Inositides in the nucleus: further developments on phospholipase C beta 1 signalling during erythroid differentiation and IGF-I induced mitogenesis.

Inositol lipids originally shown to be metabolized in the cytosol have been detected also in the nucleus, where they are both synthesized and hydrolyzed. In the case of erythroid differentiation of murine erythroleukemia cells (Friend cells) it has been previously shown that PLC beta 1, which is the major nuclear PLC, undergoes down-regulation upon treatment with DMSO or tiazofurin which act as differentiative agents. On the contrary, i.e., during IGF-I induced mitogenesis, it has been shown that PLC beta 1 is rapidly activated and this event is essential for the onset of DNA synthesis. Even though its key role in cell growth has been shown, both the mechanism by which nuclear PLC beta 1 is activated and the direct relationship with erythroid differentiation are still unknown. We have addressed the question if PLC beta 1 expression and activity in the nucleus are directly related or not to the establishment of the differentiated state and we have checked the two main ways of activation, i.e., via G-protein or via phosphorylation, in order to establish whether nuclear PLC beta 1 is regulated the same way as the one at the plasma membrane or not. The data reported here show that nuclear PLC beta 1 is responsible for a continuous recycling of Friend cells, acting as a negative regulator of differentiation and that its activation is dependent on the phosphorylation state.

Inositides in the nucleus: taking stock of PLC beta 1.

The nucleus was shown to be a site for inositol lipid cycle which can be affected by treatment of quiescent cells with growth factors such as IGF-I. In fact, the exposure of Swiss 3T3 cells to IGF-I results in a rapid and transient increase in nuclear PLC beta 1 activity. In addition, several other reports have shown the involvement of PLC beta 1 in nuclear signalling in different cell types. Indeed, PLC beta 1 differs from the PLC gamma and della isozymes in that it has a long COOH-terminal sequence which contains a cluster of lysine residues that are critical for association with the nucleus. Although the demonstration of PtInsP and PtdInsP2 hydrolysis by nuclear PLC beta 1 established the existence of nuclear PLC signalling, the significance of this autonomous pathway in the nucleus has yet to be thoroughly clarified. By inducing both the inhibition of PLC beta 1 expression by antisense RNA and its overexpression we show that this nuclear PLC is essential for the onset of DNA synthesis following IGF-I stimulation of quiescent Swiss 3T3 cells. Moreover, using a different cell system, i.e. Friend erythroleukemia cells induced to differentiate towards erythrocytes, it has been evidenced that there is a relationship between the expression and activity of nuclear PLC beta 1 and the association of PI-PT alpha with the nucleus in that, when PLC activity ceases, in differentiated and resting cells at the same time there is a dramatic decrease of the association of PI-PT alpha with the nucleus.

Effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in goldfish cerebellum: neurochemical and immunocytochemical analysis.

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administered in goldfish for 3 consecutive days (10 mg kg-1 i.p.), caused cerebellar disappearance of dopamine-hydroxylase (DBH) immunoreactive fibres, whereas the noradrenergic cell bodies located in the medulla oblongata appeared intact. This effect was coupled with marked decreases in cerebellar noradrenaline (NA) and dopamine (DA) levels. An increase of immunostaining for glial fibrillary acidic protein (GFAP) was also observed. In the cerebellum of MPTP-treated fish, the contents of glutamate and GABA were significantly reduced, whereas glutamine was strongly increased. These modifications were concomitant with a significant increase of glutamine synthetase (GS) activity, whereas glutamic acid decarboxylase (GAD) activity was decreased. No changes in choline acetyltransferase (ChAT) and ornithine decarboxylase (ODC) activities were observed. High affinity uptake of glutamate and GABA was strongly reduced. Pretreatment of fish with either the monoamine oxidase inhibitor pargyline or the catecholamine (CA) uptake blocker mazindol largely prevented such modifications. The NMDA-sensitive glutamate receptor uncompetitive antagonist, dizocilpine maleate (MK-801), failed to protect against MPTP-induced damage. In conclusion, the neurotoxic effects of MPTP in goldfish cerebellum appear to be not specific against catecholaminergic terminals and could promote astrocytic reactions.

