ACh-induced Increase Research Articles

Abstract 6959: Acetylcholine potentiates invasion and migration of human pancreatic cancer cells under hypoxia through α7 nAChR-mediated increases in HIF-1α expression

Abstract The most significant nerve-related problem in clinical pancreatic cancer management is perineural invasion (PNI), characterized as the neoplastic invasion of cancer cells into or surrounding the nerves. It is a characteristic feature of pancreatic ductal adenocarcinoma (PDAC) associated with a poor prognosis, tumor recurrence, and emergent pain. However, why PDAC is associated with a higher incidence of PNI remains unclear. HIF-1 (hypoxia-inducible factor-1) regulates the expression of ~1000 genes involved in angiogenesis, metastasis, tumor growth, chemoresistance and radioresistance, underscoring the growing interest in targeting HIF-1 for cancer control. In the present study, we investigated the molecular mechanisms underlying neurotransmitter acetylcholine (Ach)-potentiated HIF-1α expression and subsequent increase in invasion and migration of human pancreatic cancer cells in hypoxia (2% O2). The expression of various acetylcholine receptors in six types of pancreatic cancer cell lines (MIA-PaCa-2, PANC-1, HPAF-II, Capan-1, AsPC-1, and BxPC-3 cells) was observed using qPCR, and α7 nAChRs was observed to be expressed in all pancreatic cancer cells. Therefore, we chose MIA-PaCa-2 cells, expressing high levels of α7 nAChRs, and HPAF-II cells, expressing low levels of α7 nAChRs, for loss- and gain-of-function studies, respectively. In vitro assays showed that, under hypoxic conditions, Ach caused a concentration-dependent increase in α7 nAChRs-mediated HIF-1α expression, enhancing expression of HIF-1α target genes, and promoting invasion and migration of human pancreatic cancer cells. We further found that Ach increased HIF-1α protein stability in pancreatic cancer cells under hypoxic conditions. This effect was mediated by YAP signaling, as evidenced by the fact that Ach-induced increases in HIF-1α protein stability were blocked by siRNA-mediated knockdown of YAP. Consistent with these findings, an investigation of the relationship between α7 nAChRs and clinicopathological features in pancreatic cancer patients using publicly available datasets showed that high levels of α7 nAChRs are correlated with a poorer prognosis in pancreatic cancer patients. Collectively, these findings identify novel mechanisms by which Ach potentiates invasion and migration of pancreatic cancer cells and provide further evidence that HIF-1α is a potential anticancer target in Ach-associated pancreatic cancer. Citation Format: Yunmi Cho, Eun-Taex Oh, Ha Gyeong Kim, Heon Joo Park. Acetylcholine potentiates invasion and migration of human pancreatic cancer cells under hypoxia through α7 nAChR-mediated increases in HIF-1α expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6959.

Muscarinic receptor activation in colon cancer selectively augments pro-proliferative microRNA-21, microRNA-221 and microRNA-222 expression.

Overexpression of M3 subtype muscarinic receptors (M3R) hastens colon cancer progression. As microRNA (miRNA) expression is commonly dysregulated in cancer, we used microarrays to examine miRNA profiles in muscarinic receptor agonist-treated human colon cancer cells. We used quantitative RT-PCR (qPCR) to validate microarray results and examine miRNA expression in colon cancers and adjacent normal colon. These assays revealed that acetylcholine (ACh) treatment robustly induced miR-222 expression; miR-222 levels were three-fold higher in cancer compared to normal colon. In kinetic studies, ACh induced a 4.6-fold increase in pri-miR-222 levels within 1 h, while mature miR-222 increased gradually to 1.8-fold within 4 h. To identify post-M3R signaling mediating these actions, we used chemical inhibitors and agonists. ACh-induced increases in pri-miR-222 were attenuated by pre-incubating cells with atropine and inhibitors of protein kinase C (PKC) and p38 MAPK. Treatment with a PKC agonist, phorbol 12-myristate 13-acetate, increased pri-miR-222 levels, an effect blocked by PKC and p38 MAPK inhibitors, but not by atropine. Notably, treatment with ACh or transfection with miR-222 mimics increased cell proliferation; atropine blocked the effects of ACh but not miR-222. These findings identify a novel mechanism whereby post-M3R PKC/p38 MAPK signaling stimulates miR-222 expression and colon cancer cell proliferation.

Skin Blood Flow Responses to Acetylcholine, Local Heating, and to 60% VO2max exercise with and without Nitric Oxide inhibition, in Boys vs. Girls.

