Impaired metabotropic glutamate receptor/phospholipase C signaling pathway in the cerebral cortex in Alzheimer's disease and dementia with Lewy bodies correlates with stage of Alzheimer's-disease-related changes.

The aim of the present work was to analyze the status of metabotropic glutamate receptors (mGluRs) in the frontal cortex (area 8) from ten cases with common form DLB (cDLB) and eleven cases with pure AD in comparison with five age-matched controls. mGluRs, determined by radioligand binding assays, were significantly decreased in cerebral cortex in cDLB. This decrease was already present in cases with early AD changes not involving the frontal cortex, but dramatically correlated with AD neuropathological changes, at its greatest in isocortical stages, which was associated with a decrease in the expression levels of mGluR1 detected by Western blotting. Moreover, mGluRs analyzed in pure AD were lower than those obtained in cDLB and also correlated with progression of illness. On the other hand, the expression levels of phospholipase Cbeta1 (PLCbeta1) isoform, which is the effector of group I mGluRs, was decreased in parallel in cDLB cases. Finally, the PLCbeta1 decrease was associated with reduced GTP- and l-glutamate-stimulated PLC activity in both cDLB and AD cases. These results show that group I mGluRs/PLC signaling are down-regulated and desensitized in the frontal cortex in cDLB and AD cases and that these modifications worsen with progression of AD changes in the cerebral neocortex. Therefore, group I mGluR dysfunction may be implicated in the pathogenesis of cognitive impairment and dementia in common form of DLB and pure AD.

Cloning and characterization of a phospholipase C from the C(4) plant Digitaria sanguinalis.

As a PLC activity was implicated in the light transduction pathway that controls C(4) photosynthesis in Digitaria sanguinalis, a full length PLC cDNA (DsPLC2) was cloned. The proteins encoded by the two possible open reading frames were produced in Escherichia coli; they both harbour a PLC activity but with different response to Ca(2+) concentration, and with different sensitivity to the PLC inhibitor U-73122.

Changes in phosphoinositide signalling activity and levels of the alpha subunit of G(q/11) protein in rat brain induced by E-5842, a sigma(1) receptor ligand and potential atypical antipsychotic.

Changes in the phosphoinositide (PPI) signal transduction system induced by E-5842, a new sigma(1) (sigma(1)) receptor ligand and potential atypical antipsychotic, were studied in the rat frontal cortex, hippocampus and striatum. Acute treatment with E-5842 increased phospholipase C (PLC) activity in the striatum and the hippocampus. Chronic treatment with E-5842 induced an increase in the activity of PLC in the frontal cortex and the striatum. Similar up-regulation of the activity of the enzyme was also observed in rat frontal cortex membranes in presence of GTPgammaS. After chronic treatment with E-5842, it was also observed a significant increase of the immunoreactivity levels of G(q/11)alpha in the frontal cortex. Our results suggest that part of the antipsychotic effects of E-5842 could be related to the regulation of the PPI signal transduction pathway, especially after a prolonged treatment.

Effect of estradiol and progesterone on growth of porcine myometrial smooth muscle cells and phospholipase C and adenylate cyclase signalling systems in vitro.

