Interleukin 2 does not induce phosphatidylinositol hydrolysis in activated T cells

Hydrolysis of phosphatidylinositol-4,5-bisphosphate to diacylglycerol and myoinositol-1,4,5-trisphosphate is thought to be a primary event in the activation of cells by some growth factors, mitogenic lectins, and oncogenes. The mechanism whereby interleukin 2 (IL 2) binding to its receptor on activated T lymphocytes leads to cell proliferation has not been determined. Because the mitogenic has not been determined. Because the mitogenic action of IL 2 resembles that of some growth factors, the possible role of phosphatidylinositol breakdown in the activation of T cells by IL 2 was examined. In human or murine IL 2-sensitive cells, incubation with IL 2 did not alter the rate of turnover of phosphatidylinositol, phosphatidylinositol-5-phosphate, phosphatidylinositol-4,5-bisphosphate, or phosphatidylcholine in 32PO4-loaded cells. IL 2 also did not alter either the isotopic labeling of diacylglycerol or [3H]arachidonic acid release from cells. In addition, IL 2 did not alter the rate of formation of the phosphatidylinositol breakdown products myoinositol-1,4,5-trisphosphate, myoinositol-1,4-bisphosphate, or myoinositol-1-phosphate. In contrast, under similar conditions, IL 2 induced significant increases in [3H]thymidine incorporation and cell proliferation. Mitogenic lectins such as concanavalin A and phytohemagglutinin gave significant changes in isotopic labeling of phosphoinositols, diacylglycerols, and phosphatidylinositols, indicating that phosphatidylinositol hydrolysis induced by mitogenic lectins was detectable in the assay systems. IL 2, in contrast to other growth factors, does not appear to signal cells by increasing phosphatidylinositol breakdown.     

Phospholipid biosynthesis in mature human erythrocytes

Mature human erythrocytes were tested for their ability to synthetize membrane phospholipids from simple precursors: [32P]-orthophosphate (32Pi), [U-14C] glycerol, [U-14C] glucose, [U-14C] serine, and [U-14C] choline. The incorporation of these labels into phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA), lysophosphatidylcholine (lyso-PC), phosphatidylinositol-4-phosphate (PIP), and phosphatidylinositol-4,5-bisphosphate (PIP2) was measured. All the phospholipids tested incorporated 32Pi, glycerol, and glucose in a time dependent manner. According to the rate of 32Pi incorporation, three groups of phospholipids could be distinguished: 1) PA, PIP2, PIP, lyso-PC; 2) PI and PS; 3) PC and PE, which incorporated 5 x 10(3), 40, and 6 nmol 32Pi/mmol phospholipid per 1 h, respectively. Moreover, [U-14C] serine and [U14C] choline were found to incorporate into phospholipids, and PS-decarboxylase activity could be measured. The possibility that the observed incorporation was due to contamination with bacteria or other blood cells could be ruled out. Our results bring evidence for de novo phospholipid synthesis of human red blood cells.     

Production of novel polyphosphoinositides in vivo is linked to cell transformation by polyomavirus middle T antigen.

Phosphatidylinositol 3-kinase associates with the polyomavirus middle T antigen (PyMTAg)-pp60c-src complex in polyomavirus-transformed cells. Here we show that anti-PyMTAg immunoprecipitates from PyMTAg-transformed NIH 3T3 cells have lipid kinase activities that phosphorylate phosphatidylinositol, phosphatidylinositol-4-bisphosphate, and phosphatidylinositol-4,5-bisphosphate at the D-3 position of the inositol ring to produce three new polyphosphoinositides: phosphatidylinositol-3-phosphate (PI-3-P), phosphatidylinositol-3,4-bisphosphate (PI-3,4-P2), and phosphatidylinositol trisphosphate (PIP3), respectively. PI-3-P was detected in intact parental and PyMTAg-transformed NIH 3T3 fibroblasts at both low and high cell densities. However, parental NIH 3T3 fibroblasts produced no detectable PI-3,4-P2 or PIP3 at high density. In contrast, growing, subconfluent cells and wild-type PyMTAg-transformed cells at high density had greatly enhanced incorporation of [3H]-inositol into these highly phosphorylated lipids. Cells transfected with a transformation-defective mutant of PyMTAg had undetectable levels of PI-3,4-P2 and PIP3 at high density. Thus, the synthesis of novel polyphosphoinositides by lipid kinase activity associated with PyMTAg correlates with cell growth and transformation.     

