Phosphatidylinositol-specific phospholipase C in fetal membranes and uterine decidua.

An assay procedure was developed in which phosphatidyl[2-(3)H]inositol was employed as substrate for the measurement of phosphatidylinositol-specific phospholipase C activity. Employing this assay, phosphatidylinositol-specific phospholipase C activity in human fetal membranes and uterine decidua was identified and characterized. The specific activity of this enzyme in amnion (4.4 mumol x mg(-1) protein x h(-1)) was three times that in uterine decidua and more than five times that in chorion laeve. No difference was found between the specific activity of phosphatidylinositol-specific phospholipase C in placental amnion and that in reflected amnion. The products of phosphatidylinositol hydrolysis in short-term incubations were stoichiometric amounts of diacylglycerol and inositol-1,2-cyclic-phosphate plus inositol-1-phosphate. After longer periods of incubation, monoacylglycerol also was detected. Diacylglycerol lipase activity also was demonstrated in these tissues. More than 90% of phosphatidylinositol-specific phospholipase C activity of amnion tissue was recovered in the 105,000-g supernatant fraction, and optimal enzymatic activity in vitro was observed at pH 6.5-7.5 in the presence of Ca(2+) (8 mM) and mercaptoethanol (4 mM). Phosphatidylinositol-specific phospholipase C activity was stimulated by fatty acids in low concentrations, but was inhibited by lysophosphatidylcholine and a variety of detergents. No effect of labor on the specific activity of phosphatidylinositol-specific phospholipase C in either fetal membranes or uterine decidua could be detected. The finding of an active phosphatidylinositol-specific phospholipase C activity in human fetal membranes and uterine decidua is complementary to our previous finding of a selective loss of arachidonic acid from phosphatidylinositol of human fetal membranes during labor. The action of phosphatidylinositol-specific phospholipase C, coupled to diacylglycerol lipase action, could provide a mechanism for the release of arachidonic acid for prostaglandin biosynthesis during parturition.

Hepatic insulin resistance in KKA(y) mice and its amelioration by pioglitazone do not involve alterations in phospholipase C activity.

It has been proposed that an abnormality in the regulation of cytosolic-free Ca2+ may be the cause of some forms of insulin resistance. In support of this proposition, it was reported that phospholipase C-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by liver plasma membranes from obese patients with non-insulin-dependent diabetes mellitus (NIDDM) was abnormally augmented (Thakker et al., J. Biol. Chem. 264, 7169-7175). The objective of this investigation was to determine if a novel antidiabetic agent, pioglitazone, ameliorated hepatic insulin resistance in KKA(y) mice and to identify any alterations in PIP2-phospholipase C activity of liver plasma membranes that may accompany changes in insulin sensitivity. Treatment of KKA(y) mice for 4 days with pioglitazone (20 mg/kg per day) decreased blood glucose and insulin and improved a variety of indices of hepatic insulin resistance, but did not alter the rate of PIP2 hydrolysis by liver plasma membranes. Acute treatment of isolated liver plasma membranes with pioglitazone (1-100 microM) also failed to alter PIP2-phospholipase C activity. Furthermore, the specific activity, Ca(2+)-requirement, pH-dependence and sensitivity to guanosine 5'-thiotriphosphate of the PIP2-phospholipase C in KKA(y) liver membranes were indistinguishable from those of C57BL/6J (normal) mice. Among C57BL/6J and KKA(y) mice fed either a control or pioglitazone-supplemented diet, there was no correlation between PIP2-phospholipase C activity in isolated liver membranes and either glucose or insulin concentrations in the circulation. These data indicate that an alteration in PIP2-phospholipase C activity of liver plasma membranes is neither a cause nor an obligatory consequence of insulin resistance in KKAy mice or its amelioration by pioglitazone. Alterations of liver membrane phospholipase C activity in NIDDM, therefore, may reflect diabetic pathology other than the insulin resistance associated with this disease.

CMP-dependent incorporation of [14C]Glycerol 3-phosphate into phosphatidylglycerol and phosphatidylglycerol phosphate by rabbit lung microsomes.

