Novel contraceptive targets to inhibit ovulation: the prostaglandin E2 pathway.

BACKGROUND: Prostaglandin E2 (PGE2) is an essential intrafollicular regulator of ovulation. In contrast with the one-gene, one-protein concept for synthesis of peptide signaling molecules, production and metabolism of bioactive PGE2 requires controlled expression of many proteins, correct subcellular localization of enzymes, coordinated PGE2 synthesis and metabolism, and prostaglandin transport in and out of cells to facilitate PGE2 action and degradation. Elevated intrafollicular PGE2 is required for successful ovulation, so disruption of PGE2 synthesis, metabolism or transport may yield effective contraceptive strategies. METHODS: This review summarizes case reports and studies on ovulation inhibition in women and macaques treated with cyclooxygenase inhibitors published from 1987 to 2014. These findings are discussed in the context of studies describing levels of mRNA, protein, and activity of prostaglandin synthesis and metabolic enzymes as well as prostaglandin transporters in ovarian cells. RESULTS: The ovulatory surge of LH regulates the expression of each component of the PGE2 synthesis-metabolism-transport pathway within the ovulatory follicle. Data from primary ovarian cells and cancer cell lines suggest that enzymes and transporters can cooperate to optimize bioactive PGE2 levels. Elevated intrafollicular PGE2 mediates key ovulatory events including cumulus expansion, follicle rupture and oocyte release. Inhibitors of the prostaglandin-endoperoxide synthase 2 (PTGS2) enzyme (also known as cyclooxygenase-2 or COX2) reduce ovulation rates in women. Studies in macaques show that PTGS2 inhibitors can reduce the rates of cumulus expansion, oocyte release, follicle rupture, oocyte nuclear maturation and fertilization. A PTGS2 inhibitor reduced pregnancy rates in breeding macaques when administered to simulate emergency contraception. However, PTGS2 inhibition did not prevent pregnancy in monkeys when administered to simulate monthly contraceptive use. CONCLUSION: PTGS2 inhibitors alone may be suitable for use as emergency contraceptives. However, drugs of this class are unlikely to be effective as monthly contraceptives. Inhibitors of additional PGE2 synthesis enzymes or modulation of PGE2 metabolism or transport also hold potential for reducing follicular PGE2 and preventing ovulation. Approaches which target multiple components of the PGE2 synthesis-metabolism-transport pathway may be required to effectively block ovulation and lead to the development of novel contraceptive options for women. Therapies which target PGE2 may also impact disorders of the uterus and could also have benefits for women's health in addition to contraception.

Basic mechanisms of calcific aortic valve disease.

Calcific aortic valve disease (CAVD) is the most common heart valve disorder. There is no medical treatment to prevent and/or promote the regression of CAVD. Hence, it is of foremost importance to delineate and understand the key basic underlying mechanisms involved in CAVD. In the past decade our comprehension of the underpinning processes leading to CAVD has expanded at a fast pace. Hence, our understanding of the basic pathobiological processes implicated in CAVD might lead eventually to the development of novel pharmaceutical therapies for CAVD. In this review, we discuss molecular processes that are implicated in fibrosis and mineralization of the aortic valve. Specifically, we address the role of lipid retention, inflammation, phosphate signalling and osteogenic transition in the development of CAVD. Interplays between these different processes and the key regulation pathways are discussed along with their clinical relevance.

Lipid biology of the podocyte--new perspectives offer new opportunities.

In the past 15 years, major advances have been made in understanding the role of lipids in podocyte biology. First, susceptibility to focal segmental glomerulosclerosis (FSGS) and glomerular disease is associated with an APOL1 sequence variant, is expressed in podocytes and encodes apolipoprotein L1, an important component of HDL. Second, acid sphingomyelinase-like phosphodiesterase 3b encoded by SMPDL3b has a role in the conversion of sphingomyelin to ceramide and its levels are reduced in renal biopsy samples from patients with recurrent FSGS. Furthermore, decreased SMPDL3b expression is associated with increased susceptibility of podocytes to injury after exposure to sera from these patients. Third, in many individuals with membranous nephropathy, autoantibodies against the phospholipase A2 (PLA2) receptor, which is expressed in podocytes, have been identified. Whether these autoantibodies affect the activity of PLA2, which liberates arachidonic acid from glycerophospholipids and modulates podocyte function, is unknown. Fourth, clinical and experimental evidence support a role for ATP-binding cassette sub-family A member 1-dependent cholesterol efflux, free fatty acids and glycerophospolipids in the pathogenesis of diabetic kidney disease. An improved understanding of lipid biology in podocytes might provide insights to develop therapeutic targets for primary and secondary glomerulopathies.

