Histamine H1 and Endothelin ETB Receptors Mediate Phospholipase D Stimulation in Rat Brain Hippocampal Slices

Abstract: Different neurotransmitter receptor agonists [carbachol, serotonin, noradrenaline, histamine, endothelin-1, and trans -(1 S ,3 R )-aminocyclopentyl-1,3-dicarboxylic acid ( trans -ACPD)], known as stimuli of phospholipase C in brain tissue, were tested for phospholipase D stimulation in [³²P]P_i-prelabeled rat brain cortical and hippocampal slices. The accumulation of [³²P]phosphatidylethanol was measured as an index of phospholipase D-catalyzed transphosphatidylation in the presence of ethanol. Among the six neurotransmitter receptor agonists tested, only noradrenaline, histamine, endothelin-1, and trans -ACPD stimulated phospholipase D in hippocampus and cortex, an effect that was strictly dependent of the presence of millimolar extracellular calcium concentrations. The effect of histamine (EC₅₀ 18 µ M ) was inhibited by the H₁ receptor antagonist mepyramine with a K _i constant of 0.7 n M and was resistant to H₂ and H₃ receptor antagonists (ranitidine and tioperamide, respectively). Endothelin-1-stimulated phospholipase D (EC₅₀ 44 n M ) was not blocked by BQ-123, a specific antagonist of the ET_A receptor. Endothelin-3 and the specific ET_B receptor agonist safarotoxin 6c were also able to stimulate phospholipase D with efficacies similar to that of endothelin-1, and EC₅₀ values of 16 and 3 n M , respectively. These results show that histamine and endothelin-1 stimulate phospholipase D in rat brain through H₁ and ET_B receptors, respectively.     

Modulation of neuronal phospholipase D activity under depolarizing conditions

Neuronal phospholipase D (PLD) activity was hypothesized to be involved in vesicle trafficking and endocytosis and, possibly, transmitter release. We here report that prolonged depolarization of rat hippocampal slices by potassium chloride (KCl) or 4-aminopyridine inhibited PLD activity. Similarly, PLD activity in rat cortical synaptosomes was significantly inhibited by depolarizing agents including veratridine and ouabain. Inhibition of calcium/calmodulin kinase II (CaMKII) which positively modulates synaptosomal PLD activity [Sarri et al. (1998) FEBS Lett. 440, 287-290] by KN-62 caused a further reduction of PLD activity in depolarized synaptosomes. Depolarization-induced inhibition of PLD activity was apparently not due to transmitter release or activation of other kinases. We observed, however, that KCl-induced depolarization caused an increase of inositol phosphates and a reduction of the synaptosomal pool of phosphatidylinositol-4, 5-bisphosphate (PIP(2)). Moreover, in synaptosomes permeabilized with Staphylococcus aureus alpha-toxin, PLD activation induced by calcium was abolished by neomycin, a PIP(2) chelator. We conclude that depolarizing conditions cause an inhibition of neuronal PLD activity which is likely due to breakdown of PIP(2), a required cofactor for PLD activity. Our findings suggest that neuronal PLD activity is regulated by synaptic activity.     

Effects of oxidative stress on phospholipid signaling in rat cultured astrocytes and brain slices

Although reactive oxygen species (ROS) are conventionally viewed as toxic by-products of cellular metabolism, a growing body of evidence suggests that they may act as signaling molecules. We have studied the effects of hydrogen peroxide (H(2)O(2))-induced oxidative stress on phospholipid signaling in cultured rat cortical astrocytes. H(2)O(2) stimulated the formation of phosphatidic acid and the accumulation of phosphatidylbutanol, a product of the phospholipase D (PLD)-catalyzed transphosphatidylation reaction. The effect of exogenous H(2)O(2) on the PLD response was mimicked by menadione-induced production of endogenous H(2)O(2). Oxidative stress also elicited inositol phosphate accumulation resulting from phosphoinositide phospholipase C (PLC) activation. The PLD response to H(2)O(2) was totally suppressed by chelation of both extracellular and cytosolic Ca(2+) with EGTA and BAPTA/AM, respectively. Furthermore, H(2)O(2)-induced PLD stimulation was completely abolished by the protein kinase C (PKC) inhibitors bisindolylmaleimide and chelerythrine and by PKC down-regulation. Activation of PLD by H(2)O(2) was also inhibited by the protein-tyrosine kinase inhibitor genistein. Finally, H(2)O(2) also stimulated both PLC and PLD in rat brain cortical slices. These results show for the first time that oxidative stress elicits phospholipid breakdown by both PLC and PLD in rat cultured astrocytes and brain slices.     