To determine sex-related differences in the skin blood flow (SkBF) response to exercise, local heating, and acetylcholine (ACh) in children, and to assess nitric oxide contribution to the SkBF response. Forearm SkBF during local heating (44°C), ACh iontophoresis, and exercise (30-min cycling and 60% of maximum oxygen consumption) was assessed, using laser Doppler fluxmetry, in 12 boys and 12 girls (7-13y old), with and without nitric oxide synthase inhibition, using Nω-nitro-L-arginine methyl ester iontophoresis. Local-heating-induced and ACh-induced SkBF increase were not different between boys and girls (local heating: 1445% [900%] and 1432% [582%] of baseline, P = .57; ACh: 673% [434%] and 558% [405%] of baseline, respectively, P = .18). Exercise-induced increase in SkBF was greater in boys than girls (528% [290%] and 374% [192%] of baseline, respectively, P = .03). Nω-nitro-L-arginine methyl ester blunted the SkBF response to ACh and during exercise (P < .001), with no difference between sexes. SkBF responses to ACh and local heat stimuli were similar in boys and girls, while the increase in SkBF during exercise was greater in boys. The apparent role of nitric oxide was not different between boys and girls. It is suggested that the greater SkBF response in boys during exercise was related to greater relative heat production and dissipation needs at this exercise intensity. The response to body size-related workload should be further examined.

Vasodilators stimulate SK3 channel anterograde trafficking in endothelial cells to elicit vasodilation

Endothelial cells (ECs) line the lumen of blood vessels and regulate functions, including contractility. Physiological stimuli, such as acetylcholine (ACh) and intraluminal flow, activate small conductance calcium‐activated potassium (SK3) channels in ECs, leading to hyperpolarization and vasodilation. Whether these stimuli modulate SK3 surface abundance in ECs to elicit vasodilation is unclear. Here, we investigated physiological regulation of SK3 channel surface trafficking in mesenteric artery (MA) ECs. Biotinylation experiments demonstrated that ACh or intravascular flow rapidly (2 min) increased surface SK3 protein ~ 2.0‐ and 2.1‐fold, respectively in MAs, with similar results obtained in primary‐cultured ECs. Brefeldin A, an anterograde trafficking inhibitor, removal of extracellular Ca2+, GSK2193874, a selective TRPV4 channel blocker or TRPV4 knockdown using siRNA, each inhibited the ACh‐induced increase in surface SK3 channels in MAs. Super resolution microscopy (SMLM) experiments demonstrated that ACh increased the size of surface SK3 clusters in ECs. ACh activated whole‐cell SK currents and this effect was inhibited by brefeldin A. Brefeldin A also reduced vasodilation induced by ACh and intravascular flow to between ~48 and 57 % of those in control. Brefeldin A blocked the SK channel component of ACh‐ and flow‐mediated vasodilations. In summary, our data indicate that ACh and flow stimulate anterograde trafficking of SK3 channels in ECs via a TRPV4‐dependent Ca2+ signaling mechanism to elicit vasodilation.

Cholinergic modulation of persistent inward currents is mediated by activating muscarinic receptors of serotonergic neurons in the brainstem of ePet-EYFP mice.

Persistent inward currents (PICs) play important roles in regulating neural excitability. Results from our previous studies showed that serotonergic (5-HT) neurons of the brainstem expressed PICs. However, little is known about cholinergic (ACh) modulation of PICs in the 5-HT neurons. The whole-cell patch-clamp recordings were performed in the brainstem slices of ePet-EYFP mice to investigate the electrophysiological properties of PICs with cholinergic modulation. PICs in 5-HT neurons were activated at -51.4 ± 3.7mV with the amplitude of -171.6 ± 48.9 pA (n = 71). Bath application of 20-25μM ACh increased the amplitude by 79.1 ± 42.5 pA (n = 23, p < 0.001) and hyperpolarized the onset voltage by 2.2 ± 2.7mV (n = 23, p < 0.01) and half-maximal activation by 3.6 ± 2.7mV (n = 6, p < 0.01). Muscarine mimicked the effects of ACh on PICs, while bath application of nicotine (15-20μM) did not induce substantial change in the PICs (n = 9). Muscarine enhanced the amplitude of PICs by 100.0 ± 27.4 pA (n = 28, p < 0.001) and lowered the onset voltage by 2.8 ± 1.2mV (n = 28, p < 0.001) and the half-maximal activation by 2.9 ± 1.4mV. ACh-induced increase of amplitude and hyperpolarization of onset voltage were blocked by 3-5μM atropine. Furthermore, the muscarine-induced enhancement of the PICs was antagonized by 5μM 4-DAMP, the antagonist of M3 receptor, while the antagonists of M1 (Telenzepine, 5μM) and M5 (VU6008667, 5μM) receptors did not significantly affect the PIC enhancement. This study suggested that ACh potentiated PICs in 5-HT neurons of the brainstem by activating muscarinic M3 receptor.

