Characterization of urea transport in Bufo arenarum oocytes

Xenopus laevis oocytes have been extensively used for expression cloning, structure/function relationships, and regulation analysis of transporter proteins. Urea transporters have been expressed in Xenopus oocytes and their properties have been described. In order to establish an alternative system in which urea transporters could be efficiently expressed and studied, we determined the urea transport properties of ovarian oocytes from Bufo arenarum, a toad species common in Argentina. Bufo oocytes presented a high urea permeability of 22.3 x 10(-6) cm/s, which was significantly inhibited by the incubation with phloretin. The urea uptake in these oocytes was also inhibited by mercurial reagents, and high-affinity urea analogues. The urea uptake was not sodium dependent. The activation energy was 3.2 Kcal/mol, suggesting that urea movement across membrane oocytes may be through a facilitated urea transporter. In contrast, Bufo oocytes showed a low permeability for mannitol and glycerol. From these results, we propose that one or several specific urea transporters are present in ovarian oocytes from Bufo arenarum. Therefore, these oocytes cannot be used in expression studies of foreign urea transporters. The importance of Bufo urea transporter is not known but could be implicated in osmotic regulation during the laying of eggs in water.     

Inhibition of Bufo arenarum oocyte maturation induced by cholesterol depletion by methyl-beta-cyclodextrin. Role of low-density caveolae-like membranes

The invaginated structure of caveolae seems to provide an optimal environment for hormone binding leading to oocyte meiotic maturation. We conducted a quantitative analysis of lipids and proteins of detergent-free low-density membranes isolated from Bufo arenarum oocytes and we modulated cellular cholesterol to further understand how these domains perform their regulatory functions in the amphibian system. Light membranes derive from the plasma membrane as suggested by the enrichment in the activity of 5'nucleotidase. Lipid analysis by chromatography techniques revealed that this fraction is enriched in phosphatidylserine and cholesterol and that it evidences an important level of sphingomyelin. The finding of a single 21 kDa caveolin in light membranes indicates the presence of caveolae-like structures in B. arenarum oocytes. In support of this finding, c-Src is significantly associated to this fraction. Cholesterol content of oocytes treated with methyl-beta-cyclodextrin (MbetaCD) decreased when compared to control oocytes. Drug treatment inhibited meiotic maturation in a dose-dependent manner and affected the localization of caveolin and c-Src among membrane fractions. Repletion of cholesterol showed a recovery of the ability of MbetaCD-treated oocytes to mature, particularly at the 25 mM concentration in which reversibility was close to the control level. Results highlight the importance of caveolae-like microdomains for maturation signaling in Bufo oocytes.     

The role of calcium in the nuclear maturation of Bufo arenarum oocytes

In Bufo arenarum, progesterone is the physiological maturation inducer. However, in this species, oocytes reinitiate meiosis with no need of an exogenous hormonal stimulus when deprived of their enveloping cell, a phenomenon called spontaneous maturation. We demonstrated that in Bufo arenarum spontaneous maturation occurs only in oocytes obtained during the reproductive period, which can be considered competent to mature spontaneously, in contrast to those in the non-reproductive period, which are incompetent. Interestingly, full-grown Bufo arenarum oocytes always respond to progesterone regardless of the season in which they are obtained. There is a general consensus that both a transient increase in intracellular calcium and a decrease in cAMP-dependent protein kinase activity are the first steps in the mechanisms by which progesterone induces maturation in amphibians. In the present work we analysed the role of calcium in the spontaneous and progesterone-induced maturation of Bufo arenarum oocytes. Results demonstrated that the absence of calcium in the incubation medium or the prevention of Ca(2+) influx by channel blockers such as CdCl2 or NiCl2 did not prevent meiosis reinitiation in either type of maturation. The inhibition of the Ca(2+)-calmodulin complex in no case affected the maturation of the treated oocytes. However, when the oocytes were deprived of calcium by incubation in Ca(2+)-free AR + A23187, meiosis resumption was inhibited. In brief, we demonstrated that in Bufo arenarum the reinitiation of meiosis is a process independent of extracellular calcium at any period of the year and that oocytes require adequate levels of intracellular calcium for germinal vesicle breakdown to occur.     

