Release Of Arachidonic Acid Metabolites Research Articles

Vitamin C Deficiency Exacerbates Dysfunction of Atherosclerotic Coronary Arteries in Guinea Pigs Fed a High-Fat Diet.

Vitamin C (vitC) deficiency has been associated with an increased risk of cardiovascular disease; while several putative mechanistic links have been proposed, functional evidence supporting a causal relationship is scarce. In this study, we investigated how vitC deficiency affects coronary artery vasomotor function and the development of coronary atherosclerotic plaques in guinea pigs subjected to chronic dyslipidemia by a high-fat diet regime. Female Hartley guinea pigs were fed either a control (low-fat diet and sufficient vitC) (N = 8) or a high-fat diet with either sufficient (N = 8) or deficient (N = 10) vitC for 32 weeks. Guinea pigs subjected to the high-fat diet developed significant atherosclerotic plaques in their coronary arteries, with no quantitative effect of vitC deficiency. In isolated coronary arteries, vasomotor responses to potassium, carbachol, nitric oxide, or bradykinin were studied in a wire myograph. Carbachol, bradykinin, and nitric oxide mediated relaxation in the coronary arteries of the control group. While vasorelaxation to carbachol and nitric oxide was preserved in the two high-fat diet groups, bradykinin-induced vasorelaxation was abolished. Interestingly, bradykinin induced a significant contraction in coronary arteries from vitC-deficient guinea pigs (p < 0.05). The bradykinin-induced contraction was unaffected by L-NAME but significantly inhibited by both indomethacin and vitC, suggesting that, during vitC deficiency, increased release of arachidonic acid metabolites and vascular oxidative stress are involved in the constrictor effects mediated by bradykinin. In conclusion, the present study shows supporting evidence that poor vitC status negatively affects coronary artery function.

Cyclooxygenase pathway mediates the inhibition of Na-glutamine co-transporter B0AT1 in rabbit villus cells during chronic intestinal inflammation.

In the mammalian intestine, glutamine assimilation by the absorptive villus cells is mediated by Na-glutamine co-transport, specifically by B0AT1. In a rabbit model of chronic intestinal inflammation, B0AT1 is inhibited secondary to a decrease in the number of co-transporters in the brush border membrane (BBM). This inhibition can be reversed by treatment with a broad-spectrum immune modulator such as glucocorticoid suggesting that immune inflammatory mediators may regulate B0AT1 during chronic intestinal inflammation. Arachidonic acid (AA) metabolites (AAM) are increased during chronic intestinal inflammation. However, whether AAM may regulate B0AT1 during chronic intestinal inflammation is unknown. Treatment of rabbits with ATK, to prevent the release of AAM reversed the inhibition of B0AT1. AAM are products of cyclooxygenase (COX) and/or lipoxygenase (LOX) pathways. Inhibition of COX with piroxicam, therefore reduction of prostaglandin formation in the chronically inflamed intestine, reversed the inhibition of B0AT1 to its normal levels. In contrast, inhibition of LOX with MK886, thus reduction of leukotriene formation during chronic enteritis, did not affect the inhibition of B0AT1. Kinetic studies showed that the mechanism of restoration of B0AT1 by ATK or piroxicam was secondary to the restoration of BBM co-transporter numbers. Western Blot analysis also demonstrated restoration of BBM B0AT1 co-transporter numbers. In conclusion, this study demonstrates that Na-glutamine co-transport mediated by B0AT1 in villus cells is regulated by prostaglandins rather than leukotrienes in the chronically inflamed intestine.

Cytokines as biomarkers of inflammatory response after open versus endovascular repair of abdominal aortic aneurysms: a systematic review.