Neurotoxic effects of aluminum are associated with its interference with estrogen receptors signaling

Aluminum compounds have been observed in various brain regions, and their accumulation has been associated with many neurodegenerative disorders. Neurotoxic effects of aluminum are attributed to reactive oxygen species generation, induction of apoptosis and inflammatory reactions activation. Metalloestrogen activity of aluminum has also been linked to breast cancer progression and metastasis. In this study, taking into account the anti-apoptotic and anti-oxidant activities of estrogens in neuronal cells, which are mediated by estrogen receptors, the possible estrogenic activity of aluminum in SH-SY5Y neuroblastoma cells was studied. Our results showed that aluminum in the form of aluminum chlorohydrate (ACH) exhibited no effect on estrogen receptors transcriptional activation, and differential effect on estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) protein levels. ACH caused reduction in ERβ protein levels, and increase in its mitochondrial localization. ACH-induced reduction in ERβ protein level may be linked, at least in part, to the ACH-induced increase in ERα protein level. This statement is based on our observations showing aluminum-induced reduction in the E2-induced increase in ERα S118 phosphorylation, in MCF-7 and SH-SH5Y cells. Phosphorylation at S118 residue is known to be associated with inhibition of the ubiquitin-induced proteolytic degradation of ERα, leading to its accumulation. Since it is known that ERα negatively regulate ERβ expression, increase in ERα, may contribute to reduction in ERβ levels and subsequent weakening of its anti-apoptotic and anti-oxidant activity, justified by the observed reduction in procaspase 9, mitochondrial cytochrome c, Bcl-2, Bcl-xL and mitochondrial thioredoxin protein level, as well as by the increase in proapoptotic BAX level, in ACH treated SH-SY5Y cells. In addition, increase in mitochondrial ERβ localization may also trigger mitochondrial metabolism, suppress biosynthetic process of gluconeogenesis, as indicated by the observed reduction in the phosphoenolpyruvate carboxykinase protein level, and eventually lead to increase in reactive oxygen species (ROS) generation, known to be implicated in aluminum induced neurodegeneration. This statement was verified by the observed ACH–induced increase in ERβ mitochondrial localization, induction of the mitochondrial membrane depolarization and increase in ROS production, in neuronal-like differentiated SH-SY5Y cells.

NS8593 inhibits Ca2+ permeant channels reversing mouse airway smooth muscle contraction

AimsThis study focused on investigating whether NS8593 reverses airway smooth muscle (ASM) contraction and the underlying mechanism. Main methodsASM contraction in mouse tracheal rings and lung slices was measured. Currents mediated by voltage dependent Ca2+ channels (VDCCs) and ACH-activated channels were measured using the whole-cell patch-clamp technique in single tracheal smooth muscle cells (TSMCs). Intracellular Ca2+ level and cell length were measured using an LSM 700 laser confocal microscope and a Zen 2010 software. Mouse respiratory system resistance (Rrs) was assessed using a FlexiVent FX system. Key findingsHigh K+ (80 mM K+) and ACH induced ASM contraction in mouse tracheal rings and lung slices, which was partially relaxed by nifedipine (blocker of L-type VDCCs, LVDCCs), YM-58483 (blocker of store-operated Ca2+ entry (SOCE), transient receptor potential C3 (TRPC3) and TRPC5 channels), respectively. However, the contraction was completely reversed by NS8593, whereas, slightly relaxed by formoterol. ACH activated inward currents, which displayed linear and reversed around 0 mV, indicating the currents were mediated by non-selective cation channels (NSCCs). Moreover, these currents were blocked by YM-58483. In addition, such currents were abolished by NS8593, implicating that NS8593 inhibits the same channels. Besides, NS8593 inhibited increases of intracellular Ca2+ and the associated cell shortening. Finally, NS8593 inhibited ACH-induced increases of mouse respirator system resistance (Rrs). SignificanceOur results indicate that NS8593 inhibits LVDCCs and NSCCs, resulting in decreases of intracellular Ca2+ and then leading to ASM relaxation. These data suggest that NS8593 might be a new bronchodilator.

