRs leading to a potential cerebral hypoperfusion may contribute to the limitation of ChEIs and question the validity of using a ChEI alone in treating AD. The efficacy of ChEIs may be improved by concurrent use of statins.

Oroxylin-A rescues LPS-induced acute lung injury via regulation of NF-κB signaling pathway in rodents.

Background and Purpose Successful drug treatment for sepsis-related acute lung injury (ALI) remains a major clinical problem. This study was designed to assess the beneficial effects of post-treatment of oroxylin A (OroA), a flavonoid, in ameliorating lipopolysaccharides (LPS)-induced lung inflammation and fatality. Experimental Approach Rats were injected with LPS (10 mg/kg, iv) to induce ALI, and OroA was given (15 mg/kg, iv) 1 hr or 6 hrs after LPS challenge. Twenty four hrs after LPS challenge, biochemical changes in the blood and lung tissues, and morphological/histological alterations in the lung associated with inflammation and injury were examined. Therapeutic effect of OroA was assessed by measuring the survival rate in endotoxemic mice. Key Results LPS (10 mg/kg, iv) significantly altered WBC counts, elevated plasma tumor necrosis factor (TNF)-α and nitric oxide (NO), increased pulmonary edema, thickened alveolar septa, and decreased survival rate. These changes were ameliorated by OroA (15 mg/kg, iv) administered 1 hr or 6 hrs after LPS challenge. This post-treatment also significantly attenuated LPS-induced activation of nuclear factor-κB (NF-κB) and the release of high mobility group box 1 (HMGB1) in lung tissues. Furthermore, post-treatment with OroA (60 mg/kg, ip) administered 1 hr or 6 hrs after LPS challenge in mice significantly increased survival rate. Conclusion and Implication OroA administered after induction of ALI by LPS significantly prevent and revere lung tissues injuries with increased survival rate. Positive post-treatment effects of OroA suggest that OroA is a potentially useful candidate for managing lung inflammation in LPS-induced endotoxemia and septic shock.

Statins prevent β-amyloid inhibition of sympathetic α7-nAChR-mediated nitrergic neurogenic dilation in porcine basilar arteries

The exact mechanism underlying regional cerebral hypoperfusion in the early phase of Alzheimers disease (AD) is not understood. We have shown in isolated porcine cerebral arteries that stimulation of sympathetic α7-nicotinic acetylcholine receptors (α7-nAChRs) causes release of nitric oxide in parasympathetic nitrergic nerves and vasodilation. We therefore examined if β-amyloid peptides (Aβs), which play a key role in pathogenesis of AD, blocked sympathetic α7-nAChRs leading to reduced neurogenic nitrergic dilation in isolated porcine basilar arteries, using in vitro tissue bath, calcium image, and patch clamping techniques. The results indicated that Aβ1–40, but not Aβ40–1, blocked relaxation of endothelium-denuded basilar arterial rings induced by nicotine (100μ;mol/L) and choline (1 mmol/L) without affecting that induced by sodium nitroprusside or isoproterenol. In cultured superior cervical ganglion (SCG) cells, Aβ1–40, but not Aβ40–1, blocked choline- and nicotine-induced calcium influx and inward currents. The Aβ blockade of the nitrergic vasodilation and inward currents, but not that of calcium influx, was prevented by acute pretreatment with 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors mevastatin and lovastatin. These results suggest that Aβ1–40 blocks cerebral perivascular sympathetic α7-nAChRs, resulting in the attenuation of cerebral nitrergic neurogenic vasodilation. This effect of Aβ may be responsible in part for cerebral hypoperfusion occurred in the early phase of the AD, which may be prevented by statins most likely because of their effects independent of cholesterol lowering. Statins may offer an alternative strategy in the prevention and treatment of AD.

