Phosphoinositide signal transduction pathway in rat liver mitochondria

Phosphorylation of endogeneous phosholipids of rat liver mitochondrial fractions with γ[³²P]ATP revealed formation of all the known inositol phospholipids, such as phosphatidylinositol, phosphatidylinositol phosphate and phosphatidylinositol bisphosphate. Additionally, a new inositol phospholipid was detected. Incorporation of [³H]-labelled insositol followed a similar profile. Enzymatic experiments indicated that the new lipid could possibly be phosphatidylinositol trisphosphate. The presence of phosphoinositides-generated second messengers such as diacylglycerol and inositol trisphosphate was also confirmed. Protein kinase C, which acts as mediator between second messengers and nuclear factors, was also found to be present in mitochondria in significant amount. These results suggest that phosphoinositide signal transduction pathway is operative in rat liver mitochondria.     

Lipid second messengers and cell signaling in vascular wall

Agonists of cellular receptors, such as receptor tyrosine kinases, G protein-coupled receptors, cytokine receptors, etc., activate phospholipases (C(gamma), C(beta), A(2), D), sphingomyelinase, and phosphatidylinositol-3-kinase. This produces active lipid metabolites, some of which are second messengers: inositol trisphosphate, diacylglycerides, ceramide, and phosphatidylinositol 3,4,5-trisphosphate. These universal mechanisms are involved in signal transduction to maintain blood vessel functions: regulation of vasodilation and vasoconstriction, mechanical stress resistance, and anticoagulant properties of the vessel lumen surface. Different signaling pathways realized through lipid second messengers interact to one another and modulate intracellular events. In early stages of atherogenesis, namely, accumulation of low density lipoproteins in the vascular wall, cascades of pro-atherogenic signal transduction are triggered through lipid second messengers. This leads to atherosclerosis, the general immuno-inflammatory disease of the vascular system.     

Mitogenic signal transduction in normal and transformed 32D hematopoietic cells

We studied mitogenic signal transduction in normal and oncogene-transformed 32D cells, a murine hematopoietic cell line that is normally dependent on interleukin-3 (IL3) for proliferation and survival. The formation of second messengers was measured in normal cells stimulated with IL3, and in cells transfected with foreign growth factor receptor genes and stimulated with appropriate growth factors. We also measured the steady-state level of second messengers in 32D cells transformed by erbB, abl, and src oncogenes which abrogate growth factor requirement. We found that IL3 stimulated the formation of diacylglycerol independently of inositol lipid turnover, but concomitantly with increased turnover of phosphatidylcholine. Epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) stimulated the 'classical' turnover of inositol lipids with formation of diacylglycerol and calcium-mobilizing inositol phosphates. Colony stimulating factor-1 triggered inositol lipid turnover, although to a much lower extent than EGF and PDGF. Transformed cells showed elevated levels of diacylglycerol together with increased turnover of phosphoinositides and phosphatidylcholine. Taken together these results indicate that different growth factors and oncoproteins associate with multiple signalling pathways in 32D cells.     

Alpha 1 adrenergic receptor function in senescent Fischer 344 rat aorta

There have been numerous conflicting reports concerning alpha 1 adrenergic receptor-mediated blood vessel contraction during aging and possible changes in alpha 1 receptor transduction mechanisms have not been investigated. These studies assess capacity of the aging vascular alpha 1 receptor to stimulate production of inositol phosphates, which are its intracellular second messengers, and to elicit a contractile response via this pathway. Aortic ring segments from mature adult (6 month old) and senescent (24 month old) Fischer 344 rats were incubated with [3H]myo-inositol and then stimulated with the alpha 1 agonist norepinephrine (NE, 10(-7)M-3 x 10(-5)M) in the presence of LiCl (10mM), an inhibitor of inositol phosphate metabolism. There was a substantial increase in inositol phosphate accumulation throughout the dose range in aortic rings from 24 month old rats compared to 6 month old rats. This is an alpha 1 receptor response since it is blocked by the alpha 1 antagonist prazosin but not by the alpha 2 antagonist yohimbine. Aortic inositol phosphate accumulation in response to serotonin did not change with age. To assess second messenger stimulated contraction, aortic ring segments were placed in Ca++ free buffer and then stimulated with NE. Under these conditions Ca++ influx is eliminated and contraction depends on the actions of intracellular second messengers. There is an age-related reduction in aortic contraction in Ca++ free buffer. These results suggest that aortic alpha 1 receptor-mediated formation of inositol phosphate intracellular second messengers is enhanced during aging. Despite this, the capacity of senescent arteries to elicit contraction utilizing second messenger pathways seems to be deficient.     