Effect of insulin on spontaneous and progesterone-induced GVBD on Bufo arenarum denuded oocytes

Progesterone is considered as the physiological steroid hormone that triggers meiosis reinitiation in amphibian oocytes. Nevertheless, isolated oocytes can be induced to undergo germinal vesicle breakdown (GVBD) in a saline medium by means of treatment with various hormones or inducing agents such as other steroid hormones, insulin or an insulin-like growth factor. It has been demonstrated that Bufo arenarum oocytes obtained during the reproductive period (spring-summer) resume meiosis with no need of an exogenous hormonal stimulus if deprived of their enveloping follicle cells, a phenomenon called spontaneous maturation. This study was undertaken to evaluate the participation of the purine and phosphoinositide pathway in the insulin-induced maturation of oocytes competent and incompetent to mature spontaneously, as well as to determine whether the activation of the maturation promoting factor (MPF) involved the activation of cdc25 phosphatase in Bufo arenarum denuded oocytes. Our results indicate that insulin was able to induce GBVD in oocytes incompetent to mature spontaneously and to enhance spontaneous and progesterone-induced maturation. In addition, high intracellular levels of purines such as cAMP or guanosine can reversibly inhibit the progesterone and insulin-induced maturation process in Bufo arenarum as well as spontaneous maturation. Assays of the inhibition of phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolysis and its turnover by neomycin and lithium chloride respectively exhibited a different response in insulin- or progesterone-treated oocytes, suggesting that phosphoinositide turnover or hydrolysis of PIP2 is involved in progesterone- but not in insulin-induced maturation. In addition, the inhibitory effect of vanadate suggests that an inactive pre-maturation promoting factor (pre-MPF), activated by dephosphorylation of Thr-14 and Tyr-15 on p34cdc2, is present in Bufo arenarum full-grown oocytes; this step would be common to both spontaneous and hormone-induced maturation. The data presented here strongly suggest that insulin initiates at the cell surface a chain of events leading to GVBD. However, our studies point to the existence of certain differences between the steroid and the peptide hormone pathways, although both involve the decrease in intracellular levels of cAMP, the activation of phosphodiesterase (PDE) and the activation of pre-MPF.     

Role of oviductal pars recta extract on interspecific fertilization between anurans

During their journey through the oviductal pars recta, the vitelline envelope (VE) of Bufo arenarum oocytes encounter structural alterations that make them sensitive to attack by sperm lysin and thus to penetration by sperm cells. The role of pars recta (PR) on the specificity of fertilization between amphibians was analyzed by conditioning Bufo arenarum oocytes with either homologous PR extract (PRE) or Leptodactylus chaquencis PRE. The oocytes were thereafter exposed to sperm lysin preparations from both species. Lysis of the VE only took place when the oocytes were exposed to the homologous PRE. The pattern of protein composition of PRE of these species was strikingly different as shown by Coomassie blue staining of SDS-PAGE. Moreover, antibodies against PR fluid (PRF) of Bufo arenarum produced seven bands of immunoprecipitation in electrophoresed homologous PRE and only one faint band in Leptodactylus chaquencis PRE. Here we show that: (i) the biological activity of PR from Bufo arenarum and Leptodactylus chaquencis over the VE of Bufo arenarum oocytes is species-specific; (ii) this specificity seems to be based in differences in protein structure, which was indicated by the fact that proteins from PRE of Leptodactylus chaquencis and Bufo arenarum were antigenically distinct; (iii) the specificity was solely related to PR activity and not to sperm lysin activity since sperm lysin preparations from both species showed comparable activity.     