The objective of presented studies was to investigate whether estradiol and progesterone administered in vivo and/or added in vitro can influence the primary myometrial cell culture and how these steroid hormones can affect hCG stimulated cAMP and inositol phosphate production in the porcine uterine myocytes. Myometrial smooth muscle cells were obtained from six ovariectomized gilts pretreated (n = 4) or not (n = 2) with oestradiol benzoate and progesterone for 5 consecutive days. Immunocytochemical staining proved that the pattern of filamentous actin in the cytoplasm of the myometrial fibroblasts (basketlike network) was different from that of myometrial smooth muscle cells (long parallel fibres). The myocytes derived from steroid treated pigs and supplemented with estradiol and progesterone in vitro formed a hillock pattern on days 4-5 day of culture whereas cells obtained from not steroid pretreated gilts were smaller and did not create confluent form. Myocytes were treated in vitro with two doses of estradiol/progesterone (low - 0.2 nM/50 nM and high - 2 nM/500 nM, respectively) and two doses of hCG - 0.1 mU and 1000 mU/ml to study hCG action on the second messenger system in myocytes. The myometrial smooth muscle cells treated with low dose of estradiol and progesterone in vivo responded with much higher accumulation of inositol phosphates to strong (1000 mU/ml) hCG stimulation when compared with those receiving high dose of both steroids. The different doses of estradiol and progesterone caused a similar increase in basal cAMP accumulation as compared to control cells cultured without steroid hormones. hCG (0.1 mU/ml) had usually the additive effect on cAMP production in porcine myometrial cells. The presented paper shows that estradiol and progesterone administration in vivo followed by steroid hormone treatment in vitro affects the primary myometrial cells culture and that both steroid hormones modify the basal accumulation of the second messengers: cAMP and IP3 and their answer to hCG stimuli in pigs.

Effect of membrane lipid alteration on the growth, phospholipase C activity and G protein of HT-29 tumor cells.

The objective of the present study was to examine the effect of modifying the fatty acid composition of membranes on cell growth and phosphoinositide specific phospholipase C (PLC) activity in HT-29 colon cancer cells. Cells were seeded at a density of 12 x 10(3) cells/cm2 and supplemented with 30 microM of either 18:0, 18:2 (n6) or 18:3 (n3) complexed to bovine serum albumin (BSA) in DMEM medium. Cell growth was followed for 12 days. The 18:0 supplemented cells (control) reached maximum growth at day nine which was greater than either 18:2 (n6) or 18:3 (n3) supplemented cells. There was no difference between the latter two groups in their growth. To investigate the fatty acid incorporation of the supplemented fatty acid and how they may influence composition in the cell membrane, we examined the fatty acid composition of each phospholipid (PL) species. Both phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were significantly influenced by the type of fatty acid supplemented. Supplementation with 18:0 resulted in HT-29 cell membranes having more monounsaturated fatty acids than the cells grown in the other fatty acids. Polyunsaturated fatty acid (PUFA) supplementation (both 18:2 and 18:3) resulted in the enrichment of PUFA in the PL fractions. Cells supplemented with 18:3 (n3) had the highest unsaturation index in membrane PE as compared to the other phospholipid species. PLC activity of the membranes was measured using PIP2 as a substrate in the presence of 15 micrograms alamethicin and 42 microM free calcium. The contribution of G protein to the activity of the enzyme was assessed using GTP gamma(S). PLC activity of HT-29 cells was 16% higher in the presence of GTP gamma(S) response. GTP gamma(S)-activated PLC activity of 18:3 (n3) supplemented cells was 81% of those supplemented with either 18:0 or 18:2 (n6) cells. It is concluded that the decrease in cell proliferation with supplementation with 18:3 (n3) may be mediated through its inhibitory effect on PLC, which provides the second messengers for protein kinase C (PKC) activation. PLC may be influenced by an increased unsaturation index of the PE fraction of the HT-29 tumor cell membranes.

Imipramine stimulates phospholipase C activity in rat brain.

