Effect of lipid composition changes on carbocyanine dye fluorescent response

Egg yolk phosphatidyl choline liposomes containing variable amounts of phosphatidyl ethanolamine, phosphatidyl inositol or phosphatidyl serine demonstrated important variations in the fluorescence of 3.3' dipropylthiodicarbocyanine. When the membrane contained no cholesterol, fluorescence was not correlated with membrane fluidity as measured by diphenyl hexatriene polarization. Increasing cholesterol concentration in valinomycin containing liposome membranes decreased the potassium induced apparent membrane potential and prevented sorption of dye to the membrane. Discontinuity in the apparent potential occurred at 30 mol% cholesterol but could not be correlated with changes in microviscosity. These results indicate that great care should be taken when correlating rapid variations of fluorescence to changes in membrane potential. We propose that changes in phospholipid metabolism could well explain fluorescent changes when monitoring the fluorescence of cyanine dye molecules sorbed to biological membranes.     

Phospholipidic second messengers and calcium

A number of signal molecules bind to surface receptors of target cells and generate intracellular messengers from inositol-containing phospholipids. The phosphatidyl inositol (4, 5) bisphosphate is hydrolyzed into inositol (1, 4, 5) trisphosphate and diacylglycerol. These messengers, via changes in the concentrations of cytosolic Ca2+ and H+ and/or protein phosphorylations, couple the signal to a variety of responses including activation of metabolism, secretion, aggregation, phototransduction, cell proliferation and possibly contraction.     

Phospholipid metabolism and protein kinase C mediated protein phosphorylation in dietary protein deficiency in rat lung

Nutritional deprivation of proteins decreases the protein kinase C (PKC) activity in rat lung. The activity of (PKC) is influenced by lipid metabolism. Changes in PKC activity may influence phosphorylation of its substrate proteins in the tissues. Therefore, alterations in phospholipid metabolism and PKC mediated protein phosphorylation in dietary protein deficiency in rat lung were envisaged. The study was conducted on rats fed on three different types of diet viz., casein (20% protein), deficient (4% protein, rice flour as source of protein) and supplemented (deficient diet supplemented with L-lysine and DL-threoning). Feeding of protein deficient diet caused reduction in incorporation of [3H] myo-inositol in the total phosphoinositides in lungs and an increase in total inositol phosphate pool. There was a significant reduction in the contents and turnover rate of phosphatidyl inositol and phosphatidyl inositol monophosphate. Supplementation of diet with L-lysine and DL-threonine had a reversing effect on total pool of phosphoinositides and, the metabolism of phosphatidyl inositol bisphosphate and phosphatidyl inositol. In phosphatidyl choline metabolism, the dietary protein deficiency led to a decrease in incorporation of [14C-methyl] choline-chloride in total phospholipids. In contrast, its incorporation increased in phosphatidyl choline pool. The contents of phosphatidyl choline and residue, incorporation of [14C-methyl] choline-chloride in them and their turnover rate also increased. Supplementation of diet had a reversal effect on most of these parameters. Phosphorylation of proteins of 84, 47, 35 and 16 kDa was identified to be mediated by PKC. In dietary protein deficiency, phosphorylation of all these proteins, except that of 47 kDa, increased. Supplementation of diet reversed the pattern except that of 84 kDa. The findings suggest that changes in phospholipid metabolism in dietary protein deficiency may effect the activity of PKC thereby influencing the phosphorylation of its substrate proteins and hence associated functions that may lead to pathophysiology of lung.     

Metabotropic receptor activation,desensitization and sequestration-II: modelling the dynamics of the pleckstrin homology domain

Recent observations have been made regarding the generation of inositol 1,4,5-trisphosphate (IP(3)), using chimeras of green fluorescent protein and the pleckstrin homology domain of phospholipase C-delta. In this paper a model is presented giving the quantitative relations between the green fluorescent protein-pleckstrin homology domain (GFP-PHD) construct and membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)) levels as well as the concentration of IP(3), the product of hydrolysis of PIP(2). The model can correctly reproduce the dependence of cytosolic GFP-PHD fluorescence on IP(3) concentration. This model extends a previous one (Metabotropic receptor activation, desensitization and sequestration-I: modelling calcium and inositol 1,4,5-trisphosphate dynamics following receptor activation, in this issue) dealing with the processes governing the production of IP(3) and the subsequent calcium (Ca2+) changes in cells following activation of metabotropic receptors. This model is applied to the case of purinergic P(2)Y(2) receptor activation in Madin-Darby Canine Kidney (MDCK) cells with adenosine triphosphate (ATP) (Science 284 (1999) 1527). It is shown that it can correctly reproduce the dependence of GFP-PHD fluorescence on the concentration of P(2)Y(2) receptor ligand, as well as the temporal changes of GFP-PHD fluorescence following application of ligand.     

