Phorbol ester binding to a human lymphoblastoid B-cell line, LA350, stimulates 32P incorporation into selected phospholipids and immunoglobulin secretion

Anti-mu antibody binds to surface IgM on LA350, a transformed human B-cell line, and causes the immediate (5 min) hydrolysis of phosphatidylinositol (PI) into inositol 1,4,5-triphosphate (IP3) and diacylglycerol followed by a subsequent (48-72 hr) increase in immunoglobulin M (IgM) production. Phorbol myristate acetate (PMA) in a dose-dependent fashion inhibited completely the anti-mu-stimulated hydrolysis of PI and its resynthesis (PI cycle) from phosphatidic acid (PA) (P less than 0.001). Phorbol dibutyrate (PD), but not the inactive methyl ester derivative of PMA (PMA-ME), inhibited the anti-mu stimulation of the PI cycle (P less than 0.001). Conversely, PMA and PD, but not PMA-ME, stimulated in a dose-dependent fashion the metabolic events consistent with an activation of a putative phosphatidylcholine (PC) cycle. For example, at 10(-8) M PMA there was a 300% increase in the acute (1 hr) incorporation of [3H]choline into PC (P less than 0.001), a 680% increase in the acute (1 hr) incorporation of 32P into PC (P less than 0.001), but no net synthesis of PC as measured by the lack of PMA-stimulated incorporation of 32P into PC in LA350 prelabeled for 24 hr. Also in cells labeled to equilibrium with [3H]choline and in pulse-chase experiments we established that PMA produces a rapid incorporation of choline phosphate into PC and a rapid breakdown of PC, yielding choline metabolites released as choline itself into external medium surrounding the cell. Binding studies with [3H]PD demonstrated a dissociation constant of 20 mM and 5.3 x 10(5) total binding sites per cell. PMA was as effective as cold PD in inhibiting [3H]PD binding (P less than 0.001), but PMA-ME was ineffective. PMA and PD, but not PMA-ME, produced a similar dose-dependent (maximal at 10(-8) M) increase (300%) in immunoglobulin production as measured by either an ELISA assay or a reverse hemolytic plaque assay (P less than 0.001). Thus, activation of either the PI or the PC cycle results in significant enhancement in immunoglobulin production in LA350. Although PMA turns off the PI cycle, it turns on the PC cycle. A common mechanism to explain these findings might be the activation of protein kinase C, indirect via diacylglycerol release in the PI cycle stimulation by anti-mu and direct in the PC cycle stimulation by PMA by virtue of direct binding to protein kinase C.     

Antibody to human lymphocyte actin regulates immunoglobulin secretion by an EBV-transformed human B-cell line

Antibody against actin isolated from a human EBV-transformed lymphoblastoid B-cell line exerted an inhibitory effect on in vitro IgM secretion by a different lymphoblastoid B-cell line, LA350. This effect was dose dependent showing from 24-40% inhibition at a dilution of 1:100 and 68-80% inhibition at a dilution of 1:50. This effect was noted in the absence of changes in either total cell count or [3H]-thymidine incorporation and was reversed by co-incubation with purified rabbit thymus actin (100 micrograms/ml) but not bovine serum albumin at the same concentration. These data demonstrate regulation of immunoglobulin synthesis by antibodies against human lymphocyte derived actin in a lymphoblastoid B-cell line.     

The incorporation of [myo-2-3H] inositol into phosphatidyl inositol of stimulated rat pancreas

The 'phospholipid effect' involves agonist induced breakdown of phosphatidyl inositol (PI) or its phosphorylated derivates with increased incorporation of 32P or [myo-2-3H] inositol during resynthesis. In rat pancreas pancreozymin and bethanecol resulted in the standard dose dependent increased incorporation of 32P into PI which was paralleled by increased amylase secretion. By contrast the incorporation of [myo-2-3H] inositol into PI was significantly decreased by pancreozymin whereas bethanecol had no effect. However, pancreozymin caused a 30% decrease in labelled PI irrespective of whether it was prelabelled with 32P or [myo-2-3H] inositol. Thus in rat pancreas, pancreozymin resulted in the standard agonist induced breakdown of pre-labelled PI but inhibited the incorporation [2-3H-myo] inositol during the resynthetic phase.     