Partial characterization of proapoptotic action of biliary deteriorated lipids on biliary epithelial cells in pancreaticobiliary diseases.

BACKGROUND: Lysophosphatidylcholine (LPC), a derivative of phosphatidylcholine (PC) hydrolyzed by phospholipase A2 (PLA2), is reported to be increased in bile of the patients with pancreaticobiliary maljunction or intrahepatic cholelithiasis, both of which are major risk factors for biliary tract cancers with undefined etiology. METHODS: To investigate the influence of LPC on biliary epithelial cells (BECs), a human cholangiocarcinoma cell line HuCCT-1 and an immortalized human BECs line MMNK-1 were treated by LPC in vitro. RESULTS: The treatment of LPC exhibited cytotoxicity with significant induction of apoptosis. In addition to upregulation of Fas receptor mRNA, the activities of caspase-8 and -3 were significantly increased by LPC treatment. We also observed upregulation of Bax mRNA and significant activation of caspase-9. Interestingly, LPC significantly upregulated G2A, a member of transmembrane G protein-coupled receptor family at mRNA and protein levels, and 9-hydroxyoctadecaduenoic acid (9HODE), an oxidized free fatty acid that functions as a ligand for G2A dramatically reduced cell viability when treated together with LPC. CONCLUSIONS: These data suggest that PLA2, which catalyzes the hydrolysis of PC to yield LPC and free fatty acid, is supposed to be an important etiological factor in BECs injury in pancreaticobiliary maljunction or intrahepatic cholelithiasis.

Deficiency of phospholipase A2 group 7 decreases intestinal polyposis and colon tumorigenesis in Apc(Min/+) mice.

Platelet-activating factor (PAF) is a naturally occurring phospholipid that mediates diverse effects such as physiological and pathological inflammation, immunosuppression, and cancer. Several lines of evidence support both positive and negative roles for PAF in carcinogenesis. PAF stimulates cell growth, oncogenic transformation, and metastasis, but can also limit proliferation and induce apoptosis. The biological context and microenvironment seem to define whether PAF has pro- or anticarcinogenic effects. To investigate the role of exacerbated PAF signaling in colon cancer, we conducted cell-based and in vivo studies using genetically engineered mice lacking expression of phospholipase A2 group 7 (PLA2G7), an enzyme that specifically metabolizes PAF and structurally related glycerophospholipids. Absence of Pla2g7 robustly decreased intestinal polyposis and colon tumor formation in Apc(Min)(/+) mice, suggesting an antitumorigenic role for PAF in settings characterized by aberrant function of the tumor suppressor Adenomatous polyposis coli (Apc). In colonic epithelial cells, exposure to a PAF analog led to dephosphorylation of Akt at serine-473 and induction of apoptosis. The mechanism of this response involved formation of a complex between ß-arrestin 1 and the Akt phosphatase PHLPP2, and activation of the intrinsic pathway of apoptosis. Our results suggest that strategies based on inhibiting PLA2G7 activity or increasing PAF-mediated signaling hold promise for the treatment of intestinal malignancies that harbor mutations in APC.

Role of patatin-like phospholipase domain-containing 3 on lipid-induced hepatic steatosis and insulin resistance in rats.

