COMT-Catalyzed Palmitic Acid Methyl Ester Biosynthesis in Perivascular Adipose Tissue and its Potential Role Against Hypertension.

Decreased release of palmitic acid methyl ester (PAME), a vasodilator, from perivascular adipose tissue (PVAT) might contribute to hypertension pathogenesis. However, the PAME biosynthetic pathway remains unclear. In this study, we hypothesized that PAME is biosynthesized from palmitic acid (PA) via human catechol-O-methyltransferase (COMT) catalysis and that decreased PAME biosynthesis plays a role in hypertension pathogenesis. We compared PAME biosynthesis between age-matched normotensive Wistar Kyoto (WKY) rats and hypertensive spontaneously hypertensive rats (SHRs) and investigated the effects of losartan treatment on PAME biosynthesis. Computational molecular modeling indicated that PA binds well at the active site of COMT. Furthermore, in in vitro enzymatic assays in the presence of COMT and S-5'-adenosyl-L-methionine (AdoMet), the stable isotope [13C16]-PA was methylated to form [13C16]-PAME in incubation medium or the Krebs-Henseleit solution containing 3T3-L1 adipocytes or rat PVAT. The adipocytes and PVATs expressed membrane-bound (MB)-COMT and soluble (S)-COMT proteins. [13C16]-PA methylation to form [13C16]-PAME in 3T3-L1 adipocytes and rat PVAT was blocked by various COMT inhibitors, such as S-(5'-adenosyl)-L-homocysteine, adenosine-2',3'-dialdehyde, and tolcapone. MB- and S-COMT levels in PVATs of established SHRs were significantly lower than those in PVATs of age-matched normotensive WKY rats, with decreased [13C16]-PA methylation to form [13C16]-PAME. This decrease was reversed by losartan, an angiotensin II (Ang II) type 1 receptor antagonist. Therefore, PAME biosynthesis in rat PVAT is dependent on AdoMet, catalyzed by COMT, and decreased in SHRs, further supporting the role of PVAT/PAME in hypertension pathogenesis. Moreover, the antihypertensive effect of losartan might be due partly to its increased PAME biosynthesis. SIGNIFICANCE STATEMENT: PAME is a key PVAT-derived relaxing factor. We for the first time demonstrate that PAME is synthesized through PA methylation via the S-5'-adenosyl-L-methionine-dependent COMT catalyzation pathway. Moreover, we confirmed PVAT dysfunction in the hypertensive state. COMT-dependent PAME biosynthesis is involved in Ang II receptor type 1-mediated blood pressure regulation, as evidenced by the reversal of decreased PAME biosynthesis in PVAT by losartan in hypertensive rats. This finding might help in developing novel therapeutic or preventive strategies against hypertension.

Long-Term Exposure to Oroxylin A Inhibits Metastasis by Suppressing CCL2 in Oral Squamous Cell Carcinoma Cells.

Oroxylin A (Oro-A), the main bioactive flavonoid extracted from Scutellaria radix, has been reported to inhibit migration in various human cancer cell models. In this study, we further explored the anti-migration effects of Oro-A on oral squamous cell carcinoma (OSCC) cells and investigated the underlying mechanisms. A 24-h (short-term) exposure of OSCC cells to non-cytotoxic concentrations (5⁻20 µM) of Oro-A significantly suppressed cell migration according to a wound-healing assay. Furthermore, a 30-day exposure (long-term) to Oro-A (20 µM), which did not exhibit a cytotoxic effect on OSCC cells, significantly suppressed cell migration more than short-term Oro-A exposure. To uncover the molecular mechanisms underlying the inhibitory effect of long-term Oro-A exposure on OSCC migration, a cDNA microarray and the Ingenuity software were used. Overall, 112 upregulated and 356 downregulated genes were identified in long-term Oro-A-exposed cells compared with untreated OSCC cells. Among them, 75 genes were reported to be associated with cancer cell migration. Consistent with the cDNA microarray results, we found that the expression levels of several cell migration-related genes, such as LCN2, ID-1, MDK, S100A9 and CCL2, were significantly decreased in long-term Oro-A-exposed OSCC cells using a quantitative real-time polymerase chain reaction (Q-PCR) assay. The Western blotting and enzyme-linked immunosorbent assay (ELISA) results also demonstrated that CCL2 expression at the mRNA and protein levels was significantly decreased in long-term Oro-A-exposed OSCC cells compared with untreated OSCC cells. Moreover, the expression levels of downstream CCL2 targets, including p-ERK1/2, NFκB, MMP2, and MMP9, were also decreased in long-term Oro-A-exposed OSCC cells. Further, Oro-A treatment suppressed in vivo metastasis. These results suggest that long-term Oro-A treatment inhibits metastasis via CCL2 signaling in OSCC cells.