The repair of an abdominal aortic aneurysm (AAA) is a high-risk surgical procedure related to hormonal and metabolic stress-related response with an ensuing activation of the inflammatory cascade. In contrast to open repair (OR), endovascular aortic aneurysm repair (EVAR) seems to decrease the postoperative stress by offering less extensive incisions, dissection, and tissue manipulation. However, these beneficial effects may be offset by the release of cytokines and arachidonic acid metabolites during intra-luminal manipulation of the thrombus using catheters in endovascular repair, resulting in systemic inflammatory response (SIR), which is clinically called post-implantation syndrome. In this systematic review we compared OR with EVAR in terms of the post-interventional inflammatory response resulting from alterations in the circulating cytokine levels. We sought to summarize all the latest evidence regarding post-implantation syndrome after EVAR. We searched Medline (PubMed), ClinicalTrials.gov and the Cochrane library for clinical studies reporting on the release of cytokines as part of the inflammatory response after both open/conventional and endovascular repair of the AAA. We identified 17 studies examining the cytokine levels after OR versus EVAR. OR seemed to be associated with a greater SIR than EVAR, as evidenced by the increased cytokine levels, particularly IL-6 and IL-8, whereas IL-1β, IL-10 and TNF-α showed conflicting results or no difference between the two groups. Polyester endografts appear to be positively correlated with the incidence of post-implantation syndrome after EVAR. Future large prospective studies are warranted to delineate the underlying mechanisms of the cytokine interaction in the post-surgical inflammatory response setting.

Recent advances in oxygen sensing and signal transduction in hypoxic pulmonary vasoconstriction.

Hypoxic pulmonary vasoconstriction (HPV) is a physiological reaction, which adapts lung perfusion to regional ventilation and optimizes gas exchange. Impaired HPV may cause systemic hypoxemia, while generalized HPV contributes to the development of pulmonary hypertension. The triggering mechanisms underlying HPV are still not fully elucidated. Several hypotheses are currently under debate, including a possible decrease as well as an increase in reactive oxygen species as a triggering event. Recent findings suggest an increase in the production of reactive oxygen species in pulmonary artery smooth muscle cells by complex III of the mitochondrial electron transport chain and occurrence of oxygen sensing at complex IV. Other essential components are voltage-dependent potassium and possibly L-type, transient receptor potential channel 6, and transient receptor potential vanilloid 4 channels. The release of arachidonic acid metabolites appears also to be involved in HPV regulation. Further investigation of the HPV mechanisms will facilitate the development of novel therapeutic strategies for the treatment of HPV-related disorders.

Neutrophil to lymphocyte ratio in diagnosing acute cholecystitis: a retrospective cohort study in a tertiary rural hospital

Background:Acute cholecystitis accounts for most of the hospital admissions related to gastrointestinal diseases. In approximately 90% of patients, inflammation develops due to obstruction of the cystic duct by one or more gall-tones. Delayed management can lead to increased morbidity, due to progression to severe cholecystitis, such as gangrenous change, abscess formation, and gallbladder perforation. The neutrophil to lymphocyte ratio is derived from the counts of circulating neutrophils and lymphocytes, both of which are major leukocyte subpopulations. The inflammation triggered release of arachidonic acid metabolites and platelet-activating factors results in neutrophilia, and cortisol induced stress results in relative lymphopenia, and thus, the neutrophil to lymphocyte ratio accurately represents the underlying inflammatory process. The objective of this study was to evaluate the utility of the neutrophil to lymphocyte ratio (NLR) as a prognostic indicator in patients with cholecystitis. And to identify a relevant NLR value that discriminates between simple and severe cholecystitis.Methods: A retrospective analysis of patient data compiled by assessment of operative findings documented and patient follow up recorded over the period encompassing three years (2015-2012) at RL Jalappa Hospital and Research Centre, Tamaka, Kolar, Karnataka, India.Results:Our study population comprised of 250 patients with simple cholecystitis (69.63%) and 109 patients with severe cholecystitis (30.36%). The NLR of 3.0 could predict severe cholecystitis with 70.5% sensitivity and 70.0% specificity. A higher NLR (≥3.0) was significantly associated with older age (p = 0.001), male gender (p = 0.001), admission via the emergency department (p <0.001), longer operation time (p <0.001), higher incidence of postoperative complications (p = 0.056), and prolonged length of hospital stay (LOS) (p <0.001).Conclusions:NLR ≥3.0 was significantly associated with severe cholecystitis and prolonged LOS in patients undergoing cholecystectomy. Therefore, preoperative NLR in patients undergoing cholecystits due to cholecystitis seemed to be a useful surrogate marker for severe cholecystitis.

Binaural blood flow control by astrocytes: listening to synapses and the vasculature.