UNLABELLED: Genome-wide array studies have associated the patatin-like phospholipase domain-containing 3 (PNPLA3) gene polymorphisms with hepatic steatosis. However, it is unclear whether PNPLA3 functions as a lipase or a lipogenic enzyme and whether PNPLA3 is involved in the pathogenesis of hepatic insulin resistance. To address these questions we treated high-fat-fed rats with specific antisense oligonucleotides to decrease hepatic and adipose pnpla3 expression. Reducing pnpla3 expression prevented hepatic steatosis, which could be attributed to decreased fatty acid esterification measured by the incorporation of [U-(13) C]-palmitate into hepatic triglyceride. While the precursors for phosphatidic acid (PA) (long-chain fatty acyl-CoAs and lysophosphatidic acid [LPA]) were not decreased, we did observe an ∼20% reduction in the hepatic PA content, ∼35% reduction in the PA/LPA ratio, and ∼60%-70% reduction in transacylation activity at the level of acyl-CoA:1-acylglycerol-sn-3-phosphate acyltransferase. These changes were associated with an ∼50% reduction in hepatic diacylglycerol (DAG) content, an ∼80% reduction in hepatic protein kinase Cε activation, and increased hepatic insulin sensitivity, as reflected by a 2-fold greater suppression of endogenous glucose production during the hyperinsulinemic-euglycemic clamp. Finally, in humans, hepatic PNPLA3 messenger RNA (mRNA) expression was strongly correlated with hepatic triglyceride and DAG content, supporting a potential lipogenic role of PNPLA3 in humans. CONCLUSION: PNPLA3 may function primarily in a lipogenic capacity and inhibition of PNPLA3 may be a novel therapeutic approach for treatment of nonalcoholic fatty liver disease-associated hepatic insulin resistance.

Differential participation of phospholipase A2 isoforms during iron-induced retinal toxicity. Implications for age-related macular degeneration.

Both elevated iron concentrations and the resulting oxidative stress condition are common signs in retinas of patients with age-related macular degeneration (AMD). The role of phospholipase A(2) (PLA(2)) during iron-induced retinal toxicity was investigated. To this end, isolated retinas were exposed to increasing Fe(2+) concentrations (25, 200 or 800 µM) or to the vehicle, and lipid peroxidation levels, mitochondrial function, and the activities of cytosolic PLA(2) (cPLA(2)) and calcium-independent PLA(2) (iPLA(2)) were studied. Incubation with Fe(2+) led to a time- and concentration-dependent increase in retinal lipid peroxidation levels whereas retinal cell viability was only affected after 60 min of oxidative injury. A differential release of arachidonic acid (AA) and palmitic acid (PAL) catalyzed by cPLA(2) and iPLA(2) activities, respectively, was also observed in microsomal and cytosolic fractions obtained from retinas incubated with iron. AA release diminished as the association of cyclooxygenase-2 increased in microsomes from retinas exposed to iron. Retinal lipid peroxidation and cell viability were also analyzed in the presence of cPLA(2) inhibitor, arachidonoyl trifluoromethyl ketone (ATK), and in the presence of iPLA(2) inhibitor, bromoenol lactone (BEL). ATK decreased lipid peroxidation levels and also ERK1/2 activation without affecting cell viability. BEL showed the opposite effect on lipid peroxidation. Our results demonstrate that iPLA(2) and cPLA(2) are differentially regulated and that they selectively participate in retinal signaling in an experimental model resembling AMD.

Investigation on signal transduction pathways after H(1) receptor activated by histamine in C6 glioma cells: involvement of phosphatidylinositol and arachidonic acid metabolisms.