Evidence for a platelet-activating factor receptor on human lymphoblastoid B cells: activation of the phosphatidylinositol cycle and induction of calcium mobilization

In this report we demonstrate evidence which strongly suggests that a receptor for platelet-activating factor (PAF) exists on a lymphoblastoid B cell line, LA350. PAF ranging in concentration from 10(-6)-10(-9)M initiated the incorporation of 32P into phosphatidic acid (PA) and phosphatidylinositol (PI) with no change in phosphatidylethanolamine (PE) and phosphatidylcholine (PC) over baseline. Lyso-PAF, the inactive precursor, at 10(-7)M had no effect on membrane phospholipid metabolism. In addition, PAF from 10(-6)-10(-8)M when added to Fura-2 containing B cells induced a rapid and significant rise of calcium within the cell, with lyso-PAF having no effect. These data suggest that PAF binds to a receptor on B cells and induces the hydrolysis of PI and a subsequent increase of intracellular calcium.     

Platelet-activating factor induces an increase in intracellular calcium and expression of regulatory genes in human B lymphoblastoid cells.

Platelet-activating factor (PAF) has recently been demonstrated to be metabolized by B lymphocytes and to cause enhancement of Ig synthesis by Ig-secreting B lymphoblastoid cell lines. We have now examined some of the early activation events triggered by PAF binding to three Ig-secreting B cell lines, LA350 (IgM secreting), HSCE- (IgG secreting), and U266 (IgE secreting). After addition of 10(-7) to 10(-11) M PAF, but not equimolar concentrations of the inactive metabolite lyso-PAF, all three cell lines demonstrated rapid dose-dependent increases in free cytosolic Ca2+ concentrations ([Ca2+]i). The increases in [Ca2+]i resulted from both the release of Ca2+ from internal stores as well as transmembrane Ca2+ uptake. Addition of PAF triggered the rapid hydrolysis of phosphatidylinositol bisphosphate and accumulation of inositol phosphates. PAF also increased expression of the cell cycle-active genes c-fos and EGR2 in a dose-dependent fashion. The stimulated increases in [Ca2+]i and phosphatidylinositol bisphosphate hydrolysis and the increases in gene expression were all inhibited by the specific PAF receptor antagonist Web 2086. The LA350 cell line (which expresses surface IgM) was also shown to increase [Ca2+]i after addition of anti-IgM antibodies. Sequential addition of PAF or anti-IgM antibody in either order failed to reveal any evidence for heterologous desensitization. Furthermore, the PAF receptor antagonist did not affect anti-IgM induced changes in [Ca2+]i. These data provide evidence for the presence of functional PAF receptors on B lymphoblastoid cells and indicate a potential role for PAF in the regulation of B cell activation.     

Biochemical analysis of inhibitory effects of a lymphokine suppressive B-cell factor on the activation process of resting B cells

Suppressive B-cell factor (SBF) is elaborated by FcR gamma (Fc receptor for IgG)-bearing small, resting B cells after the stimulation of immune complexes and is known to inhibit humoral immune responses by acting on resting B cells. In order to elucidate where and how SBF interferes with B-cell activation in the course of transmembrane signaling, we examined the effect of SBF on the several sequential events which B cells undergo after crosslinking surface immunoglobulin (sIg). Hyper-Ia expression, plasma membrane depolarization, and activation of phosphatidylinositol (PI) hydrolysis of resting B cells, all of which were induced by the stimulation with anti-mu antibody, were significantly suppressed by the pretreatment of cells with SBF. However, SBF had no effect on the intracytoplasmic cyclic AMP level of either activated or resting B cells. Another inhibitory effect of SBF on the activation process of resting B cells by anti-mu antibody was to suppress the transient elevation of intracytoplasmic free Ca2+ only in the initial phase after triggering with anti-mu antibody. This seems to be due to a decrease in the release of inositol triphosphate into the cytoplasm by suppressing the activation of PI hydrolysis. Considering all the data, the suppressive effect of SBF on the transmembrane signaling by sIg crosslinking is ascribed to the selective suppression of the activation of PI hydrolysis. This provides a concept on a molecular basis that feedback regulation of humoral immune response is, at least partly, regulated by SBF.     