Chronic Electroconvulsive Seizures Down–Regulate Expression of the Immediate-Early Genes c-fos and c-jun in Rat Cerebral Cortex

Acute seizures and other stimuli that increase neuronal activity cause a rapid induction of the immediate-early genes c-fos and c-jun, also referred to as nuclear proto-oncogenes, in the nervous system. In the present study, rats were administered one or more electroconvulsive seizures (ECS) and the responsiveness of c-fos and c-jun to an acute, "test" seizure was examined. Four hours after a single ECS, the induction of c-fos mRNA by a test seizure was blocked, in agreement with earlier findings, but by 18 h the levels of c-fos mRNA could be reinduced by the test seizure, suggesting that 1 day is sufficient to "reset" the responsiveness of this system. However, it was found that chronic, daily ECS treatments resulted in a time-dependent decrease in the expression of c-fos mRNA in response to a test seizure administered 18 h after the last daily ECS; this effect was maximal after 8-10 days of treatment, at which time the induction of c-fos mRNA by the test seizure was blocked dramatically. Chronic ECS also blocked the induction of c-jun in response to an acute, test seizure. The effect of chronic ECS on levels of Fos protein was also investigated. It was found that basal levels of Fos protein were reduced after chronic (10 days) ECS and were not induced by a test seizure. Because levels of Fos protein remain elevated 4 h after a single seizure this finding suggests that the mechanisms by which acute (4 h) and chronic (8-10 days) ECS block the induction of c-fos may differ.     

Role of the plasma membrane in signal transduction in human polymorphonuclear leukocytes

To more closely examine the role of the cell surface in transmembrane signal transduction in human neutrophils, sealed right-side-membrane vesicles free of organellar membrane components were used as models of the plasma membrane. These vesicles, incubated with a fluorescent analogue of the chemotactic peptide fMLP, bound this ligand similarly in extent and kinetics to intact neutrophils. Vesicles responded to this stimulation with a slow increase in internal [Ca⁺⁺] which was inhibited by EGTA but not by verapamil; the cytosolic Ca⁺⁺ transient seen in intact cells within 10 sec of stimulation was absent in vesicles. The vesicles also maintained a transmembrane potential (ψ) and were depolarized by the K⁺ ionophore valinomycin. However, unlike intact cells which hyperpolarized and then depolarized in response to fMLP, the vesicles demonstrated only a sustained hyperpolarization. Vesicles also differed from intact cells by not producing superoxide (O₂^?) in response to fMLP. Finally, fMLP caused dramatic alterations in membrane vesicle lipid metabolism: at early time points (within 5–10 sec), there was a transient production of diacylglycerol (DAG) concomitant with inositol lipid breakdown, with no apparent hydrolysis of non-inositol phospholipids. For up to 5 min after stimulation, there was no increase in the levels of phosphatidic acid or of inositol lipids. Thus, a significant portion of the signalling pathway in neutrophils is located at the cell surface or in the plasma membrane and functions independently of intracellular components. Furthermore, the plasma membrane is intimately involved in events occurring during both the early (DAG generation) and late (slow, prolonged rise in [Ca⁺⁺]) phases of cellular response. In contrast, several of the responses to fMLP (the Ca⁺⁺ transient, depolarization, generation of O₂^?, recycling of lipid metabolites) involve signalling machinery not constitutively resident on the neutrophil surface. © 1993 Wiley-Liss, Inc.     

Mammalian phosphoinositide-specific phospholipase C isoenzymes

Procaryotic and eucaryotic cells have evolved multiple pathways for communication with their external environment. The inositol 1,4,5-trisphosphate/diacylglycerol second messenger system is an example of such a signal transduction pathway which is present in multicellular eucaryotic organisms. Binding of an agonist to a specific cell surface receptor promotes rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate. The pivotal enzyme for this second messenger system is phosphoinositide-specific phospholipase C which hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate the two second messengers, inositol 1,4,5-trisphosphate and diacylglycerol. Recently, much progress has been made in the purification, characterization and cDNA cloning of multiple PI-PLC isoenzymes. The results of the recent studies on phosphoinositide-specific phospholipase C are reviewed.     