BACKGROUND: Information related to histamine-induced cellular responses in C6 glioma cells through second messenger pathways has not been fully studied, especially the involvement of arachidonic acid (AA) metabolism. In addition, specific labeled ligand binding to histamine receptor sites still needs to be clarified. METHODS: Labeled mepyramine ligand was used to study its binding sites; [(3)H] inositol was used to detect inositol 4-phosphate (IP(1)) formation, and fura-2/AM was used to detect intracellular free calcium ion ([Ca(2+)]i) level activated by the phosphatidylinositol-phospholipase C (PI-PLC) pathway. Also, labeled AA was used to detect the metabolism of AA and its metabolites release via the activation of phospholipase A2 in the presence of histamine. RESULTS: C6 glioma cells incubated with histamine in the presence of 10 mM LiCl for 60 minutes induced an increase of IP(1) and glycerophosphoric-inositol (GPI) accumulation. In addition, histamine caused an increase of extracellular AA with its metabolite release, eliciting a transient and sustained increase of free [Ca(2+)]i. The sustained increase of [Ca(2+)]i was almost or completely blocked by La(3+) and excess ethylene diamine tetraacetic acid. The calcium ion influx associated with the sustained phase required the presence of histamine on the receptor sites, and could be blocked by a H(1) antagonist, chlorpheniramine. CONCLUSION: C6 glioma cells possess histamine H(1) receptors that have affinity towards [(3)H]mepyramine binding, and are coupled to PI-PLC to generate inositol phosphates and to increase [Ca(2+)]i, and they are coupled to phospholipase A2 (PLA2) to generate GPI and AA with its metabolite release. The transient increase in [Ca(2+)]i can be attributed to Ca(2+) release from intracellular stores, whereas the sustained increase in [Ca(2+)]i is due to influx of extracellular calcium ions. The sustained increase in [Ca(2+)]i plays a role in the activation of histamine receptor-coupled PLA2.

Calcium around the Golgi apparatus: implications for intracellular membrane trafficking.

As with other complex cellular functions, intracellular membrane transport involves the coordinated engagement of a series of organelles and machineries; in the last couple of decades more importance has been given to the role of calcium (Ca(2+)) in the regulation of membrane trafficking, which is directly involved in coordinating the endoplasmic reticulum-to-Golgi-to-plasma membrane delivery of cargo. Consequently, the Golgi apparatus (GA) is now considered not just the place proteins mature in as they move to their final destination(s), but it is increasingly viewed as an intracellular Ca(2+) store. In the last few years the mechanisms regulating the homeostasis of Ca(2+) in the GA and its role in membrane trafficking have begun to be elucidated. Here, these recent discoveries that shed light on the role Ca(2+) plays as of trigger of different steps during membrane trafficking has been reviewed. This includes recruitment of proteins and SNARE cofactors to the Golgi membranes, which are both fundamental for the membrane remodeling and the regulation of fusion/fission events occurring during the passage of cargo across the GA. I conclude by focusing attention on Ca(2+) homeostasis dysfunctions in the GA and their related pathological implications.

Paroxetine-induced Ca2+ movement and death in OC2 human oral cancer cells.

The effect of the antidepressant paroxetine on cytosolic free Ca2+ concentrations ([Ca2+]i) in OC2 human oral cancer cells is unclear. This study explored whether paroxetine changed basal [Ca2+]i levels in suspended OC2 cells by using fura-2 as a Ca2+-sensitive fluorescent dye. Paroxetine at concentrations between 100-1,000 microM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced by 50% by removing extracellular Ca2+. Paroxetine-induced Ca2+ influx was inhibited by the store-operated Ca2+ channel blockers nifedipine, econazole and SK&F96365, and protein kinase C modulators. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin abolished paroxetine-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 did not alter paroxetine-induced [Ca2+]i rise. Paroxetine at 10-50 microM induced cell death in a concentration-dependent manner. The death was not reversed when cytosolic Ca2+ was chelated with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Propidium iodide staining suggests that apoptosis plays a role in the death. Collectively, in OC2 cells, paroxetine induced [Ca2+]i rise by causing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via store-operated Ca2+ channels in a manner regulated by protein kinase C and phospholipase A2. Paroxetine (up to 50 microM) induced cell death in a Ca2+-independent manner.

Phospholipase A2 activity-dependent and -independent fusogenic activity of Naja nigricollis CMS-9 on zwitterionic and anionic phospholipid vesicles.

