RESUMEN
Multiple lines of evidence implicate brain serotonin (5-hydroxytryptamine; 5-HT) system dysfunction in the pathophysiology of stressor-related and anxiety disorders. Here we investigate the influence of constitutively deficient 5-HT synthesis on stressor-related anxiety-like behaviors using Tryptophan hydroxylase 2 (Tph2) mutant mice. Functional assessment of c-Fos after associated foot shock, electrophysiological recordings of GABAergic synaptic transmission, differential expression of the Slc6a4 gene in serotonergic neurons were combined with locomotor and anxiety-like measurements in different contextual settings. Our findings indicate that constitutive Tph2 inactivation and consequential lack of 5-HT synthesis in Tph2 null mutant mice (Tph2-/-) results in increased freezing to associated foot shock and a differential c-Fos activity pattern in the basolateral complex of the amygdala. This is accompanied by altered GABAergic transmission as observed by recordings of inhibitory postsynaptic currents on principal neurons in the basolateral nucleus, which may explain increased fear associated with hyperlocomotion and escape-like responses in aversive inescapable contexts. In contrast, lifelong 5-HT deficiency as observed in Tph2 heterozygous mice (Tph+/-) is able to be compensated through reduced GABAergic transmission in the basolateral nucleus of the amygdala based on Slc6a4 mRNA upregulation in subdivisions of dorsal raphe neurons. This results in increased activity of the basolateral nucleus of the amygdala due to associated foot shock. In conclusion, our results reflect characteristic syndromal dimensions of panic disorder and agoraphobia. Thus, constitutive lack of 5-HT synthesis influence the risk for anxiety- and stressor-related disorders including panic disorder and comorbid agoraphobia through the absence of GABAergic-dependent compensatory mechanisms in the basolateral nucleus of the amygdala.
Asunto(s)
Amígdala del Cerebelo/fisiopatología , Ansiedad/fisiopatología , Reacción de Fuga , Trastorno de Pánico/fisiopatología , Serotonina/fisiología , Agorafobia/fisiopatología , Amígdala del Cerebelo/metabolismo , Animales , Electrochoque , Miedo , Potenciales Postsinápticos Inhibidores , Masculino , Ratones Noqueados , Núcleos del Rafe/metabolismo , Serotonina/deficiencia , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Triptófano Hidroxilasa/genética , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Plants utilize sulfate to synthesize primary and secondary sulfur-containing metabolites required for growth and survival in the environment. Sulfate is taken up into roots from the soil and distributed to various organs through the functions of membrane-bound sulfate transporters, while it is utilized as the primary substrate for synthesizing sulfur-containing metabolites in the sulfate assimilation pathways. Transporters and enzymes for the assimilative conversion of sulfate are regulated in highly organized manners depending on changes in sulfate supply from the environment and demand for biosynthesis of reduced sulfur compounds in the plant systems. Over the past few decades, the effect of sulfur nutrition on gene expression of sulfate transporters and assimilatory enzymes has been extensively studied with the aim of understanding the full landscape of regulatory networks.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Plantas/metabolismo , Sulfatos/metabolismo , Plantas/genéticaRESUMEN
BACKGROUND AND PURPOSE: Acute hydrocephalus is an early and common complication of aneurysmal subarachnoid hemorrhage (SAH). However, considerably fewer patients develop chronic hydrocephalus requiring shunt placement. Our aim was to develop a risk score for early identification of patients with shunt dependency after SAH. METHODS: Two hundred and forty-two SAH individuals who were treated in our institution between January 2008 and December 2013 and survived the initial impact were retrospectively analyzed. Clinical parameters within 72 h after the ictus were correlated with shunt dependency. Independent predictors were summarized into a new risk score which was validated in a subsequent SAH cohort treated between January and December 2014. RESULTS: Seventy-five patients (31%) underwent shunt placement. Of 23 evaluated variables, only the following five showed independent associations with shunt dependency and were subsequently used to establish the Chronic Hydrocephalus Ensuing from SAH Score (CHESS, 0-8 points): Hunt and Hess grade ≥IV (1 point), location of the ruptured aneurysm in the posterior circulation (1 point), acute hydrocephalus (4 points), the presence of intraventricular hemorrhage (1 point) and early cerebral infarction on follow-up computed tomography scan (1 point). The CHESS showed strong correlation with shunt dependency (P = 0.0007) and could be successfully validated in both internal SAH cohorts tested. Patients scoring ≥6 CHESS points had significantly higher risk of shunt dependency (P < 0.0001) than other patients. CONCLUSION: The CHESS may become a valuable diagnostic tool for early estimation of shunt dependency after SAH. Further evaluation and external validation will be required in prospective studies.
