Inositol Phospholipid Breakdown Research Articles

Sir Michael John Berridge. 22 October 1938—13 February 2020

Sir Michael Berridge was a giant in the fields of physiology and biochemistry who, by his discovery in 1983 of inositol 1,4,5-trisphosphate (IP 3 ) as the ubiquitous intracellular messenger releasing Ca 2+ from intracellular stores, revolutionized our concepts of signal transduction mechanisms. Mike, as he was universally known, discovered one of the most important regulatory mechanisms in animal cells, involved in the control of virtually all bodily functions, including secretion, contraction and memory. Mike, working at the Department of Zoology at the University of Cambridge, personally identified IP 3 as one of the two initial products of hormone- or neurotransmitter-elicited inositol phospholipid breakdown and then, in collaboration with the research group led by Irene Schulz at the Max Planck Institute for Biophysics in Frankfurt, demonstrated directly the Ca 2+ -releasing effect of IP 3 in pancreatic acinar cells. Subsequently, in collaborations with research groups in Switzerland and the USA, Mike confirmed the Ca 2 + -releasing action of IP 3 in insulin-secreting cells, hepatocytes and photoreceptors. The IP 3 –Ca 2 + -releasing pathway became accepted amazingly quickly as one of the key elements of cellular signal transduction mechanisms and is now featured in virtually all textbooks of physiology and biochemistry. Mike was also a great synthesizer, who created an entirely new field, namely calcium signalling. Through his numerous, highly cited review articles, and his insightful keynote lectures at the most important biomedical congresses, he dominated this large and increasingly important research field for more than 30 years following his momentous 1983 discovery.

Stimulation by noradrenaline of inositol phospholipid breakdown in the rat hippocampus: effect of the ambient potassium concentration.

The stimulation by noradrenaline (NA) of inositol phospholipid (PI) breakdown has been studied using rat hippocampal miniprisms. Pretreatment with the monoamine oxidase inhibitor, pargyline, potentiated the stimulation produced by NA. On the other hand, pargyline pretreatment did not affect the stimulation of PI breakdown by the alpha 1-adrenoceptor agonist phenylephrine. NA- and phenylephrine-stimulated PI breakdown were enhanced by increasing the ambient potassium concentration in the assay from 5.88 to 18.2 mM. This enhancement did not, in the case of NA, change either the EC50 value for this agonist (2-3 microM) or the pA2 value for the competitive antagonism of the stimulation by the alpha 1-antagonist prazosin (pA2 value 9.2). Time-courses of the NA-stimulated PI turnover in different brain regions indicated that the rate of stimulation was in the order frontal cortex greater than hypothalamus greater than or equal to hippocampus much greater than cerebellum.

Alpha 1-adrenoceptor function in the rat hippocampus as assessed by noradrenaline-stimulated inositol phospholipid breakdown after destruction of noradrenergic neurons by neonatal 6-hydroxydopamine treatment.

Neonatal 6-hydroxydopamine treatment of male Sprague-Dawley rats resulted in a large depletion of noradrenaline 84-87 days later, as demonstrated by an 82% reduction in the intra-synaptosomal deamination of [14C]noradrenaline within noradrenergic synaptosomes with only marginal effects upon the extrasynaptosomal deamination. The hippocampal stimulation of inositol phospholipid breakdown by noradrenaline was, however, unaffected by the neonatal 6-hydroxydopamine treatment.

Investigation into the effects in-vitro of the 5-hydroxytryptamine reuptake inhibitor, alaproclate, on carbachol-stimulated inositol phospholipid breakdown in the rat cerebral cortex.

The effect of alaproclate in carbachol-stimulated inositol phospholipid (PI) breakdown in rat cerebral cortical miniprisms has been investigated. Carbachol-stimulated PI breakdown was greatly enhanced by increasing the assay potassium concentration from 5.88 to 18.2 mM. Alaproclate, on the other hand, did not influence carbachol-stimulated PI breakdown over the concentration range tested (0-100 microM) at either assay [K+]. The elution pattern of the inositol phosphates from the Dowex-1 columns was also unaffected by alaproclate both in the absence and presence of carbachol. Thus, the potentiation by alaproclate of tremor and salivation induced by the muscarinic agonist oxotremorine in-vivo reported previously is not seen when muscarinic function is measured in-vitro using carbachol-stimulated PI breakdown.