Effect of feeding protein deficient diet on phospholipid turnover and protein kinase C mediated protein phosphorylation in rat brain

Feeding of protein deficient diet is known to alter the transmembrane signalling in brain of rat by reducing total protein kinase C (PKC) activity. Phospholipid metabolism regulates the activation of PKC through generation of second messengers and the extent of PKC activation accordingly influences the magnitude of phosphorylation of its endogenous substrate proteins. Thus it was speculated that ingestion of protein deficient diet may modify the turnover rate of membrane phospholipids and magnitude of phosphorylation of endogenous substrate proteins of PKC. The experiments were conducted on rats fed on three different types of laboratory prepared diets viz. casein (20% casein), deficient (4% protein, rice flour as source of protein) and supplemented (deficient diet supplemented with L-lysine and DL-threonine) for 28 days. The metabolism of phosphoinositides (PIs) and phosphatidyl choline (PC) was studied by equilibrium labeling with [3H] myo inositol and [14C methyl] choline chloride respectively. The phosphorylation of endogenous substrate proteins of PKC was studied by using 32P-gamma-ATP followed by SDS-PAGE and autoradiography. The results suggest that in deficient group, there is an increased incorporation of [3H] myo inositol in PIs and inositol phosphate pool in comparison to the casein group. The phosphatidyl inositol (PI) turnover reduced, although there was a marginal increase in the phosphatidyl inositol monophosphate (PIP) and phosphatidyl inositol bis phosphate (PIP2). Supplementation of diet showed a reversal of the pattern towards control to a considerable extent. In the deficient group, PC metabolism showed an increased incorporation of [14C methyl] choline in choline phospholipids but decreased incorporation in phosphoryl choline in comparison with the casein group. The increase in total PC contents was significant but marginal in residue contents. The turnover rate of PC increased only marginally and that of residue declined. Supplementation of diet reduced the total contents of PC and residue, but the turnover rate of PC and residue remained still higher. Phosphorylation of endogenous proteins showed four different proteins of 78, 46, 33 and 16 kDa to be the substrates of PKC in casein group. In deficient group, phosphorylation of these proteins increased markedly while supplementation of diet had a reversing effect rendering the values to be intermediate between casein and the supplemented group. The changes in phospholipid metabolism and in phosphorylation of endogenous substrate proteins of PKC suggest that dietary protein deficiency causes alterations in transmembrane signalling mechanism in rat brain. These effects are partially reversed by improving the quality of proteins in the diet.     

Thromboxane-induced phosphatidate formation in human platelets. Relationship to receptor occupancy and to changes in cytosolic free calcium.

The inter-relationships between receptor occupancy, inositol phospholipid metabolism and elevation of cytosolic free Ca2+ in thromboxane A2-induced human platelet activation were investigated by using the stable thromboxane A2 mimetic, 9,11-epoxymethanoprostaglandin H2, and the thromboxane A2 receptor antagonist, EPO45. 9,11-Epoxymethanoprostaglandin H2 stimulated platelet phosphatidylinositol metabolism as indicated by the rapid accumulation of [32P]phosphatidate and later accumulation of [32P]phosphatidylinositol in platelets pre-labelled with [32P]Pi. These effects of 9,11-epoxymethanoprostaglandin H2 were concentration-dependent and half-maximal [32P]phosphatidate formation occurred at an agonist concentration of 54 +/- 8 nM. With platelets labelled with the fluorescent Ca2+ indicator quin 2, resting cytosolic free Ca2+ was 86 +/- 12 nM. 9,11-Epoxymethanoprostaglandin H2 induced a rapid, concentration-dependent elevation of cytosolic free Ca2+ to a maximum of 300-700 nM. Half-maximal stimulation was observed at an agonist concentration of 80 +/- 23 nM. The thromboxane A2 receptor antagonist EPO45 selectively inhibited 9,11-epoxymethanoprostaglandin H2-induced [32P]phosphatidate formation and elevation of cytosolic free Ca2+, indicating that both events are sequelae of receptor occupancy. Human platelets contain a single class of stereospecific, saturable, high affinity (KD = 70 +/- 13 nM) binding sites for 9,11-epoxymethano[3H]prostaglandin H2. The concentration-response curve for receptor occupancy (9,11-epoxymethano-[3H]prostaglandin H2 binding) is similar to that for 9,11-epoxymethanoprostaglandin H2-induced [32P]phosphatidate formation and for elevation of cytosolic free Ca2+. These observations indicate that human platelet thromboxane A2 receptor occupation is closely linked to inositol phospholipid metabolism and to elevation of cytosolic free Ca2+. Both such events may be necessary for thromboxane A2-induced human platelet activation.     

