Projection length stimulated by oxytocin is modulated by the inhibition of calcium signaling in U-87MG cells.

Neuropeptide oxytocin contributes to the regulation of glial cell morphology. The precise mechanisms, however, are not yet fully understood. In the present study, we have investigated whether an oxytocin-induced increase of intracellular calcium is required for cell extension in astrocyte-like U-87MG cells. Oxytocin (1 µM) significantly increased the length of the cell projections measured by the green-fluorescent protein labeled microtubule-associated protein after 48 h. The knockdown of oxytocin receptors (OXTR) in U-87MG cells prevented the elongation of the projections. Incubation of U-87MG cells in the presence of oxytocin, resulted in a significant increase of intracellular calcium, specifically blocked by the OXTR antagonist L-371,257. Both quercetin, which is a phosphoinositide 3-kinase inhibitor, and the phospholipase C inhibitor U-73122 reduced oxytocin-induced elevation of intracellular calcium concentration. Conversely, neither diltiazem, an L-type voltage-gated calcium channel blocker nor tetracaine, which is a blocker of the ryanodine receptors, showed an effect on intracellular calcium levels. Treatment of cells with quercetin, U-73122 and the voltage-gated calcium channel blockers cilnidipine, ω-agatoxin and mibefradil prevented the elongation of projections stimulated by oxytocin. Oxytocin treatment resulted in a significant increase in gene and protein expression of the scaffolding protein SHANK3. Our results clearly show that activation of OXTRs contributes to the elongation of cell projections in astrocyte-like U-87MG cells and that this effect is mediated by an extracellular calcium influx accompanied by an increase in scaffolding proteins expression.

Downregulation of Oxytocin Receptor Decreases the Length of Projections Stimulated by Retinoic Acid in the U-87MG Cells.

Oxytocin is a neuropeptide widely expressed in the brain. Oxytocin plays a role in both proliferation and differentiation of various cells. Previous studies have suggested that oxytocin could affect the morphology of neuronal cells, therefore the objective of the present study was to test whether (1) oxytocin receptor stimulation/inhibition by specific ligands may change cell morphology and gene expression of selected cytoskeletal proteins (2) oxytocin receptor silencing/knockdown may decrease the length of cell projections (3) oxytocin receptor knockdown may affect human glioblastoma U-87MG cell survival. We confirmed the stimulatory effect of retinoic acid (10 µM) and oxytocin (1 µM) on projection growth. The combination of retinoic acid (10 µM) and oxytocin receptor antagonist (L-371,257, 1 µM) decreased projections length. Contrary to our assumptions, oxytocin receptor silencing did not prevent stimulation of length of projection by retinoic acid. Retinoic acid's and oxytocin's stimulation of projections length was significantly blunted in U-87MG cells with oxytocin receptor knockdown. Cell viability was significantly decreased in U-87MG cells with oxytocin receptor knockdown. Significantly higher levels of mRNA for cytoskeletal proteins drebrin and vimentin were observed in response to oxytocin incubation for 48 h. The data obtained in the present study clearly show that oxytocin induces formation and elongation of cell projections in astrocyte-like U-87MG cells. The effect is mediated by oxytocin receptors and it is accompanied by an increase in gene expression of drebrin and vimentin. Thus, oxytocin receptor signaling, particularly in the glial cells, may play an important role in native cell life, differentiation processes, and tumor progression, as well.

[Quantitative and qualitative evaluation of the interim PET/CT in lymphoma treatment in the prediction of complete metabolic response]. / Evaluación cuantitativa y cualitativa de la PET/TC a mitad del tratamiento en linfomas en la predicción de respuesta metabólica completa.

OBJECTIVE: To compare two different methods for the interpretation of interim PET/CT (PET/CT-i) in lymphomas, and to establish which one best predicts a complete metabolic response (CMR) in the PET/CT study at the end of treatment (PET/CT-et). MATERIAL AND METHODS: Retrospective longitudinal analysis of the PET/CT studies for staging (PET/CT-s), PET/CT-i and PET/CT-et of 65 patients, 35 Hodgkin's lymphoma (HL) and 30 Non-HL. The PET/CT-i was performed between the second and fourth chemotherapy cycle. It was interpreted using two different criteria: qualitative criteria (5 point visual scale), semiquantitative criteria (percentage difference between the lesion with more SUVmax in the PET/CT-s and PET/CT-i). We analyzed the likelihood of obtaining a CMR in the PET/CT-et according to the results obtained on the PET/CT-i with these two criteria. RESULTS: We obtained sensitivity (S), specificity (Sp), positive predictive values (PPV), negative predictive values (NPV) and likelihood ratio (LR) for the qualitative/semiquantitative method of 91%/80%, 76.2%/67%, 88.9%/83.3%, 80%/60.9% and 32%/7.8%, respectively, to predict a CMR in the PET/CT-et. There were no statistically significant differences between the LR of both methods (p=0.1942). CONCLUSION: We found clear differences in S, Sp, PPV and NPV between both interpretation criteria for the PET/CT-i to predict a CMR in the PET/CT-et. Nevertheless, we cannot confirm the superiority of the qualitative method over the semiqualitative method for this purpose as no statistically significance differences were found in their LR in our study.