Asunto(s)
Hidrocefalia/etiología , Hemorragia Subaracnoidea/complicaciones , Adulto , Anciano , Aneurisma Roto/diagnóstico por imagen , Infarto Cerebral/diagnóstico por imagen , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Estudios Retrospectivos , Medición de Riesgo , Factores de Riesgo , Tomografía Computarizada por Rayos XRESUMEN
Neuroblastoma is an embryonal tumor with a heterogeneous clinical course. The tumor is presumed to be derived from the neural crest, but the cells of origin remain to be determined. To date, few recurrent genetic changes contributing to neuroblastoma formation, such as amplification of the MYCN oncogene and activating mutations of the ALK oncogene, have been identified. The possibility to model neuroblastoma in mice allows investigation of the cell of origin hypothesis in further detail. Here we present the evidence that murine neural crest progenitor cells can give rise to neuroblastoma upon transformation with MYCN or ALK(F1174L). For this purpose we used JoMa1, a multipotent neural crest progenitor cell line, which is kept in a viable and undifferentiated state by a tamoxifen-activated c-Myc transgene (c-MycER(T)). Expression of MYCN or ALK(F1174L), one of the oncogenic ALK variants identified in primary neuroblastomas, enabled these cells to grow independently of c-MycER(T) activity in vitro and caused formation of neuroblastoma-like tumors in vivo in contrast to parental JoMa1 cells and JoMa1 cells-expressing TrkA or GFP. Tumorigenicity was enhanced upon serial transplantation of tumor-derived cells, and tumor cells remained susceptible to the MYC-inhibitor, NBT-272, indicating that cell growth depended on functional MYCN. Our findings support neural crest progenitor cells as the precursor cells of neuroblastoma, and indicate that neuroblastomas arise as their malignant progeny.
Asunto(s)
Células Madre Neoplásicas/patología , Cresta Neural/patología , Neuroblastoma/genética , Neuroblastoma/patología , Proteínas Nucleares/genética , Proteínas Oncogénicas/genética , Proteínas Tirosina Quinasas Receptoras/genética , Células Madre/patología , Quinasa de Linfoma Anaplásico , Animales , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Humanos , Ratones , Ratones Desnudos , Ratones Transgénicos , Proteína Proto-Oncogénica N-Myc , Células Madre Neoplásicas/metabolismo , Cresta Neural/metabolismo , Neuroblastoma/metabolismo , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/metabolismo , Proteínas Oncogénicas/biosíntesis , Proteínas Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Proteínas Tirosina Quinasas Receptoras/metabolismo , Células Madre/metabolismo , Transfección , Trasplante HeterólogoRESUMEN
A cytosolic 84 kDa Group VIA phospholipase A2 (iPLA2beta) that does not require Ca2+ for catalysis was cloned from Chinese hamster ovary (CHO) cells, murine P388D1 cells, pancreatic islet beta-cells, and other sources. Proposed iPLA2beta functions include participation in phosphatidylcholine (PC) homeostasis by degrading excess PC generated in CHO cells that overexpress CTP:phosphocholine cytidylyltransferase (CT), which catalyzes the rate-limiting step in PC biosynthesis; participation in biosynthesis of arachidonate-containing PC species in P388D1 cells by generating lysophosphatidylcholine (LPC) acceptors for arachidonate incorporation; and participation in signaling events in insulin secretion from islet beta-cells. To further examine iPLA2beta functions in beta-cells, we prepared stably transfected INS-1 insulinoma cell lines that overexpress iPLA2beta activity eightfold compared to parental INS-1 cells or to INS-1 cells transfected with an empty retroviral vector that did not contain iPLA2beta cDNA. The iPLA2beta-overexpressing cells exhibit a twofold increase in CT activity compared to parental cells but little change in rates of [3H]choline incorporation into or disappearance from PC. Electrospray ionization (ESI) tandem mass spectrometric measurements indicate that iPLA2beta-overexpressing cells have 1.5-fold higher LPC levels than parental INS-1 cells but do not exhibit increased rates of [3H]arachidonate incorporation into phospholipids, and incorporation is unaffected by a bromoenol lactone (BEL) suicide substrate inhibitor of iPLA2beta. The rate of appearance of arachidonate-containing phosphatidylethanolamine species visualized by ESI mass spectrometry is also similar in iPLA2beta-overexpressing and parental INS-1 cells incubated with supplemental arachidonic acid, and this process is unaffected by BEL. Compared to parental INS-1 cells, iPLA2beta-overexpressing cells proliferate more rapidly and exhibit amplified insulin secretory responses to a protein kinase C-activating phorbol ester, glucose, and a cAMP analog. These findings suggest that iPLA2beta plays a signaling role in beta-cells that differs from housekeeping functions in PC biosynthesis and degradation in P388D1 and CHO cells.
