A novel cognition enhancer NS-105 modulates adenylate cyclase activity through metabotropic glutamate receptors in primary neuronal culture

The effect of (+)-5-oxo-D-prolinepiperidinamide monohydrate (NS-105), a novel cognition enhancer, on adenylate cyclase activity was investigated in cultured neurons of the mouse cerebral cortex. NS-105 (10^–7 and 10^–6 M) inhibited forskolin-stimulated cyclic AMP formation, an action that was dependent on pertussis toxin-sensitive G proteins. Conversely, in pertussis toxin-pretreated neurons, NS-105 (10^–7 –10^–5 M) significantly enhanced the forskolin-stimulated cyclic AMP formation, and this action was completely reversed by cholera toxin. A metabotropic glutamate receptor agonist (1S, 3R)-1-aminocyclopentane-1, 3-dicarboxylic acid (1S, 3R-ACPD) produced similar bi-directional actions on the cyclic AMP formation. Both of these inhibitory and facilitatory actions of NS-105 and 1S, 3R-ACPD were blocked by L(+)-2-amino-3-phosphopropinoic acid (L-AP3). NS-105 (10^–6 M) and 1S, 3R-ACPD (10^–4 M) significantly enhanced isoproterenol- and adenosine-stimulated cyclic AMP formation. The enhancement of such Gs-coupled receptor agonists-stimulated cyclic AMP formation was also produced by quisqualate but not by L(+)-2-amino-4-phosphonobutanoate (L-AP4). The phosphoinositides hydrolysis was enhanced by 1S, 3R-ACPD (10^–4 M) but not by NS-105 (10^–6 M), however, 1S, 3R-ACPD-induced increase in phosphoinositides turnover was attenuated by NS-105. These findings suggest that NS-105 stimulates metabotropic glutamate receptor subclasses that are coupled both negatively and positively to adenylate cyclase, but it acts as an antagonist at the receptor subclasses that are linked to phosphoinositides hydrolysis. Received: 3 February 1997 / Accepted: 25 April 1997     

Participation of Phosphoinositides in Gastric Mucosal Protection by Colloidal Bismuth Subcitrate against Ethanol-Induced Injury

The mechanism of gastric mucosal protection by an antiulcer agent, colloidal bismuth subcitrate (CBS), against ethanol-induced injury was investigated using in vivo and in vitro systems. The experiments in vivo were conducted with groups of rats with and without indomethacin pretreatment, and the animals received either a dose of CBS (100 mg/kg) or a vehicle (saline), followed 30 min later by ethanol. In the in vitro studies, gastric mucosa segments were cultured in the presence of CBS, ethanol, or both. The results of in vivo experiments revealed that in the absence of CBS, ethanol caused extensive gastric hemorrhagic lesions which were significantly reduced following CBS pretreatment and this effect of CBS was not prevented by indomethacin. The data obtained with gastric mucosal culture established that in comparison to the controls, ethanol caused a 27% decrease in mucin synthesis, while mucin synthesis in the presence of CBS increased by 48%. The increase in mucin synthesis evoked by CBS was accompanied by the enhanced metabolism of mucosal phosphoinositides, as reflected by a decrease in PI (15%) and PIP2 (30%), and an increase in IP1 (26%) and IP3 (67%). In contrast, ethanol, which exhibited detrimental effect on mucin synthesis, caused a decrease in PIP (35%), IP2 (47%) and IP3 (38%), and an increase in PIP2 (80%), and IP1 (51%). However, when the mucosal culture was carried out in the presence of both CBS and ethanol, the detrimental changes evoked by ethanol on mucin synthesis were prevented, and the phosphoinositide and inositide phosphate distribution patterns were quite similar to those in the mucosa cultured in the presence of CBS only.(ABSTRACT TRUNCATED AT 250 WORDS)     

Maintenance of PtdIns45P2 pools under limiting inositol conditions,as assessed by liquid chromatography-tandem mass spectrometry and PtdIns45P2 mass evaluation in Ras-transformed cells

Inositol-containing molecules are involved in important cellular functions, including signalling, membrane transport and secretion. Our interest is in lysophosphatidylinositol and the glycerophosphoinositols, which modulate cell proliferation and G-protein-dependent activities such as adenylyl cyclase and phospholipase A₂. To investigate the role of glycerophosphoinositol (GroPIns) in the modulation of Ras-dependent pathways and its correlation to Ras transformation, we employed a novel liquid chromatography-tandem mass spectrometry technique to directly measure GroPIns in cell extracts. The cellular levels of GroPIns in selected parental and Ras-transformed cells, and in some carcinoma cells, ranged from 44 to 925 μM, with no consistent correlation to Ras transformation across all cell lines. Moreover, the derived cellular inositol concentrations revealed a wide range (150 μM to 100 mM) under standard [³H]-inositol-loading, suggesting a complex relationship between the inositol pool and the phosphoinositides and their derivatives. We have investigated these pools under specific loading conditions, designing a further HPLC analysis for GroPIns, combined with mass determinations of cellular phosphatidylinositol 4,5-bisphosphate. The data demonstrate that limiting inositol conditions identify a preferred pathway of inositol incorporation and retention into the polyphosphoinositides pool. Thus, under conditions of increased metabolic activity, such as receptor stimulation or cellular transformation, the polyphosphoinositide levels will be maintained at the expense of phosphatidylinositol and the turnover of its aqueous derivatives.     