An engineered arginine-rich α-helical antimicrobial peptide exhibits broad-spectrum bactericidal activity against pathogenic bacteria and reduces bacterial infections in mice.

The increase in the prevalence of antibiotic-resistant bacteria has become a major public health concern. Antimicrobial peptides (AMPs) are emerging as promising candidates addressing this issue. In this study, we designed several AMPs by increasing α-helical contents and positive charges and optimizing hydrophobicity and amphipathicity in the Sushi 1 peptide from horseshoe crabs. A neural network-based bioinformatic prediction tool was used for the first stage evaluations of peptide properties. Among the peptides designed, Sushi-replacement peptide (SRP)-2, an arginine-rich and highly α-helical peptide, showed broad-spectrum bactericidal activity against both Gram-positive and Gram-negative bacteria, including methicillin-resistant Staphylococcus aureus and multidrug-resistant Acinetobacter baumannii; nevertheless, it showed little hemolytic and cytotoxic activity against mammalian cells. Atomic force microscopy results indicated that SRP-2 should interact directly with cell membrane components, resulting in bacterial cell death. SRP-2 also neutralized LPS-induced macrophage activation. Moreover, in an intraperitoneal multidrug-resistant A. baumannii infection mouse model, SRP-2 successfully reduced the bacterial number in ascitic fluid and tumor necrosis factor-α production. Our study findings demonstrate that bioinformatic calculations can be powerful tools to help design potent AMPs and that arginine is superior to lysine for providing positive charges for AMPs to exhibit better bactericidal activity and selectivity against bacterial cells.

Associations among medication regimen complexity, medical specialty, and medication possession ratio in newly diagnosed hypertensive patients: A population-based study.

The aim of this study was to explore the associations among the medication regimen complexity index (MRCI), medical specialty, and medication possession ratio (MPR) in newly diagnosed hypertensive patients.Data from 19,859 newly diagnosed hypertensive patients were collected from 2,000,000 random samples of the National Health Insurance Research Database in Taiwan. All study participants were followed for 1 year after the first diagnosis of hypertension. MPR was defined as total days of antihypertensive drugs supplied/365 days. MRCI was calculated on the basis of the type of dosage forms, dosing frequency, and additional directions for use of antihypertensive drugs. Patients were further restricted to those who visited the same medical specialty to examine specialty-specific variations in the MRCI and MPR.The mean MPR was 54.83%, and the sample sizes for the low-, medium-, and high-MPR groups were 9806 (49.38%), 4619 (23.26%), and 5434 (27.36%), respectively. More than 50% of the patients visited the same medical specialty during the 1-year follow-up. The mean MRCI was 3.64; the cardiology specialty had the highest MRCI, and the family medicine specialty had the lowest. Multiple linear regression analyses showed that MRCI was negatively associated with MPR (ß = -7.75, P ≤ .01) whether or not the patients visited the same medical specialty. For the patients who visited the same medical specialty, those treated by endocrinology and metabolism specialists had a significantly higher MPR (ß = 9.87, P ≤ .01) than that of those treated by family medicine specialists.MRCI and medical specialty were both significantly associated with the MPR of newly diagnosed hypertensive patients.

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 α3ß2-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.