Rabbit lung microsomes were found to catalyze CMP-dependent incorporation of [14C]glycerol 3-phosphate into a total lipid extract. The radioactively labeled products in the lipid extract were identified as phosphatidylglycerol and phosphatidylglycerol phosphate. CMP-dependent incorporation of [14C]glycerol 3-phosphate by lung microsomes proceeded optimally at pH 7.4 and required Mn2+. The apparent Km value for CMP in this reaction was calculated to be 0.19 mM. No other cytidine nucleotide could substitute completely for CMP in supporting [14C]glycerol 3-phosphate incorporation into lipid. Cytosine-beta-D-arabinofuranoside-5'-monophosphate-dependent incorporation of [14C]glycerol 3-phosphate was observed at pH 8.5 but not at pH 6.8 CMP-dependent incorporation of [14C]glycerol 3-phosphate by microsomes was inhibited by inositol. The optimal in vitro rates of CMP-dependent and CDP diacylglycerol-dependent incorporation of [14C]glycerol 3-phosphate into lipid were similar (approximately 1 nmol . mg-1 protein . h-1) and were not additive. Both CMP -dependent and CDP diacylglycerol-dependent incorporation of [14C]glycerol 3-phosphate by lung microsomes appeared to involve CDPdiacylglycerol:glycerol-3-phosphate phosphatidyltransferase. However, the specific activity of this enzyme in a particular subcellular fraction did not relate directly in the extent of CMP-dependent [14C]glycerol 3-phosphate incorporation in that fraction. Preincubation of lung microsomes with 5 mM CMP plus 3 mM phosphatidylinositol increased CMP-dependent incorporation of [14C]glycerol 3-phosphate. When lung microsomes were depleted specifically of phosphatidylinositol by incubating with a phosphatidylinositol-specific phospholipase C, CMP-dependent incorporation was diminished. The Mn2+ requirement for CMP-dependent incorporation of [14C] glycerol 3-phosphate, its phosphatidylinositol requirement and its inhibition by Triton X-100 (0.2%) were not features shared by CDPdiacylglycerol-dependent incorporation of [14C]glycerol 3-phosphate but were characteristics of the reverse reaction catalyzed by CDPdiacylglycerol: inositol phosphatidyltransferase. Together with the previous finding of a developmental increase in the CMP content of fetal rabbit lung, these observations are consistent with a role for CMP in the regulation of the phosphatidylinositol and phosphatidylglycerol content of lung surfactant during lung maturation.

Phosphatidylinositol-inositol exchange in a rabbit lung.

A microsomal fraction prepared from rabbit lung tissue was found to catalyze CDPdiacylglycerol-independent incorporation of [3H]inositol into phosphatidylinositol. This incorporation resulted from CMP-dependent phosphatidylinositol-inositol exchange and did not constitute a net synthesis of phosphatidylinositol. The phosphatidylinositol-inositol exchange activity was distinct from the phospholipid-base exchange enzymes and was specific for inositol. Optimal in vitro phosphatidylinositol-inositol exchange activity was observed at pH 8.5--8.8 and either Mn2+ or Mg2+ was essential for activity. Mercaptoethanol stimulated phosphatidylinositol-inositol exchange and Hg2+ inhibited this activity. In the absence of CMP, no phosphatidylinositol-inositol exchange was observed. CDP (and to a smaller extent CTP) also supported phosphatidylinositol-inositol exchange and this appeared to occur via the generation of CMP during incubations. The apparent Km values of the phosphatidylinositol-inositol exchange enzyme for CMP and inositol were 0.4 mM and 11 microM, respectively. When CDPdiacylglycerol was present at a concentration optimal for CDPdiacylglycerol : inositol transferase activity, CMP-dependent phosphatidylinositol-inositol exchange activity was still observed. However, in the presence of Hg2+ CDPdiacylglycerol inhibited phosphatidylinositol-inositol exchange activity. Several properties of the phosphatidylinositol-inositol exchange enzyme resemble those of CDPdiacylglycerol : inositol transferase, but the two enzymes appear distinct on the basis of different degrees of inhibition by either Ca2+, Hg/+ or heat, and on the basis of different changes in activity during lung development.

The effects of polyamines and aminoglycosides on phosphatidylinositol-specific phospholipase C from human amnion.