Asunto(s)
División Celular , Insulinoma/enzimología , Neoplasias Pancreáticas/enzimología , Fosfolipasas A/genética , Fosfolípidos/metabolismo , Transfección , Animales , Ácido Araquidónico/metabolismo , Células CHO , Colina/metabolismo , Citidililtransferasa de Colina-Fosfato/genética , Citidililtransferasa de Colina-Fosfato/metabolismo , Cricetinae , Expresión Génica , Vectores Genéticos , Insulinoma/metabolismo , Insulinoma/patología , Cinética , Lisofosfatidilcolinas/metabolismo , Espectrometría de Masas , Ratones , Neoplasias Pancreáticas/patología , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Retroviridae/genética , Tritio , Células Tumorales CultivadasRESUMEN
A cytosolic 84-kDa group VIA phospholipase A(2) (iPLA(2)beta) that does not require Ca(2+) for catalysis has been cloned from several sources, including rat and human pancreatic islet beta-cells and murine P388D1 cells. Many potential iPLA(2)beta functions have been proposed, including a signaling role in beta-cell insulin secretion and a role in generating lysophosphatidylcholine acceptors for arachidonic acid incorporation into P388D1 cell phosphatidylcholine (PC). Proposals for iPLA(2)beta function rest in part on effects of inhibiting iPLA(2)beta activity with a bromoenol lactone (BEL) suicide substrate, but BEL also inhibits phosphatidate phosphohydrolase-1 and a group VIB phospholipase A(2). Manipulation of iPLA(2)beta expression by molecular biologic means is an alternative approach to study iPLA(2)beta functions, and we have used a retroviral construct containing iPLA(2)beta cDNA to prepare two INS-1 insulinoma cell clonal lines that stably overexpress iPLA(2)beta. Compared with parental INS-1 cells or cells transfected with empty vector, both iPLA(2)beta-overexpressing lines exhibit amplified insulin secretory responses to glucose and cAMP-elevating agents, and BEL substantially attenuates stimulated secretion. Electrospray ionization mass spectrometric analyses of arachidonic acid incorporation into INS-1 cell PC indicate that neither overexpression nor inhibition of iPLA(2)beta affects the rate or extent of this process in INS-1 cells. Immunocytofluorescence studies with antibodies directed against iPLA(2)beta indicate that cAMP-elevating agents increase perinuclear fluorescence in INS-1 cells, suggesting that iPLA(2)beta associates with nuclei. These studies are more consistent with a signaling than with a housekeeping role for iPLA(2)beta in insulin-secreting beta-cells.
Asunto(s)
Ácido Araquidónico/metabolismo , Insulina/metabolismo , Fosfolipasas A/metabolismo , Fosfolípidos/biosíntesis , Transducción de Señal/fisiología , 1-Metil-3-Isobutilxantina/farmacología , Adenilil Ciclasas/metabolismo , Animales , Colforsina/farmacología , Inhibidores Enzimáticos/farmacología , Glucosa/farmacología , Fosfolipasas A2 Grupo VI , Humanos , Secreción de Insulina , Insulinoma , Cinética , Ratones , Naftalenos/farmacología , Neoplasias Pancreáticas , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfolipasas A/genética , Fosfolipasas A2 , Pironas/farmacología , Ratas , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Especificidad por Sustrato , Transfección , Células Tumorales CultivadasRESUMEN
The Zucker diabetic fatty (ZDF) rat is a genetic model of type II diabetes mellitus in which males homozygous for nonfunctional leptin receptors (fa/fa) develop obesity, hyperlipidemia, and hyperglycemia, but rats homozygous for normal receptors (+/+) remain lean and normoglycemic. Insulin resistance develops in young fa/fa rats and is followed by evolution of an insulin secretory defect that triggers hyperglycemia. Because insulin secretion and insulin sensitivity are affected by membrane phospholipid fatty acid composition, we have determined whether metabolic abnormalities in fa/fa rats are associated with changes in tissue phospholipids. Electrospray ionization mass spectrometric analyses of glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE) molecular species from tissues of prediabetic (6 wk of age) and overtly diabetic (12 wk) fa/fa rats and from +/+ rats of the same ages indicate that arachidonate-containing species from heart, aorta, and liver of prediabetic fa/fa rats made a smaller contribution to GPC total ion current than was the case for +/+ rats. There was a correspondingly larger contribution from species with sn-2 oleate or linoleate substituents in fa/fa heart and aorta. The relative contributions of arachidonate-containing GPC species increased in these tissues as fa/fa rats aged and were equal to or greater than those for +/+ rats by 12 wk. For heart and aorta, relative contributions from GPE species with sn-2 arachidonate or docosahexaenoate substituents to the total ion current increased and those from species with sn-2 oleate or linoleate substituents fell as fa/fa rats aged, but these tissue lipid profiles changed little with age in +/+ rats. GPC and GPE profiles for brain, kidney, sciatic nerve, and red blood cells were similar among fa/fa and +/+ rats at 6 and 12 wk of age, and pancreatic islets from fa/fa and +/+ rats exhibited similar GPC and GPE profiles at 12 wk of age. Under-representation of arachidonate-containing GPC and GPE species in some fa/fa rat tissues at 6 wk could contribute to insulin resistance, but depletion of islet arachidonate-containing GPC and GPE species is unlikely to explain the evolution of the insulin secretory defect that is well-developed by 12 wk of age.