Novel Role of Cold/Menthol-sensitive Transient Receptor Potential Melastatine Family Member 8 (TRPM8) in the Activation of Store-operated Channels in LNCaP Human Prostate Cancer Epithelial Cells

Recent cloning of a cold/menthol-sensitive TRPM8 channel (transient receptor potential melastatine family member 8) from rodent sensory neurons has provided the molecular basis for the cold sensation. Surprisingly, the human orthologue of rodent TRPM8 also appears to be strongly expressed in the prostate and in the prostate cancer-derived epithelial cell line, LNCaP. In this study, we show that despite such expression, LNCaP cells respond to cold/menthol stimulus by membrane current (I(cold/menthol)) that shows inward rectification and high Ca(2+) selectivity, which are dramatically different properties from "classical" TRPM8-mediated I(cold/menthol). Yet, silencing of endogenous TRPM8 mRNA by either antisense or siRNA strategies suppresses both I(cold/menthol) and TRPM8 protein in LNCaP cells. We demonstrate that these puzzling results arise from TRPM8 localization not in the plasma, but in the endoplasmic reticulum (ER) membrane of LNCaP cells, where it supports cold/menthol/icilin-induced Ca(2+) release from the ER with concomitant activation of plasma membrane (PM) store-operated channels (SOC). In contrast, GFP-tagged TRPM8 heterologously expressed in HEK-293 cells target the PM. We also demonstrate that TRPM8 expression and the magnitude of SOC current associated with it are androgen-dependent. Our results suggest that the TRPM8 may be an important new ER Ca(2+) release channel, potentially involved in a number of Ca(2+)- and store-dependent processes in prostate cancer epithelial cells, including those that are important for prostate carcinogenesis, such as proliferation and apoptosis.

Endocytosis Function of a Ligand-Gated Ion Channel Homolog in Caenorhabditis elegans

Ligand-gated ion channels are transmembrane proteins that respond to a variety of transmitters, including acetylcholine, gamma-aminobutyric acid (GABA), glycine, and glutamate [1 and 2]. These proteins play key roles in neurotransmission and are typically found in the nervous system and at neuromuscular junctions [3]. Recently, acetylcholine receptor family members also have been found in nonneuronal cells, including macrophages [4], keratinocytes [5], bronchial epithelial cells [5], and endothelial cells of arteries [6]. The function of these channels in nonneuronal cells in mammals remains to be elucidated, though it has been shown that the acetylcholine receptor alpha7 subunit is required for acetylcholine-mediated inhibition of tumor necrosis factor release by activated macrophages [4]. We show that cup-4, a gene required for efficient endocytosis of fluids by C. elegans coelomocytes, encodes a protein that is homologous to ligand-gated ion channels, with the highest degree of similarity to nicotinic acetylcholine receptors. Worms lacking CUP-4 have reduced phosphatidylinositol 4,5-bisphosphate levels at the plasma membrane, suggesting that CUP-4 regulates endocytosis through modulation of phospholipase C activity.

Extracellular matrix stimulates production and breakdown of inositol phospholipids.

Adhesion of rat glomerular epithelial cells (GEC) to collagen stimulates production of D-myo-inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol. This process is mediated via beta 1-integrins, and it modulates GEC proliferation. In this study, we address the changes in inositol-lipid turnover induced by GEC adhesion to extracellular matrix (ECM). The masses of both phosphatidylinositol 4,5-bisphosphate (PIP2) and IP3, as well as [3H]inositol phosphates, were increased in GEC adherent to collagen, compared with plastic substratum. Phosphatidylinositol-4-phosphate (PIP) 5-kinase activity was predominantly membrane associated and was enhanced in GEC on collagen. Phospholipase C (PLC) activity and PLC-gamma 1 protein were increased in membrane fractions of GEC adherent to collagen, compared with plastic. Stable overexpression of PLC-gamma 1 in GEC amplified the effect of ECM on the production of [3H]inositol phosphates. In addition, the PLC-gamma 1 that was membrane associated in collagen-adherent GEC was tyrosine phosphorylated. Thus production of IP3 in GEC adherent to ECM is associated with increased production of PIP2. Moreover, adhesion to ECM increases tyrosine phosphorylation and membrane association of PLC-gamma 1, which may facilitate PIP2 hydrolysis by increasing the catalytic activity of PLC-gamma 1 and the proximity of PLC-gamma 1 and its substrate. Understanding the process of ECM-induced inositol lipid production and breakdown in GEC may provide insights into the regulation of GEC proliferation and differentiated functions in normal conditions and during glomerular injury.