Relationship of ATP Turnover, Polyphosphoinositide Metabolism, and Protein Phosphorylation in Sciatic Nerve and Derived Peripheral Myelin Subfractions from Normal and Streptozotocin Diabetic Rats

Sciatic nerve from streptozotocin-induced diabetic rats has previously been shown to incorporate more 32P into phosphatidylinositol-4,5-bisphosphate (PIP2) and the principal myelin proteins than normal nerve. In the present study, labeling of ATP and PIP2 was compared. Using nerve segments, [gamma-32P]ATP specific activity reached a plateau after incubation for 4 h with [32P]orthophosphate, whereas the specific activity of [32P]PIP2 rose much more slowly and was still increasing after 8 h. The rate of disappearance of radioactivity from prelabeled ATP was biphasic, with 75% being lost within 30 min and the remainder declining much more slowly for several hours thereafter. In contrast, no decrease in prelabeled PIP2 radioactivity could be detected for up to 4 h. The kinetics of ATP metabolism were not appreciably different for normal and diabetic nerve. However, after incubation with [32P]orthophosphate for 2 h, the specific activity of PIP2 was 50-120% higher in diabetic nerve. This phenomenon, therefore, cannot be ascribed to altered specific activity of the ATP precursor pool. Greater labeling of PIP2 in 32P-labeled diabetic nerve was present in purified myelin isolated using a simple discontinuous sucrose density gradient, but not in a "nonmyelin" fraction. When nerve homogenate was fractionated on a more complex gradient, three myelin-enriched subfractions were obtained which were heterogeneous as judged by morphological appearance, protein profile, and lipid metabolic activity. The proportion of total lipid radioactivity accounted for by PIP2 was elevated in all the subfractions relative to the homogenate. As compared to myelin subfractions from normal nerve, an increased percentage of 32P in PIP2 was obtained only in the major myelin subfraction from diabetic nerve. The phosphorylation of P0 relative to the other myelin proteins was also enhanced in this subfraction in nerve from diabetic animals.     

Spontaneous and LH-induced maturation in Bufo arenarum oocytes: importance of gap junctions

It has been demonstrated in Bufo arenarum that fully grown oocytes are capable of meiotic resumption in the absence of a hormonal stimulus if they are deprived of their follicular envelopes. This event, called spontaneous maturation, only takes place in oocytes collected during the reproductive period, which have a metabolically mature cytoplasm. In Bufo arenarum, progesterone acts on the oocyte surface and causes modifications in the activities of important enzymes, such as a decrease in the activity of adenylate cyclase (AC) and the activation of phospholipase C (PLC). PLC activation leads to the formation of diacylglycerol (DAG) and inositol triphosphate (IP(3)), second messengers that activate protein kinase C (PKC) and cause an increase in intracellular Ca(2+). Recent data obtained from Bufo arenarum show that progesterone-induced maturation causes significant modifications in the level and composition of neutral lipids and phospholipids of whole fully grown ovarian oocytes and of enriched fractions in the plasma membrane. In amphibians, the luteinizing hormone (LH) is responsible for meiosis resumption through the induction of progesterone production by follicular cells. The aim of this work was to study the importance of gap junctions in the spontaneous and LH-induced maturation in Bufo arenarum oocytes. During the reproductive period, Bufo arenarum oocytes are capable of undergoing spontaneous maturation in a similar way to mammalian oocytes while, during the non-reproductive period, they exhibit the behaviour that is characteristic of amphibian oocytes, requiring progesterone stimulation for meiotic resumption (incapable oocytes). This different ability to mature spontaneously is coincident with differences in the amount and composition of the phospholipids in the oocyte membranes. Capable oocytes exhibit in their membranes higher quantities of phospholipids than incapable oocytes, especially of PC and PI, which are precursors of second messengers such as DAG and IP(3). The uncoupling of the gap junctions with 1-octanol or halothane fails to induce maturation in follicles from the non-reproductive period, whose oocytes are incapable of maturing spontaneously. However, if the treatment is performed during the reproductive period, with oocytes capable of undergoing spontaneous maturation, meiosis resumption occurs in high percentages, similar to those obtained by manual defolliculation. Interestingly, results show that LH is capable of inducing GVBD in both incapable oocytes and in oocytes capable of maturing spontaneously as long as follicle cells are present, which would imply the need for a communication pathway between the oocyte and the follicle cells. This possibility was analysed by combining LH treatment with uncoupling agents such as 1-octanol or halothane. Results show that maturation induction with LH requires a cell-cell coupling, as the uncoupling of the gap junctions decreases GVBD percentages. Experiments with LH in the presence of heparin, BAPTA/AM and theophylline suggest that the hormone could induce GVBD by means of the passage of IP(3) or Ca(2+) through the gap junctions, which would increase the Ca(2+) level in the oocyte cytoplasm and activate phosphodiesterase (PDE), thus contributing to the decrease in cAMP levels and allowing meiosis resumption.     

