Inositol Lipid Metabolism Research Articles

Insulin selectively stimulates nuclear phosphoinositide-specific phospholipase C (PI-PLC) β1 activity through a mitogen-activated protein (MAP) kinase-dependent serine phosphorylation

Using NIH 3T3 cells, we have investigated nuclear phosphoinositide metabolism in response to insulin, a molecule which acts as a proliferating factor for this cell line and which is known as a powerful activator of the mitogen-activated protein (MAP) kinase pathway. Insulin stimulated inositol lipid metabolism in the nucleus, as demonstrated by measurement of the diacylglycerol mass produced in vivo and by in vitro nuclear phosphoinositide-specific phospholipase C (PI-PLC) activity assay. Despite the fact that nuclei of NIH 3T3 cells contained all of the four isozymes of the β family of PI-PLC (i.e. β1, β2, β3, and β4), insulin only activated the β1 isoform. Insulin also induced nuclear translocation of MAP kinase, as demonstrated by Western blotting analysis, enzyme activity assays, and immunofluorescence staining, and this translocation was blocked by the specific MAP kinase kinase inhibitor PD98059. By means of both a monoclonal antibody recognizing phosphoserine and in vivo labeling with [ 32P]orthophosphate, we ascertained that nuclear PI-PLC-β1 (and in particular the b subtype) was phosphorylated on serine residues in response to insulin. Both phosphorylation and activation of nuclear PI-PLC-β1 were substantially reduced by PD98059. Our results conclusively demonstrate that activation of nuclear PI-PLC-β1 strictly depends on its phosphorylation which is mediated through the MAP kinase pathway.

Effects of a water-soluble antitumor ether phosphonoinositide, d- myo-inositol 4-(hexadecyloxy)-3( S)-methoxybutanephosphonate (c 4-pi), on inositol lipid metabolism in breast epithelial cancer cell lines

We have demonstrated previously that d- myo-inositol 4-(hexadecyloxy)-3( S)-methoxybutanephosphonate (C 4-PI), an isosteric phosphonate analog of phosphatidylinositol developed to inhibit inositol lipid metabolism, was unable to inhibit phosphatidylinositol (PI) 3-kinase activity. We now report the effects of the compound on other aspects of inositol metabolism. We demonstrated that C 4-PI inhibits the activity of purified recombinant PI-phospholipase C-β (PLC-β) at all concentrations tested; it enhanced the activity of PI-PLC-γ and PI-PLC-δ at low concentrations (10 μM), while severely inhibiting their activities at higher concentrations. In the breast cancer cell lines MCF-7 (estrogen receptor positive) and MDA-MB-468 (estrogen receptor negative), C 4-PI had no effect on the uptake of d- myo-inositol but severely inhibited its incorporation into PI. In spite of the drastic decrease in PI synthesis, C 4-PI did not affect the levels of inositol incorporated into phosphatidylinositol 4,5-bisphosphate (PIP 2) in the cells. In vitro assays showed that C 4-PI inhibited PI synthase activity (inhibition of 35% at 50 μM) but had little effect on PI 4-kinase activity (inhibition of 13% at 150 μM). C 4-PI inhibited the proliferation of MCF-7 and MDA-MB-468 cell lines with ic 50 values of 12 and 18 μM. Taken together, the results suggest that the accumulation of [ 3H]inositol in PIP 2 in cells incubated with C 4-PI may be due to the inhibition of PIP 2 hydrolysis in the cells with no effect on its synthesis. The role of these C 4-PI-induced effects in the mechanism of growth inhibition by C 4-PI remains to be established.

Metabolism and possible compartmentalization of inositol lipids in isolated rat-liver nuclei.