The effects of polyvalent cations (polyamines and aminoglycoside antibiotics) on Ca2+-dependent phosphatidylinositol-specific phospholipase C activity of human amnion tissue were examined. In the presence of 1 mM Ca2+, the hydrolysis of phosphatidylinositol (2 mM) by phospholipase C was increased greatly (240-560% of control) by spermine (0.4 mM), spermidine (1 mM), neomycin (0.1 mM), gentamicin (0.2 mM), kanamycin (0.4 mM) and streptomycin (0.8 mM). Putrescine and cadaverine (0.1-2.0 mM), however, stimulated phospholipase C activity only slightly. The effects of spermidine, spermine and gentamicin on phospholipase C activity were characterized and found to be dependent upon the concentrations of phosphatidylinositol, Ca2+ and the particular polyvalent cation. At low concentrations of phosphatidylinositol and Ca2+ the predominant effect of polyamines and aminoglycosides was to inhibit phospholipase C activity. When the concentrations of phosphatidylinositol and Ca2+ were increased, spermidine, spermine and gentamicin stimulated phospholipase C activity. In the presence of 16 mM Ca2+, however, phospholipase C activity was maximal and was unaffected by either polyamines or aminoglycosides. At all concentrations of Ca2+ examined, the maximal stimulation of phospholipase C activity by a given polyvalent cation occurred at a fixed molar ratio of the particular polyvalent cation to phosphatidylinositol. Polyamines and aminoglycosides appeared to modulate the Ca2+ requirement for phospholipase C activity, but could not substitute completely for Ca2+. The activities of phospholipase A2, diacylglycerol lipase, monoacylglycerol lipase and diacylglycerol kinase in amnion tissue were unaffected by any of the polyvalent cations examined. It is proposed that any in vivo influences (stimulatory or inhibitory) of polyamines and aminoglycosides on amnion phospholipase C activity would depend upon the effective concentrations of Ca2+ and phosphatidylinositol.

Characterization of the forward and reverse reactions catalyzed by CDP-diacylglycerol:inositol transferase in rabbit lung tissue.

CDPdiacylglycerol:inositol transferase activity in rabbit lung tissue has been characterized and the optimum conditions for assaying this enzyme in vitro were determined. Rabbit lung tissue CDPdiacylglycerol:inositol transferase activity was found primarily in the microsomal fraction. The pH optimum of the enzyme activity was between 8.8 and 9.4, and the reaction was dependent on either Mn2+ or Mg2+. Detergents and Ca2+ inhibited the activity of the enzyme. The apparent Km values of the enzyme for CDPdioleoylglycerol and myoinositol were 0.18 mM and 0.10 mM, respectively. The reversibility of the reaction catalyzed by CDPdiacylglycerol:inositol transferase in microsomes prepared from rabbit lung tissue was demonstrated by the synthesis of [3H]CMPdiacylglycerol when [3H]CMP and phosphatidylinositol were present in the incubation mixture. The reverse reaction was characterized and its importance in the regulation of the acidic phospholipid composition of surfactant during lung development is discussed. The pH optimum for the reverse reaction was 6.2, and the reverse reaction was also dependent on Mn2+ or Mg2+. The apparent Km value of CDPdiacylglycerol:inositol transferase for CMP was found to be 2.8 mM.

The measurement of phosphatidate phosphohydrolase in human amniotic fluid.

Phosphatidate phosphohydrolase (EC 3.1.3.4) activity can be found in late gestational human amniotic fluid and is thought to originate in type II alveolar cells of the fetal lungs where it plays an important role in lung surfactant synthesis. In the present study, phosphatidate phosphohydrolase activity was detected and characterized in a 105 000 X g pellet of amniotic fluid using either [32P]phosphatidate or a water-soluble analog, 1-O-hexadecyl-rac-[2-(3)H]glycerol 3-phosphate as substrate. With either substrate, enzyme activity was optimal at pH 6.0. The soluble analog was hydrolyzed with a Km value of 163 micrometer and a V of 30 nmole/min per mg of protein, and offered several advantages over phosphatidate as a substrate for assaying phosphatidate phosphohydrolase in amniotic fluid. Using the synthetic analog, phosphatidate phosphohydrolase activity was measured in the 700 X g supernatant fraction of 30 human amniocentesis samples and compared with another index of fetal lung maturity, the phosphatidylcholine/sphingomyelin ratio. The results suggest that the new phosphohydrolase assay may be clinically useful in the assessment of fetal lung development.

