Understanding structure-function relationships in the Aequorea victoria green fluorescent protein.
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Biomedical subjects
A B Cubitt
Publications and source records attributed to A B Cubitt.
Coexpression of a constitutively active plasma membrane calcium pump with GFP identifies roles for intracellular calcium in controlling cell sorting during morphogenesis in Dictyostelium.
To examine the potential role of calcium in regulating Dictyostelium development, we reduced free cytosolic and total cell Ca2+ in Dictyostelium cells by expressing a constitutively active form of a human erythrocyte plasma membrane calcium pump. The pump-expressing cells lacked a thapsigargin-mediated increase in cytoplasmic calcium, consistent with a reduced level of total cellular Ca2+. During aggregation, the cells initially formed a large number of aggregation centers, many of which coalesced to form mounds that were smaller than those of wild-type cells, and the cells did not exhibit the normal formation of elongated aggregation streams. The majority of the mounds either arrested at this stage with the formation of small protrusions or formed very aberrant finger-like structures, indicating an essential role for cellular calcium in morphogenesis. We used pump and wild-type cells differentially labeled by expressing different wavelength (green and blue) forms of green fluorescent protein and three-dimensional (3-D) reconstruction of serial fluorescent imaging to visualize the movement of pump and wild-type cells within the aggregate. The results showed that the pump cells exhibited very aberrant cell movement and sorting within the forming mound, suggesting that the reduced cytosolic calcium affects movement required for tip formation. When allowed to form chimeric organisms with wild-type cells, pump cells preferentially localized to two bands, one at the prestalk/prespore boundary and the other in the very posterior of the organism, suggesting that pump cells are unable to properly sort. Expression of the calcium pump had little effect on the induction of prestalk- or prespore-specific genes, whereas extended treatment with EGTA blocked induction of both classes of cell-type-specific genes. Our results suggest a role for intracellular Ca2+ in controlling cell sorting and morphogenesis in Dictyostelium.
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On/off blinking and switching behaviour of single molecules of green fluorescent protein.
Optical studies of individual molecules at low and room temperature can provide information about the dynamics of local environments in solids, liquids and biological systems unobscured by ensemble averaging. Here we present a study of the photophysical behaviour of single molecules of the green fluorescent protein (GFP) derived from the jellyfish Aequorea victoria. Wild-type GFP and its mutant have attracted interest as fluorescent biological labels because the fluorophore may be formed in vivo. GFP mutants immobilized in aereated aqueous polymer gels and excited by 488-nm light undergo repeated cycles of fluorescent emission ('blinking') on a timescale of several seconds-behaviour that would be unobservable in bulk studies. Eventually the individual GFP molecules reach a long-lasting dark state, from which they can be switched back to the original emissive state by irradiation at 405 nm. This suggests the possibility of using these GFPs as fluorescent markers for time-dependent cell processes, and as molecular photonic switches or optical storage elements, addressable on the single-molecule level.
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Crystal structure of the Aequorea victoria green fluorescent protein.
The green fluorescent protein (GFP) from the Pacific Northwest jellyfish Aequorea victoria has generated intense interest as a marker for gene expression and localization of gene products. The chromophore, resulting from the spontaneous cyclization and oxidation of the sequence -Ser65 (or Thr65)-Tyr66-Gly67-, requires the native protein fold for both formation and fluorescence emission. The structure of Thr65 GFP has been determined at 1.9 angstrom resolution. The protein fold consists of an 11-stranded beta barrel with a coaxial helix, with the chromophore forming from the central helix. Directed mutagenesis of one residue adjacent to the chromophore, Thr203, to Tyr or His results in significantly red-shifted excitation and emission maxima.
Amino Acid Sequence↗
Understanding, improving and using green fluorescent proteins.
Green fluorescent proteins (GFPs) are presently attracting tremendous interest as the first general method to create strong visible fluorescence by purely molecular biological means. So far, they have been used as reporters of gene expression, tracers of cell lineage, and as fusion tags to monitor protein localization within living cells. However, the GFP originally cloned from the jellyfish Aequorea victoria has several nonoptimal properties including low brightness, a significant delay between protein synthesis and fluorescence development, and complex photoisomerization. Fortunately, the protein can be re-engineered by mutagenesis to ameliorate these deficiencies and shift the excitation and emission wavelengths, creating different colors and new applications.