Rapid decreases in phosphatidylinositol in isolated luteal plasma membranes after stimulation by luteinizing hormone

Phospholipid concentrations were determined in plasma membrane preparations from porcine corpora lutea after incubation for 15 to 120 s without or with 0.5 microgram/ml luteinizing hormone (LH) or 2 microM dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP). Treatment with LH caused a dramatic loss of 9 nmol in plasma membrane phosphatidylinositol (PI)/mg protein after 15 s of incubation, but no significant changes in other measurable phospholipids. Also, phospholipid concentrations were unchanged in untreated and dbcAMP-treated plasma membranes. The nature of the LH-induced decrease in PI was studied by incubating plasma membrane preparations for 15 s with [gamma 32P] adenosine 3',5'-triphosphate (ATP). 32P was incorporated only into three phospholipids: phosphatidic acid, phosphatidylinositol 4'-phosphate (PIP), and phosphatidylinositol 4',5'-bisphosphate (PIP2). Although LH generated small but significant increases in labeling of PIP and PIP2, less than 0.5 nmol of total phospholipids/mg protein were radiolabeled in 15 s. Phosphatidylinositol kinase activity, the enzyme that converts PI into PIP, was not affected by LH or dbcAMP treatment. However, incubation of luteal plasma membranes for 15 s with LH resulted in an increase of approximately 2 nmol 1,2-diacylglycerol/mg protein more than that observed in untreated or dbcAMP-treated plasma membranes. In summary, these experiments suggest that LH may stimulate hydrolysis of PI (and possibly PIP and PIP2) in isolated luteal plasma membranes.     

Effect of chlorpromazine on inositol-lipid signalling system in human thrombocytes

The effect of 0.5 mmol/l chlorpromazine (CPZ) on phospholipid metabolism, ATP content, and protein phosphorylation was studied in isolated human platelets. After 30 min incubation CPZ reduced the ATP content of the cells to 17% of the control. At the same time, the radioactivity in 32P prelabelled inositol lipids--phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol (PI), and phosphatidic acid (PA) decreased to 30, 51, and 61% of the controls, respectively, whereas an increase up to 188% of the control was observed in phosphatidylinositol 4-phosphate (PIP). A massive dephosphorylation of proteins was found. Thrombin, added to 32P prelabelled platelets for 90 s, increased the levels of radioactivity in phosphoinositides and PA. When added to CPZ--pretreated 32P prelabelled platelets, thrombin decreased the radio-activity in PIP2, PIP, and PA to 4, 86, and 10% of the control, respectively. We assume that the pharmacological effect of CPZ might be connected with the decreased ATP content, decreased PIP2 pool and with the impairment of protein phosphorylation.     

Lipid metabolic pathways operating in amphibian full-grown oocytes

The utilization of (2-3H)-glycerol in lipid biosynthesis was analyzed in Bufo arenarum and Xenopus laevis full-grown oocytes. The precursor was more actively incorporated in Xenopus laevis oocytes. Neutral glycerides were the lipids displaying the highest levels of radioactivity followed by phosphatidylcholine and phosphatidylethanolamine. After reincubation of prelabeled oocytes in a saline buffer solution, a net fall in labeled phosphatidic acid concomitant with an increase in phosphatidylcholine were detected. The present findings establish that glycerolipid biosynthesis is operative in full-grown oocytes. In addition, Xenopus laevis oocytes seem to be metabolically more active.     

Phosphatidic acid is a specific activator of phosphatidylinositol-4-phosphate kinase.