Nuclear lipid-dependent signal transduction in human osteosarcoma cells.

The enzymes and substrates involved in phosphoinositide signal transduction which have been detected in the nucleus of several cell types have been demonstrated to be responsive to agonists. The complexity of this aspect of inositide function has been previously analyzed in some cell models characterized by a mitogenic or differentiating response to specific factors. An interesting experimental model is represented by human derived osteosarcoma Saos-2 cells, characterized by the expression of high affinity receptors for interleukin 1 alpha (IL-1 alpha), which is one of the most potent stimulators of bone resorption. In particular, we investigated the earliest intracellular events following the binding of IL-1 alpha to its receptor, involving the inositide signal transduction pathway. Saos-2 cells present a partitioning of the phosphoinositidase (PLC) isoforms; in fact, the nucleus contains both PLC beta 1 and gamma 1, while the cytoplasm contains almost exclusively the gamma 1 isoform. IL-1 alpha evokes a rapid and transient increase of the PLC beta 1 activity in the nucleus, which causes the hydrolysis of phosphatidylinositol mono- and bis-phosphate. In response to IL-1 alpha, not only the canonical inositol lipid pathway appears to be involved; also the 3'-phosphorylated lipids generated by phosphatidylinositol 3-kinase (PI 3-K), which may act as second messengers, appear to be affected. In fact, Saos-2 cells present a nuclear PI 3-K activity which can be enhanced by the IL-1 alpha treatment. Among the possible targets of the second messengers released by the nuclear PLC beta 1 activation, we found that some protein kinase C isoforms, namely the epsilon and zeta, which are present within the nucleus, are activated after IL-1 alpha exposure. These activated PKC isoforms, in turn, could modulate the activity of the transcription factor NFkB, which, 5 min after IL-1 alpha treatment, has already translocated to the nucleus and bound to DNA to promote gene activation. The actual role of the inositide pathway in the Saos-2 cell function has also been investigated by utilizing cell clones transfected with the mouse sequence of the PLC beta 1.

Inositol lipid cycle and autonomous nuclear signalling.

The involvement of phospholipids and in particular polyphosphoinositides in cellular signalling has been documented in detail in the last 20 years. In addition to the plasma membrane localization also the nucleus is shown to be a site for both synthesis and hydrolysis of the phosphorylated forms of phosphatidylinositol. Previous observation have established that the nucleus possesses a specific PLC for inositol lipids, i.e., the PLC beta 1 isoform, which undergoes rapid and transient activation after IGF-I stimulation of quiescent Swiss 3T3 cells and is down-regulated after treatment of Friend erythroleukemia cells with DMSO. Here we have reviewed: (i) the potential of nuclear PLC beta 1 to be a target for anti-cancer drug, (ii) the capability of this PLC isoform, when activated by IGF-I, to be a key signalling molecule in the onset of DNA synthesis, via DAG generation and PKC alpha translocation to the nucleus, (iii) the chromosome mapping of PLC beta 1 gene. The differentiation program of Friend cells can be activated by other agents besides DMSO including tiazofurin, an anti-tumor drug, also capable of affecting the nuclear inositol lipid cycle. Tiazofurin induces a lowering of the activity of PLC beta 1 due to down regulation of this isoform as revealed by both Western blotting and Northern blotting analyses. Using Swiss 3T3 cells stably transformed with an antisense PLC beta 1 construct, the knock-out of the PLC beta 1 gene induces both a loss of PLC beta 1 expression, as determined by Western blots, and a loss of the mitogenic responsiveness to IGF-I. These events show a direct relationship between nuclear PLC beta 1 evoked signals and IGF-I induced cell proliferation. Finally, the assignment of the PLC beta 1 gene to the band q35-36 of rat chromosome 3 paves the way for further genetic studies given the fact that the region where PLC beta 1 gene maps is a hot spot for genetic alterations in a number of experimentally induced rat tumors. Taken as a whole, these results assign a key role to the regulation of nuclear PLC activity and expression both in growth-factor activated mitogenesis and in in vitro erythroid differentiation.