NAD(P)H oxidase p22phox gene C242T polymorphism, nitric oxide production, salt sensitivity and cardiovascular risk factors in Hispanics.

Mutations in the NAD(P)H oxidase gene may be associated with abnormal superoxide generation, nitric oxide (NO) availability and cardiovascular diseases. We investigated the prevalence of the NAD(P)H oxidase p22phox gene C242T polymorphism, and its possible association with blood pressure, NO production, salt sensitivity and cardiovascular risk factors in Hispanics. Genotype frequencies were as follows: CC, 52.9%; CT, 40.3%; and TT, 6.8%. There were no significant differences in systolic blood pressure, diastolic blood pressure, age, weight, fasting and post-load glucose levels, LDL and HDL cholesterol, triglyceride and urinary albumin levels in subjects with CC, CT or the TT genotypes. Presence of the T allele was associated with increased salt sensitivity in women, but not in men. NO metabolite excretion was markedly decreased both in women and men with the TT genotype (CC: 868+/-79 micromol/day; CT: 839+/-75 micromol/day; TT: 534+/-78 micromol/day; P<0.05). In conclusion, the prevalence of the NAD(P)H oxidase p22phox gene C242T polymorphism in Venezuelans was comparable to that of Caucasians, but different from that of Chinese and Japanese. Although the T allele was not associated with cardiovascular risk factors, hyperinsulinaemia or hypertension, in women, it appeared to be a genetic susceptibility factor for salt sensitivity. Both in women and men, the p22phox gene may play a role in the genetic control of NO levels.

Endothelial nitric oxide synthase polymorphism, nitric oxide production, salt sensitivity and cardiovascular risk factors in Hispanics.

Mutations in the endothelial nitric oxide synthase (eNOS) gene may be associated with abnormal nitric oxide (NO) production and cardiovascular diseases. In this study, we investigated the prevalence of two eNOS polymorphisms, the Glu298Asp variant on exon 7, and the 4a/b variable number of tandem repeats (VNTR) on intron 4, and their association with blood pressure (BP), NO production, salt sensitivity and cardiovascular risk factors in healthy Venezuelans. The prevalence of both polymorphisms in Venezuelans was comparable to that described for Caucasians, but significantly different from that known for African-Americans and Japanese. The 4a/b genotype was associated with reduced levels of NO metabolites (25% decrease), larger BP lowering in response to salt restriction (9.0 vs 4.8 mmHg, P<0.05), greater prevalence of salt sensitivity (39% in 4a/b and 27% in 4b/b; P<0.05) and with higher LDL-cholesterol levels. The Glu298T polymorphism did not affect NO production, nor it was associated with salt sensitivity. Glu298Asp polymorphism was positively associated with higher weight, triglycerides and LDL-cholesterol. Neither polymorphism was associated with changes in fasting or postload serum glucose, BP, obesity and albuminuria. In conclusion, the prevalence of eNOS polymorphisms is strongly determined by ethnic factors. The 4a/b gene polymorphism could be a genetic susceptibility factor for the BP response to salt intake and for the genetic control of NO production. The reduced NO production in subjects with the 4a/b genotype may be responsible for the increased sensitivity of their BP to salt.

Differential blood pressure effects of oral glucose and intravenous L-arginine in healthy lean normotensive and obese hypertensive subjects.

We investigated the role of insulin and glucose in the pathophysiology of hypertension associated with obesity. The comparative effects of an oral glucose load and of an L-arginine infusion on plasma glucose, plasma insulin and blood pressures (BP) were assessed in lean normotensive and in obese hypertensive males. Oral glucose (75 g in 1-2 min) induced a small but significant lowering of BP in lean normotensives, but failed to modify BP in obese hypertensives. L-arginine infusion (30 min, 500 mg/kg total dose) reduced BP; significantly greater reductions in systolic and diastolic BP were observed in obese hypertensives than in the control group. Both oral glucose and L-arginine induced greater increases in plasma insulin in obese hypertensives than in lean normotensives. Endothelial dysfunction which accompanies the insulin resistant state of obesity, glucose intolerance and hypertension, may account for the different BP effects induced by glucose and L-arginine in obese hypertensives and lean normotensives.