The lipid dependence of phosphatidylinositol-4-phosphate (PIP) kinase purified from bovine brain membranes was investigated. In the assay used, PIP-Triton X-100 micelles containing the lipid to be tested were presented to the enzyme. Under these conditions, phosphatidic acid (PA) stimulated the enzyme activity in a concentration-dependent manner up to 20-fold when an equal molar ratio of PA to PIP was attained. Stimulation by PA was highly specific; other lipids including lyso-PA and dicetylphosphate had a relatively small effect. The activation by PA was completely suppressed by phosphatidylinositol 4,5-bisphosphate (PIP2). To investigate the effect of PA on PIP kinase activity in natural membranes, endogenous PA was generated in rat brain synaptosomal plasma membranes by incubation with phospholipase D. Subsequent phosphorylation with [gamma-32P]ATP yielded an enhanced labeling of PIP2 but not of PIP in these membranes. These results suggest that PIP kinase activity may be under control of PA levels in membranes. This may have important implications for the regulation of cellular responses by agonist-induced phosphoinositide turnover.     

Involvement of purines and phosphoinositides in spontaneous and progesterone-induced nuclear maturation of Bufo arenarum oocytes

Although progesterone is the established maturation inducer in amphibia, it has been demonstrated that Bufo arenarum oocytes resume meiosis with no need of an exogenous hormonal stimulus if deprived of their enveloping follicle cells, a phenomenon called "spontaneous maturation." The present studies were designed to evaluate the participation of purines and phosphoinositides in the spontaneous and progesterone-induced maturation in Bufo arenarum full-grown oocytes. The presented data demonstrate that high intracellular levels of purines such as cAMP or guanosine can inhibit both spontaneous and progesterone-induced maturation in full-grown denuded Bufo arenarum oocytes. Moreover, the fact that the mycophenolic acid was able to induce maturation in denuded oocytes obtained during the nonreproductive period in a manner similar to that of the progesterone and also to increase the percentages of spontaneous maturation suggests that in Bufo arenarum, inosine monophosphate dehydrogenase inhibition is an important step in the resumption of meiosis. Inhibition of the phosphatidylinositol 4,5 bisphosphate hydrolysis by treatment of denuded oocytes with neomycin totally blocks spontaneous and progesterone-induced maturation, suggesting that the products of this hydrolysis (1,2 diacylglycerol and inositol 1,4,5 trisphosphate) may be involved in the maturation process of Bufo. In addition, our results indicate that the activation of protein kinase C is also involved in both types of maturation.     

Regulation of polyphosphoinositide synthesis in cardiac membranes

The relative distribution of phosphatidylinositol (PI) and phosphatidylinositol-4-phosphate (PIP) kinase activities in enriched cardiac sarcolemma (SL), sarcoplasmic reticulum (SR), and mitochondrial fractions was investigated. PI and PIP kinase activities were assayed by measuring 32P incorporation into PIP and phosphatidylinositol 4,5-bisphosphate (PIP2) from endogenous and exogenous PI in the presence of [gamma-32P]ATP. PI and PIP kinase activities were present in SL, SR, and mitochondrial fractions prepared from atria and ventricles although the highest activities were found in SL. A similar membrane distribution was found for PI kinase activity measured in the presence of detergent and exogenous PI. PI and PIP kinase activities were detectable in the cytosol providing exogenous PI and PIP and Triton X-100 were present. Further studies focused on characterizing the properties and regulation of PI and PIP kinase activities in ventricular SL. Alamethacin, a membrane permeabilizing antibiotic, increased 32P incorporation into PIP and PIP2 4-fold. PI and PIP kinase activities were Mg2+ dependent and plateaued within 15-20 min at 25 degrees C. Exogenous PIP and PIP2 (0.1 mM) had no effect on PIP and PIP2 labeling in SL in the absence of Triton X-100 but inhibited PI kinase activity in the presence of exogenous PI and Triton X-100. Apparent Km's of ATP for PI and PIP kinase were 133 and 57 microM, respectively. Neomycin increased PIP kinase activity 2- to 3-fold with minor effects on PI kinase activity. Calmidazolium and trifluoperazine activated PI kinase activity 5- to 20-fold and completely inhibited PIP kinase activity. Quercetin inhibited PIP kinase 66% without affecting PI kinase activity. NaF and guanosine 5'-O-(3-thiotriphosphate) had no effect on PI and PIP kinase activities, indicating that these enzymes were not modulated by G proteins. The probability that PIP and PIP2 synthesis in cardiac sarcolemma is regulated by product inhibition and phospholipase C was discussed.     