Simultaneous blockade of non-NMDA ionotropic receptors and NMDA receptor-associated ionophore partially protects hippocampal slices from protein synthesis impairment due to simulated ischemia.

A large body of evidence exists to demonstrate that excitatory amino acids (EAA) and their receptors are involved in the pathophysiological mechanisms linking several acute brain insults, such as cerebral ischemia, to neuronal degeneration and death. Accordingly, the use of EAA receptor antagonists can be beneficial in attenuating or preventing the neuronal irreversible damage subsequent to various neuropathological syndromes. We have investigated the effect of 15 min of simulated ischemic conditions, i.e., oxygen/glucose deprivation, on hippocampal slices preparation measuring, as neurotoxicity indexes, both the amino acids efflux in the incubation medium, detected by HPLC, and the inhibition of protein synthesis, evaluated as 3H-Leucine incorporation into proteins. Accumulation of neurotransmitter amino acids was measured in the medium during the "ischemic" period. Glutamate increased 30-fold over the basal level while aspartate was sevenfold and GABA 12-fold higher than in normal conditions. After a reoxygenation period of 30 min, the rate of protein synthesis of hippocampal slices subjected to "ischemia" was reduced to 35-50% of controls. The non-competitive NMDA antagonist MK-801 (100 microM) and the competitive NMDA antagonist CGP 39551 (100-250 microM) as well as the non-NMDA receptor antagonists NBQX (100 microM) and AP3 (300 microM) were unable to counteract the metabolic impairment when they were present alone in the incubation fluid during simulated "ischemia." An incomplete, but highly significant (p < 0.001), protection from protein synthesis impairment was achieved in the presence of an equimolar concentration (100 microM) of MK-801 and NBQX.(ABSTRACT TRUNCATED AT 250 WORDS)

Nuclear inositol lipid cycle and differentiation.

Previous investigations from our laboratory and others have shown the existence of an autonomous intranuclear inositide cycle endowed with conventional lipid kinases and PLC which in PC12 pheochromocytoma cells, human osteosarcoma SaOS-2 cells, rat liver and Swiss 3T3 cells is the isoform beta 1, which in the latter cells is activated upon IGF-I stimulation. The behavior of the nuclear inositol lipid cycle has been investigated in nuclei of Friend erythroleukemia cells. These nuclei possess both lipid kinases and PLC. The cycle upon treatment with differentiating agents (i.e., DMSO and tiazofurin) is characterized by an accumulation of polyphosphoinositides and a decrease of DAG due to down-regulation of a specific PLC. Indeed, even if both beta 1 and gamma 1 isoforms are present in these nuclei, when Friend cells undergo terminal erythroid differentiation only the PLC beta 1 isoform is down-regulated as shown by immunochemical and immunocytochemical analysis, by direct determination of enzymatic activity and in the presence of neutralizing monoclonal antibodies as well as by Northern blot for PLC beta 1 message, whilst the amount of PLC gamma 1 and its activity are unaffected by erythroid differentiation. In conclusion, the presence of a specific nuclear PLC whose activity and expression are down-regulated during differentiation of erythroleukemia cells points out a role for nuclear phosphoinositide signalling in the processes of cell differentiation and hints at the nuclear PLC beta 1 as an important step of the cycle in relation to the erythroid differentiative commitment of murine erythroleukemia cells.

An endogenous ligand for the kainate-type binding sites from rat brain.

Extracts from the rat brain were screened to identify a putative endogenous ligand for the binding sites of the neuroexcitant kainic acid (KA). The extracted substances were separated by chromatographic techniques and tested for their ability to inhibit KA binding to fish synaptosomes and to membranes from rat brain. A substance isolated in this way (rat kainate-binding inhibitor, RKBI) display a competitive interaction with KA for the low-affinity binding sites in rat brain membranes. According to the separation behavior in the purification step, RKBI is distinct from an inhibitor formerly isolated from fish nervous tissue (KBI). The substance exhibits positive co-operativity with KA for a very-low-affinity site population, particularly concentrated in the cerebellum, and could play a physiological role in this area.

