Serotonin-induced alterations in inositol phospholipid metabolism in human platelets

When human platelets were incubated for 5 min with [32P]orthophosphate and then stimulated with serotonin, the 32P content of phosphatidylinositol (PI) increased within seconds, compared with the control. The 32P content of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) only slightly increased during the first minute after addition of serotonin and became more apparent on prolonged stimulation. These changes were not caused by serotonin-induced change in the specific activity of ATP. Using inorganic phosphate determination for the chemical quantification of different inositol phospholipid pools, we found that the platelet PI content remained nearly constant; the amount of PIP increased while that of PIP2 decreased. When the platelets were first prelabeled for 80 min with [32P]orthophosphate, the changes in 32P-labeled inositol phospholipids after addition of serotonin were similar to their changes in mass. When the platelet inositol phospholipids were labeled with myo-[2-3H]inositol, serotonin induced an increase in [3H]inositol phosphates. From these data, it is concluded in addition to the earlier-reported effects on phospholipid metabolism (de Chaffoy de Courcelles, D. et al. (1985) J. Biol. Chem. 260, 7603-7608) that serotonin induces: a very rapid formation of PI; and alterations in inositol phospholipid interconversion that cannot be explained solely as a resynthesis process of PIP2.     

Regulation of inositol phospholipid and inositol phosphate metabolism in chemoattractant-activated human polymorphonuclear leukocytes

Binding of chemoattractants to specific cell surface receptors on polymorphonuclear leukocytes (PMNs) initiates a series of biochemical responses leading to cellular activation. A critical early biochemical event in chemoattractant (CTX) receptor-mediated signal transduction is the phosphodiesteric cleavage of plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2), with concomitant production of the calcium mobilizing inositol-1,4,5-trisphosphate (IP3) isomer, and the protein kinase C activator, 1,2-diacylglycerol (DAG). The following lines of experimental evidence collectively suggest that CTX receptors are coupled to phospholipase C via a guanine nucleotide binding (G) protein. Receptor-mediated hydrolysis of PIP2 in PMN plasma membrane preparations requires both fMet-Leu-Phe and GTP, and incubation of intact PMNs with pertussis toxin (which ADP ribosylates and inactivates some G proteins) eliminates the ability of fMet-Leu-Phe plus GTP to promote PIP2 breakdown in isolated plasma membranes. Studies with both PMN particulate fractions and with partially purified fMet-Leu-Phe receptor preparations indicate that guanine nucleotides regulate CTX receptor affinity. Finally, fMet-Leu-Phe stimulates high-affinity binding of GTP gamma S to PMN membranes as well as GTPase activity. A G alpha subunit has been identified in phagocyte membranes which is different from other G alpha subunits on the basis of molecular weight and differential sensitivity to ribosylation by bacterial toxins. Thus, a novel G protein may be involved in coupling CTX receptors to phospholipase C. Studies in intact and sonicated PMNs demonstrate that metabolism of 1,4,5-IP3 proceeds via two distinct pathways: 1) sequential dephosphorylation to 1,4-IP2, 4-IP1 and inositol, or 2) ATP-dependent conversion to inositol 1,3,4,5-tetrakisphosphate (IP4) followed by sequential dephosphorylation to 1,3,4-IP3, 3,4-IP2, 3-IP1 and inositol. Receptor-mediated hydrolysis of PIP2 occurs at ambient intracellular Ca2+ levels; but metabolism of 1,4,5-IP3 via the IP4 pathway requires elevated cytosolic Ca2+ levels associated with cellular activation. Thus, the two pathways for 1,4,5-IP3 metabolism may serve different metabolic functions. Additionally, inositol phosphate production appears to be controlled by protein kinase C, as phorbol myristate acetate (PMA) abrogates PIP2 hydrolysis by interfering with the ability of the activated G protein to stimulate phospholipase C. This implies a physiologic mechanism for terminating biologic responses via protein kinase C mediated feedback inhibition of PIP2 hydrolysis.     

Effect of transforming growth factor-alpha on inositol phospholipid metabolism in human epidermoid carcinoma cells

Transforming growth factor-alpha (TGF-alpha) stimulates (in a dose-dependent manner) the incorporation of [32P]Pi into phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2), and phosphatidic acid (PA) in the human epidermoid carcinoma cell line (A431). The effect of TGF-alpha on the incorporation was found to be similar to that of EGF. On the other hand, a striking difference in the activation of diacylglycerol (DG) kinase activity was seen between TGF-alpha and EGF. At least 100 times more TGF-alpha was required to achieve maximal stimulation of DG kinase activity relative to EGF. These results suggest that the activation of DG kinase by TGF-alpha may involve a mechanism independent from or subsequent to activation of the EGF receptor.     