Asunto(s)
Hiperglucemia/metabolismo , Hiperlipidemias/metabolismo , Fosfolípidos/análisis , Espectrometría de Masa por Ionización de Electrospray/métodos , Factores de Edad , Animales , Aorta/metabolismo , Ácido Araquidónico/metabolismo , Cromatografía Líquida de Alta Presión , Ácidos Docosahexaenoicos/metabolismo , Insulina/metabolismo , Litio/metabolismo , Miocardio/metabolismo , Páncreas/metabolismo , Fosfatidilcolinas/análisis , Fosfatidiletanolaminas/análisis , Ratas , Ratas Zucker , Factores de Tiempo , Distribución TisularRESUMEN
Insulin secretion by pancreatic islet beta-cells is impaired in diabetes mellitus, and normal beta-cells are enriched in phospholipids with arachidonate as sn-2 substituent. Such molecules may play structural roles in exocytotic membrane fusion or serve as substrates for phospholipases activated by insulin secretagogues. INS-1 insulinoma cells respond to secretagogues and permit the study of effects of culture with free fatty acids on phospholipid composition and secretion. INS-1 cell glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE) lipids are demonstrated here by electrospray ionization mass spectrometry to contain a lower fraction of molecules with arachidonate and a higher fraction with oleate as sn-2 substituent than native islets. Palmitic acid supplementation induces little change in these INS-1 cell lipids, but supplementation with linoleate or arachidonate induces a large rise in the fraction of INS-1 cell GPC species with polyunsaturated sn-2 substituents and a fall in oleate-containing species to yield a GPC profile similar to native islets. The fraction of GPE lipids comprised of plasmenylethanolamine species with polyunsaturated sn-2 substituents in early-passage INS-1 cells is similar to that of islets, but declines on serial passage. Such molecules might participate in exocytotic membrane fusion, and late-passage INS-1 cells have reduced insulin secretory responses. Arachidonate supplementation induces a rise in the fraction of INS-1 cell GPE lipids with polyunsaturated sn-2 substituents and partially restores responses to insulin secretagogues by late-passage INS-1 cells, but does not further amplify secretion by early-passage cells. Effects of extracellular free fatty acids on beta-cell phospholipid composition and secretory responses could be involved in changes in beta-cell function during the period of hyper-free fatty acidemia that precedes diabetes mellitus.
Asunto(s)
Ácidos Grasos/farmacología , Insulina/análisis , Islotes Pancreáticos/química , Fosfolípidos/química , Animales , Insulinoma , Islotes Pancreáticos/metabolismo , Espectrometría de Masas/métodos , Neoplasias Pancreáticas , Ratas , Células Tumorales CultivadasRESUMEN
Upon differentiation, U937 promonocytic cells gain the ability to release a large fraction of arachidonate esterified in phospholipids when stimulated, but the mechanism is unclear. U937 cells express group IV phospholipase A(2) (cPLA(2)), but neither its level nor its phosphorylation state increases upon differentiation. A group VI PLA(2) (iPLA(2)) that is sensitive to a bromoenol lactone inhibitor catalyzes arachidonate hydrolysis from phospholipids in some cells and facilitates arachidonate incorporation into glycerophosphocholine (GPC) lipids in others, but it is not known whether U937 cells express iPLA(2). We confirm that ionophore A23187 induces substantial [(3)H]arachidonate release from differentiated but not control U937 cells, and electrospray ionization mass spectrometric (ESI/MS) analyses indicate that differentiated cells contain a higher proportion of arachidonate-containing GPC species than control cells. U937 cells express iPLA(2) mRNA and activity, but iPLA(2) inhibition impairs neither [(3)H]arachidonate incorporation into nor release from U937 cells. Experiments with phosphatidate phosphohydrolase (PAPH) and phospholipase D (PLD) inhibitors coupled with ESI/MS analyses of PLD-PAPH products indicate that differentiated cells gain the ability to produce diacylglycerol (DAG) via PLD-PAPH. DAG promotes arachidonate release by a mechanism that does not require DAG hydrolysis, is largely independent of protein kinase C, and requires cPLA(2) activity. This may reflect DAG effects on cPLA(2) substrate state.
Asunto(s)
Diferenciación Celular , Diglicéridos/metabolismo , Espectrometría de Masas/métodos , Lípidos de la Membrana/química , Fosfolipasas A/metabolismo , Fosfolípidos/metabolismo , Ácido Araquidónico/metabolismo , Diferenciación Celular/efectos de los fármacos , Diglicéridos/análisis , Dimetilsulfóxido/farmacología , Activación Enzimática , Humanos , Hidrólisis , Fosfatidato Fosfatasa/metabolismo , Fosfolipasas A2 , Tritio , Células U937RESUMEN
BACKGROUND: Fasting hyperglycemia has been associated with HIV protease inhibitor (PI) therapy. OBJECTIVE: To determine whether absolute insulin deficiency or insulin resistance with relative insulin deficiency and an elevated body mass index (BMI) contribute to HIV PI-associated diabetes. DESIGN: Cross-sectional evaluation. PATIENTS: 8 healthy seronegative men, 10 nondiabetic HIV-positive patients naive to PI, 15 nondiabetic HIV-positive patients receiving PI (BMI = 26 kg/m2), 6 nondiabetic HIV-positive patients receiving PI (BMI = 31 kg/m2), and 8 HIV-positive patients with diabetes receiving PI (BMI = 34 kg/m2). All patients on PI received indinavir. MEASUREMENTS: Fasting concentrations of glucoregulatory hormones. Direct effects of indinavir (20 microM) on rat pancreatic beta-cell function in vitro. RESULTS: In hyperglycemic HIV-positive subjects, circulating concentrations of insulin, C-peptide, proinsulin, glucagon, and the proinsulin/insulin ratio were increased when compared with those of the other 4 groups (p < .05). Morning fasting serum cortisol concentrations were not different among the 5 groups. Glutamic acid decarboxylase (GAD) antibody titers were uncommon in all groups. High BMI was not always associated with diabetes. In vitro, indinavir did not inhibit proinsulin to insulin conversion or impair glucose-induced secretion of insulin and C-peptide from rat beta-cells. CONCLUSIONS: The pathogenesis of HIV PI-associated diabetes involves peripheral insulin resistance with insulin deficiency relative to hyperglucagonemia and a high BMI. Pancreatic beta-cell function was not impaired by indinavir. HIV PI-associated diabetes mirrors that of non-insulin-dependent diabetes mellitus and impaired insulin action in the periphery.