Impaired neurogenesis by methylazoxymethanol in newborn rats results in transient reduction of ornithine decarboxylase and polyamines in the cerebellum, but not in the olfactory bulbs.

Polyamines and the key enzyme for their biosynthesis, ornithine decarboxylase (ODC) play an important role in the control of neuronal proliferation and differentiation. Exposure to agents that interfere with normal cell maturation is expected to result in alteration of neuronal ODC developmental pattern. We have administered to newborn rats, about 6 and 30 hr after birth, 20 mg/kg of methylazoxymethanol acetate (MAM), an agent able to selectively kill dividing cells and we have evaluated ODC activity and polyamine levels in the cerebellum and ODC activity in the olfactory bulbs at various developmental stages starting from postnatal day 4 (PD 4) until PD 28. Cerebellar weight decreased by 22-50% at the different developmental stages in MAM-treated animals. A decline in ODC specific activity was observed at PD 4 and a decrease of putrescine levels at PD 4 and PD 6 in the cerebellum. At PD 10, however, both ODC activity and putrescine level were increased in MAM-treated animals. Spermidine levels were never affected by the treatment, while spermine was significantly decreased at PD 6 and PD 8. These results demonstrate that altered ontogenetic patterns of ODC activity and polyamine levels are the consequence of disturbance of the normal process of brain maturation. No significant differences in specific ODC activity were noticed in the olfactory bulbs of MAM-treated rats. This may be related to the more widespread time-span of neurogenesis in this region, a fact that is also revealed by the higher ODC activity constitutively expressed at times in which neurogenesis has ended in the rest of the brain.

Lipid-dependent nuclear signalling: morphological and functional features.

Enzymes involved in lipid metabolism exist within the nucleus and are responsive to external stimuli. In particular, the kinases which phosphorylate phosphatidylinositol and phosphatidylinositol-4-monophosphate have been demonstrated in nuclei of both undifferentiated and differentiated Friend cells and of quiescent Swiss 3T3 cells as well as of those exposed to insulin-like growth factor I. Besides the lipid kinases, also the phosphoinositidases C (PIC) are active inside the nucleus. In Swiss 3T3 cells the nuclear PIC beta 1 is activated and its activation by IGF-I temporally precedes the translocation to the nucleus of protein kinase C. In Friend cell nuclei, on the other hand, when erythroid differentiation is induced, the PIC beta 1 activity is reduced. Another aspect of the nuclear signalling transduction system which appears quite interesting is its actual localization at subcellular level. By using electron microscope immunogold labelling, the nuclear PIC isoforms (the beta 1 isoform in Swiss 3T3 cells, the beta 1 and gamma 1 in Friend cells) are localized mainly in the interchromatin domains. This localization has been further confirmed on in situ matrix preparations of 3T3 cells in which PIC beta 1 is associated with the inner nuclear matrix but not with the nuclear pore-lamina complex. Colocalization experiments indicate that nuclear PIC beta 1 is present in sites in which both nuclear phospholipids and PKC can be detected, while the cytoplasmic PIC gamma 1 can be identified in close association with cytoskeletal filaments identified by anti-actin antibodies. The precise localization of the different PIC isoforms strongly indicates that the signal transduction system operating at the nuclear level may be part of a cross-talk between the cytoplasm and the nucleus controlling either cell proliferation or differentiation.

Functional adenosine A1 receptors in goldfish brain: regional distribution and inhibition of K(+)-evoked glutamate release from cerebellar slices.