Manganese stimulates incorporation of [3H]inositol into an agonist-insensitive pool of phosphatidylinositol in brain membranes

In a particulate preparation from rat brain, manganese ions stimulate the incorporation of [3H]inositol into inositol phospholipids in a concentration-dependent manner. Incubation with CDP-diacylglycerol (0.5 mM) alone had no effect on the incorporation of [3H]inositol but potentiated the stimulatory effect of manganese. Despite the increase in [3H]inositol incorporation into phosphatidylinositol, the carbachol-induced accumulation of [3H]inositol-1-phosphate was unaltered in membranes preincubated with manganese but when coincubated with CDP-diacylglycerol the carbachol-induced accumulation of [3H]inositol-1-phosphate was increased. These data suggest that manganese stimulates the incorporation of [3H]inositol into an agonist-insensitive pool of phosphatidylinositol.     

The rapid polyphosphoinositide metabolism may be a triggering event for thrombin-mediated stimulation of human platelets

The metabolism of polyphosphoinositides was examined in human platelets activated by thrombin. The addition of thrombin to [3H]glycerol-labeled platelets induced an initial loss and a subsequent increase of the radioactivity in phosphatidylinositol-4,5-bisphosphate (TPI) without any significant change in phosphatidylinositol-4-phosphate (DPI). A marked enhancement of [32P]Pi incorporation into TPI occurred in parallel with an increase in this lipid content, which was accompanied with a conccurent decrease in phosphatidylinositol (PI). The rate of this subsequent increase in TPI was smaller than that observed in [3H]arachidonic acid-labeled platelets, suggesting that formed TPI in activated platelets may contain much greater amount of arachidonate than preexisting TPI in resting platelets. These data indicate that thrombin causes a rapid change in TPI metabolism (initial degradation of preexisting TPI and subsequent production of arachidonate-rich TPI), which might be a primary candidate to modulate thrombin-induced function in human platelets.     

Tight coupling of thrombin-induced acid hydrolase secretion and phosphatidate synthesis to receptor occupancy in human platelets.

Human platelets incubated with [32P]Pi and [3H]arachidonate were transferred to a Pi-free Tyrode's solution by gel filtration. The labile phosphoryl groups of ATP and ADP as well as Pi in the metabolic pool of these platelets had equal specific radioactivity which was identical to that of[32P]phosphatidate formed during treatment of the cells with thrombin for 5 min. Therefore, the 32P radioactivity of phosphatidate was a true, relative measure for its mass. The thrombin-induced formation of[32P]-phosphatidate had the same time course and dose-response relationships as the concurrent secretion of acid hydrolases. 125I-alpha-Thrombin bound maximally to the platelets within 13s and was rapidly dissociated from the cells by hirudin; readdition of excess 125I-alpha-thrombin caused rapid rebinding of radioligand. This binding-dissociation-rebinding sequence was paralleled by a concerted start-stop-restart of phosphatidate formation and acid hydrolase secretion. [3H]Phosphatidylinositol disappearance was initiated upon binding but little affected by thrombin dissociation and rebinding. ATP deprivation caused similar changes in the time courses for [32P]-phosphatidate formation and acid hydrolase secretion which were different from those of [3H]phosphatidylinositol disappearance. The metabolic stress did not alter the magnitude (15%) of the initial decrease in phosphatidylinositol-4,5-bis[32P]phosphate, but did abolish the subsequent increase of phosphatidylinositol-4,5-bis[32P]-phosphate in the thrombin-treated platelets. It is concluded that in thrombin-treated platelets (1) phosphatidate synthesis, but not phosphatidylinositol disappearance, is tightly coupled to receptor occupancy and acid hydrolase secretion in platelets, (2) successive phosphorylations to phosphatidylinositol-4,5-bisphosphate is unlikely to be the main mechanism for phosphatidylinositol disappearance, and (3) only a small fraction (15%) of phosphatidylinositol-4,5-bisphosphate is susceptible to hydrolysis.     