RETRACTED: Functional characterization of a nonclassical nicotine receptor associated with inositolphospholipid breakdown and mobilization of intracellular calcium pools.

Classical nicotinic receptors are neurotransmitter-gated channels that, upon activation by acetylcholine, induce the opening of an intrinsic cationic channel. We have recently observed that, in frog pituitary melanotrophs, nicotine stimulates alpha-melanocyte-stimulating hormone (alpha-MSH) release through a noncholinergic mechanism. In the study reported here, we investigated the intracellular events that mediate the response of frog melanotrophs to nicotine. Nicotine was capable of stimulating alpha-MSH release in the absence of Ca2+ and/or Na+ in the extracellular medium. A short pulse of nicotine induced a rapid and transient increase of cytosolic free Ca2+ concentration ([Ca2+]i). The effect of nicotine on Ca2+ mobilization was not affected in the absence of Na+ and Ca2+ in the extracellular medium, indicating that the nicotine-evoked increase in [Ca2+]i did not result from Na+ or Ca2+ influx. Nicotine induced both an increase in inositol trisphosphate and a reduction in phosphaditylinositol bisphosphate concentrations but did not affect cAMP production. The present results indicate that nicotine-induced stimulation of alpha-MSH release in frog melanotrophs can be explained by activation of inositolphospholipid breakdown and mobilization of inositol triphosphate-dependent intracellular Ca2+ pools. These data provide evidence for the existence of an unusual type of noncholinergic nicotine receptor positively coupled to phospholipase C.

Dissociation between the phospholipases C and A2 activities in stimulated platelets and their involvement in the arachidonic acid liberation

In previous work we have demonstrated that platelets depleted from secretory phospholipase A2 (sPLA2) produced similar amounts of thromboxane (Tx)B2 as control platelets upon stimulation by thrombin. However, since depletion of sPLA2 was not total, this sole finding only suggested the non-involvement of sPLA2 in arachidonic acid release. In the present study we provide further evidence for the non-involvement of sPLA2 in arachidonic acid liberation during platelet activation. Thus, rabbit platelets exposed to thrombin secreted sPLA2, released free arachidonic acid and formed TxB2 and inositol phosphates. In contrast, U46619, a stable prostaglandin (PG)H2 analogue, activates phospholipase C (PLC) and induces release of sPLA2 without TXB2 generation nor arachidonic acid liberation. At each concentration tested of both agonists, stimulation of sPLA2 activity paralleled the production of inositol phosphates. These data suggest that sPLA2 is dependent on phosphoinositide hydrolysis and on the release reaction and that it is not involved in the liberation of arachidonic acid from stimulated platelets. In addition, a dissociation was observed between sPLA2 and the enzyme involved in the arachidonic acid mobilization, suggesting that the liberation of this fatty acid from membrane phospholipids was mediated by cytosolic phospholipase A2 (cPLA2). Finally, PLC does not play a major role in arachidonic acid liberation, since U46619, which induced the breakdown of inositol phospholipids, failed to release arachidonic acid. In confirmation, neomycin, which inhibits PLC activity, failed to inhibit ATP, sPLA2 and arachidonic acid release upon stimulation of platelets by fluoroaluminate. These data demonstrate that sPLA2 is not involved in the arachidonic acid release by stimulated platelets and indicate that the activations of PLC, sPLA2 and cPLA2 are independent events.

Ethanol-induced signal transducing mechanism associated with a transient antiviral state in human amniotic cells.

Short-term effects of ethanol on human amnion cells were investigated by studying the cellular signaling processes and the replication of vesicular stomatitis virus. Treatment of human amniotic cells with ethanol transiently triggers the breakdown of inositol phospholipids, stimulates intracellular [Ca2+]i mobilization and activates the translocation of protein kinase C. Activation of this signal transduction mechanism is associated with the development of an antiviral state, as proven by studying 3H-uridine incorporation into the RNA of vesicular stomatitis virus. Induction of the antiviral state in human amniotic cells correlates with the solubility of the alcohols in the lipid membrane of the cells.