Effects of prostaglandins and luteinizing hormone-releasing hormone on phosphatidic acid-phosphatidylinositol labeling in rat granulosa cells

In cultures of rat granulosa cells, luteinizing hormone-releasing hormone (LHRH) increases 32P incorporation into both phosphatidylinositol (PI) and phosphatidic acid (PA). After 20 min, the level of radioactivity was three- to four-fold (p less than 0.01) above control in the PI and PA fractions, respectively. The stimulatory effect of LHRH on 32P incorporation was limited to PI and PA. Similar to the effects of LHRH, a rapid and marked increase of 32P incorporation into both PI and PA is observed upon addition of prostaglandin F2 alpha (PGF2 alpha) (10(-5)M) to rat granulosa cells. Incorporation of radioactivity into PA was already increased (p less than 0.05) by 2 min following PGF2 alpha addition, while the increase in 32P-labeled PI became significant (p less than 0.01) by 5 min. In contrast to PGF2 alpha, the labeling of PI and PA following the addition of PGE2 (10(-5)M) was not significantly different from control levels during the entire 10 min of incubation. The sensitivity of the increased PA-PI labeling induced by LHRH and PGF2 alpha is compared in another experiment. After 20 min incubation 10(-6)M LHRH increased PI and PA labeling by six- and four-fold, respectively. Although the effect of PGF2 alpha is less than that of LHRH, 10(-5)M PGF2 alpha significantly (p less than 0.01) increased PI and PA labeling by three- and two-fold, respectively. By contrast, 10(-6)M PGE2 failed to affect 32P incorporation into the various phospholipid fractions, but a small enhancement (p less than 0.05) of PI and PA labeling was observed only at 10(-5)M PGE2.(ABSTRACT TRUNCATED AT 250 WORDS)     

AGEPC (platelet activating factor) induced stimulation of rabbit platelets: effects on phosphatidylinositol, di- and triphosphoinositides and phosphatidic acid metabolism

Stimulation of washed rabbit platelets with AGEPC (1-O-alkyl-2-acetyl-$\text{sn}$-glyceryl-3-phosphorylcholine) caused a 15–20% decrease in their phosphatidylinositol level within 15 seconds without affecting other major classes of phospholipids. In the same time frame the level of phosphatidic acid (PA) increased dramatically some four fold. LysoGEPC, which is inactive in stimulating rabbit platelets, did not cause any change in PI or PA. When [³²Pi] was present during the stimulation of platelets by AGEPC, the incorporation of radiolabel into PI-4-phosphate (DPI), PI-4,5-bis phosphate (TPI) and PA was enhanced significantly within one minute while the incorporation into PI increased only after one minute. These results clearly established that AGEPC induced stimulation of rabbit platelets was associated with the metabolism of inositol phospholipids and phosphatidic acid. The relevance of these findings to the mode of action of AGEPC and Ca²⁺ mobilization is also discussed.     

Effect of elevated potassium on phospholipid and inositol metabolism of isolated nerve endings

Synaptosomes were isolated from rat cerebra, and incubated in the presence of labelled phosphate and inositol. When the potassium concentration of the medium was increased by replacing NaCl with KCl, there was a marked increase in phosphate labeling of phosphatidic acid (PA) and phosphatidylinositol (PI). This was evident with [K⁺] above 12 mM and peaked at about 40 mM KCl. In normal calcium buffers, phosphate labeling of PI but not PA declined sharply with [KCl] above 40 mM. In low calcium buffers, the phosphate labeling response was greatly attenuated for both lipids, but PI labeling did not decline at higher [K⁺].The phosphate labeling response was confined to PA and PI, and was specific for the increase in [K⁺]₀. The same response was seen in constant (105 mM) sodium buffers, and atropine had no effect. The specific radioactivity of ATP was increased by elevated potassium, but not enough to account for the increased labeling of PA. Further, this appeared to be a result of the loss of stored ATP rather than an increase in turnover.Increasing [K⁺]₀ produced a decline in [³H]inositol incorporation into PI in parallel with the increase in its labeling by ³³PO₄. This was the same in constant sodium and in low calcium buffers. It could be attributed to an inhibition of synaptosomal uptake of labelled inositol from the medium. Synaptosomal inositol content was unaffected.Elevated potassium had a greater effect on PA labeling than on PI, and it was more effective in increasing phosphate labeling of PA than was acetylcholine (ACh). When ACh and elevated potassium were combined at their maximally effective concentration, they acted synergistically to stimulate phosphate incorporation into PA but elevated potassium blocked the increase in [³H]inositol incorporation into PI normally produced by ACh. These results indicate that elevated potassium and ACh act upon the same population of synaptosomes, but affect different biochemical steps. Elevated potassium probably effects phospholipid labeling by a calcium dependent increase in diglyceride production from lipids other than PA or PI.     