Langevin equation,Fokker-Planck equation and cell migration

Cell migration can be characterized by two independent variables: the speed,v, and the migration angle, ϕ. Each variable can be described by a stochastic differential equation—a Langevin equation. The migration behaviour of an ensemble of cells can be predicted due to the stochastic processes involved in the signal transduction/response system of each cell. Distribution functions, correlation functions, etc. are determined by using the corresponding Fokker-Planck equation. The model assumptions are verified by experimental results. The theoretical predictions are mainly compared with the galvanotactic response of human granulocytes. The coefficient characterizing the mean effect of the signal transduction/response system of the cell is experimentally determined to 0.08 mm/V sec (galvanotaxis) or 0.7 mm/sec (chemotaxis) and the characteristic time characterizing stochastic effects in the signal transduction/response system is experimentally determined as 30 sec. The temporal directed response induced by electric field pulses is investigated: the experimental cells react slower but are more sensitive than predicted by theory.     

Altered signal transduction in erbB-transformed cells. Implication of enhanced inositol phospholipid metabolism in erbB-induced transformation

To clarify the signal transduction mechanism of the erbB gene (virus oncogene) products leading to cell growth and transformation, the alteration of signal transduction induced by enhanced inositol phospholipid metabolism was studied in chick embryo fibroblast cells (CEF cells) transformed by gag-fused erbB gene-carrying virus (GEV cells). The incorporations of 32P into phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate were markedly increased in GEV cells. In GEV cells, the activities of lipid kinases such as phosphatidylinositol (PI), PIP, and diacylglycerol (DG) kinases were also increased. The activities of other important enzymes involved in inositol phospholipid metabolism, such as CDP-DG:myo-inositol transferase and phospholipase C, were not changed in GEV cells. Increased inositol phospholipid metabolism might lead to the production of second messengers, such as 1,2-DG and inositol 1,4,5-trisphosphate. Indeed, the 1,2-DG content was also increased in GEV cells. Moreover, the activity of protein kinase C (the Ca2+/phospholipid-dependent enzyme), which should be stimulated by 1,2-DG, was elevated in GEV cells; the protein kinase C activity in the membrane fraction of GEV cells was especially high. When CEF cells were treated with tetradecanoylphorbol acetate, protein kinase C activator, plus Ca2+ ionophore, [3H]thymidine incorporation was markedly stimulated, and maximal stimulation was observed with 1 nM Ca2+ ionophore A23187 plus 100 nM TPA. On the other hand, when GEV cells were treated with TPA plus Ca2+ ionophore A23187, [3H]thymidine incorporation was consistently inhibited. Next, studies were made to determine whether the erbB gene product itself had kinase activity on PI, PIP, and DG after membranes were mildly solubilized with Triton X-100 to prevent inactivation of these kinases. Immunoprecipitates of a GEV cell lysate with antisera that reacted with the erbB gene product had PI kinase activity, whereas no activity was detected in those of lysates of uninfected CEF cells. However, the activity was very weak compared with the total cellular activity. No difference in the PIP and DG kinase activities of immunoprecipitates of cell lysates of uninfected CEF cells and GEV cells was observed. These results suggest that the erbB gene product enhances inositol phospholipid metabolism and subsequent signal transduction, but that the erbB gene product is not involved directly in lipid kinases, although it is closely associated with lipid kinase.     

Incorporation of [3H]inositol into phosphoinositides and inositol phosphates by rabbit blastocysts

Preimplantation rabbit embryos collected at the early morula stage were cultured to blastocysts in the presence of [³H]inositol. The blastocysts were lysed, and both the aqueous and lipid portions were analysed for incorporated radioactivity. Thin-layer chromatographic separation of the lipid portion indicated that [³H]inositol was incorporated into phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate. HPLC anion-exchange chromatography indicated that [³H]inositol was incorporated into inositol phosphates, including the two second messengers, inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate, and also inositol monophosphate and inositol 1,4-bisphosphate. These results provide evidence that rabbit blastocysts may have an active phosphatidylinositol second messenger system, which may be responsive to intrauterine factors or intraembryonic paracrine factors. © 1993 Wiley-Liss, Inc.     