Asunto(s)
Fármacos Anti-VIH/efectos adversos , Diabetes Mellitus Tipo 1/complicaciones , Infecciones por VIH/complicaciones , Inhibidores de la Proteasa del VIH/efectos adversos , Indinavir/efectos adversos , Resistencia a la Insulina , Adulto , Animales , Fármacos Anti-VIH/uso terapéutico , Péptido C/metabolismo , Células Cultivadas , Estudios Transversales , Glucagón/metabolismo , Infecciones por VIH/tratamiento farmacológico , Inhibidores de la Proteasa del VIH/uso terapéutico , Humanos , Indinavir/uso terapéutico , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Masculino , Fosfolipasas A/metabolismo , Proinsulina/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
An 84-kDa group VI phospholipase A2 (iPLA2) that does not require Ca2+ for catalysis has been cloned from Chinese hamster ovary cells, murine P388D1 cells, and pancreatic islet beta-cells. A housekeeping role for iPLA2 in generating lysophosphatidylcholine (LPC) acceptors for arachidonic acid incorporation into phosphatidylcholine (PC) has been proposed because iPLA2 inhibition reduces LPC levels and suppresses arachidonate incorporation and phospholipid remodeling in P388D1 cells. Because islet beta-cell phospholipids are enriched in arachidonate, we have examined the role of iPLA2 in arachidonate incorporation into islets and INS-1 insulinoma cells. Inhibition of iPLA2 with a bromoenol lactone (BEL) suicide substrate did not suppress and generally enhanced [3H]arachidonate incorporation into these cells in the presence or absence of extracellular calcium at varied time points and BEL concentrations. Arachidonate incorporation into islet phospholipids involved deacylation-reacylation and not de novo synthesis, as indicated by experiments with varied extracellular glucose concentrations and by examining [14C]glucose incorporation into phospholipids. BEL also inhibited islet cytosolic phosphatidate phosphohydrolase (PAPH), but the PAPH inhibitor propranolol did not affect arachidonate incorporation into islet or INS-1 cell phospholipids. Inhibition of islet iPLA2 did not alter the phospholipid head-group classes into which [3H]arachidonate was initially incorporated or its subsequent transfer from PC to other lipids. Electrospray ionization mass spectrometric measurements indicated that inhibition of INS-1 cell iPLA2 accelerated arachidonate incorporation into PC and that inhibition of islet iPLA2 reduced LPC levels by 25%, suggesting that LPC mass does not limit arachidonate incorporation into islet PC. Gas chromatography/mass spectrometry measurements indicated that BEL but not propranolol suppressed insulin secretagogue-induced hydrolysis of arachidonate from islet phospholipids. In islets and INS-1 cells, iPLA2 is thus not required for arachidonate incorporation or phospholipid remodeling and may play other roles in these cells.
Asunto(s)
Ácido Araquidónico/metabolismo , Ácidos Grasos/metabolismo , Insulinoma/metabolismo , Islotes Pancreáticos/metabolismo , Lisofosfatidilcolinas/metabolismo , Fosfolipasas A/fisiología , Fosfolípidos/metabolismo , Animales , Cricetinae , Inhibidores Enzimáticos/farmacología , Etilmaleimida/farmacología , Fosfolipasas A2 Grupo VI , Interleucina-1/farmacología , Islotes Pancreáticos/efectos de los fármacos , Masculino , Naftalenos/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Fosfolipasas A2 , Propranolol/farmacología , Pironas/farmacología , Ratas , Ratas Sprague-Dawley , Células Tumorales Cultivadas , omega-N-Metilarginina/farmacologíaRESUMEN
Opiates are commonly abused substances, and forensic urine drug-testing for them requires gas chromatographic-mass spectrometric (GC-MS) confirmation. There are also medical reasons to test urine for opiates, and confirmation procedures other than GC-MS are often used for medical drug-testing. A thin-layer chromatographic (TLC) method distinguishes morphine, acetylmorphine, hydromorphone, oxymorphone, codeine, dihydrocodeine, hydrocodone, and oxycodone in clinical specimens. In certain clinical circumstances, GC-MS confirmation is requested for opiates identified by TLC, but, to our knowledge, no previous report examines all of the above opiates in a single GC-MS procedure. We find that they can be distinguished by GC-MS analyses of trimethylsilyl (TMS) ether derivatives, and identities of 6-keto opiates can be further confirmed by GC-MS analysis of methoxime (MO)-TMS derivatives. Inclusion of deuterium-labeled internal standards permits identification of the opiates in urine at concentrations below the TLC cutoff level of 600 ng/ml, and the GC-MS assay is linear over a concentration range that spans that level. This GC-MS procedure has proved useful as a third-stage identification step in a medical drug-testing sequence involving prior immunoassay and TLC.