Neurochemicals involved in medullary control of common carotid blood flow.

The common carotid artery (CCA) supplies intra- and extra-cranial vascular beds. An area in the medulla controlling CCA blood flow is defined as the dorsal facial area (DFA) by Kuo et al. in 1987. In the DFA, presynaptic nitrergic and/or glutamatergic fibers innervate preganglionic nitrergic and/or cholinergic neurons which give rise to the preganglionic fibers of the parasympathetic 7th and 9th cranial nerves. Released glutamate from presynaptic nitrergic and/or glutamatergic fibers can activate N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors on preganglionic nitrergic and/or cholinergic neurons. By modulating this glutamate release, several neurochemicals including serotonin, arginine, nitric oxide, nicotine, choline and ATP in the DFA regulate CCA blood flow. Understanding the neurochemical regulatory mechanisms can provide important insights of the physiological roles of the DFA, and may help develop therapeutic strategies for diseases involving CCA blood flow, such as migraine, hypertensive disease, Alzheimer's disease and cerebral ischemic stroke.

Oroxylin a, but not vasopressin, ameliorates cardiac dysfunction of endotoxemic rats.

The mortality in septic patients with myocardial dysfunction is higher than those without it. Beneficial effects of flavonoid oroxylin A (Oro-A) on endotoxemic hearts were evaluated and compared with that of arginine vasopressin (AVP) which is used to reverse hypotension in septic patients. Endotoxemia in rats was induced by one-injection of lipopolysaccharides (LPS, 10 mg/kg, i.p.), and hearts were isolated 5-hrs or 16-hrs later. Isolated hearts with constant-pressure or constant-flow mode were examined by Langendorff technique. Rate and force of contractions of isolated atrial and ventricular strips were examined by tissue myography. Isolated endotoxemic hearts were characterized by decreased or increased coronary flow (CF) in LPS-treated-for-5hr and LPS-treated-for-16-hr groups, respectively, with decreased inotropy in both groups. Oro-A-perfusion ameliorated while AVP-perfusion worsened the decreased CF and inotropy in both preparations. Oro-A and AVP, however, did not affect diminished force or rate of contraction of atrial and ventricular strips of endotoxemic hearts. Oro-A-induced CF increase was not affected following coronary endothelium-denudation with saponin. These results suggest that Oro-A ameliorates LPS-depressed cardiac functions by increasing CF, leading to positive inotropy. In contrast, AVP aggravates cardiac dysfunction by decreasing CF. Oro-A is a potentially useful candidate for treating endotoxemia complicated with myocardial dysfunction.

Memantine inhibits α3ß2-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 α3ß2-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 α3ß2-, α7- or α4ß2-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 α3ß2-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.

Bimodal effects of fluoxetine on cerebral nitrergic neurogenic vasodilation in porcine large cerebral arteries.

Fluoxetine-induced relaxation of the smooth muscle of small cerebral arteries is thought beneficial in treating mental disorders. The present study was designed to examine effect of fluoxetine on neurogenic nitrergic vasodilation in large cerebral arteries, using in vitro tissue myography, techniques of electrophysiology, calcium imaging and biochemistry. In isolated porcine endothelium-denuded basilar arteries in the presence of U-46619-induced active muscle tone, fluoxetine in low concentration (<0.03 µM) significantly enhanced nicotine- and choline-induced relaxations. The vasorelaxation, however, was blocked by higher concentration of fluoxetine (>0.3 µM) with maximum inhibition at 3 µM. At this concentration, fluoxetine did not affect the basal tone or vasorelaxations induced by transmural nerve stimulation, sodium nitroprusside, or isoproterenol. Furthermore, fluoxetine exclusively blocked nicotine-induced inward currents and calcium influx in cultured neurons of rat superior cervical ganglion and Xenopus oocytes expressing human α7-, α3ß2-, or α4ß2-nicotinic acetylcholine receptors (nAChRs). In addition, fluoxetine at 0.03 µM and 3 µM significantly enhanced and blocked, respectively, nicotine-induced norepinephrine (NE) release from cerebral perivascular sympathetic nerves. These results indicate that fluoxetine via axo-axonal interaction mechanism exhibits bimodal effects on nAChR-mediated neurogenic nitrergic dilation of basilar arteries. Fluoxetine in high concentrations decreases while in low concentrations it increases neurogenic vasodilation. These results from in vitro experimentation suggest that optimal concentrations of fluoxetine which increase or minimally affect neurogenic vasodilation indicative of regional cerebral blood flow may be important consideration in treating mental disorders.