Effects of eicosatetraynoic acid (ETYA) on cultured pig thyroid cells. Relationships between the inhibition of the phosphatidate-phosphatidyl inositol cycle, the iodination and the cyclic AMP responsiveness to thyrotropin

The arachidonate inhibition of the adenylate-cyclase system of cultured pig thyroid cells was not mediated by cyclooxygenase, lipoxygenase or peroxidase metabolites. Indeed ETYA, an inhibitor of cyclooxygenase and lipoxygenase, and methimazole, an inhibitor of peroxidase and iodination were without effect on the arachidonate inhibition. Moreover the effect of arachidonate was amplified by a combination with ETYA. In 32P incorporation experiments we observed a modification of the labelling of individual phospholipids of cultured pig thyroid cells resulting in a decrease into phosphatidylinositol (PI) and an increase into phosphatidate (PA) of arachidonate and ETYA-treated cells. These results may be explained by an inhibition of CDP-diacylglycerol: inositol transferase and conversely a stimulation of PI specific phospholipase C yielding a decrease in PI and an increase in PA, which inhibits in turn adenylate cyclase activity possibly by Ca2+ translocation.     

Signaling through surface IgM in tolerance-susceptible immature murine B lymphocytes. Developmentally regulated differences in transmembrane signaling in splenic B cells from adult and neonatal mice.

During the course of B lymphocyte development, newly emerging surface Ig+ B cells pass through a stage when Ag-Ag receptor interactions lead not to immune responsiveness but to a state of functional tolerance. We have explored the molecular basis of antigenic nonresponsiveness and tolerance susceptibility using tolerance-susceptible surface Ig+ splenic B lymphocytes from neonatal mice and anti-mu chain antibodies as a polyclonal ligand. In this population of cells, surface IgM is uncoupled from the inositol phospholipid (PI)-hydrolysis pathway at a point proximal to the receptor; anti-mu antibodies did not stimulate inositol phosphate generation despite the fact that PI-hydrolysis was observed after treatment with A1F4, implicating the existence of a functional G protein and phospholipase C. Further evidence for a difference early in the signal transduction pathway stems from the finding that anti-mu stimulation does not induce the expression of two immediate/early PKC-linked genes egr-1 and c-fos. This appears to be the primary signaling difference between the mature and immature B cells from the neonatal mouse splenic population, as these cells undergo a G0-G1 cell cycle phase transition when surface IgM is bypassed using phorbol diester and calcium ionophore. Interestingly, despite undetectable levels of PI-hydrolysis, we observed equivalent receptor-mediated changes in intracellular calcium when comparing the immature and mature populations. These results indicate incomplete coupling of surface IgM to the signal transduction machinery operative in mature, immunocompetent B cells and suggests a molecular mechanism accounting for the differential processing of surface IgM signals into activation vs tolerogenic responses observed in these two stages of B cell development.     