We previously demonstrated that antidepressant drugs (ADs) cause Ca2+ release from inositol 1,4,5-trisphosphate-sensitive Ca2+ stores in cultured neurons of rat frontal cortex. The present study examines the mechanism by which tricyclic ADs activate phospholipase C (PLC) in rat frontal cortex. Using an exogenous substrate to measure PLC activity, we demonstrated that a tricyclic AD, imipramine, stimulated PLC activity of the frontal cortex membrane in a concentration-dependent manner. Two tricyclic ADs, desipramine and amitriptyline, also stimulated PLC activity, while Li+ or pargyline had no effect on PLC activity. Although imipramine did not activate PLC in the membrane in the absence of Ca2+, imipramine synergistically activated PLC in the presence of Ca2+. This result indicates that the mechanism of PLC activation by imipramine is different from its activation by Ca2+. Imipramine stimulated PLC activity in the cytosol of rat frontal cortex as well as in the membrane. Preincubation of the cytosol with anti-PLC-beta 1 antibody prevented the imipramine-mediated activation of PLC. However, preincubation with anti-PLC-gamma 1 or anti-PLC-delta 1 did not prevent activation of PLC. These results suggest that imipramine activates PLC-beta 1 directly without receptor or guanine nucleotide binding protein mediation.

Effects of Eimeria brunetti infection and dietary zinc on experimental induction of necrotic enteritis in broiler chickens.

Broilers infected with Eimeria brunetti and given dietary zinc were examined for experimental induction of necrotic enteritis. Inoculation with sporulated E. brunetti oocysts at 7 days of age was followed by 5 consecutive days of oral inoculation with cultured Clostridium perfringens. Feed was supplemented with zinc at 1000 ppm. Upon necropsy of broilers 6 days after coccidial inoculation, necrotic enteritis was found in 20% (2/10) of birds given both organisms and dietary zinc. Coccidial lesion scores were also highest in that group. Birds infected with E. brunetti and C. perfringens with no dietary zinc had significantly higher coccidiosis lesion scores (P less than 0.05) than groups inoculated with E. brunetti only, regardless of zinc supplementation. Alpha toxin levels in intestinal contents were low in groups infected with both organisms, regardless of zinc supplementation. Zinc was tested for effects of alpha toxin production in vitro. In the mid-log phase (6 hours incubation), a high level of alpha toxin was produced in zinc-supplemented media, but this was lost quickly in the presence of trypsin. Addition of zinc partly protected the toxin from the action of trypsin.

Phospholipase-C and Na-K ATPase activation by cyclosporine and FK506 in LLC-PK1, cells. Possible implications in blood pressure regulation.

Enhanced Na+ and water reabsorption by proximal tubular epithelial cells plays an important role in the development of systemic hypertension associated with cyclosporine immunosuppression. Since such Na+ reabsorption is subserved by sodium-potassium adenosine triphosphatase (Na-K ATPase), the current study compared the acute effects of hydrocortisone (H), cyclosporine, and FK506 on cultured LLC-PK1 cell viability and on Na-K ATPase activity. Phospholipase-C (PL-C) activity was also investigated because of its possible regulatory effect on Na-K ATPase activity. Culture medium containing low (5 nM, 4.1 ng/ml) or high (10 nM) concentrations of FK506 plus cyclosporine at 415 microM (500 ng/ml) resulted in cell death, whereas cyclosporine concentrations of 83 microM plus 5 nM or 10 nM FK506, or isolated use of the two drugs at high dosages, did not affect cell viability. As compared with controls, cyclosporine increased Na-K ATPase activity, particularly with addition of H (P less than 0.01). In contrast, FK506 reduced the specific activity of both PL-cyclosporine and Na-K ATPase (P less than 0.001-0.01); addition of H to FK506 resulted in an even greater fall in both the enzyme activities (P less than 0.001). Na-K ATPase activity increased in cell homogenates briefly incubated with cyclosporine in the ATPase reaction mixture (P less than 0.05) while FK506 reduced such enzyme activity (P less than 0.05), suggesting a direct effect of these agents on pump activity. These data in LLC-PK1 cells pocessing proximal tubular epithelial cell characteristics indicate that the combined use of cyclosporine plus FK506 may be very deleterious to viability in such cells. The opposing effects of cyclosporine and FK506 on PL-cyclosporine and Na-K ATPase activities and the possible potentiating effect of H on such responses are speculated to affect Na+ and water homeostasis in a manner that may explain differences in systemic blood pressure due to these agents.