Sympathetic α3ß2-nAChRs mediate cerebral neurogenic nitrergic vasodilation in the swine.

The α(7)-nicotinic ACh receptor (α(7)-nAChR) on sympathetic neurons innervating basilar arteries of pigs crossed bred between Landrace and Yorkshire (LY) is known to mediate nicotine-induced, ß-amyloid (Aß)-sensitive nitrergic neurogenic vasodilation. Preliminary studies, however, demonstrated that nicotine-induced cerebral vasodilation in pigs crossbred among Landrace, Yorkshire, and Duroc (LYD) was insensitive to Aß and α-bungarotoxin (α-BGTX). We investigated nAChR subtype on sympathetic neurons innervating LYD basilar arteries. Nicotine-induced relaxation of porcine isolated basilar arteries was examined by tissue bath myography, inward currents on nAChR-expressing oocytes by two-electrode voltage recording, and mRNA and protein expression in the superior cervical ganglion (SCG) and middle cervical ganglion (MCG) by reverse transcription PCR and Western blotting. Nicotine-induced basilar arterial relaxation was not affected by Aß, α-BGTX, and α-conotoxin IMI (α(7)-nAChR antagonists), or α-conotoxin AuIB (α(3)ß(4)-nAChR antagonist) but was inhibited by tropinone and tropane (α(3)-containing nAChR antagonists) and α-conotoxin MII (selective α(3)ß(2)-nAChR antagonist). Nicotine-induced inward currents in α(3)ß(2)-nAChR-expressing oocytes were inhibited by α-conotoxin MII but not by α-BGTX, Aß, or α-conotoxin AuIB. mRNAs of α(3)-, α(7)-, ß(2)-, and ß(4)-subunits were expressed in both SCGs and MCGs with significantly higher mRNAs of α(3)-, ß(2)-, and ß(4)-subunits than that of α(7)-subunit. The Aß-insensitive sympathetic α(3)ß(2)-nAChR mediates nicotine-induced cerebral nitrergic neurogenic vasodilation in LYD pigs. The different finding from Aß-sensitive α(7)-nAChR in basilar arteries of LY pigs may offer a partial explanation for different sensitivities of individuals to Aß in causing diminished cerebral nitrergic vasodilation in diseases involving Aß.

Nicotine activation of neuronal nitric oxide synthase and guanylyl cyclase in the medulla increases blood flow of the common carotid artery in cats.

Individual activation of nicotinic acetylcholine receptor (nAChR) or nitric oxide (NO) synthase in the dorsal facial area (DFA) increases blood flow of common carotid artery (CCA) supplying intra- and extra-cranial tissues. We investigated whether the activation of nAChR initiated the activation of NO synthase and guanylyl cyclase to increase CCA blood flow in anesthetized cats. Microinjections of nicotine (a non-selective nAChR agonist), or choline (a selective α7-nAChR agonist) in the DFA produced increases in CCA blood flow ipsilaterally. These increases were significantly reduced by pretreatment with NG-nitro-arginine methyl ester (l-NAME, a non-specific NO synthase inhibitor), 7-nitroindazole (7-NI, a relatively selective neuronal NO synthase inhibitor) or methylene blue (MB, a guanylyl cyclase inhibitor) but not by that with N5-(1-iminoethyl)-l-ornithine (l-NIO, a potent endothelial NO synthase inhibitor). Control microinjection with d-NAME (an isomer of l-NAME), artificial cerebrospinal fluid or DMSO (a solvent for 7-NI) did not affect resting CCA blood flow, nor did they affect nicotine- or choline-induced response. In conclusion, activation of nAChR, at least α7-nAChR, led to the activation of neuronal NO synthase and guanylyl cyclase in the DFA, which induced an increase in CCA blood flow.