Bradykinin-stimulated release of arachidonate from phosphatidyl inositol in mouse fibrosarcoma cells

We recently proposed a new pathway by which arachidonate is released from platelet phosphatidyl inositol after stimulation by either thrombin or calcium ionophore A23187. The initial step in arachidonate liberation involves hydrolysis of phosphatidyl inositol to form 1,2-diacylglycerol which is subsequently hydrolyzed by a diacylglycerol lipase to liberate arachidonate for the prostaglandin and lipoxygenase pathways. Whether this pathway is unique to platelets or accounts for arachidonate release from other tissues has not been previously studied. Thus we have now investigated arachidonate metabolism in mouse fibrosarcoma cells (HSDM₁C₁) grown in culture. These cells contain approximately 7.6% of their total phospholipid as phosphatidyl inositol in the resting state (range 6.5–8.3%). When bradykinin (12 μM) is added to the fibrosarcoma cells, there is a rapid depletion of membrane phosphatidyl inositol reaching 62 ± 8% S.D. of baseline values by 15 seconds, falling to 36 ± 6% by 15 minutes. The drop in membrane phosphatidyl inositol is accompanied by release of arachidonate and PGE₂ into the culture medium. The time course of phosphatidyl inositol breakdown and PGE₂ formation supports the idea that phosphatidyl inositol breakdown provides the arachidonate for prostaglandin synthesis in mouse fibrosarcoma cells. Crude extracts of HSDM₁C₁ cells contained sufficient phosphatidyl inositol-specific phospholipase C activity and diacylglycerol lipase activity to account for arachidonate release in these cells.     

Thrombin's effects on osteoblastic cells. I. Cytosolic calcium and phosphoinositides

Thrombin, a blood coagulation factor, has been shown to be a very effective in vitro bone resorbing agent whose mechanism of action on osteoblastic cells remains to be elucidated. In the present study, the effects of highly purified human thrombin on Saos-2 and G292 cells, two human osteoblast-like osteosarcoma cell lines, were investigated. Thrombin (0.6-16 U/ml) caused a significant, dose-dependent increase in osteoblastic cell proliferation. Thrombin also elicited a dose-dependent increase in cytosolic calcium concentration in both Saos-2 and G292 cells (maximal increases were 38% and 200% over baseline, respectively). Addition of thrombin to the osteoblast-like cells resulted in significant time- and dose-dependent changes in phosphoinositide levels: the percentage of inositol monophosphate levels were decreased, whereas the percentage of inositol bisphosphate, inositol trisphosphate and inositol tetrakisphosphate levels were increased. The relative magnitude of the changes in phosphoinositide levels was similar to the changes in cytosolic calcium concentration. These results suggest that thrombin's mechanism of action on bone cells may involve increases in cytosolic calcium levels and in phosphoinositide metabolism.     

Phospholipid composition of oligodendroglial cells during normal development and in 18 day old hyperthyroid and malnourished rats.

The phospholipid composition of isolated oligodendroglial cell perikarya was studied in normal rats during development and in 18 day old malnourished and hyperthyroid rats. Phosphatidyl choline and phosphatidyl ethanolamine were found to be the major phospholipid constituents of oligodendroglial cells. Phospholipid content increased during development, mainly due to an increase of the above mentioned phospholipids. The major changes were observed in sphingomyelin, phosphatidyl serine, phosphatidyl inositol and phosphatidyl ethanolamine between 18 and 30 days of age. The phospholipid and protein content per cell was significantly decreased in the oligodendroglial cells isolated from malnourished rats as compared to controls. When data were expressed as a function of total proteins, the composition was similar to that of normal animals. In the hyperthyroid rats on the other hand, there were no changes in the amount of phospholipids per cell, while phospholipids per milligram of total oligodendroglial cell protein were markedly decreased. The changes in myelin composition produced by hyperthyroidism that we have previously described, do not follow closely those produced by this experimental condition in oligodendroglial cells, suggesting that the metabolism of myelin might be to a certain extent, independent of that in the parent cell.     