(1) The removal of the nuclear envelope from isolated rat-liver nuclei by washing with Triton X-100 (TX-100) was assessed by electron microscopy. All the envelope was removed by 0.04% (w/v) TX-100. (2) After this removal, phosphorylation of inositol lipids and diacylglycerol (DAG) from [gamma-32P]ATP still occurs, despite the near complete absence of detectable (by mass assay) DAG and PtdIns. This suggests that the majority of these two lipids in nuclei are present in the nuclear membrane, but the small amounts remaining after extraction, defined as intranuclear, are available for phosphorylation by lipid kinases (36% for DAG and 24% for PtdIns respectively, when expressed as a percentage of incorporation of intact nuclei). (3) PtdIns(4,5)P2 did not follow the same pattern as PtdIns and DAG; after removal of the nuclear membrane, 40% of the mass of this lipid was left in the nucleus. Moreover, a similar amount of PtdIns(4,5)P2 was also resistant to extraction with even higher concentrations of detergent, suggesting that PtdIns(4,5)P2 has a discrete intranuclear location, probably bound to nuclear proteins. (4) Addition of exogenous substrates, PtdIns, PtdIns(4)P and DAG, to membrane-depleted nuclei resulted in reconstitution of the majority of lipid phosphorylations from [gamma-32P]ATP (70%, 90% and 94% of intact nuclei respectively), suggesting a predominantly intranuclear location for the respective kinases. (5) Nuclei also showed phosphomonoesterase and phosphatidic acid hydrolase activity; dephosphorylation of pre-radiolabelled PtdIns(4)P, PtdIns(4,5)P2 and phosphatidic acid was observed when [gamma-32P]ATP was removed. However, some of the radioactivity was apparently resistant to these enzymes, suggesting the existence of multiple pools of these lipids. (6) Addition of excess non-radiolabelled ATP to nuclei pre-labelled with [gamma-32P]ATP resulted in an initial increase in the label in PtdIns(4,5)P2, implying a precursor-product relationship between the radiolabelled pools of PtdIns(4)P and PtdIns(4,5)P2. This was confirmed by analysis of the incorporation of 32P into the 4'-phosphate group of PtdIns(4)P and the individual 4'- and 5'-phosphate groups of PtdIns(4,5)P2. The data from these experiments also indicated that PtdIns(4,5)P2 can be produced from a pre-existing pool of PtdIns(4)P, as well as de novo from PtdIns. (7) Taken together our data suggest that isolated rat-liver nuclei have an intranuclear inositol lipid metabolism mechanism utilizing enzymes and substrates equivalent to those found in cytosol and plasma membrane, and that there may be some, but not complete, compartmentalization of the components of the nuclear inositol cycle.

Integrin-dependent signal transduction.

Integrins are receptor molecules for extracellular matrix molecules (e.g., the beta(1) family), serum components (alpha(v) family) and immunoglobulin family adhesion molecules (beta(2) family). Integrin-dependent adhesion has also been shown to have metabolic consequences. Adhesion to a variety of extracellular matrix proteins, such as fibronectin, collagen, and laminin, is a potent regulator of cell growth, differentiation, and gene expression. Ligand binding or aggregation of integrin receptors initiates a number of metabolic changes including activation of serine/threonine and tyrosine kinases, increased Ca2+ influx, increased cytoplasmic alkalinization, and altered inositol lipid metabolism. In some instances activation of transcription factors and induction of gene expression have also been demonstrated. Components of key signaling pathways involving integrins are beginning to be identified. Some studies have shown that integrins form multi-component complexes with signal transduction molecules. Elucidating the interactions of the signal transduction molecules with each other and with the integrin cytoplasmic domains will be key to understanding the initial events of signal transduction through the integrins.

Purification and Biochemical Characterization of a Mammalian Phosphatidylinositol 3,4,5-Trisphosphate 5-Phosphatase

Characterization of the enzymes involved in metabolism of 3-phosphorylated inositol lipids and their subcellular localization revealed that in vitro a 5-phosphatase activity was responsible for the degradation of phosphatidylinositol 3,4,5-trisphosphate, whereas a 3-phosphatase activity hydrolyzed phosphatidylinositol 3-phosphate and/or phosphatidylinositol 3,4-bisphosphate. All these activities were localized in the cytosol. No phospholipase activities were detected. The cytosolic phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase activity was purified to near homogeneity using ion exchange, affinity, and size exclusion chromatography. Characterization of the purified phosphatase revealed that it is a magnesium-dependent 5-phosphatase that is able to hydrolyze phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate. The enzyme is only partially inhibited by neomycin and vanadate but is strongly inhibited by phosphatidylinositol 4,5-bisphosphate and to a slightly lesser extent by phosphatidylinositol 4-phosphate.