Modulation of inositol phospholipid metabolism by polyamines.

At low concentrations of Mg2+, incorporation of 32P from [gamma-32P]ATP into phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) in plasma membranes isolated from human polymorphonuclear leucocytes was enhanced 2-4-fold by the polyamines spermidine and spermine. Polyamines had no effects on inositol phospholipid phosphorylation at high concentrations of Mg2+. At 1 mM-Mg2+, [32P]PIP2 synthesis was maximally enhanced by 2 mM-spermine and 5 mM-spermidine, whereas putrescine only slightly enhanced synthesis. Spermine decreased the EC50 (concn. for half-maximal activity) for Mg2+ in [32P]PIP2 synthesis from 5 mM to 0.5 mM. Spermine did not modulate the Km for ATP for [32P]PIP or [32P]PIP2 synthesis. Spermine also decreased the EC50 for PI in [32P]PIP synthesis. In contrast, spermine elevated the apparent Vmax, without affecting the EC50 for PIP, for [32P]PIP2 synthesis. Spermine and spermidine also inhibited the hydrolysis of [32P]PIP2 by phosphomonoesterase activity. Therefore polyamines appear to activate inositol phospholipid kinases by eliminating the requirements for super-physiological concentrations of Mg2+. Polyamine-mediated inhibition of polyphosphoinositide hydrolysis would serve to potentiate further their abilities to promote the accumulation of polyphosphoinositides in biological systems.     

Putative role of inositol phospholipid metabolism in neurons

Inositol phospholipids play a crucial role in the intracellular signal transduction in most cell types. Activation of an enzyme called phospholipase C or PIP2-phosphodiesterase (PIP2-PDE) leads to the production of two second messenger molecules, diacylglycerol (DG) and inositol 1,4,5-triphosphate (IP3). DG activates a kinase called protein kinase C, whereas IP3 mediates the release of Ca2+ from intracellular storage sites. The measurement of IP3 and its degradation products, inositol diphosphate (IP2) and inositol monophosphate (IP1) provides a way of assessing the extent to which this complex system has been activated. In the central nervous system (CNS) most of the studies on the neurotransmitter stimulated formation of inositol phosphates (IPs) have been performed on brain slices, a mixture of mainly neurons and glial cells. The recent development of pure neuronal cultures provides a means of determining which of these responses were of neuronal origin. The purpose of this review is to summarize the results obtained in neurons in primary culture together with a brief appraisal of the possible function of this second messenger system in neurons.     

Activation of V1-receptors by vasopressin stimulates inositol phospholipid hydrolysis and arachidonate metabolism in human platelets.

The effects of Mg2+ on the activity of pyruvate dehydrogenase phosphate phosphatase within intact mitochondria prepared from control and insulin-treated rat epididymal adipose tissue was explored by incubating the mitochondria in medium containing the ionophore A23187. The apparent Ka for Mg2+ was approximately halved in the mitochondria derived from insulin-treated tissue in both the absence and the presence of Ca2+. In this system, the major effect of Ca2+ was also to decrease the apparent Ka for Mg2+, rather than to change the Vmax. of the phosphatase. Damuni, Humphreys & Reed [(1984) Biochem. Biophys. Res. Commun. 124, 95-99] have reported that spermine activates ox kidney pyruvate dehydrogenase phosphate phosphatase. Studies were carried out on phosphatase from pig heart and rat epididymal adipose tissue which confirm and extend this observation. The major effect of spermine is shown to be a decrease in the Ka for Mg2+, which is apparent in both the presence and the absence of Ca2+. Spermine did not affect the sensitivity of the phosphatase to Ca2+ at saturating concentrations of Mg2+. Other polyamines tested were not as effective as spermine. No alteration in the maximum activity or Mg2+-sensitivity of pyruvate dehydrogenase phosphate phosphatase was apparent in extracts of mitochondria from insulin-treated tissue. The close similarity of the effects of spermine and the changes in kinetic properties of pyruvate dehydrogenase phosphate phosphatase within mitochondria from insulin-treated adipose tissue suggests that insulin may activate pyruvate dehydrogenase by increasing the concentration of spermine within the mitochondria. However, it is concluded that insulin is more likely to alter the interaction of the pyruvate dehydrogenase system with some other polybasic intramitochondrial component whose action can be mimicked by spermine.     