Regulation of lamellar body acidic glycerophospholipid biosynthesis in fetal rabbit lung in organ culture.

To study the regulation of lamellar body acidic glycerophospholipid biosynthesis, fetal rabbit lung tissue obtained on day 23 of gestation was maintained in vitro. Tissues were cultured in serum-free medium with and without the addition of cortisol, thyroxine or a combination of both hormones. The addition of cortisol plus thyroxine to the medium resulted in the formation of lamellar bodies containing increased amounts of phosphatidylglycerol and decreased amounts of phosphatidylinositol. The addition of myo-inositol to culture medium containing cortisol plus thyroxine suppressed the incorporation of [14C]glycerol into both phosphatidylglycerol and bis(monoacylglycero)phosphate and enhanced the incorporation of [14C]glycerol into phosphatidylinositol. The effect of myo-inositol on the radioactive labeling of these lamellar body acidic glycerophospholipids was rapid, and was half-maximal at myo-inositol concentrations of approximately 0.10 mM.

Gonadotropin-releasing hormone-stimulated intracellular Ca2+ fluctuations and luteinizing hormone release can be uncoupled from inositol phosphate production.

In order to study the dependence of GnRH-stimulated LH release on inositol phosphate (IP) turnover, this study used an inhibitor of phospholipase C activity, 1-[6-[[17 beta-3- methoxyestra-1,3,5(10)-triene-17-yl]amino]hexyl]-1H-pyrrole-dione (U-73122) and an inactive analog 1-[6[[17 beta-3-methoxyestra-1,3,5(10)- triene-17-yl]amino]hexyl]2,5-pyrrolidine-dione (U-73343). U-73122 (10 microM) decreased GnRH-provoked (1 microM, 45 min) IP accumulation from 873 +/- 61 dpm to 365 +/- 50 dpm (basal accumulation also was decreased from 420 +/- 18 dpm to 207 +/- 16 dpm) while LH release was not inhibited (30.2 +/- 1.4% of cellular LH in control compared to 30.3 +/- 1.1% in U-73122 pretreated cells). GnRH provoked increased IP3 accumulation (123% of basal) after 15 sec of stimulation, IP2 accumulation (131% of basal) after 30 sec, and IP1 (121% of basal) after 1 min. Pretreatment with U-73122 blocked accumulation of IPs at these early timepoints. Sodium fluoride (NaF)-stimulated IP accumulation was also inhibited by U-73122 (from 1539 +/- 132 dpm to 414 +/- 21 dpm) while LH release increased from 22.9 +/- 1.4% total cellular LH to 28.0 +/- 2.2%. In contrast, GnRH- and NaF-stimulated IP accumulation were not significantly decreased in U-73343 pretreated cells (GnRH: 817 +/- 43 dpm compared to 873 +/- 61 dpm in control; NaF: 1133 +/- 74 dpm compared to 1539 +/- 132 dpm in control cells). Results of a perifusion study showed that U-73122 did not block the initial phase of GnRH-stimulated LH release or interfere with the development of desensitization to the releasing hormone. In addition, GnRH-stimulated intracellular Ca2+ fluctuations were similar in magnitude and duration in U-73122-pretreated compared to U-73343-pretreated cells. These results demonstrate that GnRH- as well as NaF-stimulated LH release can be uncoupled from IP production calling to question the role of IP3 as a second messenger for GnRH-stimulated LH release.

NPC 15669-modulated human polymorphonuclear neutrophil functional responsiveness: effects on receptor-coupled signal transduction.