Supernumerary marker chromosome 5 diagnosed by M-FISH in a child with congenital heart defect and unusual face

We describe a female patient with a small supernumerary marker chromosome (sSMC) present in mosaic and characterized in detail by fluorescence in situ hybridization (FISH) using all 24 human whole chromosome painting probes, multicolor banding (MCB) and subcentromere specific multicolor FISH (subcenM-FISH). The sSMC was demonstrated to be derived from chromosome 5 and the karyotype of our patient was as follows: 47,XX,+mar.ish r(5)(::p13.2 approximately p13.3-->q11.2::) [60%]/46,XX [40%]. Partial trisomy for the proximal 5p and q chromosomal regions is a rare event. A critical region exists at 5p13 for the phenotype associated with duplication 5p. As far as we know, eight similar cases have been published up to now. We describe a new case which, to our knowledge, is the first characterized in such detail. The role of uniparental disomy (UPD) in cases of SMC is also discussed.     

Zinc and iron bioavailability in a powder or in-bottle-sterilized infant formula estimated by in vitro and in suckling rats

Maillard reaction and lactose isomerization may be induced during the processing involved in the manufacture of infant formulas. The effects of dehydratation and sterilization in an infant formula on iron and zinc bioavailability were studied. A powder (PIF), previously reconstituted, and an in-bottle-sterilized liquid infant formula (LIF), from the same manufacturer, were evaluated using an in vitro method and in suckling rats. After in vitro digestion the dialyzed and non-dialyzed soluble, and insoluble fractions of iron and zinc were separated. Two-week-old rat pups were fed PIF or LIF in a drinking bottle for 7 days. Infant formula intake (I), body weight and the fecal and urinary excretions were monitored and the following parameters calculated: apparent absorption (A), retention (R), and the coefficients %A/I, %R/A and %R/I. Soluble iron (dialyzed) and zinc (non-dialyzed) were higher (p < 0.001) in LIF than PIF after in vitro digestion. Insoluble iron was similar in both infant formulas but insoluble zinc was lower (p < 0.05) in LIF than PIF. Food intake (p = 0.045) and body weight on day 4 (p < 0.05) and on day 7 (p < 0.001) were lower in LIF compared to PIF. A, R (p < 0.05 for both minerals), %A/I, and %R/I (p < 0.001 and p < 0.05 for iron and zinc, respectively) were significantly lower in rats fed LIF. Similarly, the %R/A of iron was lower (p < 0.001) in this group. Hematocrit and hemoglobin did not show significant differences. Iron and zinc levels in liver, spleen and erythrocytes were similar in both groups, but skin iron concentration was higher in LIF. Therefore, in contrast with the in vitro results, consumption of the in-bottle-sterilized formula determines lower iron and zinc bioavailability compared to the reconstituted powder infant formula.     

Validity and Reproducibility of a Spanish Dietary History

Objective

To assess the validity and reproducibility of food and nutrient intake estimated with the electronic diet history of ENRICA (DH-E), which collects information on numerous aspects of the Spanish diet.

Methods

The validity of food and nutrient intake was estimated using Pearson correlation coefficients between the DH-E and the mean of seven 24-hour recalls collected every 2 months over the previous year. The reproducibility was estimated using intraclass correlation coefficients between two DH-E made one year apart.

Results

The correlations coefficients between the DH-E and the mean of seven 24-hour recalls for the main food groups were cereals (r = 0.66), meat (r = 0.66), fish (r = 0.42), vegetables (r = 0.62) and fruits (r = 0.44). The mean correlation coefficient for all 15 food groups considered was 0.53. The correlations for macronutrients were: energy (r = 0.76), proteins (r = 0.58), lipids (r = 0.73), saturated fat (r = 0.73), monounsaturated fat (r = 0.59), polyunsaturated fat (r = 0.57), and carbohydrates (r = 0.66). The mean correlation coefficient for all 41 nutrients studied was 0.55. The intraclass correlation coefficient between the two DH-E was greater than 0.40 for most foods and nutrients.

Conclusions

The DH-E shows good validity and reproducibility for estimating usual intake of foods and nutrients.     

Assessing reproductive cycles and pregnancy in cheetahs (Acinonyx jubatus) by vaginal cytology

Vaginal cytology was used to monitor ovarian cycles, two pregnancies, and three pseudopregnancies. Vaginal smears were collected two or three times per week from three adult females; smears plus blood samples were collected once per week from a fourth, adolescent female. Mean cycle lengths, based on intervals between onset of leukocyte infusions, were 11.9 ± 4.9 days (n = 43 cycles), 10.8 ± 5.1 days (n = 49), and 12.3 ± 6.3 days (n = 7) for the three females. Weekly hormone data from the adolescent female revealed a correlation between serum estradiol and percent anuclear cells, suggesting that these cells may be indicative of estrus. The fourth female experienced two sustained, 6-week increases in serum progesterone, one spontaneous and the other following follicle-stimulating hormone (FSH) administration. Leukocyte infusions continued during these periods of increased progesterone secretion. However, leukocyte infusions ceased during the two pregnancies of one adult female and during two FSH-induced pseudopregnancies of another. © 1992 Wiley-Liss, Inc.     