Astrocytes are the most common glial cells in the brain with fine processes and endfeet that intimately contact both neuronal synapses and the cerebral vasculature. They play an important role in mediating neurovascular coupling (NVC) via several astrocytic Ca2+ -dependent signalling pathways such as K+ release through BK channels, and the production and release of arachidonic acid metabolites. They are also involved in maintaining the resting tone of the cerebral vessels by releasing ATP and COX-1 derivatives. Evidence also supports a role for astrocytes in maintaining blood pressure-dependent change in cerebrovascular tone, and perhaps also in blood vessel-to-neuron signalling as posited by the 'hemo-neural hypothesis'. Thus, astrocytes are emerging as new stars in preserving the intricate balance between the high energy demand of active neurons and the supply of oxygen and nutrients from the blood by maintaining both resting blood flow and activity-evoked changes therein. Following neuropathology, astrocytes become reactive and many of their key signalling mechanisms are altered, including those involved in NVC. Furthermore, as they can respond to changes in vascular pressure, cardiovascular diseases might exert previously unknown effects on the central nervous system by altering astrocyte function. This review discusses the role of astrocytes in neurovascular signalling in both physiology and pathology, and the impact of these findings on understanding BOLD-fMRI signals.

Ferromagnetic particles as a rapid and robust sample preparation for the absolute quantification of seven eicosanoids in human plasma by UHPLC-MS/MS.

We used ferromagnetic particles as a novel technique to deproteinize plasma samples prior to quantitative UHPLC-MS/MS analysis of seven eicosanoids [thromboxane B2 (TXB2), prostaglandin E2 (PGE2), PGD2, 5-hydroxyeicosatetraenoic acid (5-HETE), 11-HETE, 12-HETE, arachidonic acid (AA)]. A combination of ferromagnetic particle enhanced deproteination and subsequent on-line solid phase extraction (on-line SPE) realized quick and convenient semi-automated sample preparation-in contrast to widely used manual SPE techniques which are rather laborious and therefore impede the investigation of AA metabolism in larger patient cohorts. Method evaluation was performed according to a protocol based on the EMA guideline for bioanalytical method validation, modified for endogenous compounds. Calibrators were prepared in ethanol. The calibration curves were found to be linear in a range of 0.1-80ngmL(-1) (TXB2, PGE2, PGD2), 0.05-40ngmL(-1) (5-HETE, 11-HETE), 0.5-400ngmL(-1) (12-HETE) and 25-9800ngmL(-1) (AA). Regarding all analytes and all quality controls, the resulting precision data (inter-assay 2.6 %-15.5 %; intra-assay 2.5 %-15.1 %, expressed as variation coefficient) as well as the accuracy results (inter-assay 93.3 %-125 %; intra-assay 91.7 %-114 %) were adequate. Further experiments addressing matrix effect, recovery and robustness, yielded also very satisfying results. As a proof of principle, the newly developed LC-MS/MS assay was employed to determine the capacity of AA metabolite release after whole blood stimulation in healthy blood donors. For this purpose, whole blood specimens of 5 healthy blood donors were analyzed at baseline and after a lipopolysaccharide (LPS) induced blood cell activation. In several baseline samples some eicosanoids levels were below the Lower Limit of Quantification. However, in the stimulated samples all chosen eicosanoids (except PGD2) could be quantified. These results, in context with those obtained in validation, demonstrate the applicability of ferromagnetic particles for the sample preparation for eicosanoids in human plasma. Thus, we conclude that ferromagnetic particle enhanced deproteination is a promising novel tool for sample preparation in LC-MS/MS, which is of particular interest for automation in clinical mass spectrometry, e.g. in order to further address eicosanoid analysis in larger patient cohorts.

IGF‐1 Deficiency Promotes Neurovascular Uncoupling: Implications for Cerebromicrovascular Aging and Age‐Related Cognitive Decline

Aging is associated with marked deficiency in circulating IGF-1, which was shown to importantly contribute to age-related cognitive decline. Impairment of moment-to-moment adjustment of cerebral blood flow (CBF) via neurovascular coupling (NVC) is thought to play a critical role in the genesis of age-related cognitive impairment. To establish the link between IGF-1 deficiency and cerebromicrovascular impairment, we compared NVC in IGF-1 deficient (Igf f/f-TBG-iCre-AAV8) and control mice. We found that glutamate (Glu)-dependent CBF responses to whisker stimulation were significantly decreased in IGF-1 deficient mice. IGF-1 deficiency did not affect neuronal activity-induced cerebral Glu release. Expression of Glu receptors (Grin1, Grin2A, Grm2, Grm4, Grm5) was down-regulated in astrocytes isolated from IGF-1 deficient mice. Glu-stimulated release of astrocyte-derived vasodilator arachidonic acid metabolites (EETs) and EET-dependent CBF responses elicited by whisker stimulation were significantly decreased in IGF-1 deficient mice. IGF-1 deficiency also increased production of the vasoconstrictor 20-HETE. Collectively, IGF-1 deficiency promotes neurovascular uncoupling mimicking the aging phenotype, which is likely causally linked to cognitive impairment.