Proposal for a pathway to mediate the metabolic effects of insulin

This review seeks to assemble recent discoveries about insulin receptor/kinase, guanine nucleotide-binding proteins, phosphatidyl inositol metabolism, and protein phosphatases to provide a mechanistic pathway by which insulin would alter carbohydrate and fat metabolism. It proposes a hypothetical chain of events that leads from the insulin receptor to protein phosphatase-1. The sequence starts with insulin binding to its receptor, activating the intrinsic receptor/kinase activity. The insulin receptor phosphorylates a guanine nucleotide-binding protein, which activates a particular phospholipase C. This in turn stimulates the production of two lipid-derived messengers: inositol-phospho-glucosamine and diacylglycerol. These messengers trigger the effects of insulin. The diacylglycerol produced by insulin is thought to be analogous to the diacylglycerol produced by alpha-adrenergic stimulation, which activates protein kinase C. Activation of this kinase could account for increases in phosphorylation of certain proteins. The inositol-phospho-glucosamine is the cytosolic messenger for insulin. One of the enzymes activated by insulin is protein phosphatase type-1. It is known that the phosphatase decreases phosphorylation of certain target enzymes. In response to insulin, activation of protein phosphatase type-1 occurs with a stable conformational change that may involve rearrangement of disulfide bonds. Rearrangement is either directly in response to the cytosolic messenger or is catalyzed by an isomerase activated by the insulin messenger. Ultimately, protein phosphatase type-1 and/or the disulfide isomerase may together mediate the pleiotropic effects of insulin on carbohydrate and fat metabolism.     

Effects of tumor necrosis factor α (TNFα) on the phospholipid metabolism of Tetrahymena pyriformis

The effect of (0·05 ng ml⁻¹ and 0·1 ng ml⁻¹) TNFα on the phospholipid metabolism of Tetrahymena pyriformis was studied. The amount of phosphatidyl choline (PC), phosphatidyl inositol (PI), phosphatidic acid (PA), phosphatidyl ethanolamine (PE), diacylglycerol (DAG), arachidonic acid (AA) and ceramide was higher, but the phosphatidyl inositol 4 phosphate (PIP) and phosphatidyl inositol bis-phosphate (PIP₂) as well, as sphingomyelin (SM) content was lower in TNFα-treated cells than in the controls. In the culture medium (secreted forms) this situation was reversed. There were differences in the results gained by incorporation of [³H]-palmitic acid or ³²P into the phospholipids. To control the functional effects of TNFα in Tetrahymena, the rate of cell division, the condensation of chromatin, the viability of cells and morphometrical values have been studied. The cytokine reduced cell growth, altered morphometric indices and increased chromatin condensation, however cell viability was not influenced. The results demonstrate the effects of TNFα at a low level of evolution, what is realized by changes in the phospolipid metabolism participating in signalling pathways. © 1998 John Wiley & Sons, Ltd.     

Maitotoxin-Induced Intracellular Calcium Rise in PC 12 Cells: Involvement of Dihydropyridine-Sensitive and ω-Conotoxin-Sensitive Calcium Channels and Phosphoinositide Breakdown