Sister-chromatid exchange in highly purified human CD4+ and CD8+ lymphocytes.

Sister-chromatid exchange (SCE) frequencies were determined in human peripheral blood CD₄⁺ and CD₈⁺ T lymphocyte subpopulations which were rapidly and highly purified from pooled T lymphocytes by immunological methods. The purified lymphocytes were stimulated with phytohemagglutinin (PHA) for 4 days. CD₄⁺ lymphocytes showed significantly higher SCE frequencies than autologous CD₈⁺ lymphocytes when measured simultaneously after identical bromodeoxyuridine (BrdU) incubation times. Differences in SCE frequencies between CD₄⁺ and CD₈⁺ lymphocytes were also detected when mitomycin C (MMC) was added to the cultures. Higher SCE frequencies in CD₄⁺ lymphocytes were associated with lower proliferating rate indices (PRI) as compared to autologous CD₈⁺ lymphocytes. Abnormalities in CD₄⁺ T lymphocyte function and number in peripheral blood have been observed in several diseases characterized by immunological disorders. Thus, our data may suggest a link between some immunological disturbances and abnormal SCE frequencies in T lymphocyte subsets.     

Intracellular Ca2+ stores regulate muscarinic receptor stimulation of phospholipase C in cerebellar granule cells

Muscarinic receptor activation of phosphoinositide phospholipase C (PLC) has been examined in rat cerebellar granule cells under conditions that modify intracellular Ca2+ stores. Exposure of cells to medium devoid of Ca2+ for various times reduced carbachol stimulation of PLC with a substantial loss (88%) seen at 30 min. A progressive recovery of responses was observed following the reexposure of cells to Ca2+-containing medium (1.3 mM). However, these changes did not appear to result exclusively from changes in the cytosolic Ca2+ concentration ([Ca2+]i), which decreased to a lower steady level (approximately 25 nM decrease in 1-3 min after extracellular omission) and rapidly returned (within 1 min) to control values when extracellular Ca2+ was restored. Only after loading of the intracellular Ca2+ stores through a transient 1-min depolarization of cerebellar granule cells with 40 mM KCl, followed by washing in nondepolarizing buffer, was carbachol able to mobilize intracellular Ca2+. However, the same treatment resulted in an 80% enhancement of carbachol activation of PLC. In other experiments, partial depletion of the Ca2+ stores by pretreatment of cells with thapsigargin and caffeine resulted in an inhibition (18 and 52%, respectively) of the PLC response. Furthermore, chelation of cytosolic Ca2+ with BAPTA/AM did not influence muscarinic activation of PLC in either the control or predepolarized cells. These conditions, however, inhibited both the increase in [Ca2+]i and the PLC activation elicited by 40 mM KCl and abolished carbachol-induced intracellular Ca2+ release in predepolarized cells. Overall, these results suggest that muscarinic receptor activation of PLC in cerebellar granule cells can be modulated by changes in the loading state of the Ca2+ stores.     

Metabolic Profile in Early Pregnancy Is Associated with Offspring Adiposity at 4 Years of Age: The Rhea Pregnancy Cohort Crete,Greece

ContextMaternal pre-pregnancy obesity may increase the risk of childhood obesity but it is unknown whether other metabolic factors in early pregnancy such as lipid profile and hypertension are associated with offspring cardiometabolic traits.ObjectiveOur objective was to investigate whether fasting lipid, glucose, and insulin levels during early pregnancy and maternal pre-pregnancy weight status, are associated with offspring adiposity measures, lipid levels and blood pressure at preschool age.ResultsPre-pregnancy overweight/obesity was associated with greater risk of offspring overweight/obesity (RR: 1.83, 95%CI: 1.19, 2.81), central adiposity (RR: 1.97, 95%CI: 1.11, 3.49), and greater fat mass by 5.10mm (95%CI: 2.49, 7.71) at 4 years of age. These associations were more pronounced in girls. An increase of 40mg/dl in fasting serum cholesterol levels in early pregnancy was associated with greater skinfold thickness by 3.30mm (95%CI: 1.41, 5.20) at 4 years of age after adjusting for pre-pregnancy BMI and several other confounders. An increase of 10mmHg in diastolic blood pressure in early pregnancy was associated with increased risk of offspring overweight/obesity (RR: 1.22, 95%CI: 1.03, 1.45), and greater skinfold thickness by 1.71mm (95% CI: 0.57, 2.86) at 4 years of age.ConclusionsMetabolic dysregulation in early pregnancy may increase the risk of obesity at preschool age.