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Narcóticos/orina , Codeína/análogos & derivados , Codeína/orina , Cromatografía de Gases y Espectrometría de Masas , Humanos , Hidrocodona/orina , Hidromorfona/orina , Estructura Molecular , Morfina/orina , Derivados de la Morfina/orina , Narcóticos/química , Oxicodona/orina , Oximorfona/orinaRESUMEN
Stimulation of pancreatic islets with glucose induces phospholipid hydrolysis and accumulation of nonesterified arachidonic acid, which may amplify the glucose-induced Ca2+ entry into islet beta-cells that triggers insulin secretion. Ca2+ loss from beta-cell intracellular compartments has been proposed to induce both Ca2+ entry and events dependent on arachidonate metabolism. We examine here effects of inducing Ca2+ loss from intracellular sequestration sites with ionophore A23187 and thapsigargin on arachidonate hydrolysis from islet phospholipids. A23187 induces a decline in islet arachidonate-containing phospholipids and release of nonesterified arachidonate. A23187-induced arachidonate release is of similar magnitude when islets are stimulated in Ca2+-replete or in Ca2+-free media or when islets loaded with the intracellular Ca2+ chelator BAPTA are stimulated in Ca2+-free medium, a condition in which A23187 induces no rise in beta-cell cytosolic [Ca2+]. Thapsigargin also induces islet arachidonate release under these conditions. A23187- or thapsigargin-induced arachidonate release is prevented by a bromoenol lactone (BEL) inhibitor of a beta-cell phospholipase A2 (iPLA2), which does not require Ca2+ for catalytic activity and which is negatively modulated by and physically interacts with calmodulin by Ca2+-dependent mechanisms. Agents that cause Ca2+ loss from islet intracellular compartments thus induce arachidonate hydrolysis from phospholipids by a BEL-sensitive mechanism that does not require a rise in cytosolic [Ca2+], and a BEL-sensitive enzyme-like iPLA2 or a related membranous activity may participate in sensing Ca2+ compartment content.
Asunto(s)
Ácido Araquidónico/metabolismo , Calcio/metabolismo , Islotes Pancreáticos/metabolismo , Lípidos de la Membrana/metabolismo , Fosfolípidos/metabolismo , Animales , Calcimicina/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Hidrólisis , Masculino , Espectrometría de Masas , Ratas , Ratas Sprague-Dawley , Tapsigargina/farmacologíaRESUMEN
The sphingolipid sulfatide is a component of myelin and some non-neuronal cells. Antibodies to sulfatide occur in some patients with autoimmune neuropathies and in patients with insulin-dependent diabetes mellitus (IDDM) caused by immunologic destruction of insulin-secreting pancreatic islet beta-cells. Distinct sulfatide molecular species may differ in immunogenicity, and facile means to identify sulfatide species in islets and other tissues obtainable in only small amounts could be useful. Electrospray ionization mass spectrometry (ESI/MS) permits structural determination of small quantities of phospholipids and is applied here to sulfatide analysis. We find that sulfatide standards are readily analyzed by negative ion ESI/MS, and tandem mass spectra of individual species exhibit some ions common to all species and other ions that reflect distinct fatty acid substituents in different sulfatide molecules. A signature ion cluster resulting from cleavage directed by the alpha-hydroxy group of sulfatide species with a hydroxylated fatty acid substituent identifies such species. Sulfatide profiles in tissue lipid extracts can be obtained by ESI/MS/MS scanning for common sulfatide ions and for ions reflecting fatty acid substituents. Islets are demonstrated to contain sulfatide and to exhibit a profile of species different from that of brain.
Asunto(s)
Química Encefálica , Islotes Pancreáticos/química , Espectrometría de Masas/métodos , Sulfoglicoesfingolípidos/análisis , Animales , Bovinos , Ácidos Grasos/análisis , Cromatografía de Gases y Espectrometría de Masas , Hidrogenación , Hidroxilación , Estructura Molecular , Ratas , Sulfoglicoesfingolípidos/químicaRESUMEN
Glucose-induced insulin secretion from pancreatic islets involves hydrolysis of arachidonic acid from phospholipids as an intermediary event. Accumulation of nonesterified arachidonate in islet membranes may influence both ion fluxes that trigger insulin secretion and fusion of secretory granule and plasma membranes. Recent findings indicate that plasmenylethanolamine species may also participate in fusion of such membranes, but high-performance liquid chromatographic (HPLC) and gas chromatographic/mass spectrometric (GC/MS) analyses of islet secretory granule phospholipids suggested that they contain little plasmenylethanolamine. Here, electrospray ionization mass spectrometry (ESI/MS) of intact phospholipid molecules is used to demonstrate that the most prominent components of all major glycerophospholipid headgroup classes in islets are arachidonate-containing species. Such species contribute the majority of the ESI/MS negative ion current from rat and human islet glycerophosphoethanolamine (GPE), and the fraction of GPE negative ion current contributed by plasmenylethanolamine species in rat islets is higher than that for rat liver or heart and similar to that for brain. The most prominent sn-2 substituent of plasmenylethanolamine species in brain is docosahexaenoate and in islets is arachidonate. Arachidonate-containing plasmenylethanolamine species are also prominent components of GPE from islet secretory granules and plasma membranes. Fusion of islet secretory granule and plasma membranes is demonstrated to be catalyzed by cytosolic components from insulinoma cells and rat brain with chromatographic similarities to a rabbit brain factor that specifically catalyzes fusion of plasmenylethanolamine-containing membranes.