1. The effect of NPC 15669, N-carboxy-L-leucine, N-[(2,7-dimethylfluoren-9-yl)methyl]ester), an inhibitor of human polymorphonuclear neutrophil (PMN) adhesion, on granule exocytosis and the oxidative burst was investigated in PMN activated with receptor-specific pathophysiological stimuli. 2. NPC 15669 caused a concentration-dependent (1-30 microM) inhibition of the extracellular release of azurophil (myeloperoxidase) and specific (vitamin B12-binding protein) granule constitutents from PMN exposed to N-formyl-methionyl-leucyl-phenylalanine (FMLP), leukotriene B4 (LTB4), platelet activating factor (PAF), C5a and interleukin-8 (IL-8). 3. The receptor agonist-triggered PMN oxidative burst, measured as superoxide anion (O2-) production, was suppressed by NPC 15669. 4. Phorbol myristate acetate (PMA)-stimulated degranulation and O2-) production were unaffected by NPC 15669. 5. NPC 15669 (0.1-10 microM) inhibited receptor-triggered inositol 1,4,5-trisphosphate (IP3) production and the IP3-triggered increase in cytosolic-free calcium ([Ca2+]i) in FMLP-activated PMN, but not in cells exposed to the other receptor agonists. 6. NPC 15669 suppressed FMLP but not PMA-stimulated redistribution of protein kinase C (PKC) in PMN. 7. The specific binding of [3H]-FMLP but not [125I]-C5a to PMN was inhibited by NPC 15669. 8. NPC 15669 suppressed O2- production and the rise in [Ca2+]i in PMN treated with the guanine nucleotide-binding protein (G-protein) activators, sodium fluoride (NaF) and mastoparan, respectively. 9. The results show that NPC 15669 inhibits PMN responsiveness to various receptor agonists, and suggest interference with receptor-coupled signal transduction in this inflammatory cell at both the receptor and post-receptor level in a stimulus-specific manner.

Effect of LHRH on incorporation of [32P]-orthophosphate into phosphatidylinositol by dispersed anterior pituitary cells.

Stimulated release of luteinizing hormone (LH) from the anterior pituitary in response to luteinizing hormone releasing hormone (LHRH), is apparently controlled by Ca2+-mediated events. In many tissues, when an involvement of Ca2+ in secretion has been found, there is an associated increased metabolic turnover of phosphatidylinositol. The objective of this investigation was to determine the effect of LHRH on the incorporation of [32P]orthophosphate into phosphatidylinositol by anterior pituitary cells maintained in vitro. When anterior pituitary cells were incubated for 5-40 min in the presence of [32P]orthophosphate, phosphatidylinositol and phosphatidylcholine were the most rapidly radiolabelled phospholipids. The addition of LHRH to the cell culture medium at concentrations previously demonstrated to release LH, increased the incorporation of 32P]orthophosphate into phosphatidylinositol in a dose-dependent manner. Incorporation of [32P]orthophosphate into other phospholipids was unaffected by LHRH at all concentrations employed. These results are consistent with the hypothesis that LHRH causes a receptor-mediated increase in turnover of phosphatidylinositol and this may be among the early metabolic events in the mechanism of LHRH-stimulated LH secretion from the anterior pituitary gland.

The cell surface progesterone receptor which stimulates calcium influx in human sperm is unlike the A ring reduced steroid site on the GABAA receptor/chloride channel.

Progesterone elicits a rapid, transient calcium influx in sperm that is a prerequisite for the progesterone-induced acrosome reaction. The possibility that the GABAA receptor/chloride channel was the receptor that mediated the progesterone-induced calcium influx in human sperm was examined. A-ring reduced 3 alpha-hydroxy pregnane steroids (e.g. alfaxalone, allopregnanolone, pregnanolone), which are active on the GABAA receptor/chloride channel, were found to be much weaker than progesterone at stimulating Ca2+ influx in sperm. The effects of a variety of progesterone metabolites and analogs and other steroids were compared for their ability to (i) stimulate GABA-induced 36Cl- uptake in synaptoneurosomes, (ii) stimulate GABA-induced Cl- currents in HEK-293 cells transfected with alpha 1, beta 2, and gamma 2 subunits of the GABAA receptor/chloride complex, and (iii) elicit a rapid Ca2+ influx in sperm. No correlation was observed between the ability of a given steroid to stimulate Ca2+ influx and efficacy in eliciting either 36Cl- uptake or chloride currents. Importantly, the action of progesterone to stimulate Ca2+ influx was not modified by GABA, diazepam, picrotoxin and pentobarbitol (known regulators of the GABAA receptor/chloride channel). It is concluded from these studies that the cell surface progesterone binding site on human sperm that mediates progesterone-induced changes in [Ca2+]i is unlike the steroid binding site on the GABAA receptor/chloride channel.(ABSTRACT TRUNCATED AT 250 WORDS)

Pioglitazone promotes insulin-induced activation of phosphoinositide 3-kinase in 3T3-L1 adipocytes by inhibiting a negative control mechanism.