Paracetamol inhibits Ca2+ permeant ion channels and Ca2+ sensitization resulting in relaxation of precontracted airway smooth muscle

The purpose of this study was to screen a bronchodilator from old drugs and elucidate the underlying mechanism. Paracetamol (acetaminophen) is a widely used analgesic and antipyretic drug. It has been reported that it inhibits the generation of prostaglandin and histamine, which play roles in asthma. These findings led us to explore whether paracetamol could be a potential bronchodilator. Paracetamol inhibited high K+- and acetylcholine (ACH)-induced precontraction of mouse tracheal and bronchial smooth muscles. Moreover, the ACH-induced contraction was partially inhibited by nifedipine (selective blocker of LVDCCs), YM-58483 (selective inhibitor of store-operated Ca2+ entry (SOCE), canonical transient receptor potential 3 (TRPC3) and TRPC5 channels) and Y-27632 (selective blocker of ROCK, a linker of the Ca2+ sensitization pathway). In single airway smooth muscle cells, paracetamol blocked the currents sensitive to nifedipine and YM-58483, and inhibited intracellular Ca2+ increases. In addition, paracetamol inhibited ACH-induced phosphorylation of myosin phosphatase target subunit 1 (MYPT1, another linker of the Ca2+ sensitization pathway). Finally, in vivo paracetamol inhibited ACH-induced increases of mouse respirator system resistance. Collectively, we conclude that paracetamol inhibits ASM contraction through blocking LVDCCs, SOCE and/or TRPC3 and/or TRPC5 channels, and Ca2+ sensitization. These results suggest that paracetamol might be a new bronchodilator.

Endogenous acetylcholine regulates neuronal and astrocytic vascular endothelial growth factor expression levels via different acetylcholine receptor mechanisms

Vascular endothelial growth factor (VEGF), a signaling molecule involved in angiogenesis, plays an important role in neuroprotection and neurogenesis. In the present study, we aimed to elucidate the mechanisms underlying endogenous acetylcholine (ACh)-induced VEGF expression in neurons and astrocytes, and identify the neuronal cells contributing to its expression in the medial septal area, a nuclear origin of cholinergic neurons mainly projecting to the hippocampus. The mRNA expression and secretion of VEGF were measured by RT-PCR and ELISA using mouse primary cultured cortical neurons and astrocytes. VEGF expression in the medial septal area was assessed by RT-PCR and immunostaining using mice treated with tacrine [9-amino-1,2,3,4-tetrahydro-acridine HCl (THA); 2.5 mg/kg, i.p.] once daily for 7 days. The THA treatment increased VEGF mRNA expression in neurons in a manner that was reversed by mecamylamine, a nicotinic ACh receptor (AChR) antagonist, whereas in mouse primary cultured astrocytes, carbachol, but not THA dose-dependently increased VEGF mRNA expression and secretion in a manner that was inhibited by scopolamine, a muscarinic AChR inhibitor. In in vivo studies, the administration of THA significantly increased the expression of VEGF in medial septal cholinergic neurons and the effects of THA were significantly blocked by mecamylamine. THA also significantly increased the expression levels of a phosphorylated form of VEGF receptor 2 (p-VEGFR2), an activated form of VEGFR2. The present results suggest that endogenous ACh plays an up-regulatory role for VEGF expression in neurons and astrocytes via different mechanisms. Moreover, endogenous ACh-induced increases in VEGF levels appear to activate VEGFR2 on medial septal cholinergic neurons via an autocrine mechanism.

Acetylcholine Activates Cystathionine γ‐Lyase Production of H2S in Aortic Endothelial Cells

Hydrogen sulfide (H2S) is the most recently described endothelium-derived vasodilator. In the endothelium, H2S is predominantly produced by cystathionine ©-lyase (CSE). We and others have previously shown CSE inhibition diminishes acetylcholine (ACh)-induced dilation in aortic ring segments. However, ACh regulation of CSE is not well-defined. The goal of this study was to identify endogenous regulators of CSE in aortic endothelial cells. Previous reports show that [Ca2+] regulates CSE activity. However, previous studies were conducted using 1–2 mM [Ca2+] and purified CSE. Because normal intracellular [Ca2+] is 100 nM to 3 μM and activity of purified enzymes may not reflect in vivo activity, it is necessary to verify regulation of endogenous CSE with physiological [Ca2+]. Thus, we hypothesized that ACh-induced increases in intracellular [Ca2+] elevates CSE production of H2S. Rat aortic endothelial cells (passage 5–6) were loaded with the H2S fluorescence indicator, sulfide fluor-7 acetoxymethylester (SF7-A...