Turnover of protein-bound phosphorylserine in membrane preparations from ox brain catalysed by intrinsic kinase and phosphatase activity

1. The turnover of protein-bound phosphorylserine in preparations of membrane fragments from ox brain cortex was studied. 2. Turnover was considered to arise from the action of intrinsic protein kinases and phosphatases on a membrane protein or proteins. 3. Properties of the kinase system were studied by measuring the rate of incorporation of (32)P from gamma-labelled ATP into protein-bound phosphorylserine isolated from partial acid hydrolysates of membrane proteins. 4. Properties of the phosphatase system were studied by observing the rate of loss of (32)P from membrane preparations pre-labelled with [(32)P]ATP. 5. Net phosphorylation and dephosphorylation of membrane protein was observed during incubation of membrane preparations with and without ATP. 6. The rate of turnover was about 4nmol of P/h per mg of protein at 20 degrees C; dephosphorylation was considered to be the rate-limiting step.     

脂多糖增加小鼠巨噬细胞多磷酸肌醇脂代谢转换

多磷酸肌醇脂（这里指ＰＩＰ和ＰＩＰ２）的代谢在细胞信息传递和膜运转中起着重要的作用．脂多糖（ｌｉｐｏｐｏｌｙｓａｃｃｈａｒｉｄｅ,ＬＰＳ）于激活小鼠腹腔巨噬细胞（Ｍφ）初期（０．５～１ｈ）和后期（１６ｈ）明显增加来自［γ－３２Ｐ］ＡＴＰ的３２Ｐ参入Ｍφ的ＰＩＰ和ＰＩＰ２,３２Ｐ参入ＰＩＰ２的显著性大于ＰＩＰ．ＬＰＳ的这种作用在其激活Ｍφ的初期不受酪氨酸蛋白激酶抑制剂ｇｅｎｉｓｔｅｉｎ、蛋白激酶Ａ激动剂（ｆｏｒｓｋｏｌｉｎ）及百日咳毒素的影响;但佛波酯（ＰＭＡ）长时间预处理的Ｍφ（其ＰＫＣ活性被耗竭）再受ＬＰＳ刺激,［３２Ｐ］ＰＩＰ２水平较ＬＰＳ刺激未受ＰＭＡ预处理的Ｍφ明显降低．结果表明,ＬＰＳ于激活Ｍφ的初期和后期更显著增加ＰＩ４Ｐ－５激酶的活性,导致ＰＩＰ２合成增加,ＰＩＰ２合成的增加可能与Ｍφ激活时不同时期所表达的功能相关．     

Metabolic heterogeneity of phosphoglyceride classes and subfractions during cell cleavage and early embryogenesis: Model for cell membrane biogenesis

During the early developmental stages of the toad, Bufo arenarum, Hensel. up to the stage of gill circulation (150 hr of development at 20–25°C) the total phospholipids content as well as that of phosphoglycerides of choline and of ethanolamine were found unchanged. The subfraction of both phosphoglycerides were separated according to the number of double bonds on silver-ion chromatography and were also found to be unchanged up to the tail bud stage. The distribution of non-polar side chains in the subfractions varied in both phosphoglycerides showing a structural heterogeneity. In the phosphatidylethanolamines predominate the polyenoic containing subfractions. In contrast with the constant concentration of polar lipids, during early embryogenesis a steady increase in ³²P incorporation into phospholipids takes place when oocytes labeled during oogenesis are used. These changes were also correlated with the DNA content up to gill circulation stage. It is proposed that most of the nascent membrane polar lipids during early embryogenesis may be derived from a storage site through an active and specific intracellular redistribution process. At the arrival of the polar lipid to the nascent membrane a change in their covalent structure by introduction of a phosphorylbase from a highly labeled pool may explain the raise in specific activity. This change may be necessary to make possible the assembly of the lipid into the membrane structure.     