The biological activities of maitotoxin are strictly dependent on the extracellular calcium concentration and are always associated with an increase of the free cytosolic calcium level. We tested the effects of voltage-sensitive calcium channel blockers (nicardipine and omega-conotoxin) on maitotoxin-induced intracellular calcium increase, membrane depolarization, and inositol phosphate production in PC12 cells. Maitotoxin dose dependently increased the cytosolic calcium level, as measured by the fluorescent probe fura 2. This effect disappeared in a calcium-free medium; it was still observed in the absence of extracellular sodium and was enhanced by the dihydropyridine calcium agonist Bay K 8644. Nicardipine inhibited the effect of maitotoxin on intracellular calcium concentration in a dose-dependent manner. The maitotoxin-induced calcium rise was also reduced by pretreating cells with omega-conotoxin. Pretreatment of cells with maitotoxin did not modify 125I-omega-conotoxin and [3H]PN 200-110 binding to PC12 membranes. Nicardipine and omega-conotoxin inhibition of maitotoxin-evoked calcium increase was reduced by pertussis toxin pretreatment. Maitotoxin caused a substantial membrane depolarization of PC12 cells as assessed by the fluorescent dye bisoxonol. This effect was reduced by pretreating the cells with either nicardipine or omega-conotoxin and was almost completely abolished by the simultaneous pretreatment with both calcium antagonists. Maitotoxin stimulated inositol phosphate production in a dose-dependent manner. This effect was reduced by pretreating the cells with 1 microM nicardipine and was completely abolished in a calcium-free EGTA-containing medium. The findings on maitotoxin-induced cytosolic calcium rise and membrane depolarization suggest that maitotoxin exerts its action primarily through the activation of voltage-sensitive calcium channels, the increase of inositol phosphate production likely being an effect dependent on calcium influx. The ability of nicardipine and omega-conotoxin to inhibit the effect of maitotoxin on both calcium homeostasis and membrane potential suggests that L- and N-type calcium channel activation is responsible for the influx of calcium following exposure to maitotoxin, and not that a depolarization of unknown nature causes the opening of calcium channels.     

Altered signal transduction secondary to surface IgM cross-linking on B-chronic lymphocytic leukemia cells. Differential activation of the phosphatidylinositol-specific phospholipase C

To further study the mechanisms by which surface Ig triggering activates the inositol phospholipid signaling pathway, we have used B cells from chronic lymphocytic leukemia patients which, as previously described, display two patterns of response upon sIg cross-linking: in one group this cross-linking induces an inositol phosphate release, an intracellular free Ca2+ concentration elevation and a subsequent cell proliferation; in a second group none of these events occur although there is an increased class II Ag expression following anti-mu stimulation as in the first group. We have been able to demonstrate that the phosphatidyl inositol specific phospholipase C (PI-PLC) can be activated in permeabilized B cells from the first group by direct stimulation, with GPT gamma S, of a guanine nucleotide binding (G) protein. In addition, since anti-mu + GTP gamma S stimulate an increased inositol phosphate production in these cells, this suggests that surface Ig cross-linking activates PI-PLC via a G protein. However, in cells from the second group no inositol phosphate is released after GTP gamma S stimulation although PI-PLC can be directly activated by high Ca2+ concentrations. This reflects in these cells, an interruption of the signaling cascade sIg/G protein/PI-PLC at the level of the G protein or at the G protein/PI-PLC coupling. In cells from both groups PMA treatment, which is known to alter phosphatidyl inositol metabolism in B cells, completely inhibits PI-PLC activation even by high Ca2+ concentrations. These studies show that the phosphatidyl inositol-dependent signaling cascade after surface Ig triggering can be altered at different levels in B cells.     

Signal transduction in normal and pathological thrombin-stimulated human platelets

Human blood platelets stimulated by thrombin undergo very rapid morphological changes, the most characteristic of which are pseudopod formation and granule centralization. These early changes in shape are accompanied by a transient decrease (30%) in phosphatidyl inositol 4,5-bisphosphate (PIP2) which occurs in the first 10 s after thrombin addition. Transient decreases in phosphatidyl inositol 4-phosphate (PIP) and phosphatidyl inositol (PI) occur later (20-30 s). These events lead to the formation of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DG) and hence phosphatidate (PA). Two polypeptides are phosphorylated during the same time span: the myosin light chain (P20) and a 43 kDa protein (P43). Concomitant with these molecular changes, platelet 'release reaction' occurs, i.e., liberation of the different granule constituents into the external medium: the earliest concerns dense bodies which liberate adenine nucleotides, calcium and serotonin; alpha-granules then liberate adhesive and specific proteins and are followed by lysosomes which liberate hydrolases. Pathological platelets from patients with inherited disorders, presenting well-characterized and specific defects of either the platelet membrane (GT) or storage granules (GPS and HPS), have also been studied. The results obtained lead to the following conclusions: (1) the transducing system is normal in platelets unable to aggregate; (2) phosphorylation of P20 and P43 proteins can be complete with impaired release; and (3) when platelets lack alpha-granules the transducing system as well as the release of other granule populations are impaired. These results evidence the relationship between the absence of intraplatelet components and metabolic events.     