CMS-9, a phospholipase A(2) (PLA(2)) from Naja nigricollis venom, induced the death of human breast cancer MCF-7 cells accompanied with the formation of cell clumps without clear boundaries between cells. Annexin V-FITC staining indicated that abundant phosphatidylserine appeared on the outer membrane of MCF-7 cell clumps, implying the possibility that CMS-9 may promote membrane fusion via anionic phospholipids. To validate this proposition, fusogenic activity of CMS-9 on vesicles composed of zwitterionic phospholipid alone or a combination of zwitterionic and anionic phospholipids was examined. Although CMS-9-induced fusion of zwitterionic phospholipid vesicles depended on PLA(2) activity, CMS-9-induced fusion of vesicles containing anionic phospholipids could occur without the involvement of PLA(2) activity. Membrane-damaging activity of CMS-9 was associated with its fusogenicity. Moreover, CMS-9 induced differently membrane leakage and membrane fusion of vesicles with different compositions. Membrane fluidity and binding capability with phospholipid vesicles were not related to the fusogenicity of CMS-9. However, membrane-bound conformation and mode of CMS-9 depended on phospholipid compositions. Collectively, our data suggest that PLA(2) activity-dependent and -independent fusogenicity of CMS-9 are closely related to its membrane-bound modes and targeted membrane compositions.

Multiple phospholipase A2 enzymes participate in the inflammatory process in osteoarthritic cartilage.

OBJECTIVE: The aim of this study was to determine the involvement of pro-inflammatory phospholipase A2 (PLA2) enzymes in human chondrocytes from patients with osteoarthritis (OA). METHODS: PLA2 involvement in OA chondrocytes was analysed by (a) arachidonic acid (AA) and oleic acid release, (b) PLA2 mRNA analysis, and (c) prostaglandin E2 (PGE2) production in cultured OA chondrocytes in response to various cytokines and platelet activating factor (PAF). RESULTS: Pro-inflammatory cytokines and PAF stimulation led to increased AA release, interleukin (IL)-1ß and tumour necrosis factor (TNF) being the strongest inducers. The pattern of oleic acid release was similar to but less prominent than AA release, suggesting that predominantly arachidonyl selective enzymes were activated. IL-1ß, TNF, IL-6, and IL-8 upregulated secretory group IIA, IID, and V phospholipase A2 (sPLA2-IIA, -IID, -V) and cytosolic group IVA phospholipase A2 (cPLA2-IVA) expression, where induction of chondrocyte sPLA2-IID is a novel finding. Furthermore, IL-1ß, TNF, and IL-6 also induced COX2 expression. PAF induced expression of group IIA, IID and IVA PLA2, and COX2. In line with its anti-inflammatory properties, IL-4 was unable to induce either AA release or expression of PLA2s or COX2. IL-1ß and TNF strongly increased PGE2 production, with IL-1ß as the most prominent inducer. CONCLUSION: Multiple PLA2 isoforms are expressed and influenced by pro-inflammatory stimuli in OA chondrocytes. Hence, several PLA2 enzymes may contribute to chondrocyte function by their upregulation and activation, and increased AA release and PGE2 production may therefore be important effectors in OA pathophysiology. PLA2 enzymes and cPLA2-IVA in particular are thus possible therapeutic targets in OA.

[Lipid networks in mast cell biology].

Tissue-resident mast cells are derived from circulating committed progenitors, which are originated from pluripotential hematopoietic stem cells in bone marrow. These progenitors migrate into extravascular tissues, where they undergo differentiation and maturation into tissue-specific mature phenotypes. When activated by IgE/antigen, stem cell factor, neuropeptides, or other stimuli, mature mast cells release three classes of biologically active products, including pre-formed mediators stored in secretory granules, newly transcribed cytokines and chemokines, and de novo synthesized lipid mediators. Therefore, these cells have been implicated as major effector cells in acute and chronic inflammatory diseases. In recent years, it has become clear that lipid mediators including arachidonic acid metabolites (prostaglandins and leukotrienes) and lysophospholipid-derived products play crucial roles in mast cell-associated pathology. In this article, we will provide an overview of the roles of various lipid mediators in allergic diseases fueled by studies of their biosynthetic enzymes or receptors. In the latter part, we will make a particular focus on phospholipase A(2) enzymes, which are placed at the bottleneck (rate-limiting) step of the lipid mediator-biosynthetic pathways.