Inositol lipid phosphorylation and breakdown in rat liver nuclei is affected by hydrocortisone blood levels

The possibility that inositol lipid metabolism is related to nuclear events accompanying steroid hormone action has been investigated by comparing lipid phosphorylation and breakdown in normal rat liver nuclei and in hypo- and hypercortisolemic conditions. Lipid phosphorylation in vitro showed the presence of diacylglycerol (DAG)-, phosphatidylinositol (PI)- and phosphatidylinositol-4-phosphate (PIP)-kinase activity, with differences between total tissue homogenates and isolated nuclei, relevant to the treatment in vivo. Administration of hydrocortisone (HC) produced a marked decrease in the phosphorylated nuclear products without influencing the homogenate kinase activity. Under conditions which were optimal for the kinase activities, nuclear PIP-kinase was strongly increased in presence of a high blood level of HC whereas PI-kinase activity was reduced. From these observations it appears that the observed differences were due to specific modulation of kinase activities rather than to changes in the availability of substrates. The phosphoinositide-specific phospholipase C (PLC) activity was also investigated. In the presence of a high HC blood level, the phosphodiesteratic cleavage of PIP strongly increased, while that of phosphatidylinositol bisphosphate (PIP2) was similar in normal and hypercortisolemic conditions. Nuclear phosphoinositide hydrolysis was affected by PLC, beta and gamma isoforms, which were equally represented in all the conditions investigated, indicating that the observed changes of activity were due to a modulation rather than to a change in the amount of enzyme. These results suggest that inositol lipid metabolism plays a role in the nuclear modifications accompanying steroid hormone induction of transcriptional activity.

Production of Second Messengers Following Chemotactic and Mitogenic Urokinase-Receptor Interaction in Human Fibroblasts and Mouse Fibroblasts Transfected with Human Urokinase Receptor

We studied urokinase-type plasminogen activator (u-PA)-dependent chemotaxis and DNA synthesis in both human fibroblasts and LB6 mouse fibroblasts transferred with human u-PA receptor (u-PAR) gene (LB6 clone 19). Both cell lines have receptors for the amino-terminal fragment of u-PA (u-PA-ATF). We observed that u-PA and u-PA-ATF stimulated chemotactic migration of both LB6 clone 19 cells and human fibroblasts, which could be impaired by down-regulation of protein kinase C (PKC) with phorbol myristate acetate (PMA). While LB6 clone 19 cells were unable to undergo mitosis following exposure to either u-PA or u-PA-ATF, human fibroblasts were stimulated to mitosis by exogenous addition of native u-PA, and u-PA-ATF was ineffective. The mitogenic activity of u-PA on human fibroblasts could also be impaired by down-regulation of PKC with PMA. We studied second messenger formation following u-PAR stimulation. Neither inositol lipid metabolism nor intracellular Ca 2+ content were affected, while an increase of diacylglycerol (DAG) generation was observed. Such DAG formation was related to de novo synthesis from glucose and was dependent on ligand-receptor interaction. Both u-PA-ATF and the native u-PA molecule were able to stimulate DAG formation, u-PA being from three to fourfold more efficient than ATF. These data suggest that u-PAR stimulation per se is sufficient to trigger DAG formation. The native molecule confers on the cell an additional stimulus, possibly related with the activation of a u-PA-catalytic site-dependent substrate. Such stimulation allows the cell to reach the DAG treshold level required to trigger DNA synthesis.

Protein Kinase C Activity Is Reduced in Epidermal Cells from Energy-Restricted SENCAR Mice ,

Female SENCAR mice were pre-fed a control or 40% energy-restricted (ER) diet with energy removed from fat and carbohydrate, or a control, balanced high fat (BHF, with similar energy from fat and carbohydrate), 35% energy restricted from fat (HCR) or 35% energy restricted from carbohydrate (HFR) diet. Epidermal cells were isolated by trypsin digestion for measurement of protein kinase C (PKC) activity, lipid composition or lipid metabolism. Dietary restriction of fat or carbohydrate energy (HFR or HCR group) reduced particulate PKC activity in epidermal cells compared with cells from control mice. The ratio of soluble particulate PKC activity was higher in epidermal cells from mice fed the HCR diet compared with those fed the HFR diet. Diet did not affect soluble PKC activity. Inositol accumulation was measured in the water- or lipid-soluble fractions of prelabeled ([3H]inositol) epidermal cells following a 1-h incubation in media with LiCl. Phosphatidylinositol, inositol biphosphate and inositol triphosphate fractions were more heavily labeled in cells from mice fed the ER diet. Energy restriction did not modify epidermal total lipid or phospholipid composition, but 1,2-diacylglycerol levels were elevated in relation to cell number in epidermal cells from mice fed the ER diet. These data suggest that dietary energy restriction modified PKC activity through a pathway other than alteration in membrane lipid composition or inositol lipid metabolism.