Inhibitory effect of prostaglandin E2, forskolin, and dibutyryl cAMP on arachidonic acid release and inositol phospholipid metabolism in guinea pig neutrophils

The effect of prostaglandin E2 (PGE2), forskolin, and dibutyryl cAMP on arachidonic acid release, inositol phospholipid metabolism, and Ca2+ mobilization was investigated. The chemotactic tripeptide (formylmethionyl-leucyl-phenylalanine (fMLP))-induced arachidonic acid release in neutrophils was significantly inhibited by PGE2, forskolin, and dibutyryl cAMP. Among them, PGE2 was found to be the most potent inhibitor. However, when neutrophils were stimulated by Ca2+ ionophore A23187, such inhibitory effect by these agents was less marked. PGE2 also suppressed the enhanced incorporation of [32P]Pi into phosphatidic acid (PA) and phosphatidylinositol in a dose-dependent manner in fMLP-stimulated neutrophils. Also in this case, Ca2+ ionophore-induced alterations were hardly inhibited by PGE2. As well, PGE2 inhibited the fMLP-induced decrease of [3H]arachidonic acid in phosphatidylcholine and phosphatidylinositol and the increase in PA very significantly. But the inhibitory effect by PGE2 was found to be weak in Ca2+ ionophore-stimulated neutrophils. These results suggest that a certain step from receptor activation to Ca2+ influx is mainly inhibited by PGE2. Concerning polyphosphoinositide breakdown, PGE2 did not affect the fMLP-induced decrease of [32P]phosphatidylinositol 4,5-bisphosphate which occurred within 10 s but inhibited the subsequent loss of [32P]phosphatidylinositol 4-phosphate and [32P]phosphatidylinositol, suggesting that the compensatory resynthesis of phosphatidylinositol 4,5-bisphosphate was inhibited. On the other hand, fMLP-induced diacylglycerol formation was suppressed for the early period until 1 min, but with further incubation, diacylglycerol formation was rather accelerated by PGE2. Moreover, the inhibition of PA formation by PGE2 became evident after a 30-s time lag, suggesting that the conversion of diacylglycerol to PA is inhibited by PGE2. The formation of water-soluble products of inositol phospholipid degradation by phospholipase C, such as inositol phosphate, inositol 1,4-bisphosphate, and inositol 1,4,5-trisphosphate, was also suppressed by PGE2 treatment. However, the inhibition was not so marked as that of arachidonic acid release and PA formation. Thus, PGE2 appeared to inhibit not only initial events such as polyphosphoinositide breakdown but also turnover of inositol phospholipids. PGE2, forskolin, and dibutyryl cAMP did not block the rapid elevation of intracellular Ca2+ which was observed within 10 s in fMLP-stimulated neutrophils. However, subsequent increase in intracellular Ca2+ which was caused from 10 s to 3 min after stimulation was inhibited by PGE2, forskolin, and dibutyryl cAMP.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effect of ischaemia and reperfusion on sarcolemmal inositol phospholipid and cytosolic inositol phosphate metabolism in the isolated perfused rat heart

In this study the mass of polyphosphoinositides as well as the turnover of [³H]inositol phospholipids and [³H]inositol phosphates during ischaemia and short periods of reperfusion were studied in the isolated perfused rat heart. Since the phosphoinositides located within the sarcolemma are precursors for release of inositoltrisphosphate (InsP₃) and diacylglycerol, sarcolemmal membranes (rather than whole tissue) isolated at the end of the experimental procedure, were used. Hearts were prelabelled with [³H]inositol and subsequently perfused with 10 mM LiCI to block the phosphatidylinositol (PI) pathway. The results showed that 20 min of global ischaemia depressed the amount of [³H]inositol present in both sarcolemmal phosphatidylinositol-4-phosphate (PI-4-P) and phosphatidylinositol-4,5-bisphosphate (PI-4,5-P₂), as well as in the cytosolic [³H]inositol phosphates, [³H]InsP₂ and [³H]InsP₃. The mass of the sarcolemmal inositol phospholipids remained unchanged during ischaemia. Reperfusion caused an immediate (within 30 sec) increase in the amount of [³H]inositol in sarcolemmal PI, PI-4-P and PI-4,5-P₂. PI-4-P levels showed a transient increase after 30 seconds postischaemic reperfusion, while the mass of the other sarcolemmal inositol phospholipids, PI and PI-4,5-P₂, remained unchanged. [³H]Insp, [³H]InsP₂ and [³H]InsP₃ also increased significantly in comparison to ischaemic hearts after only 30 sec postischaemic reperfusion.In summary, the results obtained indicate inhibition of the PI pathway during ischaemia with an immediate significant stimulation upon reperfusion. In view of the capacity of InsP3 to mobilize Ca²⁺ the possibility exists that stimulation of this pathway during reperfusion may play a role in the intracellular Ca²⁺ overload, characteristic of postischaemic reperfusion.     