Claude H. Organ, Jr. memorial lecture: splanchnic hypoperfusion provokes acute lung injury via a 5-lipoxygenase-dependent mechanism.

Postinjury multiple organ failure (MOF) is the net result of a dysfunctional immune response to injury characterized by a hyperactive innate system and a suppressed adaptive system. Acute lung injury (ALI) is the first clinical manifestation of organ failure, followed by renal and hepatic dysfunction. Circulatory shock is integral in the early pathogenesis of MOF, and the gut has been invoked as the motor of MOF. Mesenteric lymph is recognized as the mechanistic link between splanchnic ischemia/reperfusion and distant organ dysfunction, but the specific mediators remain to be defined. Current evidence suggests the lipid fraction of postshock mesenteric lymph is central in the etiology of ALI. Specifically, our recent work suggests that intestinal phospholipase A2 generated arachidonic acid and its subsequent 5-lipoxygenase products are essential in the pathogenesis of ALI. Proteins conveyed via postshock mesenteric lymph also may have an important role. Elucidating these mediators and the timing of their participation in pulmonary inflammation is critical in translating our current knowledge to new therapeutic strategies at the bedside.

Nicotine inhibits Fc epsilon RI-induced cysteinyl leukotrienes and cytokine production without affecting mast cell degranulation through alpha 7/alpha 9/alpha 10-nicotinic receptors.

Smokers are less likely to develop some inflammatory and allergic diseases. In Brown-Norway rats, nicotine inhibits several parameters of allergic asthma, including the production of Th2 cytokines and the cysteinyl leukotriene LTC(4). Cysteinyl leukotrienes are primarily produced by mast cells, and these cells play a central role in allergic asthma. Mast cells express a high-affinity receptor for IgE (FcepsilonRI). Following its cross-linking, cells degranulate and release preformed inflammatory mediators (early phase) and synthesize and secrete cytokines/chemokines and leukotrienes (late phase). The mechanism by which nicotine modulates mast cell activation is unclear. Using alpha-bungarotoxin binding and quantitative PCR and PCR product sequencing, we showed that the rat mast/basophil cell line RBL-2H3 expresses nicotinic acetylcholine receptors (nAChRs) alpha7, alpha9, and alpha10; exposure to exceedingly low concentrations of nicotine (nanomolar), but not the biologically inactive metabolite cotinine, for > or = 8 h suppressed the late phase (leukotriene/cytokine production) but not degranulation (histamine and hexosaminidase release). These effects were unrelated to those of nicotine on intracellular free calcium concentration but were causally associated with the inhibition of cytosolic phospholipase A(2) activity and the PI3K/ERK/NF-kappaB pathway, including phosphorylation of Akt and ERK and nuclear translocation of NF-kappaB. The suppressive effect of nicotine on the late-phase response was blocked by the alpha7/alpha9-nAChR antagonists methyllycaconitine and alpha-bungarotoxin, as well as by small interfering RNA knockdown of alpha7-, alpha9-, or alpha10-nAChRs, suggesting a functional interaction between alpha7-, alpha9-, and alpha10-nAChRs that might explain the response of RBL cells to nanomolar concentrations of nicotine. This "hybrid" receptor might serve as a target for novel antiallergic/antiasthmatic therapies.

Synaptic mechanism for functional synergism between delta- and mu-opioid receptors.