Role of Preferential Cyclooxygenase-2 Inhibition by Meloxicam in Ischemia/Reperfusion Injury of the Rat Liver

Background: Ischemia/reperfusion injury (IRI) is one of the major clinical problems in liver and transplant surgery. Livers subjected to warm ischemia in vivo often show a severe dysfunction and the release of numerous inflammatory cytokines and arachidonic acid metabolites. Cyclooxygenase (COX)-2 is the inducible isoform of an intracellular enzyme that converts arachidonic acid into prostaglandins. The aim of the study was to evaluate the effect of COX-2 inhibition and the role of Kupffer cells in IRI of the liver. Methods: Male Wistar rats [250- 280 g body weight (BW)] were anesthetized and subjected to 30-min warm ischemia of the liver (Pringle's maneuver) and 60-min reperfusion after median laparotomy. The I/R group received no additional treatment. In the COX-2 inhibitor (COX-2I) group, the animals received 1 mg/kg BW meloxicam prior to operation. Gadolinium chloride (GdCl₃) (10 mg/kg BW) was given 24 h prior to operation in the GdCl₃ and GdCl₃ + COX-2I groups for the selective depletion of Kupffer cells. The GdCl₃ + COX-2I group received both GdCl₃ and meloxicam treatment prior to operation. Blood and liver samples were obtained at the end of the experiments for further investigations. Results: After 30 min of warm ischemia in vivo, severe hepatocellular damage was observed in the I/R group. These impairments could be significantly prevented by the selective COX-2 inhibition and the depletion of Kupffer cells. Alanine aminotransferase was significantly reduced upon meloxicam and GdCl₃ treatment compared to the I/R group: I/R, 3,240 ± 1,262 U/l versus COX-2I, 973 ± 649 U/l, p < 0.001; I/R versus GdCl₃, 1,611 ± 600 U/l, p < 0.05, and I/R versus GdCl₃ + COX-2I, 1,511 ± 575 U/l, p < 0.01. Plasma levels of tumor necrosis factor alpha (TNF-α) were significantly reduced in the COX-2I treatment group compared to I/R (3.5 ± 1.5 vs. 16.3 ± 11.7 pg/ml, respectively; p < 0.05). Similarly, the amount of TxB₂, a marker for COX-2 metabolism, was significantly reduced in the meloxicam treatment groups compared to the I/R group: I/R, 22,500 ± 5,210 pg/ml versus COX-2I, 1,822 ± 938 pg/ml, p < 0.001, and I/R versus GdCl₃ + COX-2I, 1,530 ± 907 pg/ml, p < 0.001. All values are given as mean ± SD (n = 6). Conclusion: These results suggest that the inhibition of COX-2 suppressed the initiation of an inflammatory cascade by attenuating the release of TNF-α, which is an initiator of the inflammatory reaction in hepatic IRI. Therefore, we conclude that preferential inhibition of COX-2 is a possible therapeutic approach against warm IRI of the liver.

Role of long-chain acyl-coenzyme A synthetases in the regulation of arachidonic acid metabolism in interleukin 1β-stimulated rat fibroblasts