Epinephrine stimulates inositol phospholipid metabolism by activating alpha-2 adrenergic receptors in human platelets

The metabolism of inositol phospholipids in response to epinephrine was investigated in intact human platelets. In platelets prelabelled with [3H]-myo-inositol in Ca2+-free HEPES buffer containing 10 mM LiCl, epinephrine caused an accumulation of inositol-1-phosphate in a concentration-dependent manner. The EC50 value for epinephrine was 5 microM. Yohimbine (1 microM), a selective alpha-2 adrenergic receptor antagonist, inhibited 88% of the epinephrine (10 microM) response, whereas prazosin (1 microM), a selective alpha-1 adrenergic receptor antagonist, failed to inhibit the response. Yohimbine inhibited the epinephrine (10 microM) response in a concentration-dependent manner. The inhibition constant (Ki) value for yohimbine was 60.3 nM. These data indicate that epinephrine stimulates phosphoinositide (PI) turnover by activating adrenergic receptors of the alpha-2 type in human platelets. In addition, this PI response elicited by epinephrine was found to be inhibited in a concentration-dependent manner by treatment of platelets with dibutyryl cyclic AMP and 8-bromo-cyclic GMP which are known as potent inhibitors for platelet activation, and may therefore be a useful biochemical index for the study of the function of human alpha-2 adrenergic receptors.     

Specific phospholipid recognition by human immunodeficiency virus type-1 neutralizing anti-gp41 2F5 antibody

HIV-1 neutralizing monoclonal antibody (Mab) 2F5 recognizes a membrane-partitioning gp41 sequence. Just recently its capacity to react with cardiolipin has been demonstrated. Here, we have studied the specificity of Mab2F5-phospholipid interactions comparing partitioning into lipid bilayers with recognition of molecular species dispersed in solution. Using a liposome-based ELISA we demonstrate a preferential association with cardiolipin bilayers. When different soluble lysoderivatives were compared in their capacity to inhibit Mab2F5 binding to immobilized HIV-1 peptide epitope, only dilysocardiolipin resulted effective in blocking the process. Dilyso-cardiolipin also competed with native-functional gp41 for 2F5 recognition. Thus, our data support specific cardiolipin recognition by 2F5 that is not dependent on lipid bilayer assembly and involves the epitope-binding site. These findings might be of relevance for understanding the molecular basis of HIV-1 immune evasion.     

Adenovirus E1A proteins stimulate inositol phospholipid metabolism in PC12 cells.

To study the influence of nuclear oncogenes on inositol phospholipid metabolism, we examined the various parameters of inositol phospholipid metabolism in PC12 cells expressing adenovirus type 12 or adenovirus type 5 E1A. Although the inositol 1,4,5-trisphosphate content was increased only slightly, the diacylglycerol content was 2.4-fold higher in E1A-expressing PC12 cells. Furthermore, we found that the activity of phospholipase C, one of the key enzymes in inositol phospholipid metabolism, was increased at least five- to eightfold. Diacylglycerol kinase activity in the membrane fraction was 10 to 15% of that in parental PC12 cells. Overall protein kinase C activities in E1A-expressing PC12 cells were decreased, but the activity of membrane-bound protein kinase C was significantly increased. These observations clearly indicate that inositol phospholipid metabolism is stimulated in cells producing E1A and suggest that nuclear oncogene E1A has the ability to stimulate inositol phospholipid metabolism.     

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.     

Functional comparison of transactivation by simian immunodeficiency virus from rhesus macaques and human immunodeficiency virus type 1.