In goldfish brain, [3H]cyclohexyladenosine binding sites are ubiquitously distributed with a maximum in the hypothalamus and a minimum in the spinal cord. The binding parameters measured in cerebellar membranes (Kd = 0.88 +/- 0.08 nM; Bmax = 59.65 +/- 2.62 fmol/mg protein) are not significantly different from those of the whole brain. In perfused goldfish cerebellar slices, stimulation of cyclic AMP accumulation by 10(-5) M forskolin was markedly reduced (58.7%) by treatment with 10(-4) M cyclohexyladenosine, an adenosine A1 receptor agonist, and the reduction was reversed in the presence of 10(-4) M 8-cyclopentyltheophylline, a selective A1 receptor antagonist. In the same brain preparation, 30 mM K+ stimulated the release of glutamate, glutamine, glycine and GABA in a Ca(2+)-dependent manner, whereas the aspartate and taurine release was Ca(2+)-independent. Cyclohexyladenosine inhibited the 30 mM K(+)-evoked release of glutamate in a dose-related manner. This effect was reversed by 8-cyclopentyltheophylline. These results support the hypothesis that adenosine A1 receptors present in goldfish cerebellum are involved in the modulation of glutamate transmitter release.

Nuclear phosphoinositidase C during growth factor stimulation.

The existence of phosphoinositidase C beta is demonstrated in the nucleus of Swiss 3T3 cells. Moreover, we show that this isoform is specific for the nucleus while the gamma isoform is confined to the cytoplasm of these cells. When Swiss 3T3 fibroblasts are treated with Insulin-like Growth Factor I a rapid and transient activation of the beta isoform occurs at the nucleus whilst the cytoplasmic phosphoinositidase C is unaffected. These results seem to explain the mechanism by which the decrease in the mass of polyphosphoinositol lipids occurs in the nucleus after stimulation with Insulin-like Growth Factor I and actually point out the existence of a nuclear polyphosphoinositide signalling system distinct from the plasma membrane localized system and constituted not only by the previously demonstrated lipid kinases but also by a specific phosphoinositidase C.

Spontaneous recovery of MPTP-damaged catecholamine systems in goldfish brain areas.

In goldfish, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administered for 3 consecutive days (10 mg/kg), produced a marked decrease in dopamine (DA) and noradrenaline (NA) levels in telencephalon, diencephalon and medulla oblongata, without affecting the serotonin (5-HT) content. Furthermore the neurotoxin decreased either [3H]DA high affinity uptake or K(+)-stimulated DA release from synaptosomal (P2) preparations, with concomitant up-regulation of D2 postsynaptic receptors as well. No significant changes of choline acetyltransferase and glutamic acid decarboxylase activity or [3H]glutamate uptake were observed. Moreover the pretreatment with deprenyl (1 mg/kg) or mazindol (10 mg/kg) but not with clorgyline (5 mg/kg) prevented catecholamine depletion. Added in vitro to synaptosomal preparations both MPTP and more potently MPP+, in a concentration-dependent manner, inhibited [3H]DA uptake. Time course study revealed that MPTP-induced alteration of neurochemical parameters in goldfish brain areas were almost completely reversed within 6 weeks, suggesting that catecholamine systems in goldfish brain show a remarkable power of recovery after MPTP lesion.

Antagonists of the NMDA receptor and allopurinol protect the olfactory cortex but not the striatum after intra-cerebral injection of kainic acid.

Overstimulation of the NMDA receptor, as well as generation of excessive amounts of free radicals, has been implicated in excitotoxic brain injuries. We report here that two antagonists of the NMDA receptor and an inhibitor of the free radical-generating enzyme, xanthine oxidase, protect the olfactory cortex but not the striatum after intrastriatal injection of kainic acid. Our results suggest the existence of a precise link between excitotoxic activation of the NMDA receptor and neuropathology related to excessive amounts of free radicals. The focal point of this link may be the entry of Ca2+ through the NMDA receptor and the consequent activation of proteases and free radical-generating systems.

Induction of brain ornithine decarboxylase after systemic or intrastriatal administration of kainic acid.