Lithium and the brain: a psychopharmacological strategy to a molecular basis for manic depressive illness

Lithium, an effective treatment for mania and the prevention of recurrent episodes of both mania and depression in patients with manic depressive illness, exerts multiple biochemical effects. However, any clinically relevant site of action of lithium must occur at therapeutic concentrations attained in the brain of patients and must account for the lag period accompanying onset of action as well as effects persisting beyond discontinuation of treatment. This monovalent cation acts as a potent uncompetitive inhibitor in the receptor-coupled breakdown of inositol phospholipids, resulting in a relative depletion of inositol and an alteration in the generation of diacylglycerol, an endogenous activator of protein kinase C. In our laboratory, we are examining the action of chronically administered lithium on posttranslational modification of specific phosphoproteins involved in regulating signal transduction in the brain. We have found that chronic, but not acute, administration of lithium in rats markedly reduces a major phosphoprotein substrate of protein kinase C in the hippocampus, an effect that persists beyond the cessation of lithium treatment. This protein, myristoylated alanine-rich C kinase substrate ("MARCKS"), is implicated in synaptic neurotransmission, calcium regulation, and cytoskeletal restructuring. These findings have relevance for the long-term action of lithium in stabilizing an underlying dysregulation in the brain and may move us closer to formulating a molecular basis of manic depressive illness.

Protein kinase C down-regulation in astrocytes: Differential effects on agonist-stimulated inositol phosphate accumulation

Prolonged phorbol ester treatment abolished protein kinase C (PKC) activity for over 48 h in cortical astrocyte cultures. Receptor-stimulated inositol phospholipid breakdown in these cultures subsequent to PKC depletion produced either enhanced, depressed or unchanged responses depending upon the agonist used. Noradrenaline-, ATP-, histamine- and glutamate-evoked [3H]-inositol phosphate accumulations were potentiated to varying degrees in PKC-depleted cultures whilst those evoked by carbachol and NaF were reduced and unchanged respectively. Analysis of the individual metabolites of inositol phospholipid metabolism formed in response to noradrenaline in PKC-depleted astrocytes revealed potentiated accumulations of radiolabelled glycerophosphoinositol (GPI), inositol monophosphate (IP1) and inositol bisphosphate (IP2) but not inositol trisphosphate (IP3) when compared to controls. Under the same conditions, accumulations of radiolabelled IP1 and IP2 were reduced and those of GPI and IP3 unchanged in carbachol-treated cultures. These results suggest that astrocyte receptors coupled to inositol phospholipid metabolism are differentially regulated by PKC.

Quantification of inositol phospholipid breakdown in isolated rat hepatocytes

The hydrolysis of inositol phospholipids induced by vasopressin in hepatocytes during 60 min was quantified chemically. There was a large release of myo-inositol which was abolished by Li+, indicating that it was derived from inositol phosphates and not from phospholipase D action on PtdIns. There was also a large release of inositol phosphates which was increased approx. 2-fold by Li+ at 30 min, but then remained constant, suggesting that inositol phospholipid breakdown declined substantially beyond this time. In cells prelabelled with myo-[3H]inositol and treated with Li+, [3H]PtdIns(4,5)P2 decreased maximally (50%) at 15 s and then recovered to a level at 5 min that was maintained at 25% below control for 40 min. [3H]PtdIns4P and [3H]PtdIns showed slower decreases to approx. 30% below control at 15 min, but with no further changes. Labelled Ins(1,4,5)P3 and Ins(1,3,4)P3 showed 2-4-fold increases within 30 s and then declined to values that were maintained at a constant level above the control, except for [3H]Ins(1,3,4)P3, which showed a second increase. [3H]Ins(1,4)P2 showed a very large increase over 10 min, whereas [3H]Ins4P and [3H]Ins1P showed little change before 6 and 15 min respectively. The total [3H]inositol phosphates showed little further increase after 20 min. These data are consistent with a rapid, but not sustained, hydrolysis of PtdIns-(4,5)P2, but not of PtdIns, by phospholipase C, but do not exclude PtdIns4P as a substrate. Phosphatidate was rapidly increased by vasopressin, whereas diacylglycerol was increased after a 1-2 min lag. Both were maintained at levels 2-3-fold above control for 60 min. The vasopressin-induced increase in inositol phosphates plus myo-inositol (approx. 120 nmol/100 mg) was greater than the increase in diacylglycerol plus phosphatidate (approx. 60 nmol/100 mg) between 10 and 40 min. This indicates that there was substantial further metabolism of these lipids. Addition of 75 mM ethanol resulted in rapid production of phosphatidylethanol in response to vasopressin and a 35% reduction in phosphatidate, but no decrease in diacylglycerol. In summary, the results indicate that inositol phospholipid hydrolysis by phospholipase C can account for most of the diacylglycerol and phosphatidate that accumulate during 60 min of vasopressin action, but that these phospholipids are probably not the major source of the phosphatidate that is formed during the first 2 min by phospholipase D, or of the diacylglycerol and phosphatidate that are formed beyond 30 min.