Use of intravenous microdialysis to monitor changes in serotonin release and metabolism induced by cisplatin in cancer patients: comparative effects of granisetron and ondansetron.

Serotonin [5-hydroxytryptamine (5-HT)] is involved in the production of emesis associated with cisplatin treatment. Serotonin released from intestinal enterochromaffin cells may act either directly on vagal afferents and/or pass to the circulation and stimulate central emetic centers. However, the role for circulating 5-HT has not been determined. In this study, i.v. microdialysis probes were used to investigate 1) cisplatin-induced changes in 5-HT release and metabolism assessed through changes in blood dialysate levels of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA), 2) whether free 5-HT in blood increases after cisplatin, and 3) whether granisetron and ondansetron exert different effects on cisplatin-induced 5-HT release and metabolism. Control experiments conducted in 10 healthy volunteers revealed stable 5-HT and 5-HIAA dialysate levels for a period of 6 h. In patients with cancer (n = 16), baseline blood dialysate 5-HIAA concentrations averaged 2.98 +/- 0.38 ng/ml, which were equivalent to a total of 94 +/- 10 pg in the 30-min collection period at a flow rate of 1 microl/min. Cisplatin (89 +/- 2.9 mg of cisplatin/m(2)) produced a gradual increase in blood dialysate 5-HIAA levels (104 +/- 4% increase at 4 h). Increases in dialysate 5-HIAA were associated with increases in the urinary excretion of this metabolite. After cisplatin, dialysate 5-HIAA levels increased to 5.89 +/- 0.5 ng/ml in granisetron and to 5.27 +/- 0.9 ng/ml in ondansetron-treated patients (P >.1). Similar time courses and percentages of increase in blood dialysate and urinary 5-HIAA levels were observed in ondansetron- and granisetron-treated patients. Contrary to 5-HIAA, no significant increases in dialysate 5-HT were observed from 2 to 8 h after cisplatin either for the total group or for each of the groups separately. In conclusion, i.v. microdialysis probes coupled to HPLC-EC allowed the continuous monitoring of free-5-HT and 5-HIAA in blood. Cisplatin-induced increases in blood 5-HIAA were not associated with increases in 5-HT blood dialysates. These results argue against a possible action of free 5-HT in plasma on the chemoreceptor trigger zone (unprotected from the blood brain barrier) but support the view that 5-HT released within the intestinal wall triggers emesis after cisplatin. Our results argue against the view that at clinically effective doses, granisetron and ondansetron exert different actions on cisplatin-induced 5-HT release and metabolism.

Level of punishment determines anticonflict activity of ondansetron in pigeons: comparison with buspirone and diazepam.

The anticonflict effect of the selective 5-HT3 receptor antagonist, ondansetron, was investigated employing an operant conflict task in pigeons. Behavior (key pecking) was stimulated by food presentation. A fixed-interval program of alternated punished (electrical shocks) and unpunished responding was employed. The effects of drugs were evaluated at two levels punishment intensity; i.e., baseline responding during the punished interval was 5% (higher punishment) or 10% (lower punishment) of the unpunished responding rate. Ondansetron released responding suppressed by punishment only when pigeons were working at the lower levels of punishment. Under these conditions, ondansetron (100 microg/kg, i.v.), increased key pecking by 119% above control and vehicle values, and doubled the number of shocks received by the pigeons during the punished intervals. Similarly to ondansetron, the anticonflict effects of buspirone (0.3 and 1 mg/kg) and diazepam (1 and 1.5 mg/kg) were strongly dependent on the intensity of the punishing stimulus. When punished responding was suppressed to 5% of unpunished responding by applying shocks of higher intensity, diazepam and buspirone had negligible anticonflict action. However, at lower levels of punishment, diazepam and buspirone produced much greater anticonflict effects than ondansetron (p < 0.001). These results indicate that ondansetron exhibits a modest effect in releasing behaviors suppressed by punishment (anxiolytic-like action), which was highly dependent on the intensity of punishment applied. It is proposed that the anxiogenic response to punishment is less sensitive to 5-HT3 antagonists than the behavior induced by aversive, unpunished situations, where 5-HT3 antagonists have shown comparable efficacy to benzodiazepines.