Different signal transduction by epidermal growth factor may be responsible for the difference in modulation of amino acid transport between fetal and adult hepatocytes

[1-¹⁴C]-2-aminoisobutyric acid (AIB) uptake and signal transduction pattern after epidermal growth factor (EGF) stimulation were examined in freshly isolated hepatocytes from 20-day-old fetuses and 3-month-old rats. EGF induced a transient increase of AIB transport after 10 min only in adult animals; the observed unresponsiveness of fetal liver is not dependent on a lack of EGF receptors which are present though to a lesser extent on the plasma membrane in this period. As far as the production of the second messengers, inositol trisphosphate (IP₃) and calcium, is concerned, substantial differences were found: EGF increased IP₃ production in adult hepatocytes, whereas it had no effect in fetal ones. Moreover, the addition of EGF induced a calcium transient in hepatocytes from adult animals, while there was no increase in fetal cells. The lack of EGF effect on amino acid transport in fetal cells could be due to its inability to produce both IP₃ and calcium transients, suggesting that this transduction pathway is not activated during fetal life.     

Second messenger signalling in olfaction.

The primary reactions of the chemo-electrical signal transduction pathway in olfactory receptor neurons are mediated by two alternative second messengers, cAMP and inositol 1,4,5-trisphosphate. The rapid and transient intracellular signalling is terminated by the action of negative-feedback loops which uncouple the reaction cascades (desensitization). Recent evidence suggests that secondary reactions in olfaction (adaptation) may also be controlled by second messengers.     

Effect of phosphate on aluminium-inhibited growth and signal transduction pathways in Coffea arabica suspension cells

In acid soils, aluminium (Al) toxicity and phosphate (Pi) deficiency are the most significant constraints on plant growth. Al inhibits cell growth and disrupts signal transduction processes, thus interfering with metabolism of phospholipase C (PLC), an enzyme involved in second messenger production in the cell. Using a Coffea arabica suspension cell model, we demonstrate that cell growth inhibition by Al toxicity is mitigated at a high Pi concentration. Aluminium-induced cell growth inhibition may be due to culture medium Pi deficiency, since Pi forms complexes with Al, reducing Pi availability to cells. Phosphate does not mitigate inhibition of PLC activity by Al toxicity. Other enzymes of the phosphoinositide signal transduction pathway were also evaluated. Aluminium disrupts production of second messengers such as inositol 1,4,5-trisphosphate (IP₃) and phosphatidic acid (PA) by blocking PLC activity; however, phospholipase D (PLD) and diacylglycerol kinase (DGK) activities are stimulated by Al, a response probably aimed at counteracting Al effects on PA formation. Phosphate deprivation also induces PLC and DGK activity. These results suggest that Al-induced cell growth inhibition is not linked to PLC activity inhibition.     

Evidence for the involvement of PI-signaling and diacylglycerol second messengers in the initiation of metamorphosis in the hydroid Hydractinia echinata Fleming

Metamorphosis of the planula larvae into polyps does not occur spontaneously but depends on the reception of external trigger stimuli. Artificially, metamorphosis can be initiated by a pulse-type application of Cs⁺ or tumor-promoting phorbol esters (W. A. Müller (1985) Differentiation 29, 216–222). In the present study we examined the putative involvement of the phosphatidylinositol system in signal transduction. Planulae of Hydractinia echinata were preincubated with [³H]-inositol. Upon exposure of the larvae to Cs⁺ the label in inositol trisphosphate (InsP₃) increased twofold as early as 15 sec after addition of Cs⁺. Within the first 60 sec the levels of inositol monophosphate (InsP₁) and inositol bisphosphate (InsP₂) were also elevated compared to the values in nonstimulated larvae. After 1 and 3 hr, respectively, of incubation with Cs⁺, only the label in InsP₂ was increased. When applied to saponin-permeabilized larvae, InsP₃ did not induce metamorphosis. But 1,2-dioctanoyl-rac-glycerol (diC₈) was effective in inducing metamorphosis with a half-maximal effective concentration of 9 μM. The percentage of metamorphosed animals after the application of 5 μM diC₈ (30 mM Cs⁺) was increased by the simultaneous application of 1 μM (0.1 μM) of the diacylglycerol kinase inhibitor R 59022. The results are interpreted as evidence for the involvement of the PI-signaling/diacylglycerol transduction system in the initiation of metamorphosis of planula larvae of H. echinata.     

Atrial natriuretic factor inhibits angiotensin-induced aldosterone secretion: not through cGMP or interference with phospholipase C

ANF did not prevent the formation of [3H] inositol trisphosphate in response to AII but inhibited aldosterone secretion in calf adrenal glomerulosa cells. 8-bromo cGMP did not affect either inositol phosphate formation or aldosterone secretion. Changes in cytosolic Ca++ concentration induced by AII, as measured by Quin 2 fluorescence, were also unaffected by ANF. No difference in adrenal cell protein phosphorylation with AII or AII + ANF was observed. The results suggest that ANF may inhibit aldosterone secretion through a non-guanyl cyclase linked receptor system not involving the formation of phosphoinositide-derived second messengers. Interference with protein kinase C activity cannot be ruled out.     

Inositol phospholipid turnover in platelets of schizophrenic patients.

Abnormalities in blood cell membrane phospholipid composition and metabolism from schizophrenic patients have been reported by many groups of investigators. Among membrane phospholipids, inositol phospholipids are of special importance as they are involved in transduction system that generates second messengers such as inositol trisphosphate and diacylglycerol. Our studies on platelet inositol phospholipid turnover suggest a significant increase in platelet phosphatidylinositol 4,5-bisphosphate levels, an increased production of inositol trisphosphate in neuroleptic-treated and neuroleptic-free schizophrenic patients platelets and a reduced calcium release by thrombin in neuroleptic-treated schizophrenic patients platelets. The enhanced production of inositol trisphosphate may be due to an increase in its precursor phosphatidylinositol 4,5-bisphosphate with an associated desensitisation of the intracellular inositol trisphosphate receptor by neuroleptics, which may explain the diminished calcium response to thrombin in schizophrenic patients platelets.     

Morituri te salutant? Olfactory signal transduction and the role of phosphoinositides

During the past 150 years, researchers have investigated the cellular, physiological, and molecular mechanisms underlying the sense of smell. Based on these efforts, a conclusive model of olfactory signal transduction in the vertebrate's nose is now available, spanning from G-protein-mediated odorant receptors to ion channels, which are linked by a cyclic adenosine 3',5'-monophosphate-mediated signal transduction cascade. Here we review some historical milestones in the chronology of olfactory research, particularly emphasising the role of cyclic nucleotides and inositol trisphosphate as alternative second messengers in olfactory cells. We will describe the functional anatomy of the nose, outline the cellular composition of the olfactory epithelium, and describe the discovery of the molecular backbone of the olfactory signal transduction cascade. We then summarize our current model, in which cyclic adenosine monophosphate is the sole excitatory second messenger in olfactory sensory neurons. Finally, a possible significance of microvillous olfactory epithelial cells and inositol trisphosphate in olfaction will be discussed.     

Nuclear inositol lipid signaling and its potential involvement in malignant transformation

Growth, differentiation, and apoptosis of eukaryotic cells are mediated by extremely complex signaling pathways and a high degree of coordination is required for regulating cell proliferation.In multicellular organisms homeostasis is achieved through signal transduction events. If these homeostatic mechanisms are interrupted, a disease, such as cancer, may ensue. Lipid second messengers, particularly those derived from polyphosphoinositide cycle, play a pivotal role in several cell signaling networks. Evidence accumulated over the past 15 years has highlighted the presence of an autonomous nuclear inositol lipid metabolism, and suggests that lipid signaling molecules are important components of signaling pathways operating within the nucleus. Recent findings are starting to elucidate how the nuclear phosphoinositide cycle is regulated and what down-stream molecules are targeted through this cycle. In this review, we shall summarize the most updated data about inositol lipid-dependent nuclear signaling pathways that might have a relevance for the development of cancer. In the near future, this knowledge might also prove to have relevance for the diagnosis and treatment of the neoplastic disease.     

Signal transduction in vascular plants

We review current evidence for the presence and activity in plants of several paradigmatic components of transmembrane signal transduction systems. Components considered include the second messengers calcium, inositol 1,4,5-trisphosphate, and cyclic AMP; protein kinases and protein phosphatases; and G-proteins. At the current stage of development of the field of plant signal transduction, broad similarities between plant and the more well-studied animal systems are apparent. However, there also exist considerable differences in detail.