Nitric Oxide Increases Endothelial Cell Membrane Permeability via S-Nitrosylation-Mediated Opening of Cx43 Hemichannels

Endothelial connexin-formed gap junction channels play a central role in the coordination of vasomotor tone, but the contribution of connexin as hemichannels to endothelial cell function has not been determined. We evaluated the participation of Cx43 hemichannels in the increase in [Ca+2]i that triggers the NO-mediated vasodilation induced by acetylcholine (ACh), an endothelium-dependent vasodilator. Hemichannel activation and Cx43 S-nitrosylation were detected in mesenteric resistance arteries by assessing ethidium uptake, and through biotin switch, respectively. Changes in [Ca+2]i were recorded in primary cultures of mesenteric endothelial cells using the fluorescent Ca2+ indicator Fluo-4. Stimulation with ACh or SNAP, a nitric oxide (NO) donor, induced an increase in ethidium uptake and [Ca+2]i that was inhibited by the connexin hemichannel blocker La3+ and the connexin mimetic peptides 37,43Gap27, and 43Gap26, a Cx43 blockers. The increment in ethidium uptake and [Ca+2]i was not affected by the mimetic peptide 40Gap27, a Cx40 blocker. In addition, ACh enhanced the S-nitrosylation of Cx43. The changes in membrane permeability observed in response to ACh were inhibited by the NO synthase inhibitor NG-nitro-L-arginine, supporting the involvement of a NO-mediated pathway. To directly demonstrate that NO induces opening of Cx43 hemichannel, we measured Cx43 hemichannel currents using two-electro voltage technique; NO donors (SNAP and DENOO) promotes hemichannel currents in oocytes expressing Cx43, but not in control oocytes devoid of Cx43. Cx43 hemichannel currents were sensitive to La3+ and ascorbic acid, a scavenger of S-nitrosylated sites. Consistently, Cx43 was significantly S-nitrosylated in the presence of NO donors in oocytes. Taken together, our results indicate that NO-mediated Cx43 hemichannel opening by S-nitrosylation play a key role in the ACh-induced increase in endothelial cell [Ca+2]i.FONDECYT 1150530/RO1GM0994990/AT21120178.

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See related article, pp 802–808 Vascular endothelial cells importantly contribute to the regulation of vascular smooth muscle tone through the production of a variety of vasoactive substances and by direct electric communication through myoendothelial gap junctions.1,2 Moncada et al3 discovered the first endothelium-derived vasoactive substance, prostacyclin, in the mid 1970s and rapidly identified its structure and chemical composition.4 This was followed by the landmark observations of Furchgott and Zawadzki.5 and the discovery of endothelium-derived relaxing factor in 1980. Seven years later, the endothelium-derived relaxing factor of Furchgott and Zawadzki was positively identified as NO.6 The existence of a nonprostaglandin, non-NO endothelium-derived hyperpolarizing factor (EDHF) was postulated by Komori et al7 in 1988. Twenty-three years later, we know that there is no one EDHF, but rather multiple pathways by which endothelial cells can produce hyperpolarization-induced relaxation of overlying vascular smooth muscle cells, independent from prostacyclin and NO (Figure).1,2 Figure. Pathways for nonprostacyclin-, non-NO–mediated, endothelium-dependent hyperpolarization of vascular smooth muscle in resistance arteries. Schematic diagram of a longitudinal section through a resistance artery showing smooth muscle and endothelial cell cross-sections and depicting several potential pathways by which ACh can produce endothelium-dependent smooth cell hyperpolarization and vasodilatation independent from prostaglandins and NO. On the left, ACh-induced increases in intracellular Ca2+ activate endothelial small conductance (sKCa) and intermediate conductance (IKCa) Ca2+-activated K+ channels. The released K+ ions can then act as an EDHF through activation of smooth muscle inward rectifier K+ channels (KIR) or Na+/K+ ATPase. Smooth muscle hyperpolarization then closes voltage-gated Ca2+ channels (VGCC), leading to …

Scavenging free radicals improves endothelial dysfunction in human coronary arteries in vivo