By sustained activation of mu-opioid receptors (MORs), chronic opioids cause analgesic tolerance, physical dependence, and opioid addiction, common clinical problems for which an effective treatment is still lacking. Chronic opioids recruit delta-opioid receptors (DORs) to plasma membrane through exocytotic trafficking, but the role of this new DOR and its interaction with existing MOR in brain functions and in these clinical problems remain largely unknown. In this study, we investigated the mechanisms underlying synaptic and behavioral actions of chronic morphine-induced DORs and their interaction with MORs in nucleus raphe magnus (NRM) neurons important for opioid analgesia. We found that the emerged DOR inhibited GABAergic IPSCs through both the phospholipase A(2) (PLA(2)) and cAMP/protein kinase A (PKA) signaling pathways. MOR inhibition of IPSCs, normally mediated predominantly by the PLA(2) pathway, was additionally mediated by the cAMP/PKA pathway, with MOR potency significantly increased after chronic morphine treatment. Isobologram analysis revealed a synergistic DOR-MOR interaction in their IPSC inhibition, which was dependent on upregulated activities of both the PLA(2) and cAMP/PKA pathways. Furthermore, DOR and MOR agonists microinjected into the NRM in vivo also produced a PLA(2)-dependent synergism in their antinociceptive effects. These findings suggest that the cAMP/PKA pathway, upregulated by chronic opioids, becomes more important in the mechanisms of both MOR and DOR inhibition of GABA synaptic transmission after chronic opioid exposure, and DORs and MORs are synergic both synaptically and behaviorally in producing analgesic effects in a PLA(2)-dependent fashion, supporting the potential therapeutic use of DOR agonists in pain management under chronic opioid conditions.

Hypotonicity-induced Renin exocytosis from juxtaglomerular cells requires aquaporin-1 and cyclooxygenase-2.

The mechanism by which extracellular hypotonicity stimulates release of renin from juxtaglomerular (JG) cells is unknown. We hypothesized that osmotically induced renin release depends on water movement through aquaporin-1 (AQP1) water channels and subsequent prostanoid formation. We recorded membrane capacitance (C(m)) by whole-cell patch clamp in single JG cells as an index of exocytosis. Hypotonicity increased C(m) significantly and enhanced outward current. Indomethacin, PLA(2) inhibition, and an antagonist of prostaglandin transport impaired the C(m) and current responses to hypotonicity. Hypotonicity also increased exocytosis as determined by a decrease in single JG cell quinacrine fluorescence in an indomethacin-sensitive manner. In single JG cells from COX-2(-/ -) and AQP1(-/ -) mice, hypotonicity increased neither C(m) nor outward current, but 0.1-muM PGE(2) increased both in these cells. A reduction in osmolality enhanced cAMP accumulation in JG cells but not in renin-producing As4.1 cells; only the former had detectable AQP1 expression. Inhibition of protein kinase A blocked the hypotonicity-induced C(m) and current response in JG cells. Taken together, our results show that a 5 to 7% decrease in extracellular tonicity leads to AQP1-mediated water influx in JG cells, PLA(2)/COX-2-mediated prostaglandin-dependent formation of cAMP, and activation of PKA, which promotes exocytosis of renin.

Oxidative stress activates ADAM17/TACE and induces its target receptor shedding in platelets in a p38-dependent fashion.

AIMS: Oxidative stress accompanies inflammatory and vascular diseases. The objective of this study was to explore whether reactive oxygen species can activate shedding of platelet receptors and thus suppress platelet function. METHODS AND RESULTS: Hydrogen peroxide and glucose oxidase were chosen to model oxidative stress in vitro. We demonstrate that oxidative damage activated tumour necrosis factor-alpha-converting enzyme (TACE) and induced shedding of its targets, glycoprotein (GP) Ibalpha and GPV, in murine and human platelets. Also, 12-HpETE, a peroxide synthesized in the platelet lipoxygenase pathway, induced TACE-mediated receptor cleavage. The TACE activation was independent of platelet activation, as alpha-granule secretion, activation of alphaIIbbeta3, or phosphatidylserine expression was not observed. TACE activation induced by hydrogen peroxide was dependent on p38 mitogen-activated protein kinase signalling, whereas protein kinase C, phosphoinositide 3-kinase, and caspases were not involved. Inhibition of p38 cytoplasmic targets, phospholipase A(2) and heat shock protein 27, did not prevent shedding, whereas blocking 12-lipoxygenase or Src kinase slightly inhibited TACE activation. The loss of the GPIbalpha receptor induced by oxidative stress rendered platelets unable to incorporate into a growing thrombus in vivo. CONCLUSION: Oxidative stress can render platelets functionally less active by shedding key adhesion receptors via the activation of p38. This suggests that oxidative injury of platelets may attenuate their function.