Linoleic acid-enriched phospholipids act through peroxisome proliferator-activated receptors alpha to stimulate hepatic apolipoprotein A-I secretion

A uniquely formulated soy phospholipid, phosphatidylinositol (PI), is under development as a therapeutic agent for increasing plasma high-density lipoprotein (HDL) levels. Soy PI has been shown to increase plasma HDL and apolipoprotein A-I (apoA-I) levels in phase I human trials. Low micromolar concentrations of PI increase the secretion of apoA-I in model human hepatoma cell lines, through activation of G-protein and mitogen-activated protein (MAP) kinase pathways. Experiments were undertaken to determine the importance of the PI head group and acyl chain composition on hepatic apoA-I secretion. Phospholipids with choline and inositol head groups and one or more linoleic acid (LA) acyl chains were shown to stimulate apoA-I secretion by HepG2 cells and primary human hepatocytes. Phospholipids containing two LA groups (dilinoleoylphosphatidylcholine, DLPC) were twice as active as those with only one LA group and promoted a 4-fold stimulation in apoA-I secretion. Inhibition of cytosolic phospholipase A2 with pyrrolidine 1 (10 microM) resulted in complete attenuation of PI- and DLPC-induced apoA-I secretion. Pretreatment with the peroxisome proliferator-activated receptor alpha (PPARalpha) inhibitor MK886 (10 microM) also completely blocked PI- and DLPC-induced apoA-I secretion. Hepatic PPARalpha expression was significantly increased by both PI and DLPC. However, in contrast to that seen with the fibrate drugs, PI caused minimal inhibition of catalytic activities of cytochrome P450 and UGT1A1 enzymes. These data suggest that LA-enriched phospholipids stimulate hepatic apoA-I secretion through a MAP kinase stimulation of PPARalpha. LA-enriched phospholipids have a greater apoA-I secretory activity than the fibrate drugs and a reduced likelihood to interfere with concomitant drug therapies.     

The role of phosphatidylinositol turnover in initiation of prostaglandin biosynthesis in concanavalin A-induced activation of rat peritoneal macrophages

When macrophages were stimulated by concanavalin A (50 micrograms/ml) in the presence of 32Pi and 3H-arachidonic acid (AA), rapid incorporation of these radiolabeled substances into phosphatidylinositol (PI) and phosphatidic acid (PA) were induced within 10 min. The pool of PI and PA incorporating 3H-AA was the same as that of PI and PA which incorporated 32P. However, significant increase in incorporation of 32P and 3H-AA did not occur in phosphatidylcholine, phosphatidylethanolamine or phosphatidylserine within this time interval as compared with control levels. These results indicate that PI turnover might play a role in the initiation of prostaglandin biosynthesis in the early stages of macrophage activation induced by concanavalin A.     

Phosphoinositides in mitogenesis: neomycin inhibits thrombin-stimulated phosphoinositide turnover and initiation of cell proliferation

Thrombin stimulates 32Pi incorporation into phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bis-phosphate (PIP2), and phosphatidylinositol (PI), and initiates DNA synthesis in hamster (NIL) fibroblasts at a half-maximal concentration of 125 ng/ml. Neomycin, which binds PIP2 and PIP, inhibits both thrombin-stimulated initiation of cell proliferation and 32P pI incorporation into at concentrations above 2 mM without affecting thrombin binding, thymidine uptake, or cellular protein synthesis. At lower concentrations, neomycin inhibits thrombin-stimulated release of inositol 1,4,5-trisphosphate (IP3), by selectively binding PIP2, but does not inhibit 32P incorporation into PI or initiation of DNA synthesis. Phosphoinositide recycling and diacylglycerol release therefore appear necessary for initiation of cell proliferation by thrombin. IP3-stimulated Ca++ mobilization may not be required for thrombin mitogenesis, however, since neomycin can block IP3 release without inhibiting initiation.     

Role of phosphatidylinositol 3-kinase in anti-IgM- and anti-IgD-induced apoptosis in B cell lymphomas