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Patterns of free calcium in multicellular stages of Dictyostelium expressing jellyfish apoaequorin.
To examine the patterns of high free cytosolic calcium or [Ca2+]i during Dictyostelium's development, we expressed apoaequorin in D. discoideum, reconstituted aequorin and observed the resultant patterns of calcium-dependent luminescence. Specific, high calcium zones are seen throughout normal multicellular development and are roughly coincident with those regions that later differentiate into stalk or stalk-like cells. A slug, for example, shows a primary high calcium zone within its front quarter and a secondary one around its tail; while a mound shows such a zone around the periphery of its base. Combined with previous evidence, our findings support the hypothesis that high [Ca2+]i feeds back to favor the stalk pathway. We also discovered several high calcium zones within the mound's base that do not coincide with any known prepatterns in D. discoideum. These include two, relatively persistent, antipodal strips along the mound's periphery. These various persistent zones of high calcium are largely made up of frequent, 10 to 30 second long, semiperiodic calcium spikes. Each of these spikes generates a correspondingly short-lived, 200 to 500 microns long, high calcium band which extends along the nearby surface. Similar, but relatively large and infrequent, spikes generate cross bands which extend across migrating slugs and just behind their advancing tips as well as across the peripheries of rotating mounds and midway between their antipodal strips. Moreover, calcium has a doubling time of about a second as various spikes rise. This last observation suggests that the calcium bands seen in Dictyostelium may be generated by so-called fast calcium waves.
Aequorin↗
Regulatory role of the G alpha 1 subunit in controlling cellular morphogenesis in Dictyostelium.
To determine the function of the Dictyostelium G alpha 1 subunit during aggregation and multicellular development, we analyzed the phenotypes of g alpha 1 null cells and strains overexpressing either wild-type G alpha 1 or two putative constitutively active mutations of G alpha 1. Strains overexpressing the wild-type or mutant G alpha 1 proteins showed very abnormal culmination with an aberrant stalk differentiation. The similarity of the phenotypes between G alpha 1 overexpression and expression of a putative constitutively active G alpha 1 subunit suggests that these phenotypes are due to increased G alpha 1 activity rather than resulting from a non-specific interference of other pathways. In contrast, g alpha 1 null strains showed normal morphogenesis except that the stalks were thinner and longer than those of wild-type culminants. Analysis of cell-type-specific gene expression using lacZ reporter constructs indicated that strains overexpressing G alpha 1 show a loss of ecmB expression in the central core of anterior prestalk AB cells. However, expression of ecmB in anterior-like cells and the expression of prestalk A-specific gene ecmA and the prespore-specific gene SP60/cotC appeared normal. Using a G alpha 1/lacZ reporter construct, we show that G alpha 1 expression is cell-type-specific during the multicellular stages, with a pattern of expression similar to ecmB, being preferentially expressed in the anterior prestalk AB cells and anterior-like cells. The developmental and molecular phenotypes of G alpha 1 overexpression and the cell-type-specific expression of G alpha 1 suggest that G alpha 1-mediated signaling pathways play an essential role in regulating multicellular development by controlling prestalk morphogenesis, possibly by acting as a negative regulator of prestalk AB cell differentiation. During the aggregation phase of development, g alpha 1 null cells display a delayed peak in cAMP-stimulated accumulation of cGMP compared to wild-type cells, while G alpha 1 overexpressors and dominant activating mutants show parallel kinetics of activation but decreased levels of cGMP accumulation compared to that seen in wild-type cells. These data suggest that G alpha 1 plays a role in the regulation of the activation and/or adaptation of the guanylyl cyclase pathway. In contrast, the activation of adenylyl cyclase, another pathway activated by cAMP stimulation, was unaffected in g alpha 1 null cells and cell lines overexpressing wild-type G alpha 1 or the G alpha 1 (Q206L) putative dominant activating mutation.(ABSTRACT TRUNCATED AT 400 WORDS)
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Developmentally regulated changes in 1,2-diacylglycerol in Dictyostelium. Regulation by light and G proteins.
