Po-Yi 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.

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.

Memantine Inhibits α3β2-nAChRs-Mediated Nitrergic Neurogenic Vasodilation in Porcine Basilar Arteries

Memantine, an NMDA receptor antagonist used for treatment of Alzheimer’s disease (AD), is known to block the nicotinic acetylcholine receptors (nAChRs) in the central nervous system (CNS). In the present study, we examined by wire myography if memantine inhibited α3β2-nAChRs located on cerebral perivascular sympathetic nerve terminals originating in the superior cervical ganglion (SCG), thus, leading to inhibition of nicotine-induced nitrergic neurogenic dilation of isolated porcine basilar arteries. Memantine concentration-dependently blocked nicotine-induced neurogenic dilation of endothelium-denuded basilar arteries without affecting that induced by transmural nerve stimulation, sodium nitroprusside, or isoproterenol. Furthermore, memantine significantly inhibited nicotine-elicited inward currents in Xenopous oocytes expressing α3β2-, α7- or α4β2-nAChR, and nicotine-induced calcium influx in cultured rat SCG neurons. These results suggest that memantine is a non-specific antagonist for nAChR. By directly inhibiting α3β2-nAChRs located on the sympathetic nerve terminals, memantine blocks nicotine-induced neurogenic vasodilation of the porcine basilar arteries. This effect of memantine is expected to reduce the blood supply to the brain stem and possibly other brain regions, thus, decreasing its clinical efficacy in the treatment of Alzheimer’s disease.

L-type calcium channels in sympathetic α3β2-nAChR-mediated cerebral nitrergic neurogenic vasodilation

Nicotine stimulation of α3β2‐nicotinic acetylcholine receptors (α3β2‐nAChRs) located on sympathetic nerves innervating basilar arteries causes calcium‐dependent noradrenaline release, leading to activation of parasympathetic nitrergic nerves and dilation of basilar arteries. This study aimed to investigate the major subtype of calcium channels located on cerebral peri‐vascular sympathetic nerves, which is involved in nicotine‐induced α3β2‐nAChR‐mediated nitrergic vasodilation in basilar arteries.

Inhibition by ketamine and amphetamine analogs of the neurogenic nitrergic vasodilations in porcine basilar arteries

The abuse of ketamine and amphetamine analogs is associated with incidence of hypertension and strokes involving activation of sympathetic activities. Large cerebral arteries at the base of the brain from several species receive dense sympathetic innervation which upon activation causes parasympathetic-nitrergic vasodilation with increased regional blood flow via axo-axonal interaction mechanism, serving as a protective mechanism to meet O2 demand in an acutely stressful situation. The present study was designed to examine effects of ketamine and amphetamine analogs on axo-axonal interaction-mediated neurogenic nitrergic vasodilation in porcine basilar arteries using techniques of blood-vessel myography, patch clamp and two-electrode voltage clamp, and calcium imaging. In U46619-contracted basilar arterial rings, nicotine (100μM) and electrical depolarization of nitrergic nerves by transmural nerve stimulation (TNS, 8Hz) elicited neurogenic nitrergic vasodilations. Ketamine and amphetamine analogs concentration-dependently inhibited nicotine-induced parasympathetic-nitrergic vasodilation without affecting that induced by TNS, nitroprusside or isoproterenol. Ketamine and amphetamine analogs also concentration-dependently blocked nicotine-induced inward currents in Xenopus oocytes expressing α3β2-nicotinic acetylcholine receptors (nAChRs), and nicotine-induced inward currents as well as calcium influxes in rat superior cervical ganglion neurons. The potency in inhibiting both inward-currents and calcium influxes is ketamine>methamphetamine>hydroxyamphetamine. These results indicate that ketamine and amphetamine analogs, by blocking nAChRs located on cerebral perivascular sympathetic nerves, reduce nicotine-induced, axo-axonal interaction mechanism-mediated neurogenic dilation of the basilar arteries. Chronic abuse of these drugs, therefore, may interfere with normal sympathetic-parasympathetic interaction mechanism resulting in diminished neurogenic vasodilation and, possibly, normal blood flow in the brainstem.

Bacterial toxins activation of abbreviated urea cycle in porcine cerebral vascular smooth muscle cells

Nitric oxide (NO) overproduction via induction of inducible nitric oxide synthase (iNOS) is implicated in vasodilatory shock in sepsis, leading to septic encephalopathy and accelerating cerebral ischemic injury. An abbreviated urea-cycle (l-citrulline-l-arginine-NO cycle) has been demonstrated in cerebral perivascular nitrergic nerves and endothelial cells but not in normal cerebral vascular smooth muscle cell (CVSMC). This cycle indicates that argininosuccinate synthase (ASS) catalyzes l-citrulline (l-cit) conversion to form argininosuccinate (AS), and subsequent AS cleavage by argininosuccinate lyase (ASL) forms l-arginine (l-arg), the substrate for NO synthesis. The possibility that ASS enzyme in this cycle was induced in the CVSMC in sepsis was examined. Blood-vessel myography technique was used for measuring porcine isolated basilar arterial tone. NO in cultured CVSMC and in condition mediums were estimated by diaminofluorescein (DAF)-induced fluorescence and Griess reaction, respectively. Immunohistochemical and immunoblotting analyses were used to examine iNOS and ASS induction. l-cit and l-arg, which did not relax endothelium-denuded normal basilar arteries precontracted by U-46619, induced significant vasorelaxation with increased NO production in these arteries and the CVSMCs following 6-hour exposure to 20μg/ml lipopolysaccharide (LPS) or lipoteichoic acid (LTA). Pre-treatment with pyrrolidine dithiocarbamate (PDTC) and salicylate (SAL) (NFκB inhibitors), aminoguanidine (AG, an iNOS inhibitor), and nitro-l-arg (NLA, a non-specific NOS inhibitor) blocked NO synthesis in the CVSMC and attenuated l-cit- and l-arg-induced relaxation of LPS- and LTA-treated arteries. Furthermore, immunohistochemical and immunoblotting studies demonstrated that expression of basal iNOS and ASS in the smooth muscle cell of arterial segments denuded of endothelium and the cultured CVSMCs was significantly increased following 6-hour incubation with LPS or LTA. This increased iNOS- and ASS-proteins expression in both preparations was inhibited by SAL, but was further increased by AG. These results indicate that LPS and LTA induce the l-cit-l-arg-NO cycle via induction of iNOS and ASS in the CVSMCs, accounting for massively increased NO-production and cerebral vasodilation in septic shock. Simultaneous inhibition of both pathways and NFκB-activation may be necessary to efficiently decrease or normalize NO production in the CVSMCs in this disease condition, and/or prevention and treatment of cerebral vessel-related brain dysfunctions. Our results further suggest to avoid using iNOS inhibitors alone which may cause upregulation of iNOS and ASS resulted from feedback-inhibition of iNOS activity. Accordingly, combined treatments with specific iNOS-activity inhibitor and inhibitor for iNOS genomic expression may provide a strategy in optimally managing brain sepsis and related encephalopathy associated with enhanced iNOS expression and NO overproduction.