The activity of ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis, dramatically increases after different types of brain injuries. The role of this induction is still unclear. We report here data on the temporal pattern of ODC induction caused by the excitotoxin kainic acid. After systemic administration, ODC activity increases severalfold peaking at 8 h in the prefrontal cortex and at 16 h in the olfactory cortex and hippocampus. After intrastriatal injection, the peak of induction is reached at 32 h, while a smaller and more transient increase is also observed in the contralateral, saline-injected striatum. We suggest that ODC induction is initially linked to overactivation of neural circuits and, later on, to the development of widespread neural damage.

Protection from kainic acid neuropathological syndrome by NMDA receptor antagonists: effect of MK-801 and CGP 39551 on neurotransmitter and glial markers.

Systemic administration of kainic acid results in the development of a characteristic convulsive syndrome, accompanied by neuropathological alterations and loss of transmitter markers in some forebrain regions. Since some of these effects appear to involve the N-methyl-D-aspartate (NMDA) subtype of excitatory amino acid receptors, the protection given by a non-competitive (MK-801) and a competitive (CGP 39551) NMDA receptor antagonist against the loss of glutamatergic and gamma-amino butyric acid (GABAergic) neurochemical markers was compared. Appropriate doses of both compounds (1 mg/kg MK-801 and 25 mg/kg CGP 39551) completely reversed the decrease of high affinity uptake of glutamate and activity of glutamate decarboxylase in the olfactory cortex, amygdala, hippocampus and lateral septum. In addition, they also essentially counteracted the increase of a glial marker, the enzyme glutamine synthetase, consequent to neuronal degeneration. The results confirmed that involvement of NMDA receptors is essential for the full expression of neuropathological effects of kainic acid. They also support the use of a competitive antagonist of the NMDA receptor, such as CGP 39551, to afford substantial protection against the excitotoxic damage, whilst giving fewer side effects and motor disturbances than MK-801.

Changes in inositol lipid metabolism and protein kinase C translocation in nuclei of mitogen stimulated Swiss 3T3 cells.

The correlation between changes in nuclear polyphosphoinositide levels preceding PKC translocation to the nucleus and the onset of DNA synthesis has been discussed. Using two different clones of Swiss 3T3 fibroblasts belonging to the same original cell line, one of which is unresponsive to mitogenic stimulation with IGF-I on its own or in combination with bombesin, it has been observed that a rapid and transient breakdown of nuclear PIP and PIP2 occurs only in responsive cells and this precedes the translocation of PKC to the nucleus, as evidenced by immunochemical analysis as well as by enzymatic activity. Therefore, it seems that a direct link exists between nuclear polyphosphoinositide metabolism, PKC translocation to the nucleus and cell division. Since IGF-I acts at the plasma membrane through a tyrosine kinase receptor it seems that the mitogenic stimulation induced by this factor utilizes different signalling pathways at the plasma membrane and at the nucleus. Because of the evidence that type I IGF receptor is expressed in both responsive and unresponsive cells and that the receptor machinery at the plasma membrane is active the lack of the transient changes in nuclear inositol lipids and of PKC translocation in unresponsive cells further suggests that the cell nucleus is capable of an autonomous signalling system based on polyphosphoinositide metabolism.

Postnatal maturation of cholinergic markers in forebrain regions of C57BL/6 mice.

The maturation of some neurochemical markers linked to cholinergic function (choline acetyltransferase, acetylcholinesterase, muscarinic binding sites) has been studied from 10 to 150 days of age in mice belonging to C57BL/6 strain. Previous studies had suggested that part of the cholinergic neurons of the basal forebrain undergo degeneration during juvenile stages of life in these rodents. Our data showed a delayed maturation of the cholinergic levels in the cortex and hippocampus, the main targets of the forebrain cholinergic neurons, but not in the striatum and superior colliculus. In none of these regions was any clear trend towards a decrement of cholinergic levels observed during the lifespan considered. In the medial septum-diagonal band area, an actual decrease of cholinergic levels was observed between 60 and 150 days of age. A side experiment based on daily administration of GM1 ganglioside during juvenile life, showed no effect of this treatment on the maturation of cholinergic markers.