The effect of a novel free radical scavenger, edaravone, on coronary endothelial dysfunction was examined in patients who had no significant stenosis of coronary arteries, to elucidate the role of free radicals on coronary endothelial dysfunction. The coronary blood flow (CBF) responses to acetylcholine (ACh) were measured by quantitative coronary arteriography and the intracoronary Doppler technique before and after the administration of edaravone. Twenty-four patients were divided into two groups on the basis of CBF responses to ACh; those with "attenuated" (%Delta CBF < 300%, n = 12) and "normal" (%Delta CBF > 300%, n = 12) flow responses. An intracoronary infusion of edaravone significantly improved ACh-induced increases in CBF in patients with attenuated flow responses; however, edaravone had no effect in those with normal flow responses (36.8% +/- 7.3% vs 0.0% +/- 5.1%, P < 0.01). The plasma levels of nitric oxide compounds (NOx) in the attenuated response group were lower than those in the normal group (35.7 +/- 2.3 vs 49.4 +/- 6.2 muM, P < 0.01) and correlated with the magnitude of CBF improvement by edaravone (r = 0.566, P < 0.01). The plasma level of malondialdehyde and 4-hydroxynonenal, which indicates the level of oxidative stress, in the attenuated group was higher than that in the normal group (6.9 +/- 0.9 vs 3.3 +/- 0.5 muM, P < 0.01) and correlated with the magnitude of CBF improvement by edaravone (r = 0.854, P < 0.01). A free radical scavenger improved the ACh-induced CBF response in patients with coronary atherosclerosis in vivo. Therefore, the current results suggest that scavenging free radicals has a beneficial effect for patients with coronary endothelial dysfunction.

Antiphospholipid Antibodies Promote Leukocyte-Endothelial Cell Adhesion by Antagonizing Endothelial NO Synthase Via b2GPI and ApoER2.

Abstract 3039Poster Board II-1015The antiphospholipid syndrome (APS) is an autoimmune disease characterized by the presence of antiphospholipid antibodies (aPL), with pathogenic aPL directed primarily against the cell surface protein β2 glycoprotein I (β2GPI), increased risk for thrombosis and cardiovascular events, and pregnancy morbidity. The endothelium is a critical direct target of aPL, which upregulate adhesion molecule expression and procoagulant activity. However, the molecular basis for aPL actions on endothelium is unknown. Nitric oxide (NO) generated by endothelial NO synthase (eNOS) has potent antiadhesive and antithrombotic properties. In this study we determined if aPL-induced alterations in endothelial cell phenotype are mediated by aPL actions on eNOS. Normal human IgG (NHIgG) and polyclonal aPL were obtained from healthy adults and APS patients, respectively. We found that aPL prevented acetylcholine (Ach)- or VEGF-mediated attenuation of monocyte adhesion to cultured endothelial cells and this was reversed by an NO donor, indicating a role for eNOS antagonism. In contrast, NHIgG did not affect adhesion. Whereas NHIgG did not alter eNOS activation, stimulation of eNOS by VEGF and other agonists was fully antagonized by aPL. In mice, NO-dependent, Ach-induced increases in carotid vascular conductance were unaffected by NHIgG but impaired by aPL, indicating that these processes are operative in vivo. Additional studies in culture showed that aPL attenuates eNOS activation by inhibiting Ser1179 phosphorylation, deprivation of β2GPI prevented aPL inhibition of eNOS, and monoclonal antibody against β2GPI mimicked the effects of aPL. Furthermore, receptor-associated protein (RAP) antagonism of LDL receptors, which are known to bind dimerized β2GPI, prevented aPL inhibition of eNOS in culture, and mice null for the LDL receptor apolipoprotein receptor 2 (apoER2) were protected from aPL inhibition of eNOS in vivo. Moreover, aPL-stimulated increases in leukocyte-endothelial adhesion were absent in both eNOS-/- and apoER2-/- mice. Thus, aPL-induced increases in leukocyte-endothelial adhesion are caused by eNOS antagonism, which is due to impaired S1179 phosphorylation mediated by β2GPI and apoER2. Novel therapies for APS can now be developed targeting these mechanisms. DisclosuresThomas:NIcox Research Institute: Research Funding. Thorpe:Peregrine Pharm.: Consultancy, sponsored research agreement.

The impact of adenosine and an A2A adenosine receptor agonist on the ACh-induced increase in intracellular calcium of the glomus cells of the cat carotid body

The carotid body (CB) is a polymodal chemosensor of arterial blood located next to the internal carotid artery. The basic chemosensing unit is composed of the neurotransmitter (NT)-containing glomus cells (GCs) and the sensory afferent fibers synapsing onto the GCs. Nicotinic and muscarinic receptors have been found on both the sensory afferent fibers and on the GCs. Neural output from the CB (CBNO) increases when arterial blood perfusing it is hypoxic, hypoglycemic, hypercapnic, or acidic. The increased CBNO due to GC release of excitatory NTs must be preceded by an entrance of calcium into the GCs. With repeated release of ACh from the GCs, cholinergic receptors could become desensitized, particularly nicotinic receptors which function as calcium channels. The purpose of the present study was to see if adenosine (ADO), known to alter receptor sensitivities, could attenuate or eliminate any desensitization of the nicotinic receptors occurring during the repeated application of ACh. Cat CBs were harvested with techniques approved by the University's Animal Care/Use Committee. The GCs were cultured and prepared for detecting [Ca ++] i with standard techniques. Repeated application of ACh produced a progressively decreasing increase in [Ca ++] i. With the use of ADO or an A2 A ADO receptor agonist the decrease was avoided. Though ADO also increased GC [Ca ++] i, the sum of ADO increase and ACh increase, when superfused separately, was less than the increase when they were both included in the same superfusion. This suggested the possible involvement of a new path in the action. Potential mechanisms to explain the phenomena are discussed.