We have measured 1,2-diacylglycerol (DG) mass during Dictyostelium development. DG levels are initially high in vegetative cells, decrease upon starvation, increase during aggregation, and rise dramatically during culmination, concomitant with SpiA (a spore cell-specific gene) expression. These results are consistent with DG being involved in culmination-stage morphological changes and cell-type differentiation. Mutant analysis shows that the rise in DG during aggregation requires cAMP signaling pathways but is not directly regulated through these processes but via developmental programs induced through cAMP. DG accumulation during aggregation is approximately 8-fold higher than would be expected from inositol lipid hydrolysis (1), suggesting that DG is produced from sources in addition to phosphatidylinositol 4,5-bisphosphate. Our data suggest that during aggregation, although some DG is formed through phospholipase D activity, other pathways (e.g. de novo synthesis) may be more important regulators of DG accumulation. During culmination, DG accumulation correlated with the formation of phosphatidic acid and phosphatidylethanol suggesting the activation of phospholipase D. During this time, the [3H]palmitate labeling of a number of phospholipids decreased rapidly, suggesting a rapid metabolism of phospholipids at this time. Exposure of slugs developed in the dark to light, which initiates culmination, causes rapid DG accumulation, suggesting the activation of phospholipid hydrolysis. The temporal pattern and level of DG accumulation is altered in G alpha 1 null and overexpressing strains, suggesting that G alpha 1 is upstream from DG formation during culmination. These results demonstrate that specific pathways of DG formation are under developmental control and suggest a possible link between light, the activation of DG production, and induction of culmination.
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Characterization of phospholipase activity in Dictyostelium discoideum. Identification of a Ca(2+)-dependent polyphosphoinositide-specific phospholipase C.
We have identified a Ca(2+)-dependent polyphosphoinositide-specific phospholipase C activity in Dictyostelium discoideum. Addition of Ca2+ (20 microM) results in the rapid formation of Ins(1,4,5)P3 within 5 s and leads to sustained inositol phosphate production for up to 40 min in membranes prepared from [3H]inositol-labelled cells. The phospholipase C activity is primarily membrane-bound under the conditions used to lyse the cells. In addition to this activity we also identified a family of Ca(2+)-regulated phospholipase activities active on a range of phospholipid substrates, using [3H]palmitate labelling. Inositol-specific phospholipase C activity is highest in vegetatively growing cells and in starved cells during the first 6 h in development, during which time Ca2+ elicited a 5-fold stimulation of inositol phosphate formation. After this time, total activity decreased progressively until 15 h, after which the activity remained constant up until 24 h. During this period, Ca2+ was able to stimulate a 2-fold increase in inositol phosphates.
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Amino acid substitutions in the Dictyostelium G alpha subunit G alpha 2 produce dominant negative phenotypes and inhibit the activation of adenylyl cyclase, guanylyl cyclase, and phospholipase C.
Previous studies have demonstrated that the Dictyostelium G alpha subunit G alpha 2 is essential for the cAMP-activation of adenylyl cyclase and guanylyl cyclase and that g alpha 2 null mutants do not aggregate. In this manuscript, we extend the analysis of the function of G alpha 2 in regulating downstream effectors by examining the in vivo developmental and physiological phenotypes of both wild-type and g alpha 2 null cells carrying a series of mutant G alpha 2 subunits expressed from the cloned G alpha 2 promoter. Our results show that wild-type cells expressing G alpha 2 subunits carrying mutations G40V and Q208L in the highly conserved GAGESG (residues 38-43) and GGQRS (residues 206-210) domains, which are expected to reduce the intrinsic GTPase activity, are blocked in multicellular development. Analysis of down-stream effector pathways essential for mediating aggregation indicates that cAMP-mediated activation of guanylyl cyclase and phosphatidylinositol-phospholipase C (PI-PLC) is almost completely inhibited and that there is a substantial reduction of cAMP-mediated activation of adenylyl cyclase. Moreover, neither mutant G alpha 2 subunit can complement g alpha 2 null mutants. Expression of G alpha 2(G43V) and G alpha 2(G207V) have little or no effect on the effector pathways and can partially complement g alpha 2 null cells. Our results suggest a model in which the dominant negative phenotypes resulting from the expression of G alpha 2(G40V) and G alpha 2(Q208L) are due to a constitutive adaptation of the effectors through a G alpha 2-mediated pathway. Analysis of PI-PLC in g alpha 2 null mutants and in cell lines expressing mutant G alpha 2 proteins also strongly suggests that G alpha 2 is the G alpha subunit that directly activates PI-PLC during aggregation. Moreover, overexpression of wild-type G alpha 2 results in the ability to precociously activate guanylyl cyclase by cAMP in vegetative cells, suggesting that G alpha 2 may be rate limiting in the developmental regulation of guanylyl cyclase activation. In agreement with previous results, the activation of adenylyl cyclase, while requiring G alpha 2 function in vivo, does not appear to be directly carried out by the G alpha 2 subunit. Our data are consistent with adenylyl cyclase being directly activated by either another G alpha subunit or by beta gamma subunits released on activation of the G protein containing G alpha 2.