Generation of Arachidonic Acid and Diacylglycerol Second Messengers from Polyphosphoinositides in Ischemic Fetal Brain

Intracerebral administration of [3H]arachidonic acid ([3H]ArA) into 19-20-day-old rat embryos, resulted in a rapid incorporation of label into brain lipids. One hour after injection, 55.6 +/- 8.2, 18.0 +/- 3.4, and 13.7 +/- 1.3% of the total radioactivity was associated with phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine, respectively. Approximately 10% of radioactivity was found acylated in neutral lipids of which free ArA comprised only 1.5 +/- 0.2% of the total radioactivity. Complete restriction of the maternal-fetal circulation for < or = 40 min did not affect the rate of [3H]ArA incorporation (t1/2 = 2 min) into fetal brain lipids, suggesting an effective acylation mechanism that proceeds irrespective of the impaired blood flow. After a short restriction period (5 min), the radioactivity in diacylglycerol was elevated by 50%. After a longer restriction period (20 min), the radioactivity in the free fatty acid and diacylglycerol fractions increased to values of 130 and 87%, respectively. Polyphosphoinositides prelabeled with either [3H]ArA or 32P were rapidly degraded after 5 min of ischemia. After 20 min, the decrease in phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate radioactivity was 47 and 70%, respectively. Double labeling of phospholipids with [14C]palmitic acid and [3H]ArA indicated a preferential loss of [3H]ArA within the polyphosphoinositide species after 20 min, but not after 5 min of ischemia. The specific activity of [14C]palmitate remained unchanged. The current data suggest phospholipase C-mediated diacylglycerol formation at the beginning of the insult followed by a phospholipase A2-mediated ArA liberation at a later time, both enzymes presumably acting preferentially on polyphosphoinositide species.     

Metabolic regulation during early frog development: flow of glycolytic carbon into phospholipids in Xenopus oocytes and fertilized eggs

32P-labeled glucose 6-phosphate, [32P]phosphoenolpyruvate, and [gamma-32P]ATP were injected into oocytes and fertilized eggs of Xenopus laevis, and the incorporation of the 32P label was followed into phospholipids. Several classes of phospholipids incorporated 32P label from the injected glycolytic intermediates, including lysophosphatidic acid, phosphatidic acid, phosphatidylinositol, and phosphatidylinositol phosphates, inferring de novo synthesis of these lipids from dihydroxyacetone phosphate or glycerol 3-phosphate. Injection of [gamma-32P]ATP into oocytes and fertilized eggs led to labeling of phosphatidylinositol phosphate and phosphatidylinositol bisphosphate, indicating an active phosphatidylinositol cycle in resting oocytes and fertilized eggs. Maturation and fertilization of the oocyte led to a qualitative change in phosphatidylinositol metabolism, increased labeling of phosphatidylinositol phosphate compared to phosphatidylinositol bisphosphate (either from glycerol 3-phosphate or from ATP). This change occurs late in the maturation process, and the new pattern of phosphatidylinositol metabolism is maintained during the rapid cleavage stages of early embryogenesis.     

Phospholipid metabolism in Ehrlich ascites tumor cells. I. Turnover rate of phosphatidylinositol.