Inhibition of inositol 1,4,5-trisphosphate 5-phosphatase by micromolar concentrations of disulfiram and its analogues

Following a preincubation period of 10 min, disulfiram and its analogues FLA 46, FLA 63, FLA 99, EWP 815 and EWP 840 inhibited the breakdown of 10 microM [3H]Ins(1,4,5)P3 by Ins(1,4,5)P3 5-phosphatase from GH3 cells, with IC50 values (in microM), for soluble/particulate enzymes respectively, of: disulfiram, 24/24; FLA 46, 23/30; FLA 63, 24/6; FLA 99, 50/48; EWP 815, 8/6; EWP 840, 11/8. The inhibition produced by FLA 99 was time-dependent in nature, although inhibition was found in the absence of a preincubation period. EWP 815 and EWP 840 were more potent inhibitors of Ins(1,4)P2 phosphatase than of Ins(1,4,5)P3 5-phosphatase. Thyrotropin-releasing hormone (TRH; 3/100 microM)-stimulated inositol phospholipid breakdown in prelabelled GH3 cells was inhibited by disulfiram (IC50 values 63/52 microM respectively), FLA 46 (89/110 microM), EWP 815 (83/71 microM) and EWP 840 (220/200 microM), without affecting basal breakdown rates. FLA 99 did not inhibit either basal or TRH-stimulated activity at any of the concentrations tested (30, 100 and 300 microM). [3H]Ins(1,4,5)P3 binding to its cerebellar receptor was not inhibited by any of the compounds over a concentration range of 3-300 microM, although an increased level of binding was seen at high concentrations. FLA 99 and EWP 840 increased the basal intracellular Ca2+ concentration in GH3 cells, but with no corresponding effect on the Ca2+ response to TRH stimulation. These compounds did not increase the cellular permeability to Trypan Blue, but did affect cell proliferation. It is concluded that disulfiram and related compounds produce dramatic effects on Ins(1,4,5)P3 metabolism in GH3 cells.

Control of the distribution of hemidesmosome components in cultured keratinocytes: Ca2+ and phorbol esters.

DJM-1 cells (a human squamous cell carcinoma cell line) grown in low Ca2+ medium did not form cell-cell junctions of desmosome-keratin intermediate filament (KIF). When they were shifted to normal (high) Ca2+ medium, rapid translocation of desmoplakins from the cytosol to the plasma membrane to form desmosomes and reorganization of 180 kd-hemidesmosome proteins were induced almost simultaneously. In correlation with these morphological responses, the Ca2+ shift caused a breakdown of inositol phospholipids, a formation of diacylglycerol (DAG) and inositol trisphosphate (IP3), protein kinase C (PKC) activation, and Ca2+ influx. 12-0-tetradecanoylphorbol-13-acetate (TPA)-treatment of low Ca(2+)-grown DJM-1 cells also caused desmosome formation in association with PKC activation. These TPA effects were cancelled with PKC inhibitors, 1-(5-isoquinolinylsulfomyl)-2-methylpiperazine (H7) and staurosporine. Treatment with other PKC-activating agents, phorbol-12,13-butyrate (PDBu) and diaoctanoylglycerol (DOG), also induced desmosome formation. TPA-treatment of normal Ca(2+)-grown cells collapsed the organized distribution of the 180 kd-hemidesmosome protein and appeared to detach this protein from the cell-matrix adhering sites. This effect was also inhibited by H7. These results suggest that PKC activation plays important roles in upregulation of cell-cell junctions and downregulation of cell-matrix junctions in association with differentiation of keratinocytes.