Presynaptic Prostaglandin E2 EP1-Receptor Facilitation of Cerebral Nitrergic Neurogenic Vasodilation

Background and Purpose— Prostaglandin E2 (PGE2) modulates autonomic transmission in the peripheral circulation. We investigated the role of endogenous PGE2 and its presynaptic EP1 receptor subtype in modulating the autonomic neurotransmission in cerebral vasculature. Methods— The standard in vitro tissue-bath technique was used for measuring changes in isolated porcine basilar arterial tone. Calcium imaging and nitric oxide estimation along with immunohistochemical analysis for cyclo-oxygenase-1, cyclo-oxygenase-2, EP1 receptor, PGE synthase, and neuronal nitric oxide synthase were done in cultured sphenopalatine ganglia and basilar artery. Results— Selective EP1 receptor antagonists (SC-19220 and SC-51322) inhibited relaxation of endothelium-denuded basilar arterial rings elicited by transmural nerve stimulation (2 and 8 Hz) without affecting that induced by nicotine or sodium nitroprusside (a nitric oxide donor). The SC-19220 inhibition of transmural nerve stimulation-elicited relaxation was blocked by cyclo-oxygenase inhibitors (salicylic acid and naproxen) but was not affected by guanethidine (a sympathetic neuronal blocker) or atropine. Perivascular cyclo-oxygenase-1- and cyclo-oxygenase-2-immunoreactive fibers were observed in basilar arteries. PGE synthase and EP1 receptor immunoreactivities were coincident with neuronal nitric oxide synthase immunoreactivities in perivascular nerves of the basilar arteries and the sphenopalatine ganglia. &ohgr;-conotoxin (an N-type calcium channel blocker) significantly blocked transmural nerve stimulation-induced relaxation, which was further attenuated by SC-19220. In cultured sphenopalatine ganglia neurons, exogenous PGE2 significantly increased calcium influx and diaminofluorescein fluorescence indicative of nitric oxide synthesis. Both responses were blocked by SC-19220. Conclusions— These results suggest that neuronal PGE2 facilitates nitric oxide release from the cerebral perivascular parasympathetic nitrergic nerve terminals by increasing neuronal calcium influx through activation of presynaptic EP1 receptors. PGE2 may play an important role in regulating the nitrergic neurovascular transmission in the cerebral circulation.

STAT1 mediates oroxylin a inhibition of iNOS and pro-inflammatory cytokines expression in microglial BV-2 cells.

Microglia-mediated inflammation is implicated in pathogenesis of neurodegenerative diseases. Oroxylin A, a flavonoid isolated from Scutellariae baicalensis, has been shown to ameliorate microglia activation-mediated neurodegeneration in vivo. The molecular mechanism underlying the inhibitory effects of oroxylin A on microglia activation, however, remains unknown. In the present study, effects of oroxylin A co-treated with lipopolysaccharide (LPS, 100 ng/ml) on LPS-induced activation of cultured microglial BV-2 cells were examined. Nitric oxide (NO) production was determined by Greiss method. Expression of inducible nitric oxide synthase (iNOS), interleukin (IL)-1β and IL-6 was assessed using real-time RT-PCR or Western blot analysis. Furthermore, activation of the nuclear factor κB (NFκB) and the signal transducer and activator of transcription 1 (STAT1) was examined by Western blot analysis and transcription factor DNA-binding activity assay. Our results indicated that oroxylin A (10–100 µM) in a concentration-dependent manner inhibited LPS-induced NO production via blocking iNOS expression at both mRNA and protein levels without affecting the degradation rate of iNOS mRNA. Moreover, oroxylin A significantly attenuated LPS-induced late expression (20 hours after LPS challenge) of IL-1β and IL-6. Furthermore, oroxylin A significantly suppressed LPS-induced JAK2-mediated STAT1 phosphorylation without affecting LPS-induced NFκB-p65 nuclear translocation or NFκB-p65 DNA-binding activity. This is consistent with the finding that AG490, a specific JAK2 inhibitor, significantly inhibited LPS-induced STAT1 phosphorylation with almost completely diminished iNOS expression. These results suggest that oroxylin A, via suppressing STAT1 phosphorylation, inhibits LPS-induced expression of pro-inflammatory genes in BV-2 microglial cells.