Acyl coenzyme A synthetase long-chain family members (ACSLs) are a family of enzymes that convert long-chain free fatty acids into their acyl-CoAs and play an important role in fatty acid metabolism. Here we show the role of ACSL isozymes in interleukin (IL)-1β-induced arachidonic acid (AA) metabolism in rat fibroblastic 3Y1 cells. Treatment of 3Y1 cells with triacsin C, an ACSL inhibitor, markedly enhanced the IL-1β-induced prostaglandin (PG) biosynthesis. Small interfering RNA-mediated knockdown of endogenous Acsl4 expression increased significantly the release of AA metabolites, including PGE2, PGD2, and PGF2α, compared with replicated control cells, whereas knockdown of Acsl1 expression reduced the IL-1β-induced release of AA metabolites. Experiments with double knockdown of Acsl4 and intracellular phospholipase A2 (PLA2) isozymes revealed that cytosolic PLA2α, but not calcium-independent PLA2s, is involved in the Acsl4 knockdown-enhanced PG biosynthesis. Electrospray ionization mass spectrometry of cellular phospholipids bearing AA showed that the levels of some, if not all, phosphatidylcholine (PC) and phosphatidylinositol species in Acsl4 knockdown cells were decreased after IL-1β stimulation, while those in control cells were not so obviously decreased. In Acsl1 knockdown cells, the levels of some AA-bearing PC species were reduced even in the unstimulated condition. Collectively, these results suggest that Acsl isozymes play distinct roles in the control of AA remodeling in rat fibroblasts: Acsl4 acts as the first step of enzyme for AA remodeling following IL-1β stimulation, and Acsl1 is involved in the maintenance of some AA-containing PC species.

Modulation of COX, LOX and NFκB activities by Xanthium spinosum L. root extract and ziniolide

Xanthium spinosum L. (Asteraceae) is a medicinal weed distributed worldwide. Many of its diverse ethnopharmacological uses – namely diarrhoea, inflammation, liver disorders, snake bite and fever – are linked – at least in part – to an uncontrolled release of arachidonic acid metabolites. The crude extract of X. spinosum roots from Jordanian origin dose-dependently inhibited the 5-LOX (IC50≅10μg/mL), COX-1(IC50≅50μg/mL), and 12-LOX (IC50≅170μg/mL) enzymatic pathways in intact pro-inflammatory cells. A direct activity at the level of PLA2 is not probable, but the extract induced the synthesis of the anti-inflammatory eicosanoid 15(S)-HETE, which may in turn inhibit this enzyme. 5-LOX bioguided fractionation of the crude extract led to the isolation of ziniolide, a known 12,8-guaianolide sesquiterpene lactone, from the hydro-alcoholic fraction of the n-hexane extract (IC50=69μM). Both the plant extract and ziniolide are in vitro inhibitors of the phorbol-induced NFκB activation, a key regulator of the arachidonic pathway.

Functional Hyperemia and Mechanisms of Neurovascular Coupling in the Retinal Vasculature

The retinal vasculature supplies cells of the inner and middle layers of the retina with oxygen and nutrients. Photic stimulation dilates retinal arterioles producing blood flow increases, a response termed functional hyperemia. Despite recent advances, the neurovascular coupling mechanisms mediating the functional hyperemia response in the retina remain unclear. In this review, the retinal functional hyperemia response is described, and the cellular mechanisms that may mediate the response are assessed. These neurovascular coupling mechanisms include neuronal stimulation of glial cells, leading to the release of vasoactive arachidonic acid metabolites onto blood vessels, release of potassium from glial cells onto vessels, and production and release of nitric oxide (NO), lactate, and adenosine from neurons and glia. The modulation of neurovascular coupling by oxygen and NO are described, and changes in functional hyperemia that occur with aging and in diabetic retinopathy, glaucoma, and other pathologies, are reviewed. Finally, outstanding questions concerning retinal blood flow in health and disease are discussed.

The Influence of Aspirin on Release of Eoxin C4, Leukotriene C4 and 15-HETE, in Eosinophilic Granulocytes Isolated from Patients with Asthma

Background: The effect of aspirin on the release of key arachidonic acid metabolites in activated eosinophils from subjects with aspirin-intolerant asthma (AIA) has not been investigated previously, despite the characteristic eosinophilia in AIA. Methods: Peripheral blood eosinophils were isolated from four groups of subjects: healthy volunteers (HV; n = 8), mild asthma (MA; n = 8), severe asthma (SA; n = 9) and AIA (n = 7). In the absence or presence of lysine-aspirin, eosinophils were stimulated with arachidonic acid or calcium ionophore to trigger the 15-lipoxygenase-1 (15-LO) and 5-lipoxygenase (5-LO) pathways, respectively. 15(S)-hydroxy-eicosatetraenoic acid (15-HETE) and eoxin C₄ (EXC₄) were measured as 15-LO products and leukotriene (LT)C₄ as a product of the 5-LO pathway. Results: Activated eosinophils from patients with SA and AIA produced approximately five times more 15-HETE than eosinophils from HV or MA patients. In the presence of lysine-aspirin, eosinophils from AIA, MA and SA patients generated higher levels of 15-HETE than in the absence of lysine-aspirin. Furthermore, in the presence of lysine-aspirin, formation of EXC₄ was also significantly increased in eosinophils from AIA patients, and LTC₄ synthesis was increased both in AIA and SA patients. Conclusions: Taken together, this study shows an increased release of the recently discovered lipid mediator EXC₄, as well as the main indicator of 15-LO activity, 15-HETE, in activated eosinophils from severe and aspirin-intolerant asthmatics, and also elevated EXC₄ and LTC₄ formation in eosinophils from AIA patients after cellular activation in the presence of lysine-aspirin. The findings support a pathophysiological role of the 15-LO pathway in SA and AIA.