The role of nitric oxide‐ and prostacyclin‐independent vasodilatation in the human cutaneous microcirculation: effect of cytochrome P450 2C9 inhibition

The component of the flow- or agonist-dependent vasodilatation, insensitive to inhibitors of nitric oxide (NO) synthases (NOS) or cyclooxygenases (COX), is suggested to reflect the production of an endothelium-dependent hyperpolarizing factor (EDHF). The identity of EDHF in humans remains controversial; in coronary arterioles, it appears to be a cytochrome P450 (CYP) 2C9-derived metabolite, whereas there are no data for human skin microcirculation. Therefore, the aim of our study was to investigate the role of the NO- and prostacyclin (PGI(2))-independent mechanism, particularly the potential involvement of CYP 2C9, in skin microcirculation. We measured skin blood flow on the volar aspect of the forearm in 12 healthy subjects by laser-Doppler fluxmetry (LDF). The inhibitors of NOS, N(omega)-monomethyl-L-arginine (L-NMMA), and cyclooxygenase (COX), diclofenac, as well as sulfaphenazole, the specific CYP 2C9 inhibitor, and saline as control, were administered to the measurement sites by an intradermal microinjection in different combinations. Afterwards, baseline LDF was assessed and iontophoresis of acetycholine (ACh) applied. Combined NOS and COX inhibition had no effect on baseline LDF, whereas it significantly reduced the ACh-induced increase in LDF (t-test, P<0.05). Sulfaphenazole did not affect baseline LDF either in the control site or in the L-NMMA- and diclofenac-pretreated site. In addition, sulfaphenazole did not attenuate the ACh-induced vasodilatation in either site. We conclude that a NO- and PGI(2)-independent vasodilator mechanism, potentially attributable to EDHF, contributes substantialy to the ACh-induced vasodilatation in human skin microcirculation and that it is probably not a CYP 2C9-derived metabolite.

Protein Restriction during Pregnancy Induces Hypertension and Impairs Endothelium-Dependent Vascular Function in Adult Female Offspring

Intrauterine undernutrition plays a role in the development of adult hypertension. Most studies are done in male offspring to delineate the mechanisms whereby blood pressure may be raised; however, the vascular mechanisms involved in female offspring are unclear. Female offspring of pregnant Sprague-Dawley rats fed either a control (C; 18%) or a low-protein (LP; 6%) diet during pregnancy were used. Birth weight and later growth were markedly lower in LP than in C offspring. LP offspring exhibited impaired estrous cyclicity with increased mean arterial pressure. Hypotensive response to acetylcholine (ACh) and the hypertensive response to phenylephrine (PE) were greater in LP than in C rats. N-nitro-L-arginine methyl ester (L-NAME) induced greater hypertensive responses in C than in LP rats. Endothelium-intact mesenteric arteries from LP offspring exhibited increased contractile responses to PE and reduced vasodilation in response to ACh. In endothelium-denuded arteries, relaxation responses to sodium nitroprusside were similar in both groups. Basal and ACh-induced increase in vascular nitrite/nitrate production was lower in LP than in C offspring. L-NAME or 1H-1,2,4-oxadiazolo-4,3-quinoxalin-1-one inhibited ACh relaxations and enhanced PE contractions in C offspring, but had minimal effect in LP rats. The decreasedNO-mediated vascular response might explainthe increased vascular contraction and arterial pressure infemale offspring with low birth weight.