Organic Solvents Activate Human Platelets Through the Inositol Lipid-linked Signal Transduction System

It has recently been proposed that occupational exposure to organic solvents in vivo may lead to platelet activation and this has been substantiated by exposure of platelets to solvents in vitro. The present work was undertaken to study the effects of organic solvents on the platelet inositol lipid signal transduction system. Human platelets that had been prelabelled with [(32)P] P, were exposed to a saturated atmosphere of the organic solvents toluene, xylene or hexane. Extracts were analyzed for metabolites of the polyphosphoinositide cycle and ATP. All solvents studied induced a decrease in radioactivity in phosphatidylinositol 4,5-bisphosphate together with an increase in radioactivities in phosphatidylinositol 4-phosphate and phosphatidic acid. This is compatible with solvent-induced activation of the cells through the inositol lipid pathway. In cells exposed to toluene or xylene we could detect an increased level in inositol trisphosphates at 3 min of exposure. The solvent-induced changes in metabolic ATP could not explain the solvent-induced effects on the inositol lipid metabolism. It is concluded that the organic solvents toluene, xylene and hexane can activate human platelets through the inositol lipid-linked transmembrane signal system.

Rat testicular myoid cells respond to endothelin: characterization of binding and signal transduction pathway

The presence of endothelin (ET), a vasoconstrictor peptide, in the testis suggests that it may regulate nonvascular target cells. We investigated binding ability, regulation of inositol phosphate metabolism, changes in cytosolic free Ca2+ concentrations ([Ca2+]i), and induction of morphological changes by ET-1 in rat primary testicular myoid cell cultures. ET-1 specifically bound to highly purified rat testicular myoid cells in a time- and temperature-dependent manner. Scatchard analysis of the binding studies indicated the presence of a single class of high affinity binding sites, with an apparent Kd of 3 x 10(-10) M and a maximal binding capacity of 10(5) sites/cell. ET-1 induced both rapid production of inositol triphosphate and mobilization of [Ca2+]i in a concentration-dependent fashion. By contrast, inositol lipid metabolism was slightly affected by ET-1 in the total peritubular cell population. Purified Sertoli cells failed to show either ET-1 binding or ET-1-induced phosphatidylinositol hydrolysis. Mobilization of [Ca2+]i mostly depended upon the release of Ca2+ from thapsigargin-sensitive intracellular Ca2+, whereas it was not affected by abolishment of the Ca2+ gradient through the plasma membrane or by inhibition of L-type voltage-sensitive Ca(2+)-channels by nifedipine. These findings together with the fact that Sertoli cells are unable to respond to and bind ET-1 indicate that ET is a specific agonist of myoid cells in the seminiferous tubule and suggest a role for ET-1 in the autocrine/paracrine regulation of testicular function.

Rat testicular myoid cells respond to endothelin: characterization of binding and signal transduction pathway.