Nicotine stimulation of the medulla increases blood flow of the common carotid artery in cats.

Microinjection of nicotine or glutamate into the dorsal facial area (DFA) of the medulla increases blood flow of common carotid artery (CCA). Whether there is a causal relationship between these two events is not known. Various agonists and antagonists for the nicotinic and glutamatergic receptors were microinjected through a four-barrel tubing into the DFA of anesthetized cats. Microinjections of nicotine [a non-selective nicotinic acetylcholine receptor (nAChR) agonist], choline (a selective alpha7-nAChR agonist), glutamate or KCl induced a modest increase in CCA blood flow. The nicotine- and choline-induced increases were reduced by alpha-bungarotoxin (an alpha7-nAChR antagonist) as well as MK-801 (a non-competitive NMDA receptor antagonist) or glutamate diethylester (a competitive AMPA/kainate receptor antagonist). The glutamate or KCl-induced increases were blocked by MK-801 and glutamate diethylester, but not by alpha-bungarotoxin. In conclusion, activation of nAChRs primarily via alpha7-nAChR caused a release of glutamate, which in turn activated NMDA and AMPA receptors, while cholinergic substance was not released into the DFA to activate the nicotinic receptor.

Presynaptic prostaglandin E2 EP1-receptor facilitation of cerebral nitrergic neurogenic vasodilation.

BACKGROUND AND PURPOSE: Prostaglandin E(2) (PGE(2)) modulates autonomic transmission in the peripheral circulation. We investigated the role of endogenous PGE(2) and its presynaptic EP(1) 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, EP(1) receptor, PGE synthase, and neuronal nitric oxide synthase were done in cultured sphenopalatine ganglia and basilar artery. RESULTS: Selective EP(1) 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 EP(1) receptor immunoreactivities were coincident with neuronal nitric oxide synthase immunoreactivities in perivascular nerves of the basilar arteries and the sphenopalatine ganglia. omega-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 PGE(2) 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 PGE(2) facilitates nitric oxide release from the cerebral perivascular parasympathetic nitrergic nerve terminals by increasing neuronal calcium influx through activation of presynaptic EP(1) receptors. PGE(2) may play an important role in regulating the nitrergic neurovascular transmission in the cerebral circulation.

Nitric oxide and glutamate in the dorsal facial area regulate common carotid blood flow in the cat.

Nitric oxide (NO) or glutamate stimulation of dorsal facial area (DFA) increases blood flow in the common carotid artery (CCA), which supplies intra-and extra-cranial tissues. Nitrergic fibers and neurons as well as preganglionic cholinergic neurons are present in the DFA. We hypothesized the presence of nitrergic-glutamatergic fibers and preganglionic nitrergic-cholinergic neurons in the DFA that are involved in the regulation of CCA blood flow. In microdialysis studies, perfusion of the DFA with S-nitroso-N-acetylpenicillamine (SNAP, an NO donor) increased the glutamate concentration in the dialysate. This effect was abolished by co-perfusion of methylene blue (a guanylyl cyclase inhibitor). Intra-DFA injection of l-arginine (an NO precursor) or glutamate increased CCA blood flow. The l-arginine-induced flow increase was reduced by prior administration of NG-nitro-arginine methyl ester (l-NAME, a non-specific NO synthase inhibitor), 7-nitroindazole (7-NI, a relatively selective neuronal NO synthase inhibitor), d-2-amino-5-phosphonopentanoate (d-AP5, a competitive NMDA receptor antagonist), or glutamate diethylester (GDEE, a competitive AMPA receptor antagonist). The glutamate-induced blood flow increase was reduced by prior administration of l-NAME, 7-NI, or methylene blue. The induced increase in CCA blood flow, however, was not affected by endothelial NO synthase inhibitor. The findings indicate that NO-signal transduction within the DFA might cause glutamate release from presynaptic nitrergic-glutamatergic fibres and that the released glutamate activates NMDA/AMPA receptors on preganglionic nitrergic-cholinergic neurons in the nucleus to activate neuronal NO synthase and guanylyl cyclase in the neurons, leading to an increase in CCA blood flow. These findings may be important for developing therapeutic strategies for the diseases associated with CCA blood flow.