Effects of Angiotensin II on NO Bioavailability Evaluated Using a Catheter-Type NO Sensor

We investigated the acute or chronic effects of angiotensin (Ang) II on the bioavailability of NO in Ang II-infused rabbits using the catheter-type NO sensor. Male New Zealand White rabbits were infused with vehicle (sham), Ang II at a rate of 200 ng/kg per minute, either alone or in combination with hydralazine, Ang II type I receptor antagonist (valsartan), or an antioxidant (tempol) for 24 hours or 14 days. Plasma NO concentration was measured using the catheter-type NO sensor located in the aorta. We then infused saline (vehicle) and acetylcholine (ACh) into the aortic arch with or without pretreatment with N(G)-methyl-l-arginine. An increase in plasma NO levels in response to ACh was significantly attenuated in the Ang II group compared wit the control group. The decrease in the basal plasma NO concentration was significantly lower in the Ang II group than in the control group. Plasma peroxynitrite concentrations in Ang II group were significantly higher than in the control group. The negative effects of Ang II, that is, the decrease in basal and ACh-induced NO production and the increase in oxidative stress, were significantly suppressed by the cotreatment with either valsartan or tempol. Short-term treatment with Ang II significantly increased the ACh-induced increase in plasma NO concentration, as well as basal NO release. Although Ang II stimulates release of NO in the short term, chronic treatment with Ang II elicits the decreased NO bioavailability in the aorta of the Ang II-infusion rabbit model.

NADPH OXIDASE‐MEDIATED O 2 −· PRODUCTION AMPLIFIES VASOCONSTRICTOR RESPONSE OF SMALL CORONARY ARTERIES TO M 1 ‐MUSCARINIC RECEPTOR ACTIVATION

The present study test the hypothesis that NADPH oxidase (Nox) mediated O2−· production amplifies coronary arterial constrictor response to M1-receptor (M1R) agonists. Fluorescence imaging analysis showed that M1R agonist, acetylcholine (Ach), simultaneously increased intracellular Ca2+ and O2−· levels in bovine coronary arterial smooth muscle cells (CASMCs) with a maximal increase of 821 + 67 nM in Ca2+ levels accompanied by 75% in O2−· production. However, when cells were transfected with siRNA of p47phox(a key subunit for Nox activation), Ach-induced increases in Ca2+ and O2−· were significantly attenuated. Similarly, p47phoxsiRNA significantly reduced oxotremorine (OXO)-induced O2−· production in CASMCs by 67%. Functionally, OXO-induced constrictor response of endothelium-denuded small bovine coronary arteries was significantly reduced by Nox inhibitor, DPI or O2−· scavenger cocktail (tempol + catalase). Moreover, introduction of siRNA for p47phox, NOX1 and NOX4 into arterial smooth muscle by ultrasound microbubble technology significantly blocked this OXO-induced contraction. We concluded that Nox-derived O2−· is involved in M1R-mediated intracellular Ca2+ release in CASMCs and thereby importantly contributes to amplification of vasoconstrictor response of coronary arteries to M1R activation (Supported by NIH grants HL057244, HL075316 and AHA predoctoral fellowship 0410061Z).

Activation of Nicotinic Acetylcholine Receptors Increases the Frequency of Spontaneous GABAergic IPSCs in Rat Basolateral Amygdala Neurons

The basolateral amygdala (BLA) is a critical component of the amygdaloid circuit, which is thought to be involved in fear conditioned responses. Using whole cell patch-clamp recording, we found that activation of nicotinic acetylcholine receptors (nAChRs) leads to an action potential-dependent increase in the frequency of spontaneous GABAergic currents in principal neurons in the BLA. These spontaneous GABAergic currents were abolished by a low-Ca2+/high-Mg2+ bathing solution, suggesting that they are spontaneous inhibitory postsynaptic currents (sIPSCs). Blockade of ionotropic glutamate receptors did not prevent this increased frequency of sIPSCs nor did blockade of alpha7 nAChRs. Among the nAChR agonists tested, cystisine was more effective at increasing the frequency of the sIPSCs than nicotine or 1,1-dimethyl-4-phenyl piperazinium iodide, consistent with a major contribution of beta4 nAChR subunits. The nicotinic antagonist, dihydro-beta-erythroidine, was less effective than d-tubocurarine in blocking the increased sIPSC frequency induced by ACh, suggesting that alpha4-containing nAChR subunits do not play a major role in the ACh-induced increased sIPSC frequency. Although alpha2/3/4/7 and beta2/4 nAChR subunits were found in the BLA by RT-PCR, the agonist and antagonist profiles suggest that the ACh-induced increase in sIPSC frequency involves predominantly alpha3beta4-containing nAChR subunits. Consistent with this, alpha-conotoxin-AuIB, a nAChR antagonist selective for the alpha3beta4 subunit combination, inhibited the ACh-induced increase in the frequency of sIPSCs. The observations suggest that nicotinic activation increases the frequency of sIPSCs in the BLA by acting mainly on alpha3beta4-containing nicotinic receptors on GABAergic neurons and may play an important role in the modulation of synaptic transmission in the amygdala.