The presence of endothelin (ET), a vasoconstrictor peptide, in the testis suggests that it may regulate nonvascular target cells. We investigated binding ability, regulation of inositol phosphate metabolism, changes in cytosolic free Ca2+ concentrations ([Ca2+]i), and induction of morphological changes by ET-1 in rat primary testicular myoid cell cultures. ET-1 specifically bound to highly purified rat testicular myoid cells in a time- and temperature-dependent manner. Scatchard analysis of the binding studies indicated the presence of a single class of high affinity binding sites, with an apparent Kd of 3 x 10(-10) M and a maximal binding capacity of 10(5) sites/cell. ET-1 induced both rapid production of inositol triphosphate and mobilization of [Ca2+]i in a concentration-dependent fashion. By contrast, inositol lipid metabolism was slightly affected by ET-1 in the total peritubular cell population. Purified Sertoli cells failed to show either ET-1 binding or ET-1-induced phosphatidylinositol hydrolysis. Mobilization of [Ca2+]i mostly depended upon the release of Ca2+ from thapsigargin-sensitive intracellular Ca2+, whereas it was not affected by abolishment of the Ca2+ gradient through the plasma membrane or by inhibition of L-type voltage-sensitive Ca(2+)-channels by nifedipine. These findings together with the fact that Sertoli cells are unable to respond to and bind ET-1 indicate that ET is a specific agonist of myoid cells in the seminiferous tubule and suggest a role for ET-1 in the autocrine/paracrine regulation of testicular function.

Inositol lipid-mediated intranuclear signalling: A comparative analysis of in vivo labelling in interferon alpha-sensitive and -resistant daudi lymphoma cells

Changes in inositol lipid and diacylglycerol metabolism have been analysed in Daudi lymphoma cells treated up to 24 h with human Dna recombinant interferon alpha. Results showing a different response of nuclear phosphoinositides and diacylglycerol, compared to whole cells, suggest that the intranuclear signalling system activated by interferon in Daudi cells involves nuclear inositol lipid metabolism. A well-characterized clone of Daudi cells selected for resistence to the antiproliferative action of interferon provided controls for the specificity of results.

Arachidonic acid release and inositol lipid metabolism in response to bradykinin and related peptides in human endometrial cells in vitro

Prostaglandins of the 2 series are known to play a role in the regulation of menstruation and implantation but, more recently, other vasoactive peptides have been considered as potential regulators of these endometrial processes. The aim of the present study was to investigate the action of the potent vasoactive peptide bradykinin and the structurally related peptide, kallidin, on endometrial function by examining their effect on phosphoinositide hydrolysis and arachidonic acid release from endometrial cells in vitro. Primary cultures of endometrial glands and stromal cells were prelabelled with [ 14C]-arachidonic acid (AA) or [ 3H]-inositol to monitor arachidonic acid release and inositol phosphate accumulation respectively. Bradykinin and kallidin stimulated a dose and time-dependent release of arachidonic acid from stromal cells which, with 100 nmol/L bradykinin, was 30–150% above basal release and maximal at 5 min. Glands were less responsive; 100 nmol/L bradykinin (at 5 min) caused a release of AA of 30–69% above basal level. Bradykinin also stimulated a dose dependent increase in inositol monophosphate production. The maximum response with stromal cells was 8- to 10-fold and with glands, 2-fold (1 and 100 nmol/L bradykinin, respectively). Kallidin was equipotent to bradykinin with respect to both AA and inositol phosphate accumulation. The bradykinin analogue des Arg bradykinin (which acts through the B 1 receptor) released AA from stromal cells but did not alter phosphoinositide hydrolysis, suggesting that these two cellular responses are mediated by different receptors (B 1 and B 2 respectively). There is as yet no evidence for a role for bradykinin in endometrial function, but our findings suggest that bradykinin (or kallidin) could regulate the availability of substrate for prostaglandin synthesis or stimulate second messenger generation and thus control a variety of cellular functions.

Light stimulates in vivo inositol lipid turnover in frog retinal pigment epithelial cells at the onset of shedding and phagocytosis of photoreceptor membranes

We have developed an experimental model to study in vivo inositol lipid metabolism in frog retinal pigment epithelial (RPE) cells, including the effect of light on phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate. RPE cells were rapidly isolated after either brief light or dark periods. Light and electron microscopy showed complete detachment of the retina from the RPE cells, and that the RPE cell suspensions were devoid of photoreceptor cell outer segments. Frog tissues were labeled in vivo for 20 hr by intravitreal injection of [ 3H]inositol (4 μCi, 4μl per eye) within a 24-hr constant illumination period. Following 1 hr of darkness (priming period), frogs were intravitreally injected with LiCl (0·5 m, 4μl per eye) 15 min before the onset of either 30-min light stimulation or an additional 30 min of darkness (controls). In order to preserve endogenous inositol phosphate pools present after dark and light exposure, the RPE cells were harvested in the shortest time possible, at low temperatures (18–20°C), and in the presence of 10 m m LiCl. Total [ 3H]inositol-labeled water-soluble products (inositol plus inositol phosphates) were increased by 86% after 30 min of light. Inositol trisphosphate (IP 3) showed the highest accumulation (a 5·5-fold increase), followed by inositol bisphosphate (1·9-fold increase) and inositol monophosphate (1·4-fold increase). Free [ 3H]inositol also accumulated (2·8-fold increase), reflecting only a partial inhibition of phosphomonoesterase by LiCl. These changes were paralleled by a 12% decrease in 3H-labeled phosphatidylinositol with no significant difference in the labeling of polyphosphoinositides. This is the first time that IP 3 accumulation, correlated with a specific function in a cell type, has been demonstrated in vivo.