Stimulation of phosphatidylcholine breakdown by thrombin and carbachol but not by tyrosine kinase receptor ligands in cells transfected with M1 muscarinic receptors. Rapid desensitization of phosphocholine-specific (PC) phospholipase D but sustained activity of PC-phospholipase C

In order to evaluate the possible contribution of phospholipase D (PLD) stimulation to the mitogenic response, a screening of a variety of different compounds, some of which are known to be potent mitogens, was performed using the well characterized Chinese hamster lung fibroblast (CCL39) cell line. In wild type CCL39 cells, or derivatives expressing high levels of either the human M1 muscarinic receptor (Hm1) or the human epidermal growth factor (EGF) receptor (39M1-81 and 39ER22 clones, respectively), thrombin, a potent mitogen for all three cell types, elicited the rapid activation of PLD (t1/2 activation, 30 s). Carbachol-mediated activation of the Hm1 receptor in the 39M1-81 clone, which is not a mitogenic signal, produced a similarly rapid although greater activation of PLD. Addition of EGF to the 39ER22 clone was able to provoke both a mitogenic response and stimulate PLD, albeit a comparatively small effect. In each case, the stimulation of PLD correlated closely with the ability to stimulate inositol phospholipid breakdown and was entirely dependent on the activation of protein kinase C. Moreover, the ability of both thrombin and carbachol to stimulate PLD was found to be rapidly desensitized, with a similar time course of desensitization (t1/2 desensitization, 90 s). It has recently been reported that an increase in phospholipase C (PLC)-mediated phosphocholine (PC) hydrolysis by either addition of agonist or by extracellular addition of PC-specific PLC enzyme constitutes a mitogenic signal. In this regard, in addition to stimulation of PLD, thrombin and carbachol were both able to stimulate the activity of a phosphocholine-specific phospholipase C (PC-PLC), which did not appear to desensitize within the time course employed. By contrast, EGF was unable to elicit the stimulation of PC-PLC. Ligands such as fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF), which bind to and activate receptors with intrinsic tyrosine kinase activity, are potent mitogens for CCL39 cells but were unable to stimulate either PLD or PC-PLC activity. Furthermore, exogenous addition of purified PC-PLC enzyme, although able to induce a strong and lasting hydrolysis of PC, was unable to produce a mitogenic signal on its own. On the basis of these results, we conclude that the activation of both PLD and PC-PLC is neither sufficient nor required to produce a mitogenic response.

Influence of chronic ethanol consumption on the inositol phospholipid fatty acid composition of human peripheral blood lymphocytes.

The breakdown of inositol phospholipids is an important event after the binding of antigens to the T-cell antigen receptor. In alcoholics, changes either in early or in late steps of lymphocyte activation have been documented, however no study on the role of phosphoinositide fatty acid composition in signal transduction has been reported. We have analyzed the fatty acid pattern of phosphatidylinositol, phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate from peripheral blood lymphocytes of alcoholic patients and healthy controls, in order to point out the possible compositional differences which could interfere with the signal transmission responsible for the early events in lymphocyte activation. In alcoholics, the arachidonic acid relative molar content in all the inositol phospholipid (PtdIns) fractions derived from lymphocytes was lower than in controls; all PtdIns classes appeared much more saturated than the corresponding fractions from control lymphocytes. The different fatty acid pattern of PtdIns in alcoholic patients could be responsible for an altered second messenger production, above all the production of a modified diacylglycerol which, in turn, could cause a different activation pattern of protein kinase C, with a consequent alteration in cell proliferation. The decrease in arachidonic acid molar content in the phosphoinositides and particularly in the phosphatidylinositol-4,5-bisphosphate fraction of PBL of alcoholic patients could lead to a reduced synthesis of prostanoids of the (n-6) series, and, as a consequence, to an alteration in the mitogenic response of the cells.

Prostacyclin is a specific effector of adipose cell differentiation. Its dual role as a cAMP- and Ca(2+)-elevating agent.