54 Mechanisms and treatment of Aβ-induced neuronal dysfunction

Amyloid(A ) peptides are widely thought to cause Alzheimer’s disease (AD), but the mechanisms remain to be elucidated. Among the diverse assembly states in which A can exist, soluble A oligomers appear to have the greatest functional impact. They cause an intriguing combination of synaptic depression and aberrant excitatory neuronal activity. If these processes are linked in a causal chain or result through parallel processes from the same trigger mechanism is unknown. Both could contribute to AD-related cognitive decline. Indeed, deficits in hippocampus-dependent cognitive functions in human amyloid precursor (hAPP) transgenic mice can be reversed by normalizing neuronal expression of the receptor tyrosine kinase EphB2, which regulates the function of synaptic NMDAtype glutamate receptors. A oligomers bind directly to EphB2, promoting EphB2 degradation in the proteasome. Blocking aberrant excitatory neuronal activity in hAPP mice can also prevent or reverse their synaptic and cognitive deficits. The most effective strategies in this regard include reduction of the microtubule-associated protein tau, which appears to be involved in the intracellular transport of factors that regulate synaptic functions; reduction of group IVA phospholipase A2, which may regulate the transport of glutamate receptors through release of arachidonic acid metabolites; reduction of the tyrosine kinase Fyn, which directly regulates the activity of specific glutamate receptors; and treatment with specific antiepileptic drugs that are able to block A -induced neuronal hypersynchrony or overexcitation, possibly by preventing overactivation of extrasynaptic NMDA receptors. Ongoing studies aim to determine whether the beneficial effects of these distinct manipulations involve convergent mechanisms, with the ultimate goal of preventing and ameliorating AD-related cognitive dysfunction.

P.3.f.001 Role of ubiquitin proteasome system in schizophrenia

Schizophrenia (SCZ) and bipolar disorder (BPD) are severe illnesses representing an enormous social, familiar and individual burden that affect 1% of the population world-wide. Several evidences indicate abnormalities of the dopamine system in both SCZ and BPD. Neuronal calcium sensor-1 (NCS-1) is a protein that has many functions in neurotransmission such as inhibition of dopamine D2 receptor desensitization, regulation of ionic channels and enhancement of exocytosis of neurotransmitters. In addition, NCS-1 protein expression and mRNA levels were found increased in pre-frontal cortex (PFC) of SCZ and BPD patients. NCS-1 expression in neural and neuroendocrine cells is well documented and, recently, it was shown that NCS-1 is also expressed in mast cells and neutrophils. NCS-1 has important functions in mast cells since it stimulates Fc epsilon RI-triggered exocytosis and the release of arachidonic acid metabolites. Then, due to the known close relation between the nervous and immune systems, we sought to investigate the NCS-1 expression in lymphocytes and monocytes (CD4+ T lymphocytes, CD56+ NK cells, CD19+ B lymphocytes and CD14+ monocytes) of SCZ and BPD patients. Using flow cytometry, our results have shown that NCS-1 expression was diminished in CD4+T lymphocytes, CD19+ B lymphocytes and CD14+ monocytes of BPD patients and also decreased in CD4+ T lymphocytes and CD56+ NK cells of SCZ patients. Results suggest that immune cells might be a cellular model for studies with SCZ and BPD patients considering NCS-1 functions. Efforts need to be done to investigate the motive of the decreased percentage of immune cells expressing NCS-1 in patients with SCZ and BPD.