1. Radioactive precursors, 32 PI, [1-14C]glycerol, and [1-14C]acetate, were individually injected into the peritoneal cavity of mice bearing Ehrlich ascites tumor, and the rates of incorporation into phospholipid fraction of Ehrlich ascites tumor cells were estimated. Although no distinct difference in specific activities was observed between phosphatidylinositol and other phospholipid classes as regards the incorporation of [1-14C]acetate of [1-14C]glycerol, a higher rate of incorporation of 32Pi into phosphatidylinositol was observed. The specific activity of phosphatidylinositol reached more than ten times that of phosphatidylcholine in the first hour. 2. The radioactivities incorporated into the phospholipids of Ehrlich ascites tumor cells and liver were estimated after simultaneous injection 32Pi and [2-3H]inositol. The incorporation of 32Pi into phosphatidylinositol of liver was similar in specific activity to those of other phospholipids. The ratio (3H/32Pi) of phosphatidylinositol only slightly in the ascites tumor cells, while an appreciable decrease of the ratio was observed in the liver during the first 3 hr. 3. These results suggest that phosphatidylinositol synthesis through pathways other than de novo synthesis is rapid in ascites tumor cells.     

Polylysine and polyamine stimulation of the phosphatidylinositol kinases of amphibian oocyte membranes

Phosphatidylinositol kinase present in Xenopus laevis oocyte membranes catalyzes the formation of phosphatidylinositol 4-phosphate using phosphatidylinositol and ATP as substrates while the activity of a second enzyme, phosphatidylinositol-4-phosphate kinase, results in the synthesis of phosphatidylinositol 4,5-bisphosphate. Large (Mr greater than 20,000) homopolymers of L-lysine or L-ornithine can stimulate the activity of both of these enzymes by at least 2-fold at 10-20 microM concentrations. Under similar conditions poly-L-arginine fails to stimulate the reaction causing a partial inhibition. Smaller polylysine (25 lysines) or lysine-rich oligopeptides such as one corresponding to the last 14 amino acids of the carboxyl end of c-Ki-ras 2 protein produce appreciable stimulation of phosphatidylinositol but at concentrations of 300-500 microM. Spermine and spermidine at millimolar concentrations also stimulate exogenous phosphatidylinositol phosphorylation. The amino-glycoside antibiotic neomycin has a biphasic effect, stimulating the phosphatidylinositol kinase at concentrations below 0.5 mM and strongly inhibiting at higher concentrations. Polylysine also moderately stimulates the loss of radioactivity of phosphatidylinositol-4-[32P] phosphate observed in oocyte membranes. Polylysine and polyornithine do not change the apparent Km for ATP of the phosphatidylinositol kinase but increase the Vmax of the reaction.     

Metabolism of [14C]arachidonic acid-labeled lipids in quiescent and OAG-stimulated ascites tumor cells.

1. A rapid uptake and esterification of [14C]arachidonic acid during the first 4 hr of cultivation of ascites cells in serum-deprived medium was observed followed by a fast turnover of the fatty acid. 2. Labeling and turnover of esterified arachidonate in individual phospholipid classes was in the order: phosphatidylcholine (PC) greater than phosphatidylinositol (PI) much greater than phosphatidylinositol-4-phosphate (PIP) and -4,5-bisphosphate (PIP2) greater than phosphatidylethanolamine (PE) greater than PE-plasmalogens. 3. In cells stimulated with 1-oleoyl-2-acetyl-sn-glycerol a transient course of arachidonic acid incorporation into PC, PI, PIP and PIP2 was determined peaking 30 min after stimulation, indicating both esterification and release under these conditions. 4. The release of arachidonate was blocked by quinacrine which is a specific inhibitor of phospholipase A2.     

Gonadotropin-releasing hormone activates a rapid Ca2+-independent phosphodiester hydrolysis of polyphosphoinositides in pituitary gonadotrophs