Simian immunodeficiency virus from rhesus macaques (SIVmac), like human immunodeficiency virus type 1 (HIV-1), encodes a transactivator (tat) which stimulates long terminal repeat (LTR)-directed gene expression. We performed cotransfection assays of SIVmac and HIV-1 tat constructs with LTR-CAT reporter plasmids. The primary effect of transactivation for both SIVmac and HIV-1 is an increase in LTR-directed mRNA accumulation. The SIVmac tat gene product partially transactivates an HIV-1 LTR, whereas the HIV-1 tat gene product fully transactivates an SIVmac LTR. Significant transactivation is achieved by the product of coding exon 1 of the HIV-1 tat gene; however, inclusion of coding exon 2 results in a further increase in mRNA accumulation. In contrast, coding exon 2 of the SIVmac tat gene is required for significant transactivation. These results imply that the tat proteins of SIVmac and HIV-1 are functionally similar but not interchangeable. In addition, an in vitro-generated mutation in SIVmac tat disrupts splicing at the normal splice acceptor site at the beginning of coding exon 2 and activates a site approximately 15 nucleotides downstream. The product of this splice variant stimulates LTR-directed gene expression. This alternative splice acceptor site is also used by a biologically active provirus with an efficiency of approximately 5% compared with the upstream site. These data suggest that a novel tat protein is encoded during the course of viral infection.     

Evidence that chlorpromazine and prostaglandin E1 but not neomycin interfere with the inositol phospholipid metabolism in intact human platelets

Human platelets that had been prelabelled with [32P]Pi were stimulated with trombin in the presence or absence of neomycin, prostaglandin E1 (PGE1) or chlorpromazine. The content of [32P]Pi in phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate and phosphatidic acid (PA) were determined. The data demonstrate that PGE1 and chlorpromazine but not neomycin interfere with the tight metabolic relationship that exists between the inositol phospholipids and PA in thrombin-stimulated platelets [(1989) Biochem. J. 263, 621-624]. Our results therefore indicate that neomycin does not inhibit signal transduction in intact platelets at the level of the inositol phospholipid metabolism.     

Inhibitory effect of 1-beta-D-arabinofuranosylthymine on synthesis of Epstein-Barr virus

The effect of 1-beta-D-arabinofuranosylthymine (ara-T) on cell growth and synthesis of Epstein-Barr virus (EBV) in human lymphoblastoid cell lines was determined. The growth of P3HR-1 cells was not inhibited by 1 microgram of the drug per ml; however, infectious virus production was strongly inhibited and was accompanied by decreased expression of early antigen (EA) and viral capsid antigen (VCA). The ability of 12-O-tetradecanoylphorbol-13-acetate or n-butyric acid to induce synthesis of VCA, but not EA, in P3HR-1 cells was inhibited by ara-T. Similarly, VCA synthesis but not EA synthesis was inhibited by ara-T in Jijoye cells superinfected with the P3HR-1 strain of EBV. The results suggest that ara-T has a specific inhibitory action against EBV replication.     

Inhibitory and enhancing effects of insertion of central polypurine tract sequence on gene expression with vectors derived from human immunodeficiency virus type 1

Reportedly, in human immunodeficiency virus type 1 (HIV) vectors, insertion of central polypurine tract (cPPT) increased expression of transgenes for a short period. To test this for a stable condition, we constructed a series of vectors carrying a Neo(r) gene as a stable marker driven by a synthetic thymidine kinase (hTK) promoter. Transduction efficiency was increased in about 2-fold and decreased in about 8-fold by insertion of the reported 178bp and our 282bp cPPTs, respectively. PCR analyses revealed that insertion of 282bp cPPT, but not 178bp cPPT, impaired integration, although it did not deteriorate nuclear transport much. Furthermore, we found that insertion of 282bp cPPT between hTK promoter and an upstream LTR sequence reduced reporter gene activity in about 5-fold. This inhibitory effect of 282bp cPPT may partly account for the observed decrease in transduction efficiency. We suggest that actual effect of cPPT insertion should be examined in each HIV vector.     

Organization of immature human immunodeficiency virus type 1

Immature retrovirus particles contain radially arranged Gag polyproteins in which the N termini lie at the membrane and the C termini extend toward the particle's center. We related image features to the polyprotein domain structure by combining mutagenesis with cryoelectron microscopy and image analysis. The matrix (MA) domain appears as a thin layer tightly associated with the inner face of the viral membrane, separated from the capsid (CA) layer by a low-density region corresponding to its C terminus. Deletion of the entire p6 domain has no effect on the width or spacing of the density layers, suggesting that p6 is not ordered in immature human immunodeficiency virus type 1 (HIV-1). In vitro assembly of a recombinant Gag polyprotein containing only capsid (CA) and nucleocapsid (NC) domains results in the formation of nonenveloped spherical particles which display two layers with density matching that of the CA-NC portion of immature HIV-1 Gag particles. Authentic, immature HIV-1 displays additional surface features and an increased density between the lipid bilayers which reflect the presence of gp41. The other internal features match those of virus-like particles.