Inhibition of aldose reductase prevents endotoxin-induced inflammation by regulating the arachidonic acid pathway in murine macrophages

The bacterial endotoxin lipopolysaccharide (LPS) is known to induce release of arachidonic acid (AA) and its metabolic products, which play important roles in the inflammatory process. We have shown earlier that LPS-induced signals in macrophages are mediated by aldose reductase (AR). Here we have investigated the role of AR in LPS-induced release of AA metabolites and their modulation using a potent pharmacological inhibitor, fidarestat, and AR siRNA ablation in RAW264.7 macrophages and AR-knockout mouse peritoneal macrophages and heart tissue. Inhibition or genetic ablation of AR prevented the LPS-induced synthesis and release of AA metabolites such as PGE2, TXB, PGI2, and LTBs in macrophages. LPS-induced activation of cPLA2 was also prevented by AR inhibition. Similarly, AR inhibition also prevented the calcium ionophore A23187-induced cPLA2 and LTB4 in macrophages. Further, AR inhibition by fidarestat prevented the expression of AA-metabolizing enzymes such as COX-2 and LOX-5 in RAW264.7 cells and AR-knockout mouse-derived peritoneal macrophages. LPS-induced expression of AA-metabolizing enzymes and their catalyzed metabolic products was significantly lower in peritoneal macrophages and heart tissue from AR-knockout mice. LPS-induced activation of redox-sensitive signaling intermediates such as MAPKs, transcription factor NF-κB, and EGR-1, a transcriptional regulator of mPGES-1, which in collaboration with COX-2 leads to the production of PGE2, was also significantly prevented by AR inhibition. Taken together, our results indicate that AR mediates LPS-induced inflammation by regulating the AA-metabolic pathway and thus provide a novel role for AR inhibition in preventing inflammatory complications such as sepsis.

Possible Mediators of Connecting Tubule Glomerular Feedback

In the renal cortex, the connecting tubule (CNT) returns to the glomerular hilum and contacts the afferent arteriole (Af-Art). Increasing Na delivery to the CNT dilates the Af-Art by activating epithelial Na channels, a process that we call connecting tubule glomerular feedback (CTGF). However, the mediator(s) of CTGF are unknown. We tested the hypothesis that Na reabsorption by the CNT induces release of arachidonic acid metabolites that diffuse to and dilate the Af-Art. Microdissected rabbit Af-Arts and adherent CNTs were simultaneously microperfused. CTGF was measured as the increase in diameter of norepinephrine-preconstricted Af-Arts in response to switching NaCl concentration in the lumen of the CNT from 10 to 80 mmol/L. Under control conditions, CTGF was repeatable and completely reversed norepinephrine-induced vasoconstriction. In the presence of 5,8,11,14-eicosatetraynoic acid, an inhibitor of arachidonic acid metabolism, CTGF was completely blocked (-0.7+/-0.3 versus 7.3+/-0.5 microm), suggesting that arachidonic acid metabolites mediate CTGF. Because both cyclooxygenase-derived prostaglandins and epoxygenase-derived epoxyeicosatrienoic acids are known vasodilatory arachidonic acid metabolites, we tested whether indomethacin or MS-PPOH (a cyclooxygenase and an epoxygenase inhibitor) could block CTGF. Both indomethacin and MS-PPOH partially blocked CTGF (2.3+/-0.8 versus 6.5+/-0.5 microm, and 2.9+/-0.8 versus 6.6+/-1.1 microm, respectively). When combined, they completely blocked CTGF (-0.4+/-0.3 versus 6.6+/-1.1 microm). We confirmed these findings by using the epoxyeicosatrienoic acid antagonist 14,15-EEZE. The combination of indomethacin plus 14,15-EEZE completely abolished CTGF (-0.3+/-0.2 versus 8.0+/-1.0 microm). We conclude that increasing Na concentrations in the CNT stimulate release of prostaglandins and epoxyeicosatrienoic acids, which mediate CTGF.