Addition of gonadotropin releasing hormone (GnRH) to pituitary cells prelabeled with [32P]Pi or with myo-[2-3H]inositol, resulted in a rapid decrease in the level of [32P]phosphatidylinositol 4,5-bisphosphate (approximately 10 s), and in [32P]phosphatidylinositol 4-phosphate (approximately 1 min), followed by increased labeling of [32P]phosphatidylinositol and [32P]phosphatidic acid (1 min). GnRH stimulated the appearance of [3H]myo-inositol 1,4,5-trisphosphate (10 s), [3H]myo-inositol 1,4-bisphosphate (15 s), and [3H]myo-inositol 1-phosphate (1 min) in the presence of Li+ (10 mM). Li+ alone stimulated the accumulation of [3H]myo-inositol 1-phosphate and [3H]myo-inositol 1,4-bisphosphate but not [3H]myo-inositol 1,4,5-trisphosphate, but had no effect on luteinizing hormone release. The effect of GnRH on inositol phosphates (Ins-P) production was dose-related (ED50 = 1-5 nM), and was blocked by a potent antagonist [D-pGlu,pClPhe,D-Trp]GnRH. Elevation of cytosolic free Ca2+ levels ([Ca2+]i), by ionomycin and A23187 from intracellular or extracellular Ca2+ pools, respectively, had no significant effect on [3H]Ins-P production. GnRH-induced [3H]Ins-P production was not dependent on extracellular Ca2+ and was noticed also after extracellular or intracellular Ca2+ mobilization by A23187 or ionomycin, respectively. The effect of GnRH on [3H]Ins-P accumulation was not affected by prior treatment of the cells with the tumor promoter phorbol ester 12-O-tetradecanoylphorbol-13-acetate or with islet-activating protein pertussis toxin. These results indicate that GnRH stimulates a rapid phosphodiester hydrolysis of polyphosphoinositides. The stimulatory effect is not mediated via an islet-activating protein-substrate, is not dependent on elevation of [Ca2+]i, neither is it negatively regulated by 12-O-tetradecanoylphorbol-13-acetate which activates Ca2+/phospholipid-dependent protein C kinase. The results are consistent with the hypothesis that GnRH-induced phosphoinositide turnover is responsible for Ca2+ mobilization followed by gonadotropin release.     

On the source of the vasopressin-induced increases in diacylglycerol in hepatocytes

In hepatocytes pre-labelled with [3H]glycerol, vasopressin increased by 20% the amount of radioactivity present in diacylglycerols. The effect of vasopressin was partially dependent on Ca2+. The magnitude of the increase in [3H]diacylglycerol was 5-times the sum of the radioactivity present in phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. No stimulation by vasopressin of the initial rate of incorporation of radioactivity into diacylglycerols was observed in cells incubated in the presence of 10 mM [3H]glycerol. Treatment of hepatocytes labelled with either [3H]ethanolamine or [3H]choline with vasopressin, ionophore A23187 or phospholipase C increased the amount of radioactivity present in trichloroacetic acid extracts of the cells. The effect of vasopressin was dependent on extracellular Ca2+. It is concluded that in hepatocytes vasopressin increases diacylglycerols by a process which does not principally involve the conversion of phosphoinositides to diacylglycerol or the de novo synthesis of diacylglycerol from glycerol 3-phosphate, but does involve the Ca2+-dependent conversion of phosphatidylethanolamine and phosphatidylcholine to diacylglycerol.     

Phosphoinositide synthesis in bovine rod outer segments

Phosphoinositide turnover has been implicated in signal transduction in a variety of cells, including photoreceptors. We demonstrate here the presence of a complete pathway for rapid synthesis of phosphoinositides in isolated bovine retinal rod outer segments (ROS) free of microsomal contaminants. Synthesis was measured by the incorporation of label from radioactive precursors, [gamma-32P]ATP and [3H]inositol. [gamma-32P]ATP also produced large amounts of labeled phosphatidic acid. Incorporation of [3H]inositol required CTP and Mn2+. Mn2+ increased 32P incorporation into phosphatidylinositol 4-phosphate, while spermine increased phosphoinositide labeling generally. ROS that had been washed to remove soluble and peripheral proteins incorporated less label than unwashed ROS into phosphatidic acid and phosphatidylinositol. No effects of light were detected. Inhibitory effects of high concentrations of nonhydrolyzable GTP analogues were probably due to competition with ATP.     