Cross-linking of surface Ig receptors with anti IgM (anti-mu heavy chain, anti-mu), but not anti-IgD (anti-delta heavy chain, anti-delta), Abs leads to growth arrest and apoptosis in several extensively characterized B cell lymphomas. By poorly understood mechanisms, both Igs transiently stimulate c-Myc protein expression. However, ultimately, only anti-mu causes a severe loss in c-Myc and a large induction of p27(Kip1) protein expression. Because phosphatidylinositol 3-kinase (PI3K) has been established as a major modulator of cellular growth and survival, we investigated its role in mediating anti-Ig-stimulated outcomes. Herein, we show that PI3K pathways regulate cell cycle progression and apoptosis in the ECH408 B cell lymphoma. Anti-mu and anti-delta driven c-Myc protein changes precisely follow their effects on the PI3K effector, p70(S6K). Upstream of p70(S6K), signaling through both Ig receptors depresses PI3K pathway phospholipids below control with time, which is followed by p27(Kip1) induction. Conversely, anti-delta, but not anti-mu stimulated PI3K-dependent phospholipid return to control levels by 4-8 h. Abrogation of the PI3K pathway with specific inhibitors mimics anti-mu action, potentiates anti-mu-induced cell death and, importantly, converts anti-delta to a death signal. Transfection with active PI3K kinase construct induces anti-mu resistance, whereas transfection with dominant negative PI3K augments anti-mu sensitivity. Our results show that prolonged disengagement of PI3K or down-regulation of its products by anti-mu (and not anti-delta) determines B cell fate.     

Stimulatory effects of estrogen on gonadotropin-releasing hormone-induced phosphoinositide turnover in granulosa cells.

Gonadotropin-releasing hormone (Gn-RH) stimulates phosphoinositide metabolism in granulosa cells by binding to its specific receptor, and suppresses gonadotropin-induced steroidogenesis. Incubation of immature rat granulosa cells with Gn-RH stimulated time-sequential [32P]phosphate incorporation into phosphatidic acid (PA) and phosphatidylinositol (PI) in a dose-dependent manner; EC50 was at 10 nM. Concurrent exposure to estradiol-17 beta (E2) (100 nM) and Gn-RH (1 microM) augmented 32P-labeling of PI by 5-fold, while Gn-RH alone induced 3.5-fold increase in PI-labeling. In cells preincubated with E2 for 48 h, Gn-RH provoked a 7-fold [32P]phosphate incorporation into PI, suggesting the induction by E2 of Gn-RH-responsible phosphoinositide turnover. E2 alone provoked a low but significant increase in basal labeling rate of PA and PI. Progesterone failed to mimic the action of E2. Essentially similar results were also obtained in mature rat granulosa cells. These results indicate that E2 augments Gn-RH-stimulated phospholipid turnover in granulosa cells, and suggest that estrogens within the microenvironment of the ovary may exert a local autoregulatory effect on their own production pathway through accelerating Gn-RH action to attenuate steroidogenesis.     

Functional studies on B cell hybridomas with B cell surface antigens. I. Effects of anti-immunoglobulin antibodies on proliferation and differentiation

B cell hybridomas with Ia and IgM molecules on the cell membrane were treated with either purified goat anti-mouse mu antibody (anti-mu) or monoclonal rat anti-mouse IgM antibody (anti-IgM). The spontaneous uptake of [3H] thymidine by these cells was markedly inhibited by both reagents. These hybrid cells could be induced to differentiate into IgM-secreting cells in the presence of these reagents at high frequency. Furthermore, the induction of IgM secretion by B cell hybridomas treated with these antibodies was completely T cell independent, and cell division was not required for the differentiative response to anti-mu. In addition, F(ab')2 fragments of anti-mu showed more effects on proliferation and differentiation of these cells than intact anti-mu. Interestingly, TH2.54, a subline of B cell hybridomas, could generate IgG2a production as well as IgM when incubated with anti-mu. These findings suggest very strongly that the interaction of either goat anti-mu or monoclonal rat anti-IgM with surface IgM molecules on the cell membrane of the B cell hybridomas inhibits in vitro spontaneous proliferation, and results in providing signals for differentiation into Ig-secreting cells without T cell factors.     