Effect of phospholipase C blockade on cerebral vasospasm.

BACKGROUND: Delayed cerebral ischemia due to cerebral vasospasm remains a major cause of morbidity and mortality following subarachnoid hemorrhage. Oxyhemoglobin (OxyHb) and vasoconstrictor prostanoids have been suggested as putative spasmogens. We have previously reported a synergistic vasoconstrictive action between thromboxane A(2) (TXA(2)) and OxyHb. In the present study we examine the effect of neomycin, a phospholipase C inhibitor, on the cerebral vasoconstriction induced by TXA(2) and OxyHb. METHODS: Using an in vitro tissue bath method, we assess the effect of neomycin in a concentration-dependent manner, on isolated porcine basilar arteries constricted by U-46619 (TXA(2) analogue) and OxyHb. RESULTS: The functional synergism between TXA(2) and OxyHb, leading to significant cerebral vasoconstriction, is attenuated in a dose-dependent manner by neomycin. CONCLUSION: Blockade of phospholipase C may provide an alternative strategy in the treatment of subarachnoid-hemorrhage-induced cerebral vasospasm.

Statins prevent beta-amyloid inhibition of sympathetic alpha7-nAChR-mediated nitrergic neurogenic dilation in porcine basilar arteries.

The exact mechanism underlying regional cerebral hypoperfusion in the early phase of Alzheimer's disease (AD) is not understood. We have shown in isolated porcine cerebral arteries that stimulation of sympathetic alpha7-nicotinic acetylcholine receptors (alpha7-nAChRs) causes release of nitric oxide in parasympathetic nitrergic nerves and vasodilation. We therefore examined if beta-amyloid peptides (Abetas), which play a key role in pathogenesis of AD, blocked sympathetic alpha7-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 Abeta(1-40), but not Abeta(40-1), blocked relaxation of endothelium-denuded basilar arterial rings induced by nicotine (100 micromol/L) and choline (1 mmol/L) without affecting that induced by sodium nitroprusside or isoproterenol. In cultured superior cervical ganglion (SCG) cells, Abeta(1-40), but not Abeta(40-1), blocked choline- and nicotine-induced calcium influx and inward currents. The Abeta 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 Abeta(1-40) blocks cerebral perivascular sympathetic alpha7-nAChRs, resulting in the attenuation of cerebral nitrergic neurogenic vasodilation. This effect of Abeta 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.

EP1- and EP3-receptors mediate prostaglandin E2-induced constriction of porcine large cerebral arteries.

Prostaglandin E2 (PGE2) has been shown to dilate and constrict the systemic vascular beds, including cerebral vessels. The exact mechanism of PGE2-induced cerebral vasoconstriction, however, is less clarified. The authors' preliminary studies showed that PGE2 exclusively constricted the adult porcine basilar arteries. The present study, therefore, was designed to examine the receptor mechanisms involved in PGE2-induced constriction of large cerebral arteries in the adult pig. Results from an in vitro tissue-bath study indicated that PGE2 and its agonists 17-phenyl trinor PGE2 (17-PGE2), sulprostone (EP1/EP3 receptor agonists), and 11-deoxy-16,16-dimethyl PGE2 (11-PGE2, an EP2/EP3-receptor agonist) induced exclusive constriction, which was not affected by endothelium denudation or cold-storage denervation of perivascular nerves. The constriction induced by PGE2, 17-PGE2, and sulprostone, but not by potassium chloride, was blocked by SC-19220 (a selective EP1-receptor antagonist), AH-6809 (an EP1/EP2-receptor antagonist), and U-73122 and neomycin (phospholipase C inhibitors). AH-6809, however, did not affect 11-PGE2-induced contraction. These results suggest that the contraction was not mediated by the EP2-receptor, but was mediated by EP1- and EP3-receptors. Furthermore, EP1-receptor immunoreactivities were found across the entire medial smooth muscle layers, whereas EP3-receptor immunoreactivities were limited to the outer smooth muscle layer toward the adventitia. Western blotting also showed the presence of EP1- and EP3-receptor proteins in cultured primary cerebral vascular smooth muscle cells. In conclusion, PGE2 exclusively constricts the adult porcine large cerebral arteries. This constriction is mediated by phosphatidyl-inositol pathway via activation of EP1- and EP3-receptors located on the smooth muscle cells. These two receptor subtypes may play important roles in physiologic and pathophysiologic control of cerebral vascular tone.