Receptor- and calcium-dependent induced inositol 1,4,5-trisphosphate increases in PC12h cells as shown by fluorescence resonance energy transfer imaging

The production and further metabolism of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] require several calcium-dependent enzymes, but little is known about subsequent calcium-dependent changes in cellular Ins(1,4,5)P3. To study the calcium dependence of muscarinic acetylcholine receptor-induced Ins(1,4,5)P3 increases in PC12h cells, we utilized an Ins(1,4,5)P3 imaging system based on fluorescence resonance energy transfer and using green fluorescent protein variants fused with the pleckstrin homology domain of phospholipase C-delta1. The intracellular calcium concentration, monitored by calcium imaging, was adjusted by thapsigargin pretreatment or alterations in extracellular calcium concentration, enabling rapid receptor-independent changes in calcium concentration via store-operated calcium influx. We found that Ins(1,4,5)P3 production was increased by a combination of receptor- and calcium-dependent components, rather than by calcium alone. The level of Ins(1,4,5)P3 induced by the receptor was found to be half that induced by the combined receptor and calcium components. Increases in calcium levels prior to receptor activation did not affect the subsequent receptor-induced Ins(1,4,5)P3 increase, indicating that calcium does not influence Ins(1,4,5)P3 production without receptor activation. Removal of both the receptor agonists and calcium rapidly restored calcium and Ins(1,4,5)P3 levels, whereas removal of calcium alone restored calcium to its basal concentration. Similar calcium-dependent increases in Ins(1,4,5)P3 were also observed in Chinese hamster ovary cells expressing m1 muscarinic acetylcholine receptor, indicating that the observed calcium dependence is common to Ins(1,4,5)P3 production. To our knowledge, our results are the first showing receptor- and calcium-dependent components within cellular Ins(1,4,5)P3.     

Differential mitochondrial calcium responses in different cell types detected with a mitochondrial calcium fluorescent indicator, mito-GCaMP2

Mitochondrial calcium plays a crucial role in mitochondrial metabolism, cell calcium handling, and cell death. However, some mechanisms concerning mitochondrial calcium regulation are still unknown, especially how mitochondrial calcium couples with cytosolic calcium. In this work, we constructed a novel mitochondrial calcium fluorescent indicator (mito-GCaMP2) by genetic manipulation. Mito-GCaMP2 was imported into mitochondria with high efficiency and the fluorescent signals co-localized with that of tetramethyl rhodamine methyl ester, a mitochondrial membrane potential indicator. The mitochondrial inhibitors specifically decreased the signals of mito-GCaMP2. The apparent K(d) of mito-GCaMP2 was 195.0 nmol/L at pH 8.0 in adult rat cardiomyocytes. Furthermore, we observed that mito-GCaMP2 preferred the alkaline pH surrounding of mitochondria. In HeLa cells, we found that mitochondrial calcium ([Ca(2+)](mito)) responded to the changes of cytosolic calcium ([Ca(2+)](cyto)) induced by histamine or thapasigargin. Moreover, external Ca(2+) (100 μmol/L) directly induced an increase of [Ca(2+)](mito) in permeabilized HeLa cells. However, in rat cardiomyocytes [Ca(2+)](mito) did not respond to cytosolic calcium transients stimulated by electric pacing or caffeine. In permeabilized cardiomyocytes, 600 nmol/L free Ca(2+) repeatedly increased the fluorescent signals of mito-GCaMP2, which excluded the possibility that mito-GCaMP2 lost its function in cardiomyocytes mitochondria. These results showed that the response of mitochondrial calcium is diverse in different cell lineages and suggested that mitochondria in cardiomyocytes may have a special defense mechanism to control calcium flux.     

Inositol 1,4,5-trisphosphate 3-kinase A associates with F-actin and dendritic spines via its N terminus

The consequences of the rapid 3-phosphorylation of inositol 1,4,5-trisphosphate (IP(3)) to produce inositol 1,3,4,5-tetrakisphosphate (IP(4)) via the action of IP(3) 3-kinases involve the control of calcium signals. Using green fluorescent protein constructs of full-length and truncated IP(3) 3-kinase isoform A expressed in HeLa cells, COS-7 cells, and primary neuronal cultures, we have defined a novel N-terminal 66-amino acid F-actin-binding region that localizes the kinase to dendritic spines. The region is necessary and sufficient for binding F-actin and consists of a proline-rich stretch followed by a predicted alpha-helix. We also localized endogenous IP(3) 3-kinase A to the dendritic spines of pyramidal neurons in primary hippocampal cultures, where it is co-localized postsynaptically with calcium/calmodulin-dependent protein kinase II. Our experiments suggest a link between inositol phosphate metabolism, calcium signaling, and the actin cytoskeleton in dendritic spines. The phosphorylation of IP(3) in dendritic spines to produce IP(4) is likely to be important for modulating the compartmentalization of calcium at synapses.     