Activation of phosphoinositide 3-kinase (PI 3-kinase) is an early event in insulin signal transduction that is blocked completely in adipocytes from insulin-resistant KKAy mice. Treatment of KKAy mice with pioglitazone, an anti-diabetic thiazolidinedione, partially restores insulin-dependent changes in PI 3-kinase. The mechanism of this effect of pioglitazone was investigated using murine 3T3-L1 cells as an experimental model. Insulin and insulin-like growth factor I (IGF-I) each elicited rapid (within 2 min) and large (2-5-fold) increases in PI 3-kinase activity that could be immunoprecipitated using anti-phosphotyrosine (pY) antibodies. Maximal insulin-induced activity of PI 3-kinase in pY-immunoprecipitates was similar in 3T3-L1 adipocytes and mouse adipocytes, but the kinetics of activation differed. Insulin- and IGF-I-induced changes in PI 3-kinase were each half-maximal at 3-5 nM of hormone and were not additive. Increases in both insulin-induced and IGF-I-induced pY-immunoprecipitable PI 3-kinase activity were observed when 3T3-L1 fibroblasts became confluent and when they adopted the adipocyte phenotype. Pioglitazone (10 microM), administered either acutely or chronically to either 3T3-L1 adipocytes or 3T3-L1 fibroblasts, did not greatly alter the kinetics, magnitude or sensitivity of changes in PI 3-kinase elicited by either insulin or IGF-I. In contrast, the attenuation by isoproterenol of insulin-induced changes in PI 3-kinase was prevented in cells pretreated with pioglitazone. This effect of pioglitazone did not involve inhibition of isoproterenol-elicited accumulation of cyclic AMP. Pioglitazone also prevented attenuation of insulin induced changes in PI 3-kinase by cell penetrating analogs of cyclic AMP. Pioglitazone, therefore, has no direct effect on insulin-stimulated PI 3-kinase activity, but interferes with a cyclic AMP-dependent mechanism that normally antagonizes this action of insulin. These data support the proposition that the facilitation of insulin action by pioglitazone involves, at least in part, an inhibition of a negative control mechanism.

Pioglitazone promotes insulin-induced activation of phosphoinositide 3-kinase in 3T3-L1 adipocytes by inhibiting a negative control mechanism.

Activation of phosphoinositide 3-kinase (PI 3-kinase) is an early event in insulin signal transduction that is blocked completely in adipocytes from insulin-resistant KKAy mice. Treatment of KKAy mice with pioglitazone, an anti-diabetic thiazolidinedione, partially restores insulin-dependent changes in PI 3-kinase. The mechanism of this effect of pioglitazone was investigated, using murine 3T3-L1 cells as an experimental model. Insulin and insulin-like growth factor I (IGF-I) each elicited rapid (within 2 min) and large (2- to 5-fold) increases in PI 3-kinase activity that could be immunoprecipitated using anti-phosphotyrosine (pY) antibodies. Maximal insulin-induced activity of PI 3-kinase in pY-immunoprecipitates was similar in 3T3-L1 adipocytes and mouse adipocytes, but the kinetics of activation differed. Insulin- and IGF-I-induced changes in PI 3-kinase were each half-maximal at 3-5 nM of hormone and were not additive. Increases in both insulin-induced and IGF-I-induced pY-immunoprecipitable PI 3-kinase activity were observed when 3T3-L1 fibroblasts became confluent and when they adopted the adipocyte phenotype. Pioglitazone (10 microM), administered either acutely or chronically to either 3T3-L1 adipocytes or 3T3-L1 fibroblasts, did not alter greatly the kinetics, magnitude or sensitivity of changes in PI 3-kinase elicited by either insulin or IGF-I. In contrast, the attenuation by isoproterenol of insulin-induced changes in PI 3-kinase was prevented in cells pretreated with pioglitazone. This effect of pioglitazone did not involve inhibition of isoproterenol-elicited accumulation of cyclic AMP. Pioglitazone also prevented attenuation of insulin induced changes in PI 3-kinase by cell penetrating analogs of cyclic AMP. Pioglitazone, therefore, has no direct effect on insulin-stimulated PI 3-kinase activity, but interferes with a cyclic AMP-dependent mechanism that normally antagonizes this action of insulin. These data support the proposition that the facilitation of insulin action by pioglitazone involves, at least in part, an inhibition of a negative control mechanism.