Long term effects of septohippocampal lesions and intrahippocampal grafts on acetylcholine concentration,muscarinic stimulated formation of inositol phospholipids and electrically evoked release of neurotransmitters in the rat hippocampus

Summary Long Evans female rats sustained aspirative lesions of the septohippocampal pathways; subsequently, they received intrahippocampal suspension grafts of fetal septal-diagonal band or hippocampal tissue. The long term (8–10 months post-surgery) effects of these treatments were examined in the hippocampus for the following variables: concentration of hippocampal acetylcholine (ACh), muscarinic-stimulated (carbachol) formation of inositol monophosphate, accumulation of tritiated choline, noradrenaline (³H-NA) and serotonin (³H-5-HT), electrically evoked release of ³H-acetylcholine (³H-ACh), ³H-NA and ³H-5-HT, and choline acetyltransferase (ChAT) activity. The lesions decreased the levels of endogeneous ACh, the accumulation of ³H-choline and ³H-5-HT and the evoked release of both ³H-ACh and ³H-5-HT as well as the ChAT activity, but they failed to significantly affect the muscarinic-stimulated formation of inositol monophosphate and the accumulation and release of ³H-NA. Grafts of hippocampal cells were found to be ineffective on all lesion-induced effects. In contrast, grafts of septal-diagonal band origin attenuated the deficit of hippocampal concentrations of ACh and accumulation of ³H-choline without, however, improving release of ³H-ACh, accumulation and release of ³H-5-HT, and ChAT activity. These observations suggest that: (i) denervation-induced hippocampal muscarinic supersensitivity might not be long-lasting or the lesions, which in some cases spared the lateral edges of the fimbria, failed to induce any muscarinic supersensitivity, (ii) intrahippocampal grafts rich in cholinergic neurons do not foster recovery from the lesion-induced noncholinergic deficits we assessed, (iii) recovery of function may be expressed by some but not all biochemical or pharmacological cholinergic variables and (iv) graft-derived hippocampal reinnervation may be less efficient than the endogenous innervation of intact rats as indicated by the restoration of only some of the variables related to cholinergic function by intrahippocampal septal-diagonal band grafts.     

Changes in extracellular calcium within the physiological range influence receptor-mediated inositol phosphate responses in brain and tracheal smooth muscle slices

Summary The effect of changes in extracellular calcium concentration ([Ca²⁺]_e) on the incorporation of myo-[2-³H]-inositol into phosphoinositides and agonist-stimulated ³H-inositol phosphates (³H-InsPs) was examined in rat cerebral cortex and bovine tracheal smooth muscle slices. In brain slices, reduction in [Ca²⁺]_e from 2.4 to 1.2 mmol/l resulted in an approximate doubling of the carbachol and noradrenaline-stimulated ³H-InsP response with no effect on the EC₅₀ values. An identical effect of varying [Ca²⁺]_e was observed for carbachol-stimulated ³H-InsP formation in tracheal smooth muscle with a further increase in ³H-InsPs evident at [Ca²⁺]_e 0.6 mmol/l. In this tissue the effect of changes in [Ca ²⁺]_e on the incorporation of myo-[2-³H]-inositol into the total phosphoinositide pool directly paralleled the changes in ³H-InsPs except in conditions of no added calcium when ³H-InsP responses were markedly impaired. Additional studies in brain slices using buffer where the added calcium varied between 0 and 2.4 mmol/l, showed that both the carbachol stimulated formation of separate inositol phosphates during short incubation periods and incorporation of myo-[2-³H]-inositol into PtdInsP and PtdInsP₂ under basal conditions was maximal at [Ca²⁺]_e 0.3 mmol/l. Omitting Ca²⁺]_e from the buffer resulted in maximal labelling of PtdIns but a decrease in PtdInsP and PtdInsP₂ labelling (compared with the level at [Ca²⁺]_e 0.3 mmol/l) and a markedly impaired inositol polyphosphate response. Alterations in [Ca²⁺]_e following ³H-inositol labelling but immediately prior to carbachol stimulation did not influence ³H-inositol polyphosphate responses. It is therefore clear that even relatively small changes in [Ca²⁺]_e markedly influence agonist-stimulated ³H-InsP responses in brain and tracheal smooth muscle slices and that these reflect changes in the labelling of substrate inositol lipids. These findings have important practical implications for studies examining ³H-InsP responses in central and peripheral tissues and the differential effect of very low [Ca²⁺]_e on PtdIns and PtdlnsP/PtdInsP₂ labelling may explain in part the severe decrease in ³H-InsPs seen under these conditions despite apparent maximal total phosphoinositide labelling. Send offprint requests to S. R. Nahorski at the above address     

Interaction of choline with muscarine receptor-stimulated phosphoinositide metabolism in the rat brain