Membrane currents in the oocyte of the toad Bufo arenarum

The amphibian oocyte cell model is widely used for heterologous expression of ionic channels and receptors. Little is known, however, about the physiology of oocyte cell models other than Xenopus laevis. In this study, the two-electrode voltage clamp technique was used to assess the most common electrical patterns of oocytes of the South American toad Bufo arenarum. Basal membrane resistance, resting potential, and ionic currents were determined in this cell model. The oocyte transmembrane resistance was 0.35 M(Omega), and the resting potential in normal saline was about -33 mV with a range between -20 mV and -50 mV. This is, to our knowledge, the first attempt to begin an understanding of the ion transport mechanisms of Bufo arenarum oocytes. This cell model may provide a viable alternative to the expression of ion channels, in particular those endogenously observed in Xenopus laevis oocytes.     

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.     

Chemoattractant receptor-induced hydrolysis of phosphatidylinositol 4,5-bisphosphate in human polymorphonuclear leukocyte membranes. Requirement for a guanine nucleotide regulatory protein

Incubation of plasma membranes from human polymorphonuclear leukocytes (PMNs) with [gamma-32P]ATP in the presence of MgCl2 resulted in the formation of 32P-labeled phosphatidic acid (PA), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2). Membranes from PMN specific and azurophil granules synthesized only PIP, suggesting that PIP2 metabolism is confined to the plasma membrane in PMNs. Further incubations of the labeled plasma membranes for 60 s in the presence of 1 mM CaCl2 resulted in the hydrolysis of approximately 40 and 50% of the labeled PIP and PIP2, respectively. In the presence of 2 microM added CaCl2, PIP and PIP2 levels were unchanged by incubation with either the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe) at 0.1 microM or by 10 microM GTP; however, addition of fMet-Leu-Phe plus GTP together resulted in a 11 and 28% decrease in PIP and PIP2, respectively. These treatments had no effect on PA levels. No additional radiolabeled organic-soluble products were detected after treatment with fMet-Leu-Phe plus GTP. Incubation of intact PMNs, with the Bordetella pertussis toxin (islet-activating protein) eliminated the ability of fMet-Leu-Phe plus GTP to promote PIP2 breakdown in the isolated plasma membranes, but did not inhibit PIP2 degradation in the presence of 1 mM CaCl2. These results provide the first direct evidence that the fMet-Leu-Phe receptor in PMN membranes is coupled to polyphosphoinositide hydrolysis through an islet-activating protein-sensitive guanine nucleotide regulatory protein.     

Mitochondrial lipids in Bufo arenarum full-grown oocytes

Both the content and composition of polar and neutral lipids from the mitochondrial fraction of ovarian full-grown Bufo arenarum oocytes were analysed in the present study. Triacylglycerols (TAG) represent 33% of the total lipids, followed by phosphatidylcholine (PC), free fatty acids (FFA) and phosphatidylethanolamine (PE). Diphosphatidylglycerol (DPG) or cardiolipin, a specific component of the inner mitochondrial membrane, represents about 4% of the total lipid content. Palmitic (16:0) and arachidonic (20:4n6) acids are the most abundant fatty acids in PC and PE, respectively. DPG is enriched in fatty acids with carbon chain lengths of 18, the principal component being linoleic acid. In phosphatidylinositol (PI), 20:4n6 and stearic acid (18:0) represent about 72 mol% of the total acyl group level. The main fatty acids in TAG are linoleic (18:2), oleic (18:1), and palmitic acids. The fatty acid composition of FFA and diacylglycerols (DAG) is similar, 16:0 being the most abundant acyl group. PE is the most unsaturated lipid and sphingomyelin (SM) has the lowest unsaturation index.