Subcellular localization and enzymatic properties of rat liver phosphatidylinositol-4-phosphate kinase

The phosphatidylinositol-4-phosphate kinase activity in rat liver showed a subcellular distribution different from that of phosphatidylinositol kinase. It was preferentially associated with plasma membrane-rich subcellular fractions, while no or minimal activity could be ascribed to mitochondria, lysosomes, Golgi membranes or the endoplasmic reticulum. The plasma membrane enzyme phosphorylated endogenous and exogenously added phosphatidylinositol 4-phosphate at comparable initial rates. The phosphorylation of endogenous substrate was strongly inhibited by Triton X-100, while the phosphorylation of added substrate was enhanced, suggesting that endogenous phosphatidylinositol 4-phosphate was readily available to the enzyme in unperturbed plasma membranes. The total activity of phosphatidylinositol-4-phosphate kinase in rat liver was only 1/20 that of phosphatidylinositol kinase. The enzyme activity showed an unusually broad pH-optimum in the neutral range. Mg2+ was the preferred divalent cation and Km towards ATP was about 3-fold higher than the corresponding value for phosphatidylinositol kinase.     

Characterization of a phosphatidylinositol 4-phosphate-specific phosphomonoesterase in rat liver nuclear envelopes

Incubation of rat liver nuclear envelopes with [gamma-32P]ATP resulted in the synthesis of phosphatidylinositol-[4-32P]phosphate (PIP). Degradation of endogenously labeled PIP was observed upon the dilution of the labeled ATP with an excess of unlabeled ATP. This degradation was most rapid in the presence of EDTA, and was inhibited by MgCl2 and CaCl2. To further characterize the degradative activity, phosphatidylinositol[4-32P]phosphate and phosphatidylinositol [4,5-32P]bisphosphate (PIP2) were synthesized and isolated from erythrocyte plasma membranes. The 32P-labeled phospholipids were then resuspended in 0.4% Tween 80, a detergent that did not inhibit degradation of endogenously labeled PIP, and mixed with nuclear envelopes. [32P]PIP and [32P]PIP2 were degraded at rates of 2.25 and 0.04 nmol min-1 mg nuclear envelope protein-1, respectively. Only 32P was released from phosphatidyl[2-3H]inositol-[4-32P]phosphate, indicating that hydrolysis of PIP was due to a phosphomonoesterase activity (EC 3.1.3.36) in nuclear envelopes. Similarly, anion-exchange chromatographic analysis of the water-soluble products released from [32P]PIP indicated that inorganic phosphate was the sole 32P-labeled product. Hydrolysis of PIP was most rapid at neutral pH, and was not affected by inhibitors of acid phosphatase or alkaline phosphatase. Hydrolysis of PIP was also not inhibited by nonspecific phosphatase substrates, such as glycerophosphate, p-nitrophenylphosphate, AMP, or glucose 6-phosphate. Hydrolysis was stimulated by putrescine, and was inhibited by inositol 2-phosphate, spermidine, spermine, and neomycin.     

12-O-Tetradecanoylphorbol 13-acetate stimulates inositol lipid phosphorylation in intact human platelets

The phorbol esters are among the most potent tumor promoters. On addition of 12-O-tetradecanoylphorbol 13-acetate (TPA) to isolated human platelets prelabelled with [32P]orthophosphate we found a rapid increase in 32P incorporation into phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. In view of similar findings with cells infected with the oncogene Rous sarcoma virus, it is suggested that inositol lipid phosphorylation might be a key event in the molecular action of phorbol esters.     

Lithium-induced accumulation of inositol 1-phosphate during cholecystokinin octapeptide- and acetylcholine-stimulated phosphatidylinositol breakdown in dispersed mouse pancreas acinar cells