Pb2+ inhibition of sympathetic alpha 7-nicotinic acetylcholine receptor-mediated nitrergic neurogenic dilation in porcine basilar arteries.

Chronic exposure to inorganic lead (Pb2+) has been shown to facilitate peripheral vasoconstriction causing hypertension. Effect of lead on cerebral vascular function has not been reported. We have suggested in isolated porcine cerebral arteries that alpha 7-nicotinic acetylcholine receptors (alpha 7-nAChRs) on perivascular sympathetic nerves mediate calcium influx in these neurons, resulting in release of norepinephrine. The released norepinephrine then acts on presynaptic beta2-adrenoceptors located on the neighboring nitrergic nerve terminals, causing nitric oxide (NO) release and vasodilation. Because Pb2+ has been shown to inhibit alpha 7-nAChR-mediated responses in the central nervous system, effects of Pb2+ on alpha 7-nAChR-mediated nitrergic neurogenic dilation in isolated porcine basilar arteries and calcium influx in cultured superior cervical ganglion (SCG) cells of the pig were examined using in vitro tissue bath and confocal microscopic techniques. The results indicated that Pb2+ (but not Cd2+, Zn2+, or Al3+) in a concentration-dependent manner blocked relaxation of endothelium-denuded basilar arterial rings induced by nicotine (100 microM) and choline (1 mM) without affecting relaxation induced by sodium nitroprusside or isoproterenol. Furthermore, significant calcium influx in cultured SCG cells induced by choline and nicotine was attenuated specifically by Pb2+ with IC50 values comparable with those from tissue bath study. These results provide evidence supporting that lead is a likely antagonist for alpha 7-nAChRs that are found on postganglionic sympathetic adrenergic nerve terminals of SCG origin. Furthermore, these results indicate that lead can attenuate dilation of cerebral arteries by blocking sympathetic nerve-mediated release of NO from the perivascular nitrergic nerves.

Ginseng, sex behavior, and nitric oxide.

In Asia, ginseng is commonly included in herbals used for the treatment of sexual dysfunction. Recent studies in laboratory animals have shown that both Asian and American forms of ginseng enhance libido and copulatory performance. These effects of ginseng may not be due to changes in hormone secretion, but to direct effects of ginseng, or its ginsenoside components, on the central nervous system and gonadal tissues. Indeed, there is good evidence that ginsenosides can facilitate penile erection by directly inducing the vasodilatation and relaxation of penile corpus cavernosum. Moreover, the effects of ginseng on the corpus cavernosum appear to be mediated by the release and/or modification of release of nitric oxide from endothelial cells and perivascular nerves. Treatment with American ginseng also affects the central nervous system and has been shown to significantly alter the activity of hypothalamic catecholamines involved in the facilitation of copulatory behavior and hormone secretion. Recent findings that ginseng treatment decreased prolactin secretion also suggested a direct nitric oxide-mediated effect of ginseng at the level of the anterior pituitary. Thus, animal studies lend growing support for the use of ginseng in the treatment of sexual dysfunction and provide increasing evidence for a role of nitric oxide in the mechanism of ginsenoside action.