Adenylyl Cyclases↗
Molecular genetic analysis of signal transduction pathways controlling multicellular development in Dictyostelium.
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5'-CMP stimulates phospholipase A-mediated hydrolysis of phosphatidylinositol in permeabilized pituitary GH3 cells.
We showed previously that 5'-CMP activates PtdIns-Ins base exchange and reversal PtdIns synthase in permeabilized rat pituitary GH3 cells. Here we report another effect of 5'-CMP on PtdIns metabolism in these cells. In permeabilized GH3 cells prelabelled with [3H]Ins and incubated in buffer with LiCl and a free Ca2+ concentration of 0.1 microM but without added Ins, 5'-CMP stimulated formation of glycerophospho[3H]inositol (GroP[3H]Ins) after a lag period of at least 5 min. This effect was concentration-dependent; the apparent Km was 0.30 +/- 0.02 mM. CDP and CTP stimulated GroPIns formation less effectively than did 5'-CMP, but cytidine, 2'-CMP, 3'-CMP, 5'-AMP and 5'-GMP had no effect. 5'-CMP stimulated formation of lysoPtdIns also. In permeabilized GH3 cells prelabelled with [3H]arachidonic acid, 5'-CMP stimulated release of [3H]arachidonic acid without a measurable lag period. These data show that 5'-CMP stimulates a phospholipase A activity in permeabilized GH3 cells that hydrolyses PtdIns.
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CMP activates reversal of phosphatidylinositol synthase and base exchange by distinct mechanisms in rat pituitary GH3 cells.
CMP is known to activate phosphatidylinositol (PtdIns)/inositol (Ins) base exchange and has been reported to activate reversal of PtdIns synthase also. Because it is possible that PtdIns synthase acting in the reverse direction, followed by re-incorporation of ambient Ins, could be responsible for base-exchange activity, we characterized these processes in rat pituitary GH3 cells. In permeabilized GH3 cells prelabelled with [3H]Ins and incubated in buffer with LiCl but without added Ins, CMP stimulated rapid accumulation of [3H]Ins and decreases in [3H]PtdIns; the Km for CMP was 1.7 mM. CDP and CTP were less effective, whereas 2'-CMP, 3'-CMP, other nucleoside monophosphates and cytidine did not influence this process. In permeabilized cells prelabelled to isotopic equilibrium with [3H]Ins and [32P]Pi, CMP stimulated decreases in both the 32P and 3H labelling of PtdIns, but did not increase that of [32P]phosphatidic acid. These findings demonstrate that in the absence of added Ins the effect of CMP is not via activation of base exchange nor via a phospholipase D, but by reversal of PtdIns synthase. In permeabilized cells prelabelled with [3H]Ins and [32P]Pi, unlabelled Ins inhibited loss of 32P labelling of PtdIns caused by CMP while markedly stimulating loss of 3H labelling of PtdIns and release of [3H]Ins. These data demonstrate that Ins inhibits reversal of PtdIns synthase, but stimulates base exchange. We conclude that in GH3 cells reversal of PtdIns synthase and PtdIns/Ins base exchange are both stimulated by CMP, but are distinct processes.