Signal transduction by the neu/ebrB-2 receptor: A potential target for anti-tumor therapy

The neu/erbB-2 protooncogene encodes a transmembrane tyrosine kinase homologous to receptors for polypeptide growth factors. The oncogenic potential of the presumed receptor is released through multiple genetic mechanisms including a point mutation, truncation of non-catalytic sequences and overexpression. The latter mechanism appears to be relevant to human cancers as elevated expression of the neu/erbB-2 gene is frequently observed in solid tumors of various adenocarcinomas. It is therefore conceivable that strategies aimed at the biochemical mechanism of action of the neu/erbB-2 tyrosine kinase may contribute to the treatment of certain human cancers. To this aim we undertook a multiple research approach consisting of the following directions: (i) The neu/erbB-2 ligand—a systematic screening of potential biological sources of the hypothetical hormone molecule, that presumably binds to the neu/erbB-2 protein, resulted in detection of a candidate activity in the medium of certain cultured transformed cells. Partial purification indicated that the factor is a 30–35 kDa glycoprotein. Further studies revealed several biochemical characteristics of the factor that may be helpful for complete purification and structural analysis of this novel hormone. (ii) Signal transduction by neu/erbB-2—using a chimeric receptor approach and various mutants we found that all the oncogenic forms of the neu/erbB-2 are constitutively coupled, both physically and functionally, to a multi-protein complex of signaling molecules. The latter includes the phosphatidylinositol-specific phospholipase Cγ and a phosphatidylinositol kinase. Thus, the metabolism of inositol lipids is probably a major biochemical pathway utilized by the neu/erbB-2 tyrosine kinase. (iii) Tumor inhibitory antibodies—we generated a panel of monoclonal antibodies to the presumed receptor. Surprisingly, some antibodies almost completely inhibited the growth of tumor cells in athymic mice, whereas one antibody significantly accelerated the rate of tumor growth in animals. Interestingly, the inhibitory antibodies conferred a mature phenotype to cultured breast cancer cells, implicating terminal differentiation in tumor retardation.

Changes in polyphosphoinositide levels in rat liver nuclei in response to prolactin, a known hepatic mitogen

The effect of prolactin action on nuclear polyphosphoinositide synthesis was investigated in isolated rat liver nuclei. An increased uptake of phosphate from [γ 32P]adenosinetrisphosphate was observed in both phosphatidylinositol 4-phosphatet and phosphatidylinositol 4,5-biphosphate with a maximum response at 10 −12 M concentration of hormone. Pulse-chase experiments in isolated nuclei following prolactin treatment indicate that the observed increase in accumulation of phosphatidyloinositol 4-phosphate and phosphatidyl-inositol 4,5-biphosphate is mainly due to a decrease in their rate of turnover possibly induced by a change in activity of polyphosphoinositide-specific monesterases. In vitro prolactin also reduces the activity of nuclear phospholipase C specific for phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-biphosphate. Moreover, this feature is stronly supported by the concomitant decrease in nuclear diacylglycerol mass. Thus these data suggest that once prolactin reaches the nucleus an intranuclear signalling is evoked through inositol lipid metabolism.