The effects of ACTH, serotonin, K+ and angiotensin analogues on 32P incorporation into phospholipids of the rat adrenal cortex: basis for an assay method using zona glomerulosa cells

The effects of various concentrations of serotonin, ACTH, K+, angiotensin II (AII), angiotensin III (AIII) and [Sar1]angiotensin II (SAII) on steroidogenesis and the incorporation of 32P (after preincubation to near equilibrium with the ATP pool) into phosphatidylinositol (PI), phosphatidic acid (PA) and phosphatidylcholine (PC) in a preparation of capsular cells from rat adrenals, consisting of 95% zona glomerulosa (z.g.) and 5% zona fasciculata plus reticularis (z.f.r.) cells, were investigated. Serotonin and ACTH stimulated steroidogenesis in the usual manner but had little or no effect on 32P incorporation into any of the three phospholipids. However, AII, AIII and SAII stimulated steroidogenesis and also 32P incorporation into PA and PI (maximally to about 280% of control values) but not into PC. These results taken together with other data on effects on the cAMP output and Ca2+ fluxes of z.g. cells suggest that stimulation by ACTH and serotonin is mediated by cAMP as second messenger. However, the angiotensins probably act through Ca2+, with associated changes in phospholipid metabolism. The 32P incorporation into PA as a function of lg concentration of AII was linear and showed a reasonable index of precision (0.36 +/- 0.03, eight experiments, 0.23 +/- 0.02 for a further eight experiments) and correlation with steroidogenesis. The corresponding incorporation into PI showed a maximum effect and a much poorer index of precision (1.02 +/- 0.30 (4.69 +/- 3.7] over the same full range of AII concentration used. The effects of AIII and SAII showed similar characteristics for 32P incorporation into both PA and PI, but, as for stimulation of steroidogenesis, at higher concentrations for AIII than for AII. The effects of different doses of AII, AIII and ACTH on the corticosterone output and 32P incorporation into PA, PI and PC of a preparation of cells, consisting of more than 98% z.f.r. cells, from rat decapsulated adrenals were also studied. ACTH, at low doses, which nevertheless markedly stimulated corticosterone output, had a small (maximally to about 125% of control values) but significant effect on 32P incorporation into PA, PI and PC. The maximum effect was usually at about 10(-10) M ACTH and was not significant at 10(-8) M.     

The de novo phospholipid effect of insulin is associated with increases in diacylglycerol, but not inositol phosphates or cytosolic Ca2+.

We have previously reported that insulin increases the synthesis de novo of phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2) and diacylglycerol (DAG) in BC3H-1 myocytes and/or rat adipose tissue. Here we have further characterized these effects of insulin and examined whether there are concomitant changes in inositol phosphate generation and Ca2+ mobilization. We found that insulin provoked very rapid increases in PI content (20% within 15 s in myocytes) and, after a slight lag, PIP and PIP2 content in both BC3H-1 myocytes and rat fat pads (measured by increases in 32P or 3H content after prelabelling phospholipids to constant specific radioactivity by prior incubation with 32Pi or [3H]inositol). Insulin also increased 32Pi incorporation into these phospholipids when 32Pi was added either simultaneously with insulin or 1 h after insulin. Thus, the insulin-induced increase in phospholipid content appeared to be due to an increase in phospholipid synthesis, which was maintained for at least 2 h. Insulin increased DAG content in BC3H-1 myocytes and adipose tissue, but failed to increase the levels of inositol monophosphate (IP), inositol bisphosphate (IP2) or inositol trisphosphate (IP3). The failure to observe an increase in IP3 (a postulated 'second messenger' which mobilizes intracellular Ca2+) was paralleled by a failure to observe an insulin-induced increase in the cytosolic concentration of Ca2+ in BC3H-1 myocytes as measured by Quin 2 fluorescence. Like insulin, the phorbol diester 12-O-tetradecanoylphorbol 13-acetate (TPA) increased the transport of 2-deoxyglucose and aminoisobutyric acid in BC3H-1 myocytes. These effects of insulin and TPA appeared to be independent of extracellular Ca2+. We conclude that the phospholipid synthesis de novo effect of insulin is provoked very rapidly, and is attended by increases in DAG but not IP3 or Ca2+ mobilization. The insulin-induced increase in DAG does not appear to be a consequence of phospholipase C acting upon the expanded PI + PIP + PIP2 pool, but may be derived directly from PA. Our findings suggest the possibility that DAG (through protein kinase C activation) may function as an important intracellular 'messenger' for controlling metabolic processes during insulin action.