Methyl Palmitate: A Potent Vasodilator Released in the Retina

PURPOSE To determine whether palmitic acid methyl ester (PAME) or methyl palmitate is the retina-derived relaxing factor (RRF). METHODS A superfusion bioassay cascade technique was used with rat isolated retina as donor tissue and rat aortic ring as detector tissue. The superfusate was analyzed with gas chromatography/mass spectrometry (GC/MS). The biochemical and pharmacologic characteristics of RRF and PAME were compared. RESULTS The authors demonstrated that the retina on superfusion with Krebs solution spontaneously released RRF (indicated by aortic ring relaxation) and PAME (measured by GC/MS). The release of RRF and PAME was calcium dependent because the release was abolished when the retinas were superfused with calcium-free Krebs solution. Furthermore, aortic relaxations induced by RRF and PAME were not affected after heating their solutions at 70 degrees C for 1 hour, suggesting that both are heat stable. Exogenous PAME concentration dependently induced aortic relaxation with EC50 of 0.82+/-0.75 pM. The aortic relaxations induced by RRF and exogenous PAME were inhibited by 4-aminopyridine (2 mM) and tetraethylammonium (TEA, 10 mM) but were not affected by TEA at 1 mM or 3 mM, glibenclamide (3 microM), or iberiotoxin (100 nM). The vasodilator activity of Krebs solution containing RRF or exogenous PAME was greatly attenuated after hexane extraction. CONCLUSIONS RRF and PAME share similar biochemical properties and react similarly to all pharmacologic inhibitors examined. Both act primarily on the voltage-dependent K+ (Kv) channel of aortic smooth muscle cells, causing aortic relaxation. These results suggest that PAME is the hydrophobic RRF.

Endogenous methyl palmitate modulates nicotinic receptor-mediated transmission in the superior cervical ganglion

Nitric oxide (NO) is identified as the endothelium-derived relaxing factor and a neurotransmitter with a superfusion bioassay cascade technique. By using a similar technique with rat superior cervical ganglion (SCG) as donor tissue and rabbit endothelium-denuded aortic ring as detector tissue, we report here that a vasodilator, which is more potent than NO, is released in the SCG upon field electrical stimulation (FES) or addition of nicotine. Release of this vasodilator was enhanced by arginine analogs, including Nω-nitro-l-arginine (a NO synthase inhibitor), suggesting that it is not NO. Analysis by gas chromatography/mass spectrometry identified 2 saturated fatty acids, palmitic acid methyl ester (PAME) and stearic acid methyl ester (SAME), being released from the SCG upon FES in the presence of arginine analogs. Exogenous PAME but not SAME induced significant aortic dilation (EC50 = 0.19 nM), indicating that PAME is the potent vasodilator. Release of PAME and SAME was significantly diminished in chronically decentralized SCG but not denervated SCG, suggesting the preganglionic origin. Furthermore, release of both fatty acids was calcium- and myosin light chain kinase-dependent, suggesting that both were released from axoplasmic vesicular stores. Electrophysiological studies further demonstrated that PAME but not SAME inhibited nicotine-induced inward currents in cultured SCG and the α7-nicotinic acetylcholine receptor-expressing Xenopus oocytes. Endogenous PAME appears to play a role in modulation of the autonomic ganglionic transmission and to complement the vasodilator effect of NO.

Axo-axonal interaction in autonomic regulation of the cerebral circulation

Noradrenaline (NE) and acetylcholine (ACh) released from the sympathetic and parasympathetic neurones in cerebral blood vessels were suggested initially to be the respective vasoconstricting and dilating transmitters. Both substances, however, are extremely weak post‐synaptic transmitters. Compelling evidence indicates that nitric oxide (NO) which is co‐released with ACh from same parasympathetic nerves is the major transmitter for cerebral vasodilation, and its release is inhibited by ACh. NE released from the sympathetic nerve, acting on presynaptic β2‐adrenoceptors located on the neighbouring parasympathetic nitrergic nerves, however, facilitates NO release with enhanced vasodilation. This axo‐axonal interaction mediating NE transmission is supported by close apposition between sympathetic and parasympathetic nerve terminals, and has been shown in vivo at the base of the brain and the cortical cerebral circulation. This result reveals the physiological need for increased regional cerebral blood flow in ‘fight‐or‐flight response’ during acute stress. Furthermore, α7‐ and α3β2‐nicotinic ACh receptors (nAChRs) on sympathetic nerve terminals mediate release of NE, leading to cerebral nitrergic vasodilation. α7‐nAChR‐mediated but not α3β2‐nAChR‐mediated cerebral nitrergic vasodilation is blocked by β‐amyloid peptides (Aβs). This may provide an explanation for cerebral hypoperfusion seen in patients with Alzheimer’s disease. α7‐ and α3β2‐nAChR‐mediated nitrergic vasodilation is blocked by cholinesterase inhibitors (ChEIs) which are widely used for treating Alzheimer’s disease, leading to possible cerebral hypoperfusion. This may contribute to the limitation of clinical use of ChEIs. ChEI blockade of nAChR‐mediated dilation like that by Aβs is prevented by statins pretreatment, suggesting that efficacy of ChEIs may be improved by concurrent use of statins.