Dispersed mouse pancreas acinar cells were prepared in which phosphatidylinositol had been labeled with myo[2-3H]inositol. During incubation with 0.3 microM cholecystokinin octapeptide (CCK-8) for 15 min, there was a loss of [3H]phosphatidylinositol radioactivity (23%) and a 3-fold gain in trichloroacetic acid-soluble radioactivity. Replacement of NaCl by up to 58 mM LiCl did not significantly affect the amount of CCK-8-stimulated [3H]phosphatidylinositol breakdown or the gain in acid-soluble radioactivity. However, in normal medium, the product of phosphatidylinositol breakdown was almost all inositol, whereas in Li+-containing medium, the product was almost all inositol 1-phosphate. Similar results were obtained with acetylcholine which, in the presence of Li+, gave a dose-responsive increase in inositol 1-phosphate over the concentration range of 0.1 to 10 microM. No increased accumulation of [3H]inositol diphosphate or [3H]inositol triphosphate was detected in stimulated cells. Time courses in the presence of Li+ indicated that the formation of inositol 1-phosphate preceded the formation of inositol. Addition of up to 50 mM myoinositol to the incubation medium showed no diluting effect on the amount of [3H]inositol 1-phosphate found. The accumulation of inositol 1-phosphate is presumably due to the known ability of Li+ to inhibit myoinositol 1-phosphatase. The results provide clear evidence that stimulated phosphatidylinositol breakdown involves a phospholipase C type of phosphodiesterase activity. 1.25 mM Li+ gave half-maximal inositol 1-phosphate accumulation. This is close to the range of plasma Li+ levels which is used therapeutically in psychiatric disorders. In unstimulated cells, [3H]inositol 1-phosphate accumulation in the presence of Li+ corresponded to a breakdown rate for [3H]phosphatidylinositol of 2 to 3%/h.     

Activation of phosphatidylinositol-4-phosphate kinase in rat liver plasma membranes by polyamines

The formation of phosphatidylinositol 4,5-bisphosphate (PIP2) from endogenous substrate in rat liver plasma membranes was stimulated approximately 3-fold by 1 mM spermine, with half-maximal effect at 0.2 mM polyamine. This effect of spermine was due to enhancement of phosphatidylinositol-4-phosphate kinase activity rather than to a decrease in degradation of PIP2 formed or the substrate phosphatidylinositol 4-phosphate (PIP). The stimulation of phosphatidylinositol-4-phosphate kinase by spermine decreased to half at physiological ionic strength, and was not affected appreciably by variations in the concentration of ATP and MgCl2. Among several di- and polyamines only spermine and spermidine were effective. Although spermine may cause aggregation of membrane vesicles, thereby potentially increasing substrate availability for phosphatidylinositol-4-phosphate kinase, our results do not support such an explanation for the enhancement in enzyme activity. Phosphatidylinositol kinase activity, contrary to phosphatidylinositol-4-phosphate kinase, was not stimulated appreciably by spermine.     

Cytidine monophosphate-dependent synthesis of phosphatidylglycerol in permeabilized type II pneumonocytes.

Results of previous investigations support the proposition that, in type II pneumonocytes, CMP is involved in integration of the synthesis of phosphatidylcholine and phosphatidylglycerol for lung surfactant. In the present investigation, the amount of CMP in rat type II pneumonocytes was altered directly and resultant changes in the synthesis of phosphatidylglycerol were examined. Type II pneumonocytes were made permeable to CMP by treatment with Ca2+-free medium, and phosphatidylglycerol synthesis was then assessed by measurement of the incorporation of a radiolabelled precursor, [14C]glycerol 3-phosphate, that was not effectively utilized by cells that resisted permeabilization. Incorporation of [14C]glycerol 3-phosphate into phosphatidylglycerol (but not into other lipids) was stimulated greatly by CMP (half-maximal stimulation at approx. 0.1 mM). CMP stimulated the incorporation of [14C]glycerol 3-phosphate into both the phosphatidyl moiety and the head group of phosphatidylglycerol. Incorporation of [14C]palmitate into phosphatidylglycerol was also stimulated by CMP. myo-Inositol, at concentrations found in foetal-rat serum (0.2-2.0 mM), inhibited CMP-dependent incorporation of [14C]glycerol 3-phosphate into phosphatidylglycerol and promoted, instead, CMP-dependent incorporation into phosphatidylinositol. These data, when extrapolated to foetal type II pneumonocytes, are consistent with the view that the developmental increase in the synthesis of phosphatidylglycerol for surfactant by foetal lungs is promoted by the increase in intracellular CMP and the declining availability of myo-inositol that were found previously to be associated with this period of development.