Asunto(s)
Exocitosis , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Fusión de Membrana , Fosfolípidos/metabolismo , Fracciones Subcelulares/metabolismo , Animales , Ácido Araquidónico/química , Encéfalo/metabolismo , Ácidos Docosahexaenoicos/química , Humanos , Técnicas In Vitro , Secreción de Insulina , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Islotes Pancreáticos/química , Islotes Pancreáticos/ultraestructura , Hígado/química , Hígado/metabolismo , Masculino , Espectrometría de Masas , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Miocardio/química , Miocardio/metabolismo , Fosfatidiletanolaminas/química , Fosfolípidos/química , Ratas , Ratas Sprague-Dawley , Fracciones Subcelulares/química , Células Tumorales CultivadasRESUMEN
Electrospray ionization (ESI) tandem mass spectrometry (MS) has simplified analysis of phospholipid mixtures, and, in negative ion mode, permits structural identification of picomole amounts of phospholipid species. Collisionally activated dissociation (CAD) of phospholipid anions yields negative ion tandem mass spectra that contain fragment ions representing the fatty acid substituents as carboxylate anions. Glycerophosphocholine (GPC) lipids contain a quaternary nitrogen moiety and more readily form cationic adducts than anionic species, and positive ion tandem mass spectra of protonated GPC species contain no abundant ions that identify fatty acid substituents. We report here that lithiated adducts of GPC species are readily formed by adding lithium hydroxide to the solution in which phospholipid mixtures are infused into the ESI source. CAD of [MLi+] ions of GPC species yields tandem mass spectra that contain prominent ions representing losses of the fatty acid substituents. These ions and their relative abundances can be used to assign the identities and positions of the fatty acid substituents of GPC species. Tandem mass spectrometric scans monitoring neutral losses of the head-group or of fatty acid substituents from lithiated adducts can be used to identify GPC species in tissue phospholipid mixtures. Similar scans monitoring parents of specific product ions can also be used to identify the fatty acid substituents of GPC species, and this facilitates identification of distinct isobaric contributors to ions observed in the ESI/MS total ion current.
Asunto(s)
Glicerilfosforilcolina/química , Litio/química , Animales , Química Encefálica , Indicadores y Reactivos , Hígado/química , Masculino , Espectrometría de Masas , Ratas , Ratas Sprague-DawleyRESUMEN
Culture of rat pancreatic islets with interleukin-1 (IL-1) results in up-regulation of the inducible isoform of nitric oxide synthase and overproduction of nitric oxide (NO). This is associated with reversible inhibition of both glucose-induced insulin secretion and islet glucose oxidation, and these effects are prevented by the inducible nitric oxide synthase inhibitor NG-monomethylarginine. IL-1 also induces accumulation of nonesterified arachidonic acid in islets by an NO-dependent mechanism, and one potential explanation for that effect would involve an IL-1-induced enhancement of islet glycolytic flux. We have therefore examined effects of IL-1 on islet glycolytic utilization of glucose and find that culture of islets with IL-1 in medium containing 5.5 mM glucose results in suppression of islet glucose utilization subsequently measured at glucose concentrations between 6 and 18 mM. The IL-1-induced suppression of islet glucose utilization is associated with a decline in islet glucokinase mRNA content, as determined by competitive reverse transcriptase-polymerase chain reaction, and in glucokinase protein synthesis, as determined by immuoprecipitation experiments, and all of these effects are prevented by NG-monomethylarginine. These findings suggest that IL-1 can down-regulate islet glucokinase, which is the primary component of the islet glucose-sensor apparatus, by an NO-dependent mechanism. Because reductions in islet glucokinase levels are known to cause a form of type II diabetes mellitus, these observations raise the possibility that factors which increase islet NO levels might contribute to development of glucose intolerance.
Asunto(s)
Glucoquinasa/metabolismo , Glucosa/metabolismo , Interleucina-1/farmacología , Islotes Pancreáticos/metabolismo , Óxido Nítrico/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Animales , Inhibidores Enzimáticos/farmacología , Glucólisis/efectos de los fármacos , Radical Hidroxilo/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Masculino , Óxido Nítrico Sintasa/antagonistas & inhibidores , Reacción en Cadena de la Polimerasa , Ratas , Ratas Sprague-Dawley , omega-N-Metilarginina/farmacologíaRESUMEN
We have previously reported that pancreatic islet beta-cells and clonal HIT insulinoma cells express an ATP-stimulatable Ca(2+)-independent phospholipase A2 (ASCI-PLA2) enzyme and that activation of this enzyme appears to participate in glucose-stimulated insulin secretion. To further examine this hypothesis, glucose-responsitivity and expression of ASCI-PLA2 activity in various insulinoma cell lines were examined. Secretagogue-stimulated insulin secretion was observed with beta TC6-f7 and early passage (EP)-beta TC6 cells. In contrast, RIN-m5f, beta TC3, and late passage (LP)-beta TC6 cells exhibited little secretagogue-induced secretion. A haloenollactone suicide substrate (HELSS) which inhibits ASCI-PLA2 activity ablated secretagogue-induced insulin secretion from beta TC6-f7 and EP-beta TC6 cells. All insulinoma cell lines studied expressed both cytosolic and membrane-associated Ca(2+)-independent PLA2 activities which were inhibited by HELSS. The cytosolic enzymatic activity in the glucose-responsive beta TC6-f7 and EP-beta TC6 cells was activated by ATP and protected against thermal denaturation by ATP, but this was not the case in the glucose-unresponsive RIN-m5f, beta TC3, or LP-beta TC6 cells. Comparison of the distribution of Ca(2+)-independent PLA2 activity revealed that membrane-associated activity was higher than cytosolic activity in beta TC6-f7 and EP-beta TC6 cells but not in RIN-m5f, beta TC3, or LP-beta TC6 cells. Insensitivity of cytosolic activity to ATP may prevent association of the PLA2 activity with membrane substrates and contribute to attenuated glucose-responsitivity in the RIN-m5f, beta TC3, or LP-beta TC6 cells. HIT insulinoma cells were also found to undergo a decline in both glucose-responsitivity and membrane-associated Ca(2+)-independent PLA2 activity upon serial passage in culture, and this was associated with a reduction in membrane content of arachidonate-containing phospholipids. These and previous results suggest that the ATP-stimulatable PLA2 enzyme may participate in glucose-induced insulin secretion.