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Thyrotropin-releasing hormone receptor occupancy determines the fraction of the responsive pool of inositol lipids hydrolysed in rat pituitary tumour cells.
We report that there are distinct thyrotropin-releasing hormone (TRH)-responsive and -unresponsive pools of inositol (Ins) lipids in rat pituitary tumour (GH3) cells, and present evidence that the size of the responsive pool is determined by the number of activated TRH-receptor complexes. By use of an experimental protocol in which cycling of [3H]Ins is inhibited and resynthesis occurs with unlabelled Ins only, we were able to measure specifically the effects of TRH on the hydrolysis of the Ins lipids present before stimulation. A maximally effective dose of TRH (1 microM) caused a time-dependent decrease in 3H-labelled Ins lipids that attained a steady-state value of 42 +/- 1% of the initial level between 1.5 and 2 h. After 2 h, even though there was no further decrease in 3H-labelled Ins lipids, and no increase in [3H]Ins or [3H]Ins phosphates, turnover of Ins lipids, as assessed as incorporation of [32P]Pi into PtdIns, continued at a rate similar to that in cells incubated without LiCl or unlabelled Ins. These data indicate that Ins lipid turnover was not desensitized during prolonged TRH stimulation. Depletion of lipid 3H radioactivity by TRH occurred at higher TRH doses on addition of the competitive antagonist chlordiazepoxide. Addition of 1 microM-TRH after 3 h of stimulation by a sub-maximal (0.3 nM) TRH dose caused a further decrease in 3H radioactivity to the minimum level (40% of initial value). We propose that the TRH-responsive pool of Ins lipids in GH3 cells is composed of the complement of Ins lipids that are within functional proximity of activated TRH-receptor complexes.
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Analysis by base exchange of thyrotropin-releasing hormone responsive and unresponsive inositol lipid pools in rat pituitary tumor cells.
We have shown that there is an inositol (Ins) lipid pool in cloned rat pituitary tumor (GH3) cells that is hydrolyzed in response to thyrotropin-releasing hormone (TRH) and an unresponsive pool. Because others have suggested that incorporation of [3H]Ins by base exchange may not occur uniformly into Ins lipids in other cell types, we established conditions using permeabilized cells under which labeling occurs by Ins-phosphatidylinositol (PI) exchange in the absence of de novo PI synthesis to further characterize these pools in GH3 cells. In permeabilized cells incubated in buffer containing 10 mM Mg2+ and 0.1 mM CMP, [3H]Ins incorporation into lipids occurred by base exchange only. This was so because: 1) [3H]Ins incorporation into lipids displayed properties similar to that for release of 3H-labeled Ins by unlabeled Ins from PI in cells prelabeled in situ prior to permeabilization; and 2) there was no change in PI mass under these conditions. In permeabilized cells incubated in buffer with 0.1 mM [3H]Ins for 60 min, incorporation was 0.61 +/- 0.05 nmol of [3H]Ins/10(6) permeabilized cells, which amounted to 35% of PI, while the level of PI, measured as nonradioactive phosphorus, was 94 +/- 8.0% of control. Permeabilized GH3 cells were responsive to TRH. In cells prelabeled in situ and then permeabilized, TRH stimulated an increase in 3H-labeled Ins phosphates (IPs) in 20 min which was 10% of 3H radioactivity initially present in lipids. This increase in 3H-labeled IPs was 6.3 times the 3H radioactivity present in phosphatidylinositol 4,5-bisphosphate prior to stimulation. When prelabeled cells were exchanged with unlabeled Ins after permeabilization there was only a 10-16% decrease in 3H-labeled IP accumulation stimulated by TRH even though 3H-labeled lipids decreased to 52% of control. TRH did not affect labeling by [3H]Ins-PI exchange. In cells labeled by base exchange after permeabilization TRH stimulated a very small increase in 3H-labeled IPs of only 0.21 +/- 0.02% of 3H-labeled lipids in 20 min or only 7% of the 3H radioactivity in phosphatidylinositol 4,5-bisphosphate. These data show that in permeabilized GH3 cells base exchange can occur in the absence of de novo PI synthesis and that lipids that are preferentially labeled by base exchange comprise a pool that is less responsive to TRH than total Ins lipids.