Expression of neuronal calcium sensor-1 (NCS-1) is decreased in leukocytes of schizophrenia and bipolar disorder patients

Schizophrenia (SCZ) and bipolar disorder (BPD) are severe illnesses representing an enormous social, familiar and individual burden that affect 1% of the population world-wide. Several evidences indicate abnormalities of the dopamine system in both SCZ and BPD. Neuronal calcium sensor-1 (NCS-1) is a protein that has many functions in neurotransmission such as inhibition of dopamine D 2 receptor desensitization, regulation of ionic channels and enhancement of exocytosis of neurotransmitters. In addition, NCS-1 protein expression and mRNA levels were found increased in pre-frontal cortex (PFC) of SCZ and BPD patients. NCS-1 expression in neural and neuroendocrine cells is well documented and, recently, it was shown that NCS-1 is also expressed in mast cells and neutrophils. NCS-1 has important functions in mast cells since it stimulates Fc epsilon RI-triggered exocytosis and the release of arachidonic acid metabolites. Then, due to the known close relation between the nervous and immune systems, we sought to investigate the NCS-1 expression in lymphocytes and monocytes (CD4+ T lymphocytes, CD56+ NK cells, CD19+ B lymphocytes and CD14+ monocytes) of SCZ and BPD patients. Using flow cytometry, our results have shown that NCS-1 expression was diminished in CD4+T lymphocytes, CD19+ B lymphocytes and CD14+ monocytes of BPD patients and also decreased in CD4+ T lymphocytes and CD56+ NK cells of SCZ patients. Results suggest that immune cells might be a cellular model for studies with SCZ and BPD patients considering NCS-1 functions. Efforts need to be done to investigate the motive of the decreased percentage of immune cells expressing NCS-1 in patients with SCZ and BPD.

Inhibition of Basic Secretagogue-Induced Signaling in Mast Cells by Cell Permeable Gαi-Derived Peptides

Background: Basic secretagogues of connective tissue mast cells act as receptor mimetic agents that trigger mast cells by activating G proteins. This leads to simultaneous propagation of two signaling pathways: one that culminates in exocytosis, while the other involves protein tyrosine phosphorylation and leads to release of arachidonic acid metabolites. We have previously shown that introduction of a peptide that comprises the C-terminal end of Gαi₃ into permeabilized mast cells inhibits basic secretagogue-induced exocytosis [Aridor et al., Science 1993;262:1569–1572]. We investigated whether cell-permeable peptides, composed of the C-terminus of Gαi₃ fused with importation sequences, affect mast cell function. Methods: Following preincubation with the fused peptides, rat peritoneal mast cells were activated by compound 48/80 and analyzed for histamine and prostaglandin D₂ release and protein tyrosine phosphorylations. Results: We demonstrate that out of three importation sequences tested only Gαi₃ peptide fused with the Kaposi fibroblast growth factor importation sequence (ALL1) inhibited release of histamine. ALL1 as well as a cell-permeable peptide that corresponds to Gαi₂ also blocked compound 48/80-stimulated protein tyrosine phosphorylation, though the latter did not block histamine release. ALL1 effect was G protein-specific, as it was incapable of blocking protein tyrosine phosphorylation stimulated by pervanadate. Conclusion: ALL1, a transducible Gαi₃-corresponding peptide, blocks the two signaling pathways in mast cells: histamine release and protein tyrosine phosphorylation. Cell permeable peptides that block these two signaling cascades may constitute a novel approach for preventing the onset of the allergic reaction.

The mast cell: where endocytosis and regulated exocytosis meet

We have investigated whether Ca(2+)-binding proteins, which have been implicated in the control of neurons and neuroendocrine secretion, play a role in controlling mast cell function. These studies have identified synaptotagmins (Syts) II, III, and IX as well as neuronal Ca(2+) sensor 1 (NCS-1) as important regulators of mast cell function. Strikingly, we find that these Ca(2+)-binding proteins contribute to mast cell function by regulating specific endocytic pathways. Syt II, the most abundant Syt homologue in mast cells, resides in an amine-free lysosomal compartment. Studying the function of Syt II-knocked down rat basophilic leukemia cells has shown a dual function of this homologue. Syt II is required for the downregulation of protein kinase Calpha, but it negatively regulates lysosomal exocytosis. Syt III, the next most abundant homologue, localizes to early endosomes and is required for the formation of the endocytic recycling compartment (ERC). Syt IX and NCS-1 localize to the ERC and regulate ERC export, NCS-1 by activating phosphatidylinositol 4-kinase beta. Finally, we show that recycling through the ERC is needed for secretory granule protein sorting as well as for the activation of the mitogen-activated protein kinases, extracellular signal-regulated kinase 1 and 2. Accordingly, NCS-1 stimulates Fc epsilon RI-triggered exocytosis and release of arachidonic acid metabolites.