Asunto(s)
Glucosa/farmacología , Insulinoma/enzimología , Neoplasias Pancreáticas/enzimología , Fosfolipasas A/metabolismo , Adenosina Trifosfato/farmacología , Animales , Calcio/farmacología , Membrana Celular/enzimología , Cricetinae , Citosol/enzimología , Activación Enzimática/efectos de los fármacos , Insulina/metabolismo , Secreción de Insulina , Insulinoma/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/enzimología , Islotes Pancreáticos/metabolismo , Ratones , Neoplasias Pancreáticas/metabolismo , Fosfolipasas A2 , Ratas , Células Tumorales CultivadasRESUMEN
Arachidonylethanolamide (AEA) isolated from porcine brain binds to cannabinoid receptors, mimics cannabinoid pharmacologic effects, and has been proposed as an endogenous cannabinoid receptor ligand. Demonstration of co-distribution of AEA and cannabinoid receptors in various brain regions could provide supportive evidence for this role. We have performed isotope dilution mass spectrometric measurements of AEA and have demonstrated AEA production by rat tissue homogenates in vitro from exogenous arachidonate and ethanolamine. No detectable endogenous AEA (<3.5 pmol/g of tissue) was observed in fresh rat brain, whether or not inhibitors of AEA hydrolysis were present during tissue processing. AEA (>1 nmol/g) was produced during saponification of brain phospholipid extracts. This appears not to reflect hydrolysis of N-arachidonylethanolamine phospholipid precursors of AEA, because Streptomyces chromfucsis phospholipase D, which is active against NAPE, failed to generate AEA from brain phospholipids despite substantial conversion of phospholipids to phosphatidic acid. Such experiments suggested that the abundance of N-arachidonylethanolamine phospholipid in fresh rat brain may be less than 1 in 10(6) phospholipid molecules. AEA generated during saponification of tissue phospholipids appears to arise from base-catalyzed aminolysis of arachidonate-containing glycerolipids, because AEA was produced from synthetic (1-stearoyl, 2-arachidonoyl)-phosphatidylethanolamine under saponification conditions, and the amount produced increased 300-fold when free ethanolamine was included in the hydrolysis solution. Although AEA was not detectable (<0.17 pmol/mg of protein) in fresh rat brain, AEA accumulated post mortem to levels of 126 pmol/mg of brain protein. These findings do not exclude the possibility that AEA is rapidly synthesized and degraded locally in vivo, but they indicate that the AEA content of fresh rat brain and of NAPE precursors from which AEA might be derived are exceedingly low and that AEA can be produced artifactually from biological materials.
Asunto(s)
Ácidos Araquidónicos/metabolismo , Encéfalo/metabolismo , Cambios Post Mortem , Animales , Ácidos Araquidónicos/análisis , Química Encefálica , Cerebelo/metabolismo , Cromatografía Líquida de Alta Presión , Deuterio , Endocannabinoides , Cromatografía de Gases y Espectrometría de Masas , Ligandos , Hígado/metabolismo , Masculino , Miocardio/metabolismo , Fosfolipasa D/metabolismo , Fosfolípidos/aislamiento & purificación , Fosfolípidos/metabolismo , Alcamidas Poliinsaturadas , Técnica de Dilución de Radioisótopos , Ratas , Ratas Sprague-Dawley , Receptores de Cannabinoides , Receptores de Droga/metabolismo , Streptomyces/enzimología , Factores de Tiempo , TritioRESUMEN
A method is described for detection and quantitation of agmatine [4-(aminobutyl)guanidine] by gas chromatography/negative-ion chemical ionization/mass spectrometry after derivatization with hexafluoroacetylacetone. The lower limit of detection of the derivative was about 25 fmol on-column. For quantitative studies of agmatine content in biological samples, a procedure utilizing an internal standard ([15N4]agmatine prepared from [15N4]arginine) and an extraction step had a lower limit of detection of about 15 pmol for total sample content. Agmatine content was measured in rat tissue samples and normalized to protein content. Kidney and spleen samples exhibited the greatest content of agmatine per unit protein mass but agmatine was also detected in pancreatic islets and brain regions (cerebellum and cerebral cortex). On the basis of these measurements, it is estimated that the pancreatic islet intracellular agmatine concentration may exceed 1 microM. The sensitive and highly specific means of detection and quantitation provided by mass spectrometry may be useful in investigating the physiological role of agmatine in mammalian systems.