Differences in regulation between nuclear and cytoplasmic Ca2+ in cultured smooth muscle cells

The free Ca2+ concentrations in the nucleus ([Ca2+]n) and cytoplasm ([Ca2+]c) of cultured smooth muscle cells were estimated using the fluorescent dye indo-1 and the ACAS 570 confocal laser microscope. In resting DDT1MF2 smooth muscle cells [Ca2+]n was found to be lower than [Ca2+]c. Both values increased transiently in response to histamine (100 microM), but during this stimulation [Ca2+]n exceeded [Ca2+]c. Maximal increase of [Ca2+]n was observed in the center of the nucleus, and a maximal increase of [Ca2+]c was observed in the immediate vicinity of the plasma membrane. A similar response was obtained with other agonists, such as carbachol or ATP. Comparable results with ATP were obtained in cultured aorta cells. The differential rise of [Ca2+]n over [Ca2+]c in DDT1MF2 cells did not occur during either spontaneous release of Ca2+ or Ca2+ release induced by caffeine (7.5 mM). The differential rise during histamine stimulation was abolished by the presence of the intercalating substance ethidium bromide. Thapsigargin, a presumed specific inhibitor of the endoplasmic reticulum Ca(2+)-Mg(2+)-adenosine-triphosphatase, abolished the Ca2+ gradient between nucleus and cytosol at rest. During subsequent histamine stimulation the Ca2+ increase was largely blocked in both compartments and attained similar levels. We propose that the lower value of [Ca2+]n at rest is dependent on an active Ca2+ extrusion system. The differential rise of [Ca2+]n over [Ca2+]c during agonist stimulation can be explained by an influx of Ca2+ from perinuclear stores and/or by a release of intranuclear Ca2+ possibly mediated by a process dependent on the inositol lipid metabolism.

Changes in cyclic strain increase inositol trisphosphate and diacylglycerol in endothelial cells.

The purpose of this study was to assess the effect of cyclic strain on phosphatidylinositol turnover in cultured bovine aortic endothelial cells (EC). Confluent EC grown on flexible membrane bottoms were deformed by vacuum to 24% maximum strain and subjected to two cyclic strain regimens. In the first set of experiments, EC were subjected to deformation at a frequency of 60 cycles/min for either 0 (stationary control), 1, 5, 10, 25, 50, or 100 cycles of stretch. In the second set of experiments, EC were preconditioned by deforming the membranes at 60 cycles/min for 24 h. The cycling frequency was then acutely increased to 100 cycles/min for 0, 1, 5, 10, 25, or 100 cycles. Inositol phosphate and diacylglycerol concentrations were determined at the end of each regimen. The results demonstrate that either the initiation of pulsatile stretch or an acute change in cyclic stretch frequency stimulates a sequential and transient generation of inositol trisphosphate, its metabolites inositol biphosphate and monophosphate, and diacylglycerol. Thus EC may respond to the initiation and change in cyclic stretch frequency by a signal transduction pathway involving inositol lipid metabolism.

Mitogenic signal transduction: A common target for oncogenes that induce resistance to ionizing radiations

We hypothesized that resistance to ionizing radiations accompanying neoplastic transformation caused by some oncogenes was due to common biochemical pathways affecting the mechanism of mitogenic signal transduction. In order to verify this hyphotesis, we studied the formation of mitogenic second messengers in cells transformed by oncogenes that induce radioresistance. We observed an increase of diacylglycerol which activates protein kinase C, an increase of phosphatidylcholine metabolism, with a concomitant decrease of inositol lipid metabolism. Our data show that sensitivity to ionizing radiations was inversely related to the intracellular level of diacylglycerol; study of signalling alterations in spontaneous tumors could provide predictive indications about the responsiveness of neoplasia to radiation therapy.

Intragonadal signalling mechanisms in the control of steroid hormone production

In the gonads, there are two recognized signal transduction mechanisms which operate in the processing of hormonal stimuli. The gonadotropins, follicle stimulating hormone and luteinizing hormone, act primarily through the generation of cyclic AMP. Several other hormonal regulators in the ovary and the testis, such as gonadotropin releasing hormone and prostaglandin F 2α stimulate inositol lipid metabolism following receptor binding. This triggers a cascading mechanism which ultimately results in the generation of increased cytosolic free calcium levels, enhanced protein kinase C activity, and liberation of arachidonic acid. There is also evidence that luteinizing hormone shares in the activation of this pathway. In this review, the significance of these signal transduction pathways is discussed in relation to the effects of various hormones on steroid biosynthesis in the gonads.