The mitogenic-adipogenic activity of carbaprostacyclin (cPGI2), a stable analogue of prostacyclin (PGI2), has been proposed to be related to its ability to elicit cAMP production and to activate the protein kinase A cascade (Négrel, R., Gaillard, D., and Ailhaud, G. (1989) Biochem. J. 257, 399-405). In the present study, cPGI2 has been compared with other activators of the cAMP pathway, namely isoproterenol, forskolin and 8-bromo-cAMP, with respect to adipose cell differentiation. Carbaprostacyclin behaved as a much more potent and efficient effector of mouse Ob1771 preadipocyte differentiation than the latter agents. Moreover, cPGI2 also exerted a specific amplifying mitogenic-adipogenic role, as compared with isoproterenol in rat as well as human adipose precursor cells in primary culture, suggesting that the prostanoid was able to generate an additional second messenger. The fact that ionomycin was able to potentiate and amplify the differentiation induced by 8-bromo-cAMP led us to give evidence, using preadipocytes preloaded with the fluorescent calcium chelator Indo-1, that cPGI2, besides its ability to activate adenyl cyclase, was also able to induce a transient increase in intracellular free calcium. This phenomenon was independent of cAMP production or inositol phospholipid breakdown and appeared to be mediated after binding to a single class of PGI2 receptor. The potential to generate simultaneously two synergistic intracellular signals allows us to ascribe to PGI2 a key and specific role in the differentiation of adipose precursor cells in vitro that would likely lead in vivo to the recruitment of "dormant" preadipocytes to become adipocytes.

The membrane IgM-associated proteins MB-1 and Ig-beta are sufficient to promote surface expression of a partially functional B-cell antigen receptor in a nonlymphoid cell line.

The B-cell antigen receptors consist of membrane immunoglobulins (mIgs) noncovalently associated with two accessory proteins, MB-1 and Ig-beta. We used transfection into a nonlymphoid cell line to test whether MB-1 and Ig-beta were sufficient to promote cell surface expression of mIgM capable of signal transduction. Expression of MB-1 and Ig-beta, but not MB-1 alone, allowed high-level surface expression of mIgM in the AtT20 endocrine cell line, which presumably lacks other B-cell-specific components. The reconstituted antigen receptor was capable of mediating some of the signaling reactions characteristic of mIgM in B lymphocytes. Crosslinking mIgM on transfected AtT20 cells stimulated tyrosine phosphorylation of MB-1 and Ig-beta and also increased the amount of phosphatidylinositol 3-kinase activity that could be precipitated with anti-phosphotyrosine antibodies. When total cell lysates were analyzed by anti-phosphotyrosine immunoblotting, however, no induced phosphorylation of more abundant proteins was detected. Moreover, crosslinking of the receptor in AtT20 cells did not stimulate inositol phospholipid breakdown. Thus, the transfected B-cell antigen receptor could initiate some signal transduction events but AtT20 cells may lack components required for other signaling events associated with mIgM.

Effects of isoflavone compounds on the activation of phospholipase C.

The effect of isoflavone compounds, genistein and daidzein, on the breakdown of inositol phospholipids in 3T3 cells was studied. Genistein (100 micrograms/ml) inhibited the stimulation of the production of inositol phosphates by bombesin. The stimulated production of inositol phosphates by AlF-4 was also inhibited by genistein (IC50 = 0.6 micrograms/ml) and daidzein (IC50 = 2 micrograms/ml). However, the catalytic activity of phospholipase-C (PLC) in 3T3 cell extracts was not inhibited by these isoflavones. These results suggest that the isoflavones inhibited the activation of PLC at the G-protein or downstream of the sequences in signal transduction. In permeabilized 3T3 cells, the inhibition of AlF-4 plus adenosine triphosphate (ATP)-dependent PLC was recovered by increasing ATP but not AlF-4. Genistein also inhibited the activity of adenosine 5'-[3-O-thiotriphosphate] (ATP[S])-dependent PLC. The effect of genistein and other inhibitors of protein tyrosine kinases and phosphatases suggests that protein tyrosine phosphorylation is not involved in the activation of PLC in 3T3 cells and that AlF-4- and ATP[S]-mediated activation of PLC involves a different mechanism from the tyrosine kinase-mediated activation of PLC. Daidzein and genistein seem to interrupt the ATP-dependent step of PLC activation by a putative G-protein.