Summary Previous receptor binding studies had shown that choline can interact with low potency with muscarine cholinoceptors. In the present study we have investigated whether choline is capable of functionally activating muscarine receptors by investigating its ability in stimulating the hydrolysis of phosphoinositides, a response believed to be coupled in brain to the M1 subtype of muscarine receptors. The results indicated that choline was only a very weak inducer of inositol phosphates (InsPs) accumulation in rat cerebral cortex slices as compared with acetylcholine or charbachol. Maximal increase of InsPs accumulation, at a choline concentration of 10 mM, was only 39±7%, as compared with the 4- to 6-fold stimulation induced by the other compounds. This effect of choline was not modified by physostigmine nor by the uptake inhibitor hemicholinium-3. At high concentrations, however, choline antagonized the stimulatory effect of acetylcholine and carbachol, suggesting that it might act as a partial agonist at this subtype of muscarine receptors, similar to what has been observed with oxotremorine. Choline had no effect on noradrenalinestimulated InsPs accumulation.     

Role of nitric oxide/cyclic GMP and cyclic AMP in beta3 adrenoceptor-chronotropic response

In this study we determine different signaling pathways involved in beta(3) adrenoceptor (beta(3)-AR) dependent frequency stimulation in isolated rodent atria. Promiscuous coupling between different G-proteins and beta(3)-AR could explain the multiple functional effects of beta(3)-AR stimulation. We examine the mechanisms and functional consequences of dual adenylate cyclase and guanylate cyclase pathways coupling to beta(3)-AR in isolated rodent atria. The beta(3)-AR selective agonists ZD 7114 and ICI 215001 stimulated in a dose-dependent manner the contraction frequency that significantly correlated with cyclic AMP (cAMP) accumulation. Inhibition of adenylate cyclase shifted the chronotropic effect to the right. On the other hand, the ZD 7114 activity on frequency was enhanced by the inhibition of nitric oxide synthase (NOS) and soluble guanylate cyclase. This countervailing negative chronotropic nitric oxide-cyclic GMP (NO-cGMP) significantly correlated with the increase on NOS activity and cGMP accumulation. Current analysis showed a negative cross talk between cAMP chronotropic and NO-cGMP effects by inhibition of phospholipase C (PLC), calcium/calmodulin (CaM), protein kinase C (PKC), NOS isoforms and Gi-protein on the effects of beta(3)-AR stimulation. RT-PCR detected both eNOS and nNOS in isolated rat atria. NOS isoforms performed independently. Only nNOS participated in limiting the effect of beta(3)-AR stimulation. In eNOS-KO (eNOS-/-) mice the chronotropic effect of beta(3)-AR agonists did not differ from wild type (WT) mice atria, but it was increased by the inhibition of nNOS activity. Our results suggest that the increase in frequency by beta(3)-AR activation on isolated rodent atria is associated to a parallel increases in cAMP. The nNOS-cGMP pathway negatively modulates beta(3)-AR activation. Multiple signal transduction pathways between G-protein and beta(3)-AR may protect myocardium from catecholamine-induced cardiotoxic effects.     

Novel role of the phosphatidylinositol phosphatase Sac1 in membrane homeostasis and polarized growth in Candida albicans

Phosphoinositides (PIPs) are one kind of membrane components functioning in many intracellular processes, especially in signaling transduction and membrane transport. Phosphatidylinositide phosphatases (PIPases) are specifically important for the PIP homeostasis in cell. In our previous study, we have identified the actin-related protein CaSac1 in Candida albicans, while its functional mechanisms in regulating membrane homeostasis has not been identified. Here, we show that the PIPase CaSac1 is a main membrane-related protein and regulates hyphal polarization by governing phosphoinositide dynamic and plasma membrane (PM) electrostatic field. Deletion of CaSAC1 resulted in large-scale abnormal redistribution of phosphatidylinositide 4-phosphate (PI4P) from the endomembrane to the PM. This abnormality further led to disturbance of the PM's negative electrostatic field and abnormally spotted distribution of phosphatidylinositide 4,5-bisphosphate (PI(4,5)P₂). These changes led to a severe defect in polarized hyphal growth, which could be diminished with recovery of the PM's negative electrostatic field by the anionic polymer polyacrylic acid (PAA). This study revealed that the PIPase CaSac1 plays an essential role in regulating membrane homeostasis and membrane traffic, contributing to establishment of polarized hyphal growth.     

Separation and detection of all phosphoinositide isomers by ESI-MS

Phosphoinositides (PIs) play fundamental roles as signalling molecules in numerous cellular processes. Direct analysis of PIs is typically accomplished by metabolic labelling with ³H-inositol or inorganic ³²P followed by deacylation, ion-exchange chromatography and flow scintillation detection. This analysis is laborious, time-consuming, and involves massive amounts of radioactivity. To overcome these limitations we established a robust, non-radioactive LC–ESI–MS assay for the separation and analysis of deacylated PIs that allows discrimination of all isomers without the need for radioactive labelling. We applied the method to various cell types to study the PI levels upon specific stimulation.