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Characterization of a salt-extractable phosphatidylinositol synthase from rat pituitary-tumour membranes.
Solubilization of phosphatidylinositol (PtdIns) synthase (CDP-diacylglycerol: myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11) from rat pituitary (GH3) tumours was investigated. PtdIns synthase activity was partially extracted from crude membranes by 3 M-KCl. Prior separation of membranes revealed that a greater proportion of plasma-membrane PtdIns synthase activity was salt-extractable than was endoplasmic reticulum activity. The activity of the salt-extracted enzyme was maximized by low concentrations of 3-(3-cholamidopropyl) dimethylammonio-1-propanesulphonate (CHAPS; 0.5 mM), Triton X-100 (0.1 mM) or a phospholipid mixture (0.05 mg/ml), but higher concentrations of detergents were inhibitory. The activity of salt-extracted PtdIns synthase was 0.25 +/- 0.08 nmol/min per mg of protein. Salt-extracted PtdIns synthase activity was dependent on Mg2+ (maximal at 0.1 mM) and Mn2+ (maximal at 5 mM), and its pH optimum was in the range 7.0-7.5. The apparent Km for myo-inositol (in the presence of 0.1 mM-CDP-diacylglycerol) was 0.06 mM, and that for CDP-diacylglycerol (at 0.1 mM-myo-inositol) was 0.21 mM. Salt-extracted PtdIns synthase activity was potently inhibited by Ca2+ (50% inhibition at 1 microM), with over 90% inhibition at 10 microM-Ca2+. These data imply the existence of two forms of membrane-associated PtdIns synthase, namely salt-extractable and salt-resistant, with different intracellular localizations. The salt-extractable form of this enzyme may be a useful preparation for further characterization and purification of mammalian PtdIns synthase.
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Dephosphorylation of myo-inositol 1,4,5-trisphosphate and myo-inositol 1,3,4-triphosphate.
We have augmented our previous studies [Storey, Shears, Kirk & Michell (1984) Nature (London) 312, 374-376] on the subcellular location and properties of Ins(1,4,5)P3 (inositol 1,4,5-trisphosphate) phosphatases in rat liver and human erythrocytes. We also investigate Ins(1,3,4)P3 (inositol 1,3,4-trisphosphate) metabolism by rat liver. Membrane-bound and cytosolic Ins(1,4,5)P3 phosphatases both attack the 5-phosphate. The membrane-bound enzyme is located on the inner face of the plasma membrane, and there is little or no activity associated with Golgi apparatus. Cytosolic Ins(1,4,5)P3 5-phosphatase (Mr 77,000) was separated by gel filtration from Ins(1,4)P2 (inositol 1,4-bisphosphate) and inositol 1-phosphate phosphatases (Mr 54,000). Ins(1,4,5)P3 5-phosphatase activity in hepatocytes was unaffected by treatment of the cells with insulin, vasopressin, glucagon or dibutyryl cyclic AMP. Ins(1,4,5)P3 5-phosphatase activity in cell homogenates was unaffected by changes in [Ca2+] from 0.1 to 2 microM. After centrifugation of a liver homogenate at 100,000 g, Ins(1,3,4)P3 phosphatase activity was largely confined to the supernatant. The sum of the activities in the supernatant and the pellet exceeded that in the original homogenate. When these fractions were recombined, Ins(1,3,4)P3 phosphatase activity was restored to that observed in unfractionated homogenate. Ins(1,3,4)P3 was produced from Ins(1,3,4,5)P4 (inositol 1,3,4,5-tetrakisphosphate) and was metabolized to a novel InsP2 that was the 3,4-isomer. Ins(1,3,4)P3 phosphatase activity was not changed by 50 mM-Li+ or 0.07 mM-Ins(1,4)P2 alone, but when added together these agents inhibited Ins(1,3,4)P3 metabolism. In Li+-treated and vasopressin-stimulated hepatocytes, Ins(1,4)P2 may reach concentrations sufficient to inhibit Ins(1,3,4)P3 metabolism, with little effect on Ins(1,4,5)P3 hydrolysis.
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