Analysis of in vitro interactions of protein tyrosine phosphatase 1B with insulin receptors.

One strategy to treat the insulin resistance that is central to type II diabetes mellitus may be to maintain insulin receptors (IR) in the active (tyrosine phosphorylated) form. Because protein tyrosine phosphatase 1B (PTP1B) binds and subsequently dephosphorylates IR, inhibitors of PTP1B-IR binding are potential insulin 'sensitizers.' A Scintillation Proximity Assay (SPA) was developed to characterize and quantitate PTP1B-IR binding. Human IR were solubilized and captured on wheat germ agglutinin (WGA)-coated SPA beads. Subsequent binding of human, catalytically inactive [35S] PTP1B Cys(215)/Ser (PTP1B(C215S)) to the lectin-anchored IR results in scintillation from the SPA beads that can be quantitated. Binding of PTP1B to IR was pH- and divalent cation-sensitive. Ca(2+) and Mn(2+), but not Mg(2+), dramatically attenuated the loss of PTP1B-IR binding observed when pH was raised from 6.2 to 7.8. PTP1B binding to IR from insulin-stimulated cells was much greater than to IR from unstimulated cells and was inhibited by either an antiphosphotyrosine antibody or treatment of IR with alkaline phosphatase, suggesting that tyrosine phosphorylation of IR is required for PTP1B binding. Phosphopeptides modeled after various IR phosphotyrosine domains each only partially inhibited PTP1B-IR binding, indicating that multiple domains of IR are likely involved in binding PTP1B. However, competitive displacement of [35S]PTP1B(C215S) by PTP1B(C215S) fitted best to a single binding site with a K(d) in the range 100-1000 nM, depending upon pH and divalent cations. PNU-200898, a potent and selective inhibitor of PTP1B whose orientation in the active site of PTP1B has been solved, competitively inhibited catalysis and PTP1B-IR binding with equal potency. The results of this novel assay for PTP1B-IR binding suggest that PTP1B binds preferentially to tyrosine phosphorylated IR through its active site and that binding may be susceptible to therapeutic disruption by small molecules.

myo-Inositol homeostasis in the human fetus.

The concentration of myo-inositol in the serum of pregnant women (21.4 microM) remained unchanged throughout pregnancy and was not significantly different from that in the serum of nonpregnant women (24.5 microM). myo-Inositol concentrations in mixed cord serum averaged 125 microM at midgestation. At term, the concentration of myo-inositol was 60.2 microM in the umbilical artery and 45.4 microM in the umbilical vein. In every umbilical cord examined, the ratio of myo-inositol concentration in umbilical artery to that in umbilical vein exceeded 1.0. Activity of a putative regulatory enzyme in the biosynthesis of myo-inositol from glucose, ie, glucose 6-P:inositol 1-P cyclase (cyclase), was measured in placenta, fetal lung, and fetal liver. In placenta at midgestation and at term, cyclase activity was 87.8 and 90.7 nmol X g-1 tissue X h-1, respectively. Cyclase activity in fetal lung and liver at midgestation was 54.6 and 54.4 nmol X g-1 tissue X h-1, respectively. The gestational decline in the concentration of myo-inositol in fetal serum may represent a decreasing availability of myo-inositol to the fetal lungs and could be important in the mechanism whereby the amounts of phosphatidylglycerol and phosphatidylinositol in lung surfactant are regulated during fetal lung development.

Phosphatidic acid metabolism, calcium ions and transmitter release from electrically stimulated synaptosomes.

Synaptosomes isolated from guinea pig brain cortex were stimulated electrically in a medium containing [32P]-orthophosphate. The electrical stimulation caused increased labelling of phosphatidic acid in a synaptic vesicle fraction prepared by osmotic shock of the incubated synaptosomes. Electrical stimulation also provokes transmitter release from the synaptosomes. Both increased phosphatidate labelling and transmitter release required calcium ions in the medium. The effects are discussed in relation to earlier work with acetylcholine and the possible involvement of membrane phosphatidic acid in transmitter release by exocytosis.