Cytosolic calcium, calcium fluxes, and regulation of alveolar macrophage superoxide anion production

The recently available compound quin-2, which acts as a high affinity fluorescent indicator for calcium in the cytosol, was used to examine the role of calcium mobilization in the alveolar macrophage during the stimulation of 0-2 production by the tripeptide N-formyl norleucyl leucyl phenylalanine (FNLLP). After preloading with quin-2, the production of 0-2 was measured in conjunction with the transfer of 45Ca+2 and changes in quin-2 fluorescence upon stimulation with FNLLP. When cells were maintained in low (10 microM) extracellular calcium medium the presence of 1.5 mM quin-2 in the cytosolic space partially inhibited the rate of 0-2 production upon stimulation by FNLLP. Addition of 1 mM Ca+2 to the medium prior to stimulation rapidly restored the cell's capability to produce 0-2 upon stimulation at rates equal to control and extended the duration of stimulated 0-2 production as well. Quin-2 fluorescence measurements indicated an increase in cytosolic Ca+2 upon stimulation with FNLLP. This increase was lowest under conditions in which 0-2 production was inhibited. The addition of 1 mM Ca+2 to the medium caused by itself a rapid but transient increase in cytosolic Ca+2 as measured with quin-2 without stimulating 0-2 production. This intracellularly redistributed calcium was determined to be the source of the greater increase in cytosolic calcium during stimulation in the presence of high extracellular calcium. Measurements of 45Ca+2 transfer demonstrated a buffering of cytosolic Ca+2 changes by quin-2, which in low calcium medium could deplete calcium stores. It is suggested that this effect, prior to stimulation, was responsible for the mitigated 0-2 response for those cells maintained in low calcium medium, wherein calcium stores could not be replenished. These results suggested that the cell's mechanism for regulating cytosolic and bound calcium concentrations may also play an integral role in its normal mechanism for stimulated 0-2 production. They further support the postulate that the commonly observed rise in the concentration of calcium in the cytosol upon formyl peptide stimulation is a concomitant but nonregulatory event only.     

Subsecond and second changes in inositol polyphosphates in GH4C1 cells induced by thyrotropin-releasing hormone.

It has been demonstrated previously that thyrotropin-releasing hormone (TRH) induces changes in inositol polyphosphates in the GH3 and GH4C1 strains of rat pituitary cells within 2.5-5.0 s. TRH also causes a rapid rise in cytosolic free calcium concentration ([Ca2+]i) in these cells which is due largely to redistribution of cellular calcium stores. Therefore, it has been concluded that TRH acts to release sequestered calcium in these cells via enhanced generation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]. If this conclusion were correct, TRH-enhanced accumulation of Ins(1,4,5)P3 should occur at least as rapidly as the increase in [Ca2+]i. We have shown previously that the rise in [Ca2+]i induced by TRH occurs within about 400 ms; thus, it was important to investigate the subsecond time-course of changes in inositol phosphates caused by TRH. Using a rapid mixing device, we have measured changes in inositol polyphosphates on a subsecond time scale in GH4C1 cells prelabelled with myo-[2-3H]inositol. Although TRH did alter inositol polyphosphate metabolism within 500 ms, the changes observed did not reveal a statistically significant increase in Ins(1,4,5)P3 within time intervals of less than 1000 ms. Thus, we have been unable to demonstrate that a TRH-induced rise in Ins(1,4,5)P3 precedes or occurs concomitantly with the rise in [Ca2+]i in GH4C1 cells. Although these results do not disprove the current view that Ins(1,4,5)P3 mediates the action of TRH on intracellular calcium redistribution, we conclude that caution should be exercised in this, and possibly other cell systems, in accepting the dogma that all of the rapid, agonist-induced redistributions of intracellular